THE ROLE OF TRIGLYCERIDES IN

ORIGINAL CONTRIBUTION Nonfasting Triglycerides and Risk of Ischemic in the General Population Jacob J. Freiberg, MD Anne Tybjærg-Hansen, MD, DMSc Jan Skov Jensen, MD, DMSc Børge G. Nordestgaard, MD, DMSc THE ROLE OF TRIGLYCERIDES IN risk of ischemic stroke remains controversial. 1-8 Two recent cohort studies reported a strong association between elevated levels of nonfasting, but not fasting, triglycerides and increased risk of myocardial infarction, ischemic heart disease, and death 9 and total cardiovascular events, 1 respectively. It is therefore possible that nonfasting triglyceride levels are also associated with increased risk of ischemic stroke. Increased levels of nonfasting triglycerides indicate the presence of increased levels of remnants from chylomicrons and very low-density lipoproteins. 9 These cholesterol-containing, triglyceride-rich lipoproteins penetrate the arterial endothelium 11,12 and may get trapped within the subendothelial space, 13-16 potentially leading to the development of atherosclerosis. 17,18 Triglyceride levels are usually measured after an 8- to 12-hour fast, 19 thus excluding most remnant lipoproteins; however, except for a few hours before breakfast, most individuals are in the nonfasting state most of the time. Therefore, by mainly studying fasting rather than nonfasting triglyceride levels, several previous studies 1,4,6,7 may have missed an association between triglycerides and ischemic stroke. Also, because former studies 1-7 mainly focused on moderately elevated levels of triglycerides, an association of very high Context The role of triglycerides in the risk of ischemic stroke remains controversial. Recently, a strong association was found between elevated levels of nonfasting triglycerides, which indicate the presence of remnant lipoproteins, and increased risk of ischemic heart disease. Objective To test the hypothesis that increased levels of nonfasting triglycerides are associated with ischemic stroke in the general population. Design, Setting, and The Copenhagen City Heart Study, a prospective, Danish population based cohort study initiated in 1976, with follow-up through July 27. were 13 956 men and women aged 2 through 93 years. A cross-sectional study included 9637 individuals attending the 1991-1994 examination of the prospective study. Main Outcome Measures Prospective study: baseline levels of nonfasting triglycerides, other risk factors at baseline and at follow-up examinations, and incidence of ischemic stroke. Cross-sectional study: levels of nonfasting triglycerides, levels of remnant cholesterol, and prevalence of ischemic stroke. Results Of the 13 956 participants in the prospective study, 1529 developed ischemic stroke. Cumulative incidence of ischemic stroke increased with increasing levels of nonfasting triglycerides (log-rank trend, P.1). with elevated nonfasting triglyceride levels of 89 through 176 mg/dl had multivariate-adjusted hazard ratios (HRs) for ischemic stroke of 1.3 (95% CI,.8-1.9; 351 events); for 177 through 265 mg/dl, 1.6 (95% CI, 1.-2.5; 189 events); for 266 through 353 mg/dl, 1.5 (95% CI,.9-2.7; 73 events); for 354 through 442 mg/dl, 2.2 (95% CI, 1.1-4.2; 4 events); and for 443 mg/dl or greater, 2.5 (95% CI, 1.3-4.8; 41 events) vs men with nonfasting levels less than 89 mg/dl (HR, 1.; 85 events) (P.1 for trend). Corresponding values for women were 1.3 (95% CI,.9-1.7; 47 events), 2. (95% CI, 1.3-2.9; 135 events), 1.4 (95% CI,.7-2.9; 26 events), 2.5 (95% CI, 1.-6.4; 13 events), and 3.8 (95% CI, 1.3-11; 1 events) vs women with nonfasting triglyceride levels less than 89 mg/dl (HR, 1.; 159 events) (P.1 for trend). Absolute 1-year risk of ischemic stroke ranged from 2.6% in men younger than 55 years with nonfasting triglyceride levels of less than 89 mg/dl to 16.7% in men aged 55 years or older with levels of 443 mg/dl or greater. Corresponding values in women were 1.9% and 12.2%. In the cross-sectional study, men with a previous ischemic stroke vs controls had nonfasting triglyceride levels of 191 (IQR, 131-259) mg/dl vs 148 (IQR, 14-214) mg/dl (P.1); corresponding values for women were 167 (IQR, 121-229) mg/dl vs 127 (IQR, 91-181) mg/dl (P.5). For remnant cholesterol, corresponding values were 38 (IQR, 26-51) mg/dl vs 29 (IQR, 2-42) mg/dl in men (P.1) and 33 (IQR, 24-45) mg/dl vs 25 (IQR, 18-35) mg/dl in women (P.5). Conclusion In this study population, nonfasting triglyceride levels were associated with risk of ischemic stroke. JAMA. 28;3(18):2142-2152 www.jama.com Author Affiliations: Department of Clinical Biochemistry, Herlev Hospital (Drs Freiberg and Nordestgaard), Copenhagen City Heart Study, Bispebjerg Hospital (Drs Tybjærg-Hansen, Jensen, and Nordestgaard), Department of Clinical Biochemistry, Rigshospitalet (Dr Tybjærg-Hansen), and Department of Cardiology, Gentofte Hospital (Dr Jensen), Copenhagen University Hospitals; and Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. Corresponding Author: Børge G. Nordestgaard, MD, DMSc, Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, Herlev Ringvej 75, DK-273 Herlev, Denmark (brno@heh.regionh.dk). 2142 JAMA, November 12, 28 Vol 3, No. 18 (Reprinted) 28 American Medical Association. All rights reserved.

levels with risk of ischemic stroke could have gone unnoticed. We tested the hypothesis that increased levels of nonfasting triglycerides are associated with risk of ischemic stroke in men and women in the general population. For this purpose, we studied 13 956 individuals from the Copenhagen City Heart Study with up to 31 years of follow-up, during which time 1529 developed ischemic stroke. METHODS The prospective and cross-sectional studies were approved by Herlev Hospital and a Danish ethical committee (Nos. 1.239/91 and 1-144/1, Copenhagen and Frederiksberg committee) and were conducted according to the Declaration of Helsinki. provided written informed consent. Prospective Study The Copenhagen City Heart Study is a prospective cardiovascular study of the Danish general population initiated in 1976. 2 We invited 19 329 white women and men of Danish descent, stratified into 5-year age groups from 2 to 8 years and older and drawn randomly from the national Danish Civil Registration System. Of those invited, 14 223 (74%) attended and 13 956 (72%) had nonfasting triglyceride levels determined using fresh plasma samples; women with triglyceride levels greater than 266 mg/dl (to convert to mmol/l, multiply by.113) and men with levels greater than 31 mg/dl were referred to their general practitioners for further evaluation. underwent follow-up from baseline at the 1976-1978 examination through July 27. Follow-up was complete; ie, we did not lose track of any individuals during the up to 31 years of follow-up. Eighty-three individuals who emigrated from Denmark during follow-up were censored at the date of emigration. The present prospective study on ischemic stroke comprised 13 956 individuals, 1529 with ischemic stroke and 12 697 without. Multiple events in the same individual were not considered in the statistical analysis, because the first event led to censoring of the individual. Cross-sectional Study We also studied cross-sectionally those participants in the Copenhagen City Heart Study who attended the 1991-1994 examination, because levels of nonfasting remnant lipoprotein cholesterol as well as a lipid profile were measured at this examination. Of the 16 563 individuals invited to this examination, 1 135 (61%) attended and 9637 (58%) had nonfasting lipid profiles measured on fresh plasma samples. End Points Diagnoses of cerebrovascular disease, including ischemic stroke (International Classification of Diseases, 8th Revision codes 431 through 438 and International Statistical Classification of Diseases, 1th Revision codes I61 through I69 G45) were gathered from the national Danish Patient Registry and the national Danish Causes of Death Registry. For each person registered with cerebrovascular disease, hospital records were requested. To also include nonhospitalized patients with nonfatal ischemic stroke, participants were asked at 3 study examinations (conducted 1976-1978, 1981-1983, and 1991-1994) whether they had previously had a stroke. If affirmative, further information was obtained from that person s general practitioner. Experienced neurologists blinded to triglyceride values reviewed all potential cases. 21 Possible stroke events (among hospitalized as well as nonhospitalized patients) were validated using the World Health Organization definition of stroke, ie, an acute disturbance of focal or global cerebral function with symptoms lasting longer than 24 hours or leading to death, with presumably no other reasons than of vascular origin. 22 To distinguish among stroke subtypes ie, infarction (ischemic stroke), intracerebral hemorrhage, and subarachnoid hemorrhage either computed tomography or magnetic resonance imaging scan, autopsy, spinal fluid examination, or surgical description was necessary. The event was diagnosed as ischemic stroke if the scan did not visualize an infarction or hemorrhage but the person had symptoms that met the criteria of the stroke definition. The diagnosis of stroke was not applied to persons in whom a scan revealed signs of prior cerebrovascular disease but who had no history of any symptoms. Cerebrovascular Risk Factors Alcohol drinkers were defined as persons consuming 4 units or more of alcohol weekly (12 g of alcohol per unit). Smokers were defined as active smokers. Hypertension was defined as use of antihypertensive medication, a systolic blood pressure of 14 mm Hg or greater, or a diastolic blood pressure of 9 mm Hg or greater. Atrial fibrillation was diagnosed from electrocardiographic recordings obtained at study examinations in 1976-1978, 1981-1983, and 1991-1994. 23 Furthermore, information on atrial fibrillation (International Classification of Diseases, Eighth Revision codes 427.93 and 427.94 and International Statistical Classification of Diseases, 1th Revision code I48.9) was gathered from the national Danish Patient Registry and the national Danish Causes of Death Registry. reported menopausal status and use of hormone therapy. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Diabetes mellitus was defined as self-reported disease, use of insulin or oral hypoglycemic agents, or nonfasting plasma glucose level greater than 198 mg/dl (to convert to mmol/l, multiply by.555). Lipids and Lipoproteins Enzymatic methods (Boehringer Mannheim, Mannheim, Germany) were used on fresh samples to measure plasma levels of nonfasting triglycerides, total cholesterol, and high-density lipoprotein cholesterol (HDL-C). Levels of HDL-C were not measured at baseline but at the 1981-1983, 1991-1994, and 21-28 American Medical Association. All rights reserved. (Reprinted) JAMA, November 12, 28 Vol 3, No. 18 2143

23 examinations. The coefficient of variation for measurement of triglycerides at the levels of 89 mg/dl and 283 mg/dl were 5% and 2%, respectively. Remnant lipoprotein cholesterol was calculated as total cholesterol minus cholesterol in high- and low-density lipoproteins. Levels of low-density lipoprotein cholesterol were calculated using the Friedewald equation if triglyceride levels were below 443 mg/dl and were measured directly if levels were 443 mg/dl or greater (Thermo Fisher Scientific, Waltham, Massachusetts). All blood samples were drawn between 8 AM and 4 PM, and 82% of participants had eaten a meal within the last 3 hours of blood sampling. The remaining 18% had eaten their most recent meal more than 3 hours prior to blood sampling. We estimated that at most 3% of participants had eaten their most recent meal more than 8 hours prior to blood sampling, ie, were fasting. Statistical Analysis Data were analyzed using Stata version 9.2 (StataCorp, College Station, Texas). Two-sided P.5 was considered significant. Analyses were stratified a priori by sex, because we previously found large risk differences between men and women for myocardial infarction by levels of nonfasting triglycerides. 9 In the prospective study, to examine the association of very high levels of nonfasting triglycerides with ischemic stroke, we preplanned stratification at each 89-mg/dL increase until the top group became too small for statistically meaningful comparison with the less than 89-mg/dL group. Thus, baseline nonfasting triglyceride levels were stratified into 6 groups: less than 89 mg/ dl, 89 through 176 mg/dl, 177 through 265 mg/dl, 266 through 353 mg/dl, 354 through 442 mg/dl, and 443 mg/dl or greater. Also, age and multivariate adjustment were prespecified; in multivariate adjustment, we included known cardiovascular risk factors not by themselves highly associated with elevated levels of nonfasting triglycerides and remnant lipoprotein cholesterol. For comparison we also performed adjustment for BMI and diabetes mellitus and for HDL-C levels. Cumulative incidence differences between strata of nonfasting triglyceride levels were determined using log-rank trend tests. Cox regression models using triglyceride levels in strata or on a continuous scale estimated hazard ratios (HRs) for ischemic stroke. Proportionality of hazards over time for nonfasting triglyceride levels was assessed by plotting ln[ ln(survival)] vs ln (analysis time). Suspicion of nonparallel lines was further tested using Schoenfeld residuals. No major violations of the proportional hazard assumption were detected. For all survival statistics, age was the time scale using left truncation (or delayed entry), which implies that age is automatically adjusted for. Hazard ratios were adjusted for age alone and for age and other traditional cerebrovascular risk factors (total cholesterol level, alcohol consumption, smoking, hypertension, atrial fibrillation, and lipidlowering therapy [in women, HRs were also adjusted for postmenopausal status and hormone therapy]). Additional adjustment for BMI and diabetes mellitus and for HDL-C levels was performed separately on both the ageadjusted model and the multivariateadjusted model. For analysis of the association between triglyceride levels (in strata and on a continuous scale) and risk of ischemic stroke, we stratified analysis on sex, age at study entry, hypertension, BMI, physical activity, and hormone therapy (in women). For each stratified analysis we tested for interactions between levels of nonfasting triglycerides on a continuous scale and the dichotomized stratifying covariate on risk of ischemic stroke. Evidence for stepwise increases in risk of ischemic stroke for increasing levels of nonfasting triglycerides was tested for by using a likelihood ratio test between models using triglyceride levels on a continuous scale and models using levels in strata of 89- mg/dl increases. Information on baseline covariates was more than 99% complete; individuals with incomplete information on covariates were excluded from multivariate analysis. Data from the 1976-1978, 1981-1983, 1991-1994, and 21-23 examinations were used as time-dependent covariates for multivariate adjustments. Because HDL-C levels were not measured at the 1976-1978 examination, adjustment for HDL-C was based only on measurements from the 1981-1983, 1991-1994, and 21-23 examinations. This reduced the number of participants from 13 956 in the main analyses to 11 416 (82%) in analyses adjusted for levels of HDL-C. Hazard ratios including confidence intervals (CIs) were corrected for regression dilution bias using a nonparametric method. 24 For this correction, we used nonfasting triglyceride values from 679 individuals without lipidlowering therapy attending both the baseline 1976-1978 examination and the 1991-1994 examination; however, the main analyses were conducted on 13 956 individuals. These 2 measurements were 15 years apart, equivalent to roughly halfway through the observation period, the ideal time difference for this correction. 24 A regression dilution ratio of.57 was computed for women and of.6 for men. Correction for regression dilution bias increases the effect size for risk estimates and increases the range of CIs but does not change significance levels. In the cross-sectional study among all 9637 participants attending the 1991-1994 examination approximately 15 years after the first examination, we compared levels of nonfasting triglycerides and lipoprotein cholesterol in those who developed and did not develop (controls) ischemic stroke between the examinations. We used general linear models adjusting for total cholesterol level, alcohol consumption, smoking, hypertension, atrial fibrillation, and lipid-lowering therapy; in women, models also adjusted for postmenopausal status and hormone therapy. receiving lipid- 2144 JAMA, November 12, 28 Vol 3, No. 18 (Reprinted) 28 American Medical Association. All rights reserved.

lowering therapy were excluded from analysis. For levels of nonfasting triglycerides on a continuous scale in the prospective study with 13 956 participants and 1529 ischemic stroke events, we had 9% statistical power at a 2-sided P.5 to detect an HR of 1.7 per 89-mg/dL increase in both men and women. RESULTS Baseline characteristics of individuals from the general population of the Copenhagen City Heart Study are shown in TABLE 1. The study included 13 956 individuals (6375 men, 7581 women) aged 2 to 93 years with up to 31 years of follow-up; of these, 1529 (779 men, 75 women) developed ischemic stroke. At the 1976-1978, 1981-1983, 1991-1994, and 21-23 examinations, %, %, 1%, and 2%, respectively, of the participants were receiving lipid-lowering therapy. Prospective Study: Nonfasting Triglycerides and Ischemic In both sexes, the cumulative incidence of ischemic stroke increased with increasing levels of nonfasting triglycerides (P.1 by log-rank tests for trend). with elevated nonfasting triglyceride levels had age-adjusted HRs for ischemic stroke ranging from 1.4 (95% CI,.9-2.1) for triglyceride levels of 89 through 176 mg/dl to 3.2 (95% CI, 1.7-6.2) for levels of 443 mg/dl or greater vs men with nonfasting triglyceride levels of less than 89 mg/dl (P.1 for trend) (FIGURE 1). Corresponding values for women ranged from 1.3 (95% CI, 1.-1.8) for triglyceride levels of 89 through 176 mg/dl to 5.1 (95% CI, 1.7-14.8) for levels of 443 mg/dl or greater vs women with nonfasting triglyceride levels of less than 89 mg/dl (P.1 for trend). After multivariate adjustment, corresponding HRs (95% CIs) ranged from 1.3 (.8-1.9) to 2.5 (1.3-4.8) in men (P.1 for trend) and 1.3 (.9-1.7) to 3.8 (1.3-11.1) in women (P.1 for trend). With adjustment for systolic or diastolic blood pressures rather than the dichotomized variable hypertension, the results were similar. Additional adjustment for BMI and diabetes mellitus attenuated risk estimates in both the age- and multivariateadjusted models (Figure 1). Additional adjustment for HDL-C levels likewise attenuated risk estimates in both the age- and multivariate-adjusted models; however, nonfasting triglyceride levels were still associated with risk of ischemic stroke (Figure 1). The HR for ischemic stroke for each 89-mg/dL increase in nonfasting triglyceride levels was 1.24 (95% CI, 1.19-1.29; 1529 events; 52 events/1 person-years) after age adjustment and 1.15 (95% CI, 1.9-1.22) after multivariate adjustment (TABLE 2). After stratifying for sex, age-adjusted HRs for ischemic stroke were 1.14 (95% CI, 1.7-1.21; 779 events; 44 events/ 1 person-years) for men and 1.33 (95% CI, 1.2-1.48; 75 events; 64 events/1 person-years) for women; corresponding multivariateadjusted HRs (95% CIs) were 1.12 (1.4-1.2) and 1.28 (1.15-1.43). There was statistical evidence for interaction between nonfasting triglyceride levels and sex on risk of ischemic stroke (ageadjusted P=.1; multivariate-adjusted P=.3), as anticipated by our a priori stratification. We found no evidence for nonlinearity between increasing levels of triglycerides and increasing risk of ischemic stroke in women (P.99) or in men (P=.14). Table 1. Baseline Characteristics of Individuals From the General Population Copenhagen City Heart Study (Prospective Study) Quartile of Nonfasting Triglyceride Level, Median (Range), mg/dl 1 2 3 4 1 2 3 4 Characteristic 85 (16-16) 127 (17-15) 18 (151-219) 298 (22-2716) 69 (19-83) 97 (84-112) 131 (113-157) 22 (158-131) observations 161 1586 16 1579 191 1931 1856 1893 No. with ischemic stroke 162 185 198 234 133 17 23 244 Age, median (IQR), y 52 (41-61) 54 (44-62) a 55 (46-62) a 54 (45-6) a 48 (39-56) 53 (44-59) a 55 (48-62) a 57 (51-64) a Total cholesterol, 28 221 232 247 216 232 244 259 median (IQR), mg/dl (183-234) (196-247) a (26-258) a (221-277) a (188-244) (25-263) a (215-277) a (23-293) a Alcohol drinker, No. (%) b 1122 (71) 136 (67) c 1114 (72) 1136 (73) 718 (38) 661 (34) c 662 (36) a 65 (32) a Smoker, No. (%) b 147 (67) 178 (7) 193 (7) c 1135 (73) a 113 (54) 199 (57) a 1113 (61) a 1127 (6) a Hypertension, No. (%) b 731 (47) 85 (52) c 875 (57) a 124 (66) a 559 (3) 764 (4) a 861 (47) a 172 (58) a Atrial fibrillation, No. (%) b 27 (2) 33 (2) 32 (2) 24 (2) 18 (1) 35 (2) 28 (2) 42 (2) c Lipid-lowering therapy, No. Postmenopausal, No. (%) 938 (5) 1228 (65) 1353 (74) 1511 (82) Postmenopausal with 276 (15) 352 (18) a 322 (18) a 364 (2) a hormone therapy, No. (%) Abbreviation: IQR, interquartile range. SI conversion factor: To convert triglyceride values to mmol/l, multiply by.113; cholesterol values to mmol/l, multiply by.259. a P.1. b See Methods for definition. c P.5 by t test or Pearson 2 test comparing individuals with those in the first quartile of nonfasting triglyceride levels. 28 American Medical Association. All rights reserved. (Reprinted) JAMA, November 12, 28 Vol 3, No. 18 2145

Figure 1. Hazard Ratios (HRs) for Ischemic by Increasing Levels of Nonfasting Triglycerides, Stratified by Adjustment Triglycerides, mg/dl (mmol/l) 95 319 141 545 238 258 85 351 189 73 4 41 /1 Person-Years (95% CI) 45 (36-55) 61 (55-68) 74 (64-85) 72 (57-9) 94 (69-128) 87 (64-118) 1.4 (.9-2.1) 1.8 (1.2-2.9) 1.9 (1.1-3.3) 2.9 (1.5-5.6) 3.2 (1.7-6.2) Age-Adjusted 1.3 (.8-1.9) 1.6 (1.-2.5) 1.5 (.9-2.7) 2.2 (1.1-4.2) 2.5 (1.3-4.8) Multivariate-Adjusted 221 3987 985 241 96 62 159 47 135 26 13 1 28 (24-33) 46 (42-5) 69 (58-81) 57 (39-84) 69 (4-119) 94 (51-175) 1.3 (1.-1.8) 2.2 (1.5-3.3) 1.6 (.8-3.3) 2.6 (1.-6.6) 5.1 (1.7-14.8) 1.3 (.9-1.7) 2. (1.3-2.9) 1.4 (.7-2.9) 2.5 (1.-6.4) 3.8 (1.3-11.1) Age, BMI, and diabetes Multivariate, BMI, and diabetes 93 312 148 544 238 258 85 351 189 73 4 41 45 (36-55) 61 (55-68) 74 (64-85) 72 (57-9) 94 (69-128) 87 (64-118) 1.3 (.8-2.) 1.6 (1.-2.5) 1.6 (.9-2.8) 2.1 (1.1-4.2) 2.4 (1.2-4.7).2 1.2 (.8-1.8) 1.4 (.9-2.2) 1.3 (.7-2.2) 1.6 (.8-3.1) 1.8 (.9-3.6).5 226 3981 984 24 96 62 158 47 134 26 13 1 28 (24-33) 46 (42-51) 68 (57-81) 57 (39-84) 69 (4-119) 94 (51-175) 1.3 (.9-1.8) 1.9 (1.3-2.8) 1.3 (.7-2.7) 2. (.8-5.2) 3.1 (1.-9.2).2 1.2 (.9-1.7) 1.7 (1.1-2.6) 1.2 (.6-2.4) 2. (.8-5.3) 2.1 (.7-6.3).1 Age and HDL cholesterol Multivariate and HDL cholesterol 729 2377 186 436 195 196 63 257 15 59 34 3 37 (29-47) 5 (44-56) 66 (56-77) 66 (51-85) 89 (63-124) 73 (51-15) 1.2 (.7-2.) 1.8 (1.-3.) 1.9 (1.-3.6) 2.9 (1.4-6.1) 2.7 (1.2-5.9) 1.1 (.7-1.9) 1.6 (.9-2.7) 1.6 (.8-3.) 2.2 (1.-4.7) 2.1 (.9-4.6).5 1962 3331 799 19 75 4 141 335 19 21 11 9 27 (23-32) 42 (38-47) 62 (51-75) 52 (34-8) 68 (38-122) 116 (6-223) 1.2 (.8-1.6) 1.8 (1.1-2.7) 1.2 (.6-2.7) 2.2 (.8-6.3) 6.7 (2.1-2.9).1 1.1 (.8-1.6) 1.7 (1.1-2.6) 1. (.5-2.3) 2.2 (.8-6.2) 5.1 (1.6-16.2).6 Values are from 13 956 individuals from the Copenhagen City Heart Study with up to 31 years of follow-up, during which time 1529 developed ischemic stroke. Multivariate adjustment was for total cholesterol level, alcohol consumption, smoking, hypertension, atrial fibrillation, and lipid-lowering therapy, with further adjustment in women for postmenopausal status and hormone therapy. P values for trend examine whether increased levels of triglycerides are associated with increased HRs (triglyceride strata were coded, 1, 2, 3, 4, and 5 for increasing triglyceride levels). BMI indicates body mass index (calculated as weight in kilograms divided by height in meters squared); CI, confidence interval (shown as error bars in the plots); HDL, high-density lipoprotein. 2146 JAMA, November 12, 28 Vol 3, No. 18 (Reprinted) 28 American Medical Association. All rights reserved.

No consistent evidence for statistical interaction on risk of ischemic stroke was found between nonfasting triglyceride levels and age at study entry ( 55 years vs 55 years), hypertension (normotensive vs hypertensive), BMI ( 25 vs 25), physical activity (active vs inactive), and hormone therapy in women (no vs yes) (Table 2). Hazard ratios for ischemic stroke for increasing strata of nonfasting triglyceride levels stratified for age at study entry, hypertension, BMI, and physical activity are shown in FIGURE 2 and FIGURE 3. Increasing levels of nonfasting triglycerides were associated with increasing risk of ischemic stroke in all subgroups examined (Table 2 and Figures 2 and 3). In both sexes, absolute 1-year risk of ischemic stroke increased with increasing levels of nonfasting triglycerides and with increasing age (TABLE 3). Absolute 1-year risk of ischemic stroke ranged from 2.6% in men younger than 55 years with nonfasting triglyceride levels less than 89 mg/dl to 16.7% in men 55 years or older with triglyceride levels of 443 mg/dl or greater. Corresponding values in women were 1.9% and 12.2%. Cross-sectional Study: Nonfasting Triglycerides, Remnant Cholesterol, and Ischemic Among individuals who participated in the 1991-1994 examination of the Copenhagen City Heart Study, men with a previous ischemic stroke vs controls had nonfasting triglyceride levels of 191 (interquartile range [IQR], 131-259) mg/dl vs 148 (IQR, 14-214) mg/dl (P.1); corresponding values for women were 167 (IQR, 121-229) mg/dl vs 127 (IQR, 91-181) mg/dl (P.5) (FIGURE 4). For remnant cholesterol, corresponding values were 38 (IQR, 26-51) mg/dl vs 29 (IQR, 2-42) mg/dl in men (P.1) and 33 (IQR, 24-45) mg/dl vs 25 (IQR, 18-35) mg/dl in women (P.5). In contrast, levels of low-density lipoprotein cholesterol and HDL-C did not differ between these groups. COMMENT By using levels of nonfasting rather than fasting triglycerides 1,4,6,7 and by having more statistical power than any previous study, 1-8 we detected a previously unnoticed association between linear increases in levels of nonfasting triglycerides and stepwise increases in risk of ischemic stroke, with no threshold effect. The notion of linear increases is supported both by the risk estimates for levels of triglycerides on a continuous scale and by the direct test for nonlinearity using increasing 89-mg/dL strata of triglyceride levels. The highest levels of nonfasting triglycerides ( 443 mg/dl) were associated with a 3- and 4-fold risk of ischemic stroke in 4% of men and 1% of women, respectively, in the general population. Even the most recent European and North American guidelines on stroke prevention do not recognize elevated triglyceride levels as a risk factor for stroke. 8,25 Table 2. Hazard Ratios for Ischemic per 89-mg/dL Increase in Nonfasting Triglyceride Levels on a Continuous Scale a Group /1 Person-Years (95% CI) Age-Adjusted Interaction P Value c Multivariate-Adjusted b All 13 956 1529 52 (5-55) 1.24 (1.19-1.29) 1.15 (1.9-1.22) Sex 6375 779 64 (6-69) 1.14 (1.7-1.21) 1.12 (1.4-1.2).1 7581 75 44 (41-47) 1.33 (1.2-1.48) 1.28 (1.15-1.43) Age at study entry, y 55 7385 525 28 (26-31) 1.24 (1.18-1.3) 1.16 (1.8-1.25).94 55 6571 14 93 (87-99) 1.25 (1.14-1.36) 1.13 (1.3-1.24) Hypertension No 778 496 3 (27-32) 1.36 (1.21-1.53) 1.16 (1.1-1.34).3 Yes 6822 126 82 (77-87) 1.17 (1.11-1.23) 1.12 (1.5-1.2) Body mass index d 25 7495 665 4 (37-43) 1.3 (1.16-1.46) 1.18 (1.4-1.35).18 25 6437 862 68 (63-72) 1.19 (1.13-1.25) 1.11 (1.4-1.19) Physical activity Active 954 14 47 (44-5) 1.23 (1.17-1.29) 1.14 (1.7-1.22).41 Inactive 445 524 65 (59-7) 1.28 (1.16-1.42) 1.18 (1.5-1.32) Hormone therapy (women) No 6247 599 42 (39-46) 1.33 (1.19-1.49) 1.29 (1.15-1.45).96 Yes 1326 149 49 (42-58) 1.35 (1.-1.82) 1.21 (.89-1.66) Interaction P Value c Abbreviations: CI, confidence interval; HR, hazard ratio. a Values are from 13 956 individuals from the Copenhagen City Heart Study with up to 31 years of follow-up, during which time 1529 developed ischemic stroke. The number of participants varies slightly from covariate to covariate due to availability of data. b Adjusted for age, total cholesterol level, alcohol consumption, smoking, hypertension, atrial fibrillation, and lipid-lowering therapy (% in 1976-1978, % in 1981-1983, 1% in 1991-1994, and 2% in 21-23); in women, also adjusted for postmenopausal status and use of hormone therapy. c Interaction between nonfasting triglyceride levels and the stratifying covariate. d Calculated as weight in kilograms divided by height in meters squared..3.29.38.56.85.8 28 American Medical Association. All rights reserved. (Reprinted) JAMA, November 12, 28 Vol 3, No. 18 2147

Mechanistically, the explanation for these findings is straightforward. Elevated levels of nonfasting triglycerides mark elevated levels of remnant from chylomicrons and very lowdensity lipoproteins that likely promote atherosclerosis. 17,18 Because all human cells can degrade triglycerides but not cholesterol, it may not be the triglyceride content of remnants that cause atherosclerosis but rather the cholesterol content of these particles. Remnant lipoproteins can penetrate into the arterial intima 11,12 and may preferentially get trapped within the subendothelial space. 13-16 Since cholesterol in remnant particles cannot be degraded when taken up by intimal macrophages, these cells are transformed into cholesterol-laden foam cells, leading to fatty streak formation and eventually the development of atherosclerosis, myocardial infarction, ischemic heart disease, 9,1 and as demonstrated in the present study ischemic stroke. Some former studies have overadjusted for potential confounders, ie, have adjusted for covariates that themselves are associated with elevated levels of nonfasting triglycerides and remnant lipoproteins. The classic example is adjustment for levels of HDL-C: when levels of triglycerides and remnant lipoproteins are elevated, levels of HDL-C are reduced and vice versa for low levels of triglycerides. As a consequence, many researchers and even several large pharmaceutical companies have focused research solely on HDL-C levels and the so-called reverse cholesterol transport and have largely ignored levels of triglycerides and remnant lipoproteins. The strong association of HDL-C with risk of cardiovascular disease may be explained in part by the association between elevated levels of nonfasting triglycerides or remnant cholesterol and cardiovascular disease. 9 In support of this idea, we recently observed that loss-of-function mutations in the adenosine triphosphate binding cassette transporter 1 gene are associated with low HDL-C levels without elevated levels of Figure 2. Hazard Ratios (HRs) for Ischemic by Increasing Levels of Nonfasting Triglycerides, Stratified by Age at Study Entry and Hypertension Age-Adjusted Multivariate-Adjusted Triglycerides, mg/dl (mmol/l) Age <55 y at Study Entry 254 3525 163 385 165 193 94 234 19 34 28 26 /1 Person-Years (95% CI) 17 (14-21) 27 (23-3) 44 (36-53) 39 (28-54) 78 (54-113) 64 (44-95) 1.8 (1.2-2.7) 4.2 (2.6-6.9) 3.6 (1.8-7.2) 12. (5.7-25.3) 9. (4.2-19.5) 1.3 (.8-2.) 2.3 (1.4-3.9) 1.6 (.8-3.4) 4.8 (2.2-1.5) 3.6 (1.6-8.) Age 55 y at Study Entry 161 3481 1332 41 169 127 15 524 215 65 25 25 78 (66-91) 9 (82-98) 16 (93-121) 19 (86-139) 98 (66-145) 144 (98-214) 1.3 (.9-1.8) 1.8 (1.3-2.6) 2.1 (1.2-3.4) 1.8 (.8-3.7) 4. (1.9-8.4) 1.2 (.9-1.7) 1.5 (1.-2.2) 1.5 (.9-2.5) 1.2 (.6-2.5) 2.2 (1.-4.8).2 Normotensive 28 3618 984 27 12 78 95 276 8 22 12 11 18 (15-22) 33 (29-37) 38 (31-47) 38 (25-58) 48 (27-85) 74 (41-133) 1.9 (1.2-2.8) 2.2 (1.3-3.7) 2.3 (1.-5.3) 3.8 (1.3-11.) 9.4 (3.1-28.5) 1.5 (1.-2.3) 1.4 (.8-2.3) 1.3 (.5-2.9) 2.1 (.7-6.2) 3.6 (1.2-11.2).6 Hypertensive 195 336 142 511 214 24 148 476 244 77 41 4 66 (56-78) 77 (7-84) 11 (89-115) 87 (69-19) 112 (82-152) 94 (69-128) 1.2 (.8-1.6) 2. (1.4-2.9) 1.9 (1.2-3.1) 2.9 (1.6-5.3) 3. (1.6-5.5) 1.1 (.8-1.5) 1.7 (1.2-2.5) 1.5 (.9-2.4) 2. (1.1-3.7) 2.1 (1.1-4.) Values are from 13 956 individuals from the Copenhagen City Heart Study with up to 31 years of follow-up during which time 1529 developed ischemic stroke. See Figure 1 legend for details on multivariate adjustment and P values. CI indicates confidence interval (shown as error bars in the plots). 2148 JAMA, November 12, 28 Vol 3, No. 18 (Reprinted) 28 American Medical Association. All rights reserved.

Figure 3. Hazard Ratios (HRs) for Ischemic by Increasing Levels of Nonfasting Triglycerides, Stratified by BMI and Physical Activity Triglycerides, mg/dl (mmol/l) BMI <25 2267 3952 921 219 79 57 151 374 16 16 8 1 Age-Adjusted /1 Person-Years (95% CI) 27 (23-32) 44 (4-49) 59 (49-71) 4 (24-65) 51 (25-11) 93 (5-172) 1.6 (1.1-2.2) 2.4 (1.6-3.8) 1.4 (.6-3.5) 1.5 (.4-5.3) 7. (2.2-21.6) 1.4 (1.-1.9) 1.8 (1.2-2.9) 1. (.4-2.6) 1.1 (.3-3.8) 3.4 (1.1-1.6) Multivariate-Adjusted.3 BMI 25 842 341 1471 565 255 263 92 384 217 83 45 41 48 (39-59) 63 (57-69) 8 (7-91) 78 (63-96) 98 (73-132) 87 (64-119) 1.3 (.9-2.) 2.1 (1.4-3.2) 2.3 (1.4-3.9) 4. (2.1-7.4) 4. (2.1-7.6) 1.1 (.7-1.7) 1.5 (1.-2.4) 1.5 (.8-2.5) 2.2 (1.2-4.3) 2.2 (1.1-4.3).1 Physically active 2284 4694 1547 536 218 225 183 481 23 66 36 35 32 (28-37) 46 (42-5) 65 (56-74) 62 (49-79) 86 (62-12) 81 (58-113) 1.4 (1.-1.9) 2.4 (1.7-3.5) 2.6 (1.6-4.2) 4.7 (2.5-8.8) 5.3 (2.8-1.) 1.1 (.8-1.5) 1.6 (1.1-2.3) 1.4 (.8-2.3) 2.3 (1.2-4.4) 2.4 (1.3-4.7) Physically inactive 831 2311 847 25 116 95 61 277 12 33 17 16 34 (27-44) 66 (59-74) 87 (73-14) 81 (58-114) 86 (53-138) 19 (67-178) 2. (1.2-3.3) 3. (1.7-5.2) 3. (1.4-6.3) 3.4 (1.3-8.9) 7.2 (2.7-19.1) 1.8 (1.1-2.9) 2.4 (1.4-4.2) 2.2 (1.-4.7) 2.4 (.9-6.2) 3.8 (1.4-1.4).2 Values are from 13 956 individuals from the Copenhagen City Heart Study with up to 31 years of follow-up during which time 1529 developed ischemic stroke. See Figure 1 legend for details on multivariate adjustment and P values. BMI indicates body mass index (calculated as weight in kilograms divided by height in meters squared); CI, confidence interval (shown as error bars in the plots). Table 3. Absolute 1-Year Risk for Ischemic for Increasing Levels of Nonfasting Triglycerides by Age and Sex a Triglycerides, mg/dl (mmol/l) Absolute 1-Year Risk, % b Absolute 1-Year Risk Difference, % Absolute 1-Year Risk, % b Absolute 1-Year Risk Difference, % Age 55 y at Study Entry 89 ( 1) 529 27 2.6 1525 67 1.9 1543 127 3.3.7 1982 17 2.4.5 676 65 4.1 1.5 387 44 2.9 1.1 29 27 3.9 1.3 95 7 2.8.9 12 21 4.8 2.2 45 7 3.5 1.6 443 ( 5) 166 25 5.5 2.9 27 1 4. 2.1 Age 55 y at Study Entry 89 ( 1) 376 58 8.1 5.5 685 92 5.8 4. 1476 224 1.2 7.6 25 3 7.4 5.5 734 124 12.5 9.9 598 91 9.1 7.2 255 46 11.9 9.3 146 19 8.6 6.8 118 19 14.6 12. 51 6 1.6 8.8 443 ( 5) 92 16 16.7 14.1 35 9 12.2 1.3 a Values are from 13 956 individuals from the Copenhagen City Heart Study with up to 31 years of follow-up, during which time 1529 developed ischemic stroke. b By Poisson regression. 28 American Medical Association. All rights reserved. (Reprinted) JAMA, November 12, 28 Vol 3, No. 18 2149

nonfasting triglycerides or remnant cholesterol but are not associated with the expected increased risk of ischemic heart disease. 26 Furthermore, torcetrapib, a cholesteryl ester transfer protein inhibitor that increases plasma levels of HDL-C, failed to protect against the progression of atherosclerosis and even increased the risk of cardiovascular disease, cardiovascular mortality, and overall mortality. 27 Likewise, adjustment for diabetes mellitus and overweight also tends to mask the association between elevated levels of nonfasting triglycerides and cardiovascular disease. 9 This is because elevated levels of remnants may contribute to the increased cardiovascular risk observed in overweight individuals, those with diabetes mellitus, or both. 18 In accordance with this, adjustment for age, BMI, and diabetes mellitus or for age and HDL-C level slightly attenuated risk estimates for ischemic stroke for increasing levels of nonfasting triglycerides. Figure 4. Levels of Nonfasting Triglycerides and Lipoprotein Cholesterol for Individuals With Previous Ischemic vs Controls mg/dl mg/dl 5 4 3 2 1 25 2 15 1 5 Observations, No. Triglycerides Controls Ischemic Controls 4136 Controls Ischemic LDL cholesterol Ischemic 69 Controls 5377 Ischemic 55 6. 5. 4. 3. 2. 1. 6. 5. 4. 3. 2. 1. mmol/l mmol/l mg/dl mg/dl 8 6 4 2 1 8 6 4 2 Remnant cholesterol Controls Ischemic Controls 4136 Controls Ischemic HDL cholesterol Ischemic 69 Controls 5377 Ischemic 55 Values were measured in 9637 individuals participating in the 1991-1994 examination of the Copenhagen City Heart Study; these individuals were not treated with lipid-lowering therapy. Boxes indicate interquartile range; horizontal lines, median; error bars, 95% confidence intervals. P values (triglycerides: P.1 for ischemic stroke vs controls in men and P.5 in women; remnant cholesterol: P.1 for ischemic stroke vs controls in men and P.5 in women) are from general linear models adjusting for age, total cholesterol level, alcohol consumption, smoking, hypertension, and atrial fibrillation, with further adjustment in women for postmenopausal status and hormone therapy. HDL indicates high-density lipoprotein; LDL, low-density lipoprotein. 2. 1.5 1..5 2.5 2. 1.5 1..5 mmol/l mmol/l Patients with familial forms of hypertriglyceridemia have increased risk of cardiovascular death, 28 while patients with remnant hyperlipidemia have premature atherosclerosis. 29 In accordance with this, those with ischemic stroke in the Copenhagen City Heart Study who attended the 1991-1994 examination had elevated levels of nonfasting triglycerides and remnant cholesterol. Also, heterozygosity for genetic defects in lipoprotein lipase, the plasma enzyme that degrades triglycerides, is associated with elevated triglyceride levels as well as with increased risk of ischemic heart disease. 3-32 Furthermore, subanalyses of 3 randomized double-blind trials suggest that among patients with elevated triglyceride levels, a 2% to 4% reduction in triglyceride levels achieved using fibrates was associated with a 3% to 4% reduction in risk of ischemic heart disease. 33-35 Finally, in the Coronary Drug Project, a 26% reduction in triglyceride levels achieved using niacin was associated with a 24% reduction in cerebrovascular events. 36 An association between elevated triglyceride levels and risk of cardiovascular disease has previously been questioned, since extremely high levels of triglycerides ( 22 mg/dl), as seen in familial lipoprotein lipase deficiency with the chylomicronemia syndrome, does not lead to accelerated atherosclerosis. 3 The simple and straightforward explanation for this apparent paradox is that at such extreme triglyceride levels, lipoproteins are very large 37 and consequently not able to penetrate the intima of arteries. 38 In support of this explanation, patients with lipoprotein lipase deficiency who, during part of their lives because of lipidlowering treatment, have triglyceride levels as low as 266 to 616 mg/dl and thus much smaller triglyceriderich lipoproteins do develop premature atherosclerosis. 39 Another argument often presented against using nonfasting triglyceride levels for assessment of cardiovascular risk is that triglyceride levels vary greatly after intake of fatty meals. How- 215 JAMA, November 12, 28 Vol 3, No. 18 (Reprinted) 28 American Medical Association. All rights reserved.

ever, this is mainly based on information from fat-tolerance tests in which participants ingest 1 g of fat per kilogram of body weight and in which triglycerides may increase up to a level of 176 to 353 mg/dl 4 hours after fat intake. 9,4 In contrast, triglyceride levels only increased to 14 mg/dl 4 hours after normal intake of food in individuals in the general population. 9 The potential difficulty in using nonfasting triglyceride levels in clinical practice without standardization of the time since the most recent meal and the content of that meal should also be considered. First, because lipid profiles usually are measured in fasting individuals, nonfasting levels would require yet another blood sampling, unless nonfasting lipid levels were to become the standard in the future. Second, our results show that even random levels of triglycerides are associated with ischemic stroke; however, whether standardization of the time since the most recent meal and the content of that meal would further improve the association between nonfasting triglyceride levels and risk of ischemic stroke needs to be studied. In support of our findings, the s Health Study showed that elevated nonfasting triglyceride levels, but not fasting levels, are associated with increased risk of ischemic stroke. 1 Another study also found an association between elevated levels of nonfasting triglycerides and increased risk of ischemic stroke, 3 while 2 others found no association. 2,5 Regarding fasting triglycerides, some inconsistency exists; for example, a large Asian-Pacific metaanalysis with fasting lipid measurements in 9% of participants reported a positive association with risk of ischemic stroke, 7 while other studies found no association. 1,4,6,1 None of these former studies on fasting as well as on nonfasting triglyceride levels attempted to associate very high levels of triglycerides with risk of ischemic stroke and therefore were not able to detect our result that nonfasting triglyceride levels of 443 mg/dl or greater are associated with a 3- and 4-fold increased risk of ischemic stroke in men and women, respectively. Limitations include that we only studied white individuals, and therefore our results may not necessarily apply to other racial groups. Also, because we did not measure levels of remnant lipoproteins at baseline, we cannot implicate these lipoproteins directly in risk of ischemic stroke; however, the finding at the 1991-1994 examination that participants with previous ischemic stroke had elevated levels of nonfasting triglycerides and remnant lipoproteins supports this hypothesis. Moreover, because 82% of our participants ate within 3 hours of the blood sampling and at least 97% ate within 8 hours of sampling, we could not compare fasting and nonfasting triglyceride levels for association with ischemic stroke. This does not diminish the finding that nonfasting triglyceride levels are associated with ischemic stroke, but it makes it difficult to know if fundamental differences exist between the fasting and nonfasting states. However, results from the s Health Study suggested that levels of nonfasting, but not fasting, triglycerides are associated with increased risk of ischemic stroke. 1 Our study has several strengths. We studied a homogeneous white general population of 1% Danes; we had up to 31 years of complete follow-up; end points were validated using records from hospitals, general practitioners, or both; nonfasting triglyceride levels were measured at baseline in 1976-1978 using fresh samples; less than 1% of study participants were given lipidlowering therapy; we corrected for regression dilution bias; and we had sufficient statistical power to examine even the association of very high levels of triglycerides with ischemic stroke in men as well as women. Our results, together with those from 2 previous studies, 9,1 suggest that elevated levels of nonfasting triglycerides and remnant lipoprotein cholesterol could be considered together with elevated levels of low-density lipoprotein cholesterol for prediction of cardiovascular risk. However, these findings require replication in other populations. Author Contributions: Drs Freiberg and Nordestgaard had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Freiberg, Tybjærg- Hansen, Jensen, Nordestgaard. Acquisition of data: Tybjærg-Hansen, Jensen, Nordestgaard. Analysis and interpretation of data: Freiberg, Tybjærg- Hansen, Nordestgaard. Drafting of the manuscript: Freiberg. Critical revision of the manuscript for important intellectual content: Tybjærg-Hansen, Jensen, Nordestgaard. Statistical analysis: Freiberg, Nordestgaard. Obtained funding: Tybjærg-Hansen, Nordestgaard. Administrative, technical, or material support: Freiberg, Nordestgaard. Study supervision: Tybjærg-Hansen, Jensen, Nordestgaard. Financial Disclosures: Dr Nordestgaard reported serving as a consultant for AstraZeneca and BG Medicine and receiving lecture honoraria from Boehringer Ingelheim, Merck, Pfizer, Sanofi-Aventis, and AstraZeneca. No other disclosures were reported. Funding/Support: This study was supported by the University of Copenhagen, the Danish Heart Foundation, the Danish Medical Research Council, the Research Fund at Rigshospitalet, Copenhagen University Hospital, and the European Union, Sixth Framework Programme Priority (FP-25-LIFESCIHEALTH-6), contract 37631. Role of the Sponsor: The study sponsors had no role in the conduct of the study; the collection, management, analysis, and interpretation of data; or the preparation, review, or approval of the manuscript. REFERENCES 1. Gordon T, Kannel WB, Castelli WP, Dawber TR. 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