C-Reactive Protein, Interleukin 6, Fibrinogen and Risk of Sudden Death in European Middle-Aged Men: The PRIME Study
Objective—To examine prospectively the association of high-sensitivity C-reactive protein, interleukin 6, and fibrinogen with sudden death in asymptomatic European men.
Methods and Results—Among the 9771 men from the Etude PRospective de l’Infarctus du Myocarde (PRIME) Study, 664 had a first coronary heart disease over 10 years, including 50 sudden deaths, 34 nonsudden coronary deaths, and 580 nonfatal coronary heart disease events. For each outcome, 2 matched controls, who were free of coronary heart disease at the index date, were randomly selected from the initial cohort (nested case control study design). There was a 3-fold increased risk (95% CI, 1.20 to 7.81) of sudden death between the upper and the lower third of interleukin 6 after adjustment for baseline confounders in conditional logistic regression analysis. Neither high-sensitivity C-reactive protein (hazard ratiothird versus first tertile=1.27; 95% CI, 0.51 to 3.17) nor fibrinogen (hazard ratiothird versus first tertile=1.90; 95% CI, 0.76 to 4.75) was associated with sudden death. For comparison, there was a 6-fold increased risk of nonsudden coronary death from the highest compared with the lowest tertile of fibrinogen and a trend toward an association with higher C-reactive protein and higher interleukin 6. All 3 inflammatory biomarkers were moderately, but significantly, associated with nonfatal coronary heart disease.
Conclusion—Interleukin 6, but not high-sensitivity C-reactive protein or fibrinogen, is an independent predictor of sudden death in asymptomatic European men.
Sudden death accounts for more than 300 000 deaths every year in the United States and for 275 000 deaths in Europe.1,2 It is usually caused by fast ventricular tachycardia or ventricular fibrillation complicating an ischemic cardiac event in adults. The identification of those in the population who are at higher risk of sudden death remains a major challenge, as nearly half of the victims are asymptomatic, and successful resuscitation outside a hospital is rare. We and others previously reported significant associations between traditional and nontraditional risk factors with sudden death,3–9 but these factors are not sufficiently sensitive to identify those who are at higher risk. Therefore, new risk markers need to be investigated.10 Inflammatory biomarkers might be useful candidates, because pathological signs of inflammation are evident in the coronary atherosclerotic and thrombotic lesions that are found in most sudden death victims.11,12 So far, the inflammatory hypothesis of sudden death in the population has been evaluated in 3 United States prospective studies with mixed results.13–15 In these studies, higher C-reactive protein (CRP) was predictive of sudden death in men from the Physicians’ Health Study but not in women from the Nurses’ Health Study.13,14 In the Atherosclerosis Risk in Communities Study, higher fibrinogen and higher white blood cell counts were associated with sudden death.15 Interleukin 6 (IL-6) is the main inducer of CRP secretion by the liver, and higher IL-6 has been associated with coronary heart disease (CHD) and especially with fatal events.16–18 However, whether IL-6 is associated with sudden death in asymptomatic individuals is currently unknown.
Therefore, our aims were (1) to confirm in a European population the association of baseline systemic levels of CRP and fibrinogen with subsequent sudden death, and (2) to test the new hypothesis that higher IL-6 is an independent predictor of sudden death, using the 10 years of follow-up in the PRIME Study.19 To put our results in perspective, we also examined in the same study and during the same study period the associations of these 3 inflammatory biomarkers with nonfatal CHD and with nonsudden coronary death, in an attempt to better discriminate their contribution to atherothrombosis and/or to arrhythmia susceptibility.
Details of the PRIME Study regarding the recruitment, design, and CHD cases ascertainments have been described elsewhere.19
The PRIME Study is a multicenter prospective cohort designed to identify risk factors for CHD. The target study size was to recruit 2500 men, aged 50 to 59 years, in each 4 collaborating World Health Organization (WHO) MONitoring Trends and Determinants in CArdiovascular Disease (MONICA) centers in Belfast (Northern Ireland, UK), Lille, Strasbourg, and Toulouse (France). Overall, 10 602 men, aged 50 to 59 years, were recruited between 1991 and 1993. Among them, 823 had coronary disease and 77 a history of stroke at baseline examination and were excluded from the present analysis, leaving a study population of 9711 men.
Briefly, a self-administered health questionnaire was completed by subjects at home and was subsequently checked by trained interviewers at the clinic. It covered a broad range of clinical information, cigarette smoking, and treatment use. Diabetes was defined by the current intake of oral hypoglycemic treatment or use of insulin. Blood pressure was measured in the sitting position with the same automatic device (Spengler SP9; Spengler). Hypertension was defined as a blood pressure higher than 140/90 mm Hg or the use of antihypertensive treatment. Height, weight, and waist circumferences were measured in a participant in light clothing. A 12-lead ECG was also recorded.
Blood was drawn after an overnight fast. A subset of biological measurements was performed in fresh plasma in the entire cohort. Plasma lipid analyses were centralized (U325 Institut National de la Santé et de la Recherche Médicale [INSERM] Institut Pasteur de Lille, France). Total-cholesterol (Total-C) and high-density lipoprotein (HDL)-cholesterol were measured by enzymatic methods using commercial kits in an automatic analyzer (Boehringer). Fibrinogen was assessed at the Laboratory of Hemostasis of La Timone Hospital (Marseilles, France), using commercially available ELISAs from Diagnostica Stago.17,19 Aliquots of serum and plasma were then frozen in liquid nitrogen until analysis of biomarkers in nested case control studies (see below).
Follow-Up and Ascertainment of Cases
All subjects were followed-up over 10 years. Subjects were contacted annually by letter and asked to complete a clinical event questionnaire. For all subjects reporting a possible event, clinical information was sought directly from the hospital or general practitioner records. All details of electrocardiograms, hospital admissions, enzymes, surgical intervention, angioplasty, and treatments were collected. Whenever possible, circumstances of death were obtained from the practitioner or the family. In the few cases where the circumstances surrounding the death were not available from the practitioner or the family, death certificates were checked for supporting clinical and postmortem information on cause of death. CHD events were defined as previously described and were validated by an independent medical committee.19 Sudden death was defined as a natural death occurring within 1 hour of symptoms onset. Extra cardiac causes, such as end stage renal disease, trauma, or cancer, as well as subjects found dead, were excluded. After 10 years of follow-up, the CHD event status was available for 95.1% of the cohort. The protocol was approved by the institutional review board of Broussais Hospital (Paris, France). Informed consent was obtained for each man who agreed to participate in the PRIME Study.
Nested Case Control Study
At the end of follow-up, a series of nested case-control studies was built within the PRIME prospective cohort for the investigation of several novel biomarkers. A total of 664 men suffered from a CHD event at the end of follow-up, 227 in Belfast and 437 in France, including 50 sudden deaths (15 in Belfast), 34 nonsudden coronary deaths (18 in Belfast), and 580 nonfatal CHD events (194 in Belfast). For each study outcome, 2 controls per case were randomly chosen from the initial cohort. Matched controls were men recruited in the same center on the same day (±3 days), of the same age (±3 years) as the corresponding case, and who were free of CHD at the time of the index date.17,20 Measurements of biomarkers were performed on baseline plasma samples blinded with respect to the case control status. Circulating levels of high-sensitivity CRP (hs-CRP) were determined with a multiplex bioassay using commercially available kits obtained from R&D Systems for hs-CRP. The multiplex assay was performed according to the manufacturer’s specifications. The plates were read on a Luminex 200 instrument system. IL-6 was determined using high sensitivity Elisa (BMS213HS; Bender MedSystems). The coefficients of variation were 8.0 and 9.7% for IL-6 and CRP, respectively.20
Due to their skewed distribution, hs-CRP and IL-6 were log transformed for statistical analyses. The baseline characteristics of cases and controls were compared using conditional logistic regression that accounted for the matching variables. Associations between hs-CRP, IL-6, and fibrinogen with the traditional risk factors were evaluated in the overall group of controls: correlations with age, body mass index (BMI), waist circumference, HDL, and Total-C were estimated by the Spearman rank test, whereas the median values of the inflammatory biomarkers were compared across baseline smoking (nonsmokers, ex-smokers, or current smokers), hypertension, and diabetes statuses using Wilcoxon–Mann–Whitney or Kruskal–Wallis tests. The hazard ratios (HRs) and their 95% CI of hs-CRP, IL-6, and fibrinogen for each study outcome were estimated from separate conditional logistic regression analyses and were given by thirds of each inflammatory variable calculated in the overall group of controls, taking the lower third as the reference category. Analysis was adjusted for BMI, smoking status, hypertension, diabetes, HDL, and Total-C, on an a priori basis. Backward elimination analysis was used to retain the most significant variables, using a cut off value of <5%. All comparisons were 2-sided, and a probability value of <0.05 indicated a statistically significant difference. Analyses were performed using SAS 9.1 (Statistical Analysis System).
The baseline characteristics of cases and controls are described in Table 1. Sudden death cases were more often hypertensive, current smokers, and had higher levels of IL-6 than their matched controls. In men with nonsudden coronary death, significant difference existed for fibrinogen only which was higher than in the controls; differences were borderline significant for hs-CRP and IL-6. Finally, nonfatal CHD cases had a worse cardiovascular risk profile than their controls, including higher levels of the inflammatory biomarkers.
Relationships Between hs-CRP, Fibrinogen, and IL-6 With Cardiovascular Risk Factors in the Controls
As shown in Table 2, hs-CRP, fibrinogen, and IL-6 correlated moderately with the traditional cardiovascular risk factors, the highest correlation being observed between hs-CRP and BMI. Fibrinogen and hs-CRP were intercorrelated (correlation coefficient, 0.43), whereas moderate correlation existed between IL-6 and hs-CRP (correlation coefficient, 0.20) or fibrinogen (correlation coefficient, 0.15). Moreover, the median levels of the 3 inflammatory biomarkers increased progressively with smoking status, whereas higher CRP was associated with hypertension and diabetes and higher IL-6 with hypertension.
HRs of hs-CRP, Fibrinogen, and IL-6 for Study Outcomes
The HRs for sudden death, nonsudden coronary death, and nonfatal CHD by third of IL-6, hs-CRP, and fibrinogen are reported in Table 3. Neither hs-CRP nor fibrinogen was associated with sudden death in unadjusted and adjusted analyses. Conversely, higher IL-6 was progressively associated with an increased risk of sudden death, and subjects in the upper third had a 3-fold increased risk compared with those from the bottom third in the fully adjusted analysis (P for trend=0.02). In this same model, hypertension was the remaining significant predictor (HR=2.91; 95% CI, 1.23 to 6.88). Additional adjustment for hs-CRP or fibrinogen did not change the HRs estimates (data not shown). Of note, when we tested for the difference between the standardized HRs, a borderline significant difference could be observed for the comparison between IL-6 and CRP only (P=0.12). Such analysis lacked statistical power however (data not shown).
Regarding the association with nonsudden coronary death, men with fibrinogen in the highest third had a significant 6-fold increased risk compared with those in the bottom third in multivariate-adjusted analysis. There was a trend toward an association with higher hs-CRP and higher IL-6. Moreover, all 3 inflammatory biomarkers were moderately, but significantly, associated with nonfatal CHD, the HRs of the highest compared with the bottom third ranging from 1.32 to 1.53 in multivariate-adjusted analysis.
We also assessed the association between IL-6 and sudden death by smoking status but found consistent associations in the never or past smokers (n=99) and in the current smokers (n=46). The HRs for 1-SD increment of the natural logarithm of IL-6 were, respectively, 1.68 (95% CI, 0.97 to 2.91) and 1.66 (0.42 to 6.49) after multivariate adjustment, and no significant interaction was detected (P=0.96) (data not shown). To assess the confounding effect of socioeconomic status, our analyses were further adjusted for the current employment status at baseline, but the associations remained unchanged. In particular, higher IL-6 remained associated with sudden death as shown by an HR of the third versus the first tertile of 2.82 (95% CI, 1.08 to 7.36) (data not shown).
Consistent results were observed when men who developed a nonfatal CHD event (angina pectoris or myocardial infarction) before the fatal event were excluded (9 men in those who had sudden death and 11 men among those who died from nonsudden coronary death). In particular, the HR for sudden death of the highest compared with the bottom third of IL-6 was 3.45 (95% CI, 1.21 to 9.79; P for trend=0.028), after adjustment for hypertension. The same applied when men with a baseline hs-CRP above 10 mg/L (possibly reflecting acute inflammation) were excluded (2 sudden death cases excluded): the HR for sudden death of the highest compared with the bottom third was 3.55 (95% CI, 1.26 to 10.05, P=0.017) after adjustment for hypertension (data not shown).
In healthy European middle-aged men who were participants in the PRIME Study, higher IL-6 was a strong predictor of sudden death. Men with a baseline IL-6 level in the upper third of the distribution had a 3-fold increased risk of sudden death over 10 years compared with men whose IL-6 levels were in the bottom third, independent of traditional risk factors. In contrast, higher hs-CRP and fibrinogen were not predictors of sudden death. For comparison and regarding nonsudden coronary death risk, there was a strong association with fibrinogen, as men from the highest third had a 6-fold increased risk, and a trend toward an association with higher hs-CRP and IL-6 levels. Moreover, all 3 inflammatory biomarkers were moderately, but significantly, associated with nonfatal CHD, with the multivariate-adjusted HR of the higher against the bottom third ranging from 1.32 to 1.53.
So far, the epidemiological evidence for an association between inflammatory biomarkers and sudden death in asymptomatic populations is scarce and inconclusive. The current lack of association between higher CRP and sudden death in the PRIME Study is consistent with a recent report from the Nurses’ Health Study, in which the moderate and borderline significant association in age-adjusted analysis disappeared after adjustment for traditional risk factors over 10 years of follow-up.14 Conversely, it differs from the results of the Physicians’ Health Study, in which a 2.65-increased risk of sudden death was reported between the highest and the lowest quartile of CRP, over an average duration of follow-up of 9 years and after multivariable adjustment.13 Differences in the age range may partly explain these apparently contradictory results, because men were aged 40 to 84 years in the Physicians Health Study compared with 50 to 59 years in the PRIME Study. Moreover, in a recent report from the Atherosclerosis Risk in Communities Study, a prospective study of men and women aged 45 to 64 years, higher fibrinogen (as well as a higher white blood cell count and lower albumin level) was predictive of sudden death over a maximum of 14 years of follow-up,15 whereas in the PRIME Study, fibrinogen was not related to sudden death.
To the best of our knowledge, this is the first published report suggesting an association between higher IL-6 and sudden death in the population. This is consistent with the recent description of an increased risk of sudden death associated with higher IL-6 in end-stage renal disease patients followed-up over 2.5 years.21 Two previous articles have suggested that inflammatory markers and IL-6 in particular were associated more with fatal than with nonfatal events.17,18 In a previous PRIME report based on the first 5 years of follow-up, we showed that higher IL-6 was predictive of a composite end point, including myocardial infarction and coronary death, but not of incident angina pectoris, and that IL-6 was a stronger predictor of such end points than higher CRP or fibrinogen.17 More recently, a post hoc analysis from the The Prospective Study of Pravastatin in the Elderly at Risk trial reported that inflammatory biomarkers and IL-6 in particular was a stronger predictor of fatal rather than nonfatal cardiovascular events.18 In these 2 previous articles, however, sudden death was not studied as a specific outcome.
The particularly strong association of IL-6 with fatal CHD and especially sudden death in the current study may suggest that in addition to contributing to atherothrombosis, IL-6 (or an unmeasured factor associated with higher plasma IL-6 concentration) is involved in mechanisms favoring susceptibility to arrhythmia. As adipocytes represent a main source of IL-6, such an association might have relevance to the risk associated with abdominal adiposity and insulin resistance syndrome. We have previously reported in the Paris Prospective Study I that abdominal adiposity, as well as the metabolic syndrome, was more specifically associated with sudden death than with fatal myocardial infarction.3,22 IL-6 might also have a direct electrophysiological effect, because a higher level has been associated with the risk of other arrhythmias, such as atrial fibrillation.23 Higher IL-6 may result from an imbalance between the sympathetic and the parasympathetic tone, leading to cardiac electric instability and ultimately to sudden death. Finally, environmental factors, such as air pollution, may increase inflammation and IL-6 in particular and indirectly contribute to the association between higher IL-6 and sudden death.24
In terms of clinical implications, future research should evaluate whether IL-6 improves the identification of asymptomatic subjects who are at higher risk of sudden death. This could be accomplished through a multimarker approach, by testing the utility of other biomarkers from different pathways.25 For instance, the incremental value of IL-6 should be assessed in comparison with that of natural terminal pro-brain natriuretic peptide, as the latter has been recently found to be predictive of sudden death in women, possibly reflecting asymptomatic structural heart disease.14 Unfortunately, brain natriuretic peptide was not measured in the current study.
The present study has some limitations. An obvious limitation is the small number of fatal events, so that the current findings should be confirmed in studies with a larger number of events. Also, this precluded us to compare HRs from each other reliably. Serial examination was not performed in the PRIME Study, so that it was not possible to account for changes in risk factor levels and therapy during follow-up. This is particularly true for IL-6, which has high within-person variability. Accounting for regression dilution bias has proved to yield stronger association between IL-6 and nonfatal myocardial infarction and coronary death, in comparison with an analysis based on a single IL-6 measurement.16 Finally, as a study of Caucasian middle-aged men, the results on IL-6 should be evaluated in other ethnicities, in women, and in the elderly.
The current study suggests that in healthy European middle-aged men, the systemic level of IL-6, but not hs-CRP or fibrinogen, is an independent and strong predictor of sudden death over 10 years. The utility of IL-6 for the identification of those subjects from the general population who are at higher risk of sudden death should be further evaluated.
The PRIME Study is organized under an agreement between INSERM and the Merck, Sharpe, and Dohme-Chibret Laboratory, with the following participating Laboratories:
The Strasbourg MONICA Project, Laboratoire d’Epidémiologie et de Santé Publique, EA 3430, Strasbourg, F-67085, France; and Université de Strasbourg, Strasbourg, F-67085, France (D. Arveiler, B. Haas).
The Toulouse MONICA Project, INSERM, U558; and Department of Epidemiology, Université Paul Sabatier-Toulouse Purpan, Toulouse, France (J. Ferrieres, J.B. Ruidavets).
The Lille MONICA project; INSERM, U744; Institut Pasteur de Lille; Université Lille Nord de France.
The United Kingdom Clinical Research Collaboration Centre of Excellence for Public Health (Northern Ireland), the Queen’s University, Belfast, United Kingdom (A. Evans, J. Yarnell, F. Kee).
The Department of Atherosclerosis, INSERM, U545, Lille; Institut Pasteur de Lille, Lille; and Faculté de Médecine, and Université Lille Nord de France, Lille, France (G. Luc, J.M. Bard).
The Laboratory of Hematology, INSERM, U626, Marseille, Hôpital La Timone, Marseille, France (I. Juhan-Vague, Pierre Morange).
The Laboratory of Endocrinology, INSERM, U563, Toulouse, France (B. Perret).
The Vitamin Research Unit, The University of Bern, Bern, Switzerland (F. Gey).
The Nutrition and Metabolism Group, Centre for Public Health, Queen’s University Belfast, Northern Ireland (Jayne Woodside, Ian Young).
The DNA Bank, INSERM/Université Pierre Marie Curie, Paris Universitas Unité Mixte de Recherche en Santé 937, Paris, F-75013 (F. Cambien).
The Coordinating Center, IFR 69, Villejuif, F-94807, France (P. Ducimetiere).
INSERM, Unit 970, Villejuif, F-94807, France; and Université Paris V, Paris Cardiovascular Research Centre, Paris, F-75015, France (A. Bingham).
We thank the following organizations, which allowed the recruitment of the PRIME subjects: the Health screening centers organized by the Social Security of Lille (Institut Pasteur), Strasbourg, Toulouse, and Tourcoing; Occupational Medicine Services of Haute-Garonne, of the Urban Community of Strasbourg; the Association Interentreprises des Services Médicaux du Travail de Lille et environs; the Comité pour le Développement de la Médecine du Travail; the Mutuelle Générale des Postes et Télécommunications du Bas-Rhin; the Laboratoire d’Analyses de l’Institut de Chimie Biologique de la Faculté de Médecine de Strasbourg; and the Department of Health and Social Services and Personal Safety (Northern Ireland), its Research and Development Office, and the Northern Ireland Chest Heart and Stroke Association. We also thank the members of the event validation committees Prof L. Guize, Dr C. Morrison, Dr M.-T. Guillanneuf, and Prof M. Giroud; and the Alliance Partnership Programme for its financial support.
Sources of Funding
The PRIME Study was funded by INSERM and the Merck, Sharpe, and Dohme-Chibret Laboratories. The PRIME Study was also supported by a 3-year research financed program by La Fondation Coeur et Artères.
Received on: April 29, 2010; final version accepted on: June 27, 2010.
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