Association of Plasma Sphingomyelin Levels and Incident Coronary Heart Disease Events in an Adult Population
Multi-Ethnic Study of Atherosclerosis
Objective— A high plasma sphingomyelin level has been associated with subclinical atherosclerosis, coronary artery disease, and worse prognosis in subjects with acute coronary syndromes. We wanted to assess the predictive value of plasma sphingomyelin levels for incident coronary heart disease (CHD) events in the Multi-Ethnic Study of Atherosclerosis.
Methods and Results— The plasma sphingomyelin level was measured in 6809 of 6814 subjects (age, mean±SD, 62.2±10.2 years) participating in the Multi-Ethnic Study of Atherosclerosis, a population-based cohort study of adults free of clinical cardiovascular disease at baseline recruited at 6 clinic sites in the United States. The subjects consisted of 52.8% females, 38.5% whites, 11.8% Chinese, 27.8% blacks, and 21.9% Hispanics. Cox proportional hazard analysis was used to examine the association between plasma sphingomyelin level and 5 years of adjudicated incident CHD events, including myocardial infarction, resuscitated cardiac arrest, angina, CHD-related death, and revascularization (coronary artery bypass grafting or percutaneous transluminal coronary angioplasty). The mean±SD plasma sphingomyelin level was 48.0±16.0 mg/dL. A total of 189 subjects had an adjudicated CHD event during the 5 years of follow-up. In the Kaplan-Meier analysis, subjects with a plasma sphingomyelin level higher than the sex-specific median had a similar event-free survival rate compared with subjects with a plasma sphingomyelin level at or less than the sex-specific median (97.16% versus 97.00%; log rank P=0.71). In the univariate Cox proportional hazard analysis, the plasma sphingomyelin level was not a predictor of an incident CHD event (hazard ratio, 0.992 [0.982–1.004]; P=0.09). In our multistage multivariate Cox proportional hazard models, a higher plasma sphingomyelin level had a modest negative association with incident CHD events when total cholesterol, high-density lipoprotein, and triglycerides were included in the model (hazard ratio, 0.985 [0.973–0.996]; P=0.008) and also in our full model after adjusting for age, sex, total cholesterol, high-density lipoprotein, triglycerides, diabetes mellitus, cigarette smoking, systolic and diastolic blood pressure, medication use for blood pressure, and 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitor use (hazard ratio, 0.984 [0.973–0.996]; P=0.002). In other models, the plasma sphingomyelin level was not associated with incident CHD events.
Conclusion— A high plasma sphingomyelin level is not associated with an increased risk of incident CHD in population-based adults free of clinical cardiovascular disease at baseline.
The role of sphingolipids in the pathogenesis and progression of atherosclerosis is an area of active research.1,2 Experimental data from cell biology and animals have suggested an association between both sphingomyelin and ceramide (a metabolite of sphingomyelin) and the development and progression of atherosclerosis.3 Sphingomyelin and ceramide have been isolated from atherosclerotic plaques in both humans and animals.4–6 Myriocin, a serine palmitoyl transferase inhibitor that reduces plasma and tissue levels of several sphingolipids (including sphingomyelin, ceramide, sphingosine-1-phosphate, and glycosphingolipids), has been shown to inhibit and even cause regression of atherosclerotic plaques in animal studies.7,8 Although most of the sphingolipids identified in atherosclerotic plaques appear to be synthesized de novo, current research9 has shown that some of these sphingolipids originate from plasma.
Despite the numerous data associating the sphingomyelin level with atherosclerosis in animals,1–5 limited data exist in humans. Plasma sphingolipids are elevated in various primary hyperlipoproteinemic states.10 Cross-sectional studies have shown an association between high plasma sphingomyelin levels and subclinical atherosclerosis11 and clinical coronary artery disease.12 High plasma sphingomyelin levels have also been associated with worse outcomes in acute coronary syndromes in a small selected cohort.13 Prospective data on the association between plasma sphingomyelin levels and incident coronary heart disease (CHD) events are lacking.
Therefore, we assessed the association between plasma sphingomyelin levels and incident CHD in participants of the Multi-Ethnic Study of Atherosclerosis (MESA).
Study Population and Data Collection
The study design for MESA has been published elsewhere.14 In brief, MESA is a prospective cohort study that began in July 2000 to investigate the prevalence, correlates, and progression of subclinical cardiovascular disease (CVD) in individuals without known CVD at baseline. The cohort includes 6814 women and men, aged 45 to 84 years, recruited from 6 US communities (Baltimore, Md; Chicago, Ill; Forsyth County, NC; Los Angeles County, Calif; northern Manhattan, New York; and St Paul, Minn). MESA cohort participants were 38.5% white (n=2624), 27.8% black (n=1895), 21.9% Hispanic (n=1492), and 11.8% Chinese (n=803). Individuals with a history of physician-diagnosed myocardial infarction (MI), angina, heart failure, stroke, or transient ischemic attack, or who had undergone an invasive procedure for CVD (ie, coronary artery bypass graft, angioplasty, valve replacement, pacemaker placement, or other vascular surgical procedures), were excluded from participation. This study was approved by the institutional review boards of each study site, and written informed consent was obtained from all participants.
Demographics, medical history, and anthropometric and laboratory data for the present study were taken from the first examination of the MESA cohort (July 2000 to August 2002). Current smoking was defined as having smoked a cigarette in the last 30 days. Diabetes mellitus was defined as a fasting glucose level of 126 mg/dL or less or the use of hypoglycemic medications. The use of antihypertensive and other medications was based on review of prescribed medication containers. Resting blood pressure was measured 3 times in the seated position, and the average of the second and third readings was recorded. Hypertension was defined as a systolic blood pressure of 140 mm Hg or greater, a diastolic blood pressure of 90 mm Hg or greater, or use of medication prescribed for hypertension. The body mass index was calculated as weight in kilograms divided by height in meters squared.
Blood specimens from fasting participants were processed within 30 minutes of phlebotomy and frozen at −70°C. Total cholesterol was analyzed using a cholesterol oxidase method (Roche Diagnostics, Indianapolis, Ind) (laboratory coefficient of variation, 1.6%). High-density lipoprotein (HDL) cholesterol was measured in ethylenediaminetetraacetic acid/plasma using the cholesterol oxidase method (Roche Diagnostics) after precipitation of non–HDL cholesterol with a combination of magnesium and dextran (laboratory coefficient of variation, 2.9%). The triglyceride level was measured in ethylenediaminetetraacetic acid and plasma using a triglyceride GB reagent (Roche Diagnostics) on a centrifugal analyzer (COBAS FARA; Roche Diagnostics) (laboratory coefficient of variation, 4.0%). Low-density lipoprotein cholesterol was calculated in plasma specimens with a triglyceride value of less than 400 mg/dL using the formula of Friedewald et al.15 The serum glucose level was measured by rate reflectance spectrophotometry on an analyzer (Vitros; Johnson & Johnson Clinical Diagnostics, Inc, Rochester, NY) (laboratory coefficient of variation, 1.1%). Enzymatic measurement of plasma sphingomyelin levels was performed at Columbia University using a novel 4-step procedure.12 In the first step, bacterial sphingomyelinase hydrolyzed sphingomyelin to phosphorylcholine and N-acylsphingosine. Thereafter, the addition of alkaline phosphatase generated choline from phosphorylcholine. The newly formed choline was used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide was used together with phenol and 4-aminoantipyrine to generate a red quinone pigment, with an optimal absorption at 505 nm. The plasma sphingomyelin levels were measured in a blinded fashion, and the interassay coefficient of variation ranged from 2.5% to 3.1%.
Ascertainment of Cardiovascular Events
At intervals of 9 to 12 months, an interviewer contacted each participant by telephone to inquire about all interim hospital admissions, cardiovascular outpatient diagnoses, and deaths. To verify self-reported diagnoses, study personnel requested copies of all death certificates and medical records for all hospitalizations and outpatient cardiovascular diagnoses. Next-of-kin interviews were performed for out-of-hospital cardiovascular deaths. Hospital records were obtained for an estimated 98% of hospitalized cardiovascular events, and some information was available for 95% of outpatient diagnostic encounters.
Hospital records that suggested possible cardiovascular events were abstracted by study personnel. The MESA coordinating center collated the abstracted or original end point records and sent them to 2 paired cardiologists, cardiovascular epidemiologists, or neurologists for independent end point classification and assignment of incidence dates. If, after review and adjudication, disagreements persisted, a full Mortality and Morbidity Review Committee made the final classification.
Reviewers assigned a diagnosis of MI based on combinations of symptoms, electrocardiographic findings, and cardiac biomarker levels. Death from CHD was classified as definite, probable, or absent based on hospital records, death certificates, and conversations with families. Definite fatal CHD required an MI within 28 days of death, chest pain within 72 hours before death, or a history of CHD; and the absence of a known nonatherosclerotic or noncardiac cause of death. If the definite fatal CHD criteria were not met, probable fatal CHD could be assigned with an underlying cause of death consistent with fatal CHD; this required the absence of a known nonatherosclerotic or noncardiac cause of death. The definition of angina was adapted from the Women’s Health Initiative criteria and was classified by reviewers as definite, probable, or absent. Definite or probable angina required clinical symptoms to be considered a MESA event, with definite angina requiring objective evidence of coronary atherosclerosis.
Definition of the Primary Outcome
For this study, a coronary heart disease event was defined as an incident MI, definite angina, coronary revascularization (coronary artery bypass grafting and percutaneous coronary intervention), resuscitated cardiac arrest, or CHD-related death as defined by the MESA protocol.
Descriptive data are presented as mean±SD for continuous variables and the frequencies of subjects in each category for categorical variables. Linear regression analysis was used to evaluate the association of plasma sphingomyelin levels and traditional cardiovascular risk factors. Kaplan-Meier analysis and log-rank tests were used to compare the event-free survival rates for incident CHD events among those with greater than versus at or less than sex-specific median plasma sphingomyelin levels. Cox proportional hazard models were used to evaluate the association between plasma sphingomyelin levels treated as a continuous variable and event-free survival after adjusting for potential confounding variables. The covariates were selected based on prior evidence of an association with CHD events from previous studies and statistical evidence of a univariate association with the primary outcome in the current study (a priori P≤0.20). The covariates included age, sex, diabetes mellitus, systolic and diastolic blood pressure, total and HDL cholesterol, triglycerides, smoking status, use of 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors, and blood pressure medication use. The extended Cox proportional hazard model, using the time-dependent variable approach, was used to test for the proportionality assumption. The predictive value of plasma sphingomyelin levels for the various components of the composite outcome was also explored.
A 2-tailed value of P<0.05 was considered significant. Statistical analysis was performed using commercially available software (SAS version 9.1; SAS Institute, Cary, NC).
All of the 6809 participants (of the total 6814 MESA participants) who had their plasma sphingomyelin level measured at baseline were included in the analysis. A total of 189 subjects (2.8%) subjects had an adjudicated CHD event during the 5 years of follow-up. The mean age of the cohort at baseline was 62.2 years; 52.8% were female; 38.5% were white, 11.8% were Chinese, 27.8% were black, and 21.9% were Hispanic. The mean±SD of plasma sphingomyelin levels for the MESA cohort was 48.0±16.0 mg/dL (range, 17–309 mg/dL). Table 1 shows the baseline characteristics of subjects with plasma sphingomyelin levels greater than and at or less than the sex-specific median. There was no significant difference between the mean plasma sphingomyelin levels of subjects who had an event and those who did not have an event after 5 years of follow-up (mean±SD, 46.03±14.40 mg/dL [n=189] versus 48.06±15.70 mg/dL [n=6620]; P=0.10).
Associations of Plasma Sphingomyelin With Traditional Cardiovascular Risk Factors
Age, body mass index, systolic blood pressure, and total cholesterol and triglycerides levels were positively associated with plasma sphingomyelin level. The diastolic blood pressure and cigarette smoking were negatively associated with plasma sphingomyelin level. Diabetes mellitus was not associated with plasma sphingomyelin level (Table 2).
In the Kaplan-Meier analysis, subjects with plasma sphingomyelin levels greater than the sex-specific median had similar event-free survival rates compared with subjects with plasma sphingomyelin levels at or less than the sex-specific median (97.16% versus 97.00%; log-rank P=0.71) (Figure). In the univariate Cox proportional hazard analysis, age, male sex, HDL cholesterol and triglycerides levels, diabetes mellitus, cigarette smoking status, systolic and diastolic blood pressure, 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitor use, and blood pressure medication use were significant predictors of an incident CHD event (Table 3). Plasma sphingomyelin level was not a significant predictor of an incident CHD event (hazard ratio, 0.992; 95% CI, 0.982 to 1.004; P=0.09).
A higher plasma sphingomyelin level was modestly associated with a lower risk of incident CHD events in the multistage multivariate Cox proportional hazard analysis when adjusted for age (hazard ratio, 0.987; 95% CI, 0.977 to 0.997; P=0.01); sex and total cholesterol, triglycerides, and HDL cholesterol levels (hazard ratio, 0.985; 95% CI, 0.973 to 0.998; P=0.008); and age, sex, total cholesterol, HDL, and triglycerides levels, diabetes mellitus, cigarette smoking, systolic and diastolic blood pressures, 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitor use, and blood pressure medication use (full model) (hazard ratio, 0.984; 95% CI, 0.973 to 0.996; P=0.002) (Table 4). Forcing race/ethnicity into our final model did not change the direction or the estimates of the hazard ratios. In other models, plasma sphingomyelin was not a significant predictor of incident CHD events after adjusting for the following variables (among others): age and sex (hazard ratio, 0.994; 95% CI, 0.984 to 1.004; P=0.24); and age, sex, and race (hazard ratio, 0.993; 95% CI, 0.983 to 1.004; P=0.20) (Table 4). No significant interactions were observed in all our models.
A higher sphingomyelin level showed modest negative associations with the components of the composite outcome, such as MI, angina, and hard CHD-related events (Table 5). A higher sphingomyelin level was not associated with increased incident CHD-related events when the full multivariate Cox proportional hazard model was stratified by race (Table 6).
The goal of this large, prospective, multiethnic study was to evaluate the hypothesis that a higher plasma sphingomyelin level is associated with high incident CHD events in population-based adults. We found no association between high plasma sphingomyelin levels and high incident CHD events. In our multivariate models, a higher plasma sphingomyelin level was associated with a modest reduction in incident CHD outcomes, contrary to prior results in humans.13
Current data from cell culture and animal studies and from human population studies support the hypothesis that plasma sphingomyelin participates in the development and progression of atherosclerosis. Numerous animal studies1,3 have associated high sphingomyelin levels with the progression of atherosclerosis. Cross-sectional data from MESA showed that high plasma sphingomyelin levels are associated with increased carotid intima media thickness, coronary calcium score, and ankle-brachial index.11 In a case-control study, Jiang et al12 showed that a high plasma sphingomyelin level is associated with angiographically defined coronary artery disease. In a subsequent publication,13 a high plasma sphingomyelin level was associated with coronary artery disease and worse outcomes in subjects with acute coronary syndromes. The finding of the present study is consistent with no association between plasma sphingomyelin levels and the progression of atherosclerosis to clinical CHD events.
Reverse causality, a hypothesis that was evoked by some researchers16,17 to explain the association between plasma homocysteine level and CVD, may help explain the findings of prior studies11–13 on the association of plasma sphingomyelin level and CVD and the result of the present study. In case-control studies by Jiang et al12 and Schlitt et al,13 subjects with coronary artery disease (a high atherosclerotic burden) had a high plasma sphingomyelin level compared with control subjects (who had a low atherosclerotic burden). In the study by Nelson et al,11 increased measures of subclinical atherosclerosis were associated with high plasma sphingomyelin levels, consistent with the findings of the case-control studies.12,13 However, the present prospective study examines the association between plasma sphingomyelin levels and incident CHD events in a population without clinical CVD at baseline. At baseline,11 even though the extent/degree of subclinical atherosclerosis was associated with plasma sphingomyelin levels in the MESA cohort, and subclinical measures of atherosclerosis are strongly associated with clinical CHD events,18 the plasma sphingomyelin level has no association with clinical CHD events. Thus, plasma sphingomyelin levels have no influence on the development or progression of atherosclerosis; rather, the burden of atherosclerosis influences plasma levels of sphingomyelin and, therefore, plasma sphingomyelin levels have no effect on clinical CHD events.
Current data also suggest that much of the sphingomyelin in human and animal atherosclerotic plaques is synthesized de novo. Almost all the current data associating sphingomyelin with atherosclerosis (clinical or subclinical) in humans used plasma sphingomyelin levels.11–13 Much of the promising data showing regression of atherosclerotic plaques in animals used inhibitors of sphingomyelin synthases. These sphingomyelin synthase inhibitors may affect both the de novo synthesis of sphingomyelin in atherosclerotic plaques and in plasma.19,20 The relationship between plasma sphingomyelin and the sphingomyelin in atherosclerotic plaques is not clearly defined. Thus, the beneficial effect of these sphingomyelin synthase inhibitors in animals may have been due to the reduction of sphingomyelin synthesis in the de novo pathway and not necessarily in plasma. Therefore, despite the null association between plasma sphingomyelin and incident CHD events in the present study, the de novo–synthesized sphingomyelin levels in the arterial wall may be associated with incident CHD events. Studies evaluating the association between plasma sphingomyelin and sphingomyelin levels in atherosclerotic plaques are needed.
The present study has the following limitations. The plasma sphingomyelin level was measured once in the MESA cohort, as in prior human studies. The intrasubject variability of plasma sphingomyelin level and the determinants of the variability of plasma sphingomyelin level over time have not been well studied. Thus, a single measurement of plasma sphingomyelin level may be a poor measure of an individual’s true biological levels and multiple measurements over time may be needed to improve its accuracy. The MESA cohort is also a relatively healthy cohort; only 2.8% of the cohort had an adjudicated event during the 5 years of follow-up. It is possible that the few events may have affected the findings of the present study. Finally, plasma sphingomyelin level is a relatively new biomarker. Therefore, not all the factors or markers that influences its biological levels are known. It is unlikely, but possible, that the findings of the present study may be the result of residual confounding.
In conclusion, the plasma sphingomyelin level is not associated with increased risk of incident CHD events in population-based adults free of clinical CVD at baseline. In our multivariate Cox proportional hazard model, a high plasma sphingomyelin level was associated with a modest reduction in incident CHD events, contrary to prior published data. Studies evaluating the de novo synthesis of sphingomyelin in arterial walls or atherosclerotic plaques and its association with clinical and subclinical CVD are needed.
Sources of Funding
This study was supported by contracts N01-HC-95159 through N01-HC-95166 and N01-HC-95169 and grant NHLBI T32 HL-07355 from the National Heart, Lung, and Blood Institute, Bethesda, Md.
Received October 20, 2009; accepted December 9, 2009.
Tabas I. Sphingolipids and atherosclerosis: a mechanistic connection? a therapeutic opportunity? Circulation. 2004; 10: 3400–3401.
Schissel SL, Tweedie-Hardman J, Rapp JH, Graham G, Williams KJ, Tabas I. Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein: proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. J Clin Invest. 1996; 98: 1455–1464.
Devlin CM, Leventhal AR, Kuriakose G, Schuchman EH, Williams KJ, Tabas I. Acid sphingomyelinase promotes lipoprotein retention within early atheromata and accelerates lesion progression. Arterioscler Thromb Vasc Biol. 2008; 28: 1723–1730.
Park TS, Panek RL, Mueller SB, Hanselman JC, Rosebury WS, Robertson AW, Kindt EK, Homan R, Karathanasis SK, Rekhter MD. Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice. Circulation. 2004; 110: 3465–3471.
Hojjati MR, Li Z, Zhou H, Tang S, Huan C, Ooi E, Lu S, Jiang XC. Effect of myriocin on plasma sphingolipid metabolism and atherosclerosis in apoE-deficient mice. J Biol Chem. 2005; 280: 10284–10289.
Mukhin DN, Chao FF, Kruth HS. Glycosphingolipid accumulation in the aortic wall is another feature of human atherosclerosis. Arterioscler Thromb Vasc Biol. 1995; 15: 1607–1615.
Nelson JC, Jiang XC, Tabas I, Tall A, Shea S. Plasma sphingomyelin and subclinical atherosclerosis: findings from the Multi-Ethnic Study of Atherosclerosis. Am J Epidemiol. 2006; 163: 903–912.
Jiang XC, Paultre F, Pearson TA, Reed RG, Francis CK, Lin M, Berglund L, Tall AR. Plasma sphingomyelin level as a risk factor for coronary artery disease. Arterioscler Thromb Vasc Biol. 2000; 20: 2614–2618.
Schlitt A, Blankenberg S, Yan D, von Gizycki H, Buerke M, Werdan K, Bickel C, Lackner KJ, Meyer J, Rupprecht HJ, Jiang XC. Further evaluation of plasma sphingomyelin levels as a risk factor for coronary artery disease. Nutr Metab (Lond). 2006; 5: 3–5.
Bild DE, Bluemke DA, Burke GL, Detrano R, Diez Roux AV, Folsom AR, Greenland P, Jacob DR Jr, Kronmal R, Liu K, Nelson JC, O'Leary D, Saad MF, Shea S, Szklo M, Tracy RP. Multi-Ethnic Study of Atherosclerosis: objectives and design. Am J Epidemiol. 2002; 156: 871–881.
Friedewald WF, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge. Clin Chem. 1972; 18: 499–502.
Boston AG, Selhub J. Homocysteine and atherosclerosis: subclinical and clinical disease association. Circulation. 1999; 99: 2361–2363.
Libby P, Theroux P. Pathophysiology of coronary artery disease. Circulation. 2005; 111: 3481–3488.
Liu J, Huan C, Chakraborty M, Zhang H, Lu D, Kuo MS, Cao G, Jiang XC. Macrophage sphingomyelin synthase 2 deficiency decreases atherosclerosis in mice. Circ Res. 2009; 105: 295–303.
Liu J, Zhang H, Li Z, Hailemariam TK, Chakraborty M, Jiang K, Qui D, Bui HH, Peake DA, Kuo MS, Wadgaonkar R, Cao G, Jiang XC. Sphingomyelin synthase 2 is one of the determinants for plasma and liver sphingomyelin levels in mice. Arterioscler Thromb Vasc Biol. 2009; 29: 850–856.