Low-density lipoprotein (LDL) cholesterol is the primary measure of atherosclerotic risk and thus a therapeutic target in clinical practice. The focus on LDL cholesterol and its control by diet, lifestyle, and pharmaceuticals has contributed to a significant and sustained reduction in blood concentrations of total and LDL cholesterol among U.S. adults since 1988. Despite population-wide improvements in lipid levels, heart disease remains the No. 1 cause of death both within the U.S. and globally. Consequently, there is an ongoing search for risk factors to help identify and treat patients prior to the development of symptomatic heart disease.
Atherosclerosis begins when lipoproteins infiltrate the vascular intima, drawing monocytes across the endothelium. Phagocytosis of lipoproteins by monocytes creates lipid-bloated foam cells, which release cytokines and effector molecules that promote myocyte migration while upregulating endothelial cell adhesion and platelet activation proteins and disrupting vasodilation mechanisms. Ceramides increase LDL infiltration and promote LDL aggregation (A), are upregulated in response to inflammatory cytokines (B), are enriched in atherosclerotic plaque (C), and increase platelet activation while disrupting endothelial function (D).
Traditional risk factors for atherosclerosis include elevated body mass index, hypertension, smoking, and increased blood cholesterol. Current guidelines endorse focusing on circulating cholesterol and nonspecific inflammatory markers as biomarkers for atherosclerosis. However, the pathophysiology of atherosclerosis is a complex intersection of dyslipidemia, inflammation, endothelial dysfunction, and platelet activation. Recent data support causal associations between each of these pathways and plasma ceramides.
Plasma Ceramides: A Multifaceted Risk Marker
"Ceramides are complex lipids that play a central role in cell membrane integrity, cellular stress response, inflammatory signaling, and apoptosis," says Jeffrey (Jeff) Meeusen, Ph.D., Co-Director of Cardiovascular Laboratory Medicine in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. Synthesis of ceramides from saturated fats and sphingosine occurs in all tissues. During dyslipidemia and caloric excess, ceramides are synthesized de novo and accumulate in tissues not suited for fat storage.
Low-density lipoprotein, infamous as the carrier of "bad" cholesterol, also transports ceramides in the blood. Ceramides promote LDL infiltration into the vessel wall and enrich LDL fifty-fold within arterial plaque. The inflammatory cytokines interferon-γ, tumor necrosis factor-α (TNFα), and interleukin-1ß all stimulate ceramide synthesis. And, ceramides are implicated in platelet activation and endothelial dysfunction via uncoupling of nitric oxide signaling pathways.
In addition to the biochemical role in atherosclerosis progression, plasma concentrations of ceramides are elevated in several heart-disease-related conditions. Plasma ceramides are significantly elevated among patients with stage 3 hypertension. Plasma ceramide concentrations correlate with an increased New York Heart Association functional class in a study of 423 patients hospitalized for heart failure. Patients with type 2 diabetes mellitus have significantly elevated plasma ceramide concentrations. Furthermore, elevated ceramides correlate positively with insulin resistance.
Ceramides Predict Clinical Outcomes
Untargeted metabolomic analysis identified three plasma ceramides as significantly linked to cardiovascular mortality in a cohort with coronary artery stenosis confirmed by angiography. A total of 258 patients suffered a fatal myocardial infarct within three years. The ceramides linked with cardiovascular mortality were N-palmitoyl-sphingosine [Ceramide (16:0)], N-stearoyl-sphingosine [Ceramide (18:0)], and N nervonoyl-sphingosine [Ceramide (24:1)]. The association was independent of age, body-mass index, smoking status, statin use, triglycerides, LDL, and total cholesterol. Additional predictive value was found when ceramides were normalized to N-lignoceroyl-sphingosine [Ceramide (24:0)], a highly abundant plasma ceramide not influenced by disease.
Independent studies performed at Mayo Clinic verified that targeted measurement of these ceramides could be performed with accuracy and precision suitable for clinical application. The clinical utility of these ceramides for predicting risk of cardiovascular disease was confirmed in a follow-up study of patients referred for coronary angiography. Cardiologists at Mayo Clinic have begun using plasma ceramides in the clinic and are continuing to investigate additional applications.
Multiple published studies have repeatedly confirmed the strong predictive value of ceramides. Risk conferred by plasma ceramides is independent of traditional risk factors including age, sex, body-mass index, smoking status, and blood cholesterol. Additionally, the predictive value remains significant after adjusting for other markers such as C-reactive protein (CRP), apolipoprotein B (ApoB), and lipoprotein-associated phospholipase A2 (Lp-PLA2).
Interpreting Elevated Ceramides
New risk factors for heart disease are proposed regularly. When evaluating the potential of a new risk factor, several aspects must be considered. The first is whether the new risk marker provides new information independent of established risk factors. Plasma ceramides are able to stratify risk among patients even after adjustment for multiple traditional and contemporary risk factors.
A second consideration for new biomarkers is their practical application in the clinic. Three plasma ceramides and each of their ratios to a fourth ceramide are all independently linked to increased hazard ratios. Thus, there are a total of six results all predictive of cardiovascular disease. While this may be intriguing on an academic level, it allows for the potential of confusion regarding risk in clinical practice. This prompted development of a ceramide risk score.
"The ceramide risk score incorporates the values from all six ceramide results into a clearly defined risk category," says Dr. Meeusen. One or two points are added to the score for each result above the median or the third quartile, respectively. Thus, the potential risk attributable to ceramides is summarized on a 12-point scale. Applying the ceramide risk score to two large observational studies (>1,500 each) revealed that patients with a score of 10–12 had a four- to six-fold increase in the rate of events compared with patients with a score ≤2 points.
Caveats Associated with Novel Testing
Some data suggest that ceramides can be elevated in response to inflammation. Therefore, it is conceivable that individuals with infections or other inflammatory diseases may have elevations for that reason, which may or may not be indicative of unstable coronary artery disease. Thus, if the inflammatory state is transient, a repeat ceramide measure after resolution may be prudent. The link between inflammatory diseases and atherosclerosis is currently being investigated, and ceramides are no exception. We will update this recommendation when more data are available.
"We have corrected our local data for a large number of other variables and biomarkers, and the increased risk conferred by ceramides is maintained," says Dr. Meeusen. "There is more still to do, and we will continue to add covariates and interrogate different populations. We are optimistic based on unpublished data being gathered that the independent predictive utility of ceramides will be maintained."
Ceramides Are Modifiable
Finally, a biomarker is only useful in the clinic when it is able to guide effective interventions. Randomized clinical trials based on ceramide measures have not been reported. However, several reports have found that ceramide concentrations are significantly decreased by caloric restriction, gastric bypass, aerobic exercise, and statin therapy. A significant decrease in plasma ceramides was observed among subjects taking simvastatin (alone or in combination with ezetimibe) and rosuvastatin. Despite the paucity of outcome studies, these data are promising in that therapies already known to be effective at reducing risk of heart disease are also able to modify plasma ceramide concentrations.
In conclusion, plasma ceramides are a promising new clinical diagnostic for the identification of patients at risk of adverse cardiovascular events. Testing for plasma ceramides is available to Mayo Clinic patients and health care providers worldwide through Mayo Clinic Laboratories, the reference laboratory of Mayo Clinic. The lab offers advanced laboratory testing and pathology services to more than 4,000 health care organizations in more than 70 countries.
This article was originally published in Mayo Clinic's Medical Professionals Clinical Updates.
Jeff Meeusen, Ph.D., is a clinical chemist and Co-Director of Cardiovascular Laboratory Medicine at Mayo Clinic, Rochester, Minnesota.