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Aims Peripheral arterial disease (PAD) and coronary artery disease (CAD) are both caused by atherosclerosis. Serum lipids and lipoproteins are predictive of the development of atherosclerosis but it is not clear if they differ in the two manifestations PAD and CAD. We tested whether a more detailed characterization of the lipid and lipoprotein patterns of PAD and CAD allows a clear differentiation between the two atherosclerotic phenotypes. Methods A cohort of 274 statin-naive patients with either newly diagnosed imaging proven PAD (n = 89) or stable CAD (n = 185) was characterized using NMR- and LC-MS/MS-based advanced lipid and lipoprotein analysis. An independent cohort of 1239 patients with PAD and CAD was used for validation. Results We found a significant difference in markers of inflammation as well as ceramide and phosphatidylcholine levels between PAD and CAD patients. In contrast, basic lipid markers including total cholesterol, low density lipoprotein cholesterol, high density lipoprotein-cholesterol, lipoprotein (a) or detailed lipoprotein profiles did not differ significantly between PAD and CAD patients. Applying ratios and scores derived from ceramides and phosphatidylcholines further improved the discrimination between PAD and CAD. These significant differences were independent from body composition, from the status of smoking or T2DM, and also from apolipoprotein C-III and other inflammatory parameters which were different between CAD and PAD. Conclusion The present study clearly suggests that PAD and CAD differ in terms of their ceramide- and phosphatidylcholine-based lipid pattern but not in lipoprotein characteristics. This article is protected by copyright. All rights reserved.

AimCholesterol-based risk prediction is often insufficient in cardiovascular disease (CVD) patients. Ceramides are a new kind of biomarkers for CVD. The Coronary Event Risk Test (CERT) is a validated cardiovascular risk predictor that uses only circulating ceramide levels, determined by coupled liquid chromatography–mass spectrometry, to allocate patients into one of four risk categories. This test has recently been modified (CERT2) by additionally including phosphatidylcholine levels.Methods and resultsIn this observational cohort study, we have recruited 999 Austrian patients with CVD and followed them for up to 13 years. We found that CERT and CERT2 both predicted cardiovascular events, cardiovascular mortality, and overall mortality. CERT2 had the higher performance compared to CERT and also to the recent cardiovascular risk score of the ESC/EAS guidelines (Systematic COronary Risk Evaluation (SCORE)) for low-risk European countries. Combining CERT2 with the ESC/EAS-SCORE, predictive capacity was further increased leading to a hazard ratio of 3.58 (2.02–6.36; P ConclusionWe conclude that CERT and CERT2 are powerful predictors of cardiovascular events, cardiovascular mortality, and overall mortality in CVD patients. Including phosphatidylcholine to a ceramide-based score increases the predictive performance and is best in combination with classical risk factors as used in the ESC/EAS-SCORE.

Serum lipids and metabolic diseases, in particular type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD) , predict the atherosclerotic diseases coronary artery disease (CAD) and peripheral arterial disease (PAD) . However, it is not known in how far a more detailed characterization including serum lipids improves discrimination of PAD from CAD. A cohort of 274 statin-naïve patients with either PAD (n = 89) or stable CAD (n = 185) were referred to metabolic screening and were characterized using nuclear magnetic resonance- and liquid chromatography-tandem mass spectrometry based advanced lipid and lipoprotein analysis. Results were validated in an independent cohort of 1239 patients with PAD or CAD. We found a significant difference in T2D prevalence and in the ceramide-based lipid profile between PAD and CAD patients. However, neither cholesterol-based markers (including LDL-C, HDL-C) and detailed lipoprotein profiles nor the NAFLD status differed significantly between PAD and CAD patients (figure) . The difference between ceramide-based lipid profiles of CAD and PAD remained significant also after adjusting for body composition, smoking, inflammatory parameters, and T2D. We conclude that PAD and CAD differ in ceramide-based lipid profiles and T2D status, but not in other lipid characteristics or metabolic diseases. Disclosure A.Leiherer: None. A.Jylha: None. M.Laaperi: None. R.Laaksonen: None. W.Maerz: None. P.Fraunberger: None. M.Kleber: None. H.Drexel: None. A.Muendlein: None. E.Brandtner: None. C.H.Saely: None. A.Vonbank: None. A.Mader: None. L.Sprenger: None. M.Maechler: None. B.Larcher: None.

Backgroundassessing cardiovascular and mortality risk with conventional biomarkers is challenging in oldest-old due to multimorbidity and polypharmacy. Ceramides are bioactive lipids shown to predict mortality in late middle-aged cohorts.Objectiveto assess whether plasma ceramides have independent prognostic value for mortality among oldest-old (85+).Designlongitudinal cohort study (Helsinki Businessmen Study, HBS) with a 3.5-year follow-up.Setting and subjectssurvivors of HBS (125 men born in 1919–1934) visited the clinic for laboratory and clinical examination.Methodsfunctional status including physical (short physical performance battery) and Montreal Cognitive Assessment (MoCA) cognitive performance was assessed and laboratory examinations included a large set of biomarkers. Plasma ceramide concentration (Cer(d18:1/16:0)) was measured using a targeted liquid chromatography–tandem mass spectrometry assay. Mortality was retrieved from national registers.Resultsmedian age was 88 years, two-thirds had multimorbidity and 59% were on statin treatment. During the follow-up, 22 (18%) men died. In a model adjusted for variables associated with mortality in the whole cohort at P Conclusionsthese data raise the hypothesis that plasma ceramide concentrations and especially Cer(d18:1/1:60) may offer a clinically useful biomarker to evaluate prognosis in very old age. Such biomarkers are needed for geriatrics, where multimorbidity and pharmacotherapies, such as statins are prevalent hampering assessment of prognosis using conventional methods.

AimsCeramide (Cer) and phosphatidylcholine (PC) lipids are associated with pathophysiological processes in cardiovascular (CV) diseases. A previously derived and validated plasma Cer-PC risk score (CERT2) was associated with CV death risk in patients with stable disease, but its prognostic value has not been evaluated in patients early post-acute coronary syndrome (ACS).Methods and resultsPrespecified plasma Cer and PC species in the CERT2 risk score were measured in 4871 subjects from SOLID-TIMI 52, which enrolled patients ≤30 days after ACS (median follow-up 2.5 years). The CERT2 score (scale 0–12 points) was calculated as previously defined. The primary outcome was CV death; Coronary heart disease death, all-cause death, hospitalization for heart failure (HF), myocardial infarction (MI) and stroke were also analyzed. Poisson models included baseline characteristics and established biomarkers. Patients with higher CERT2 risk scores were more likely to be older, female, current smokers, presenting with STEMI, and to have impaired renal function and higher LDL-C. After multivariable adjustment, patients in the highest risk score category remained at a nearly two-fold higher risk of CV death (adj relative risk [RR] 1.92, 95% CI 1.01–3.66, P = 0.047). Patients in the highest risk score category were also at higher risk of all-cause death (adj RR 2.01, 95% CI 1.21–3.35, P = 0.007), whereas the relationships with HF, MI, and stroke were attenuated with multivariable adjustment.ConclusionsA plasma ceramide and phospholipid-based risk score is associated with the risk of CV death independent of established clinical risk factors and biomarkers in patients after ACS.

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WOS:000583273800010 PubMed ID: 33122478 Objective: TCF7L2 is a repressor and transactivator of genes, and its variants are strongly associated with diabetes. This study aimed to evaluate the sex-specific relationship between the most common TCF7L2 gene variants (-98368G>T, rs12255372 and -47833C>T, rs7903146) with diabetes and coronary heart disease in Turkish Adult Risk Factor (TARF) Study. Methods: Single nucleotide variants (SNVs) have been genotyped using the TaqMan allelic discrimination assays in 2,024 (51.3% in women, age: 55 +/- 11.8) Turkish adults participating in the TARF study. Statistical analyses were used to investigate the association of genotypes with clinical and biochemical measurements. Results: Among the TARF study participants, 11.7%, 24.3%, 14.1%, and 38.3% had diabetes, hypertension, coronary heart disease (CHD), and obesity, respectively. The frequencies of T allele for -47833C>T and -98368G>T in Turkish adults were determined to be 0.35 and 0.33, respectively. -47833C>T was significantly associated with higher fasting glucose concentrations in all participants, especially in men. Both SNVs were significantly associated with diabetes and CHD in all participants (pT was associated with diabetes in women (p=0.041) and -47833C>T was associated with diabetes and CHD in men (p=0.018 and p=0.032, respectively). Also, both SNVs and the diplotypes of common haplotype (H1) remained strongly associated with type 2 diabetes after risk factors were adjusted (p

Aims Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular complications, but the molecular mechanism of action is unknown. We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA. Methods and results Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two tumour necrosis factor alpha (TNFα) transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly up-regulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients. Conclusions Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL, and Arg2 levels.

Background: Trimethylamine N-oxide (TMAO) is an amine oxide generated by gut microbial metabolism. TMAO may contribute to atherothrombosis and systemic inflammation. However, the prognostic value of circulating TMAO for risk stratification is uncertain. Methods: We assessed prospective relationships of plasma TMAO with long-term risk of all-cause, cardiovascular (CV), and non-CV mortality in the Western Norway Coronary Angiography Cohort (WECAC; 4132 patients with suspected coronary artery disease) and the Hordaland Health Study (HUSK; 6393 community-based subjects). Risk associations were examined using Cox regression analyses. Results: Mean follow-up was 9.8 and 10.5 years in WECAC and HUSK, respectively. Following adjustments for established CV risk factors and indices of renal function in WECAC, the hazard ratios (HRs) (95% confidence intervals [CIs]) per one standard deviation increase in log-transformed plasma TMAO were 1.04 (0.97–1.12), 1.06 (0.95–1.18), and 1.03 (0.93–1.13) for all-cause, CV, and non-CV mortality, respectively. Essentially similar results were obtained in patients with angiographically significant coronary artery disease and patients with reduced left ventricular ejection fraction. Corresponding HRs (95% CIs) in the HUSK cohort were 1.03 (0.96–1.10), 1.01 (0.89–1.13), and 1.03 (0.95–1.12) for all-cause-, CV, and non-CV mortality, respectively. Conclusions: Circulating TMAO did not predict long-term all-cause, CV, or non-CV mortality in patients with coronary heart disease or in community-based adults. This large study does not support a role of TMAO for patient risk stratification in primary or secondary prevention. publishedVersion

Aims A risk score, CERT2, based on distinct ceramide and phosphatidylcholine lipid species, has shown robust performance in predicting cardiovascular risk in secondary prevention. Here, our aim was to investigate the predictive value of CERT2 in primary prevention compared to classical lipid biomarkers and its compatibility with clinical characteristics used in the SCORE risk chart. Methods and results Four ceramides [Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), Cer(d18:1/24:1)] and three phosphatidylcholines [PC(14:0/22:6), PC(16:0/22:5), PC(16:0/16:0)] were analysed by targeted tandem liquid chromatography–mass spectrometry method in FINRISK 2002, which is a population-based risk factor survey investigating men and women aged 25–74 years. Primary prevention subjects (N = 7324) were followed up for 10 years for the following outcomes: incident coronary heart disease (CHD), cardiovascular disease (CVD), major adverse cardiovascular event (MACE), stroke, and heart failure. Hazard ratios per standard deviation obtained from adjusted Cox proportional hazard models were significant for all these endpoints, and the highest for fatal ones, i.e. fatal CHD [1.45 (95% confidence interval 1.07–1.97)], CVD [1.39 (1.06–1.83)], and MACE [1.39 (1.07–1.80)]. The categorical net reclassification improvement was 0.051 for the 10-year risk of incident CVD. Incidence of fatal events was over 10-fold more frequent in the highest CERT2 category compared to the lowest risk category and modified SCORE risk charts, utilizing CERT2 and diabetes mellitus, increased granularity of risk assessment compared to a chart utilizing total cholesterol. Conclusion CERT2 is a significant predictor of incident cardiovascular outcomes and risk charts utilizing this score provide an easy tool to estimate relative and absolute risk for incident CVD.

Introduction: The recently introduced Coronary Event Risk Test version 2 (CERT2) is a validated cardiovascular risk predictor score that uses circulating ceramide and phosphatidylcholine concentrations. Hypothesis: We hypothesize that CERT2 hat the power to predict cardiovascular mortality in patients with type 2 diabetes (T2DM). Methods: Prospectively, out of 280 male and 121 female patients with T2DM we recorded 55 cardiovascular deaths in men and 19 in women during a mean follow-up time of 7.6±3.6 and 8.1±3.4 years respectively. Results: Overall, cardiovascular survival decreased with increasing CERT2 risk categories (figure 1). In Cox regression models, CERT2 significantly predicted the incidence of cardiovascular mortality in male patients with T2DM (unadj. HR 1.82 [1.39-2.37] per standard deviation; p Conclusions: We conclude that sex does not significantly impact the association of CERT2 with cardiovascular mortality in patients with T2DM.

Background and aims: Ceramides have been identified as novel biomarkers for cardiovascular disease (CVD) related events and mortality but their role in etiology of subclinical atherosclerosis is unknown. We aimed to assess association between plasma ceramides and carotid intima-media thickness (CIMT) and evaluate predictive value of the ceramides for high CIMT over traditional CVD risk factors. Methods: Association between plasma ceramides and CIMT in the Young Finns Study participants was analyzed with CIMT as outcome and ceramides along with traditional risk factors as predictors with regression model. Predictive value of the ceramides and related coronary event risk test (CERT) score for high CIMT as surrogate marker of subclinical atherosclerosis was assessed by comparing logistic regression-based prediction models including, i) traditional risk factors and ceramides, ii) traditional risk factors and CERT score, iii) age, sex and ceramides or alternatively CERT score with a reference model including only traditional risk factors. The prediction models were fitted to training data (70% data) and tested on test data (30% data). The predictive models were assessed with area under the receiver operating curve (AUC). The variance of AUC was estimated by repeating the model fitting and testing for 1000 bootstraps of the original data. Results: Predictive models with plasma ceramides or alternatively with CERT score in addition to age and sex variables were able to predict high CIMT with AUC 0.726 and 0.720 respectively. However, the ceramides and CERT score did not have statistically significant added predictive value for high CIMT over traditional risk factors. Conclusions: The new systemic biomarkers, high-risk plasma ceramides and CERT score, showed promising predictive performance for high CIMT with only age and sex as additional variables. This may help in predicting subclinical atherosclerosis for primary prevention. publishedVersion

The recently introduced Coronary Event Risk Test version 2 (CERT2) is a validated cardiovascular risk predictor score that uses circulating ceramide and phosphatidylcholine concentrations. The purpose of this study was to investigate the power of CERT2 to predict cardiovascular mortality in 280 male and 121 female patients with type 2 diabetes (T2DM). Prospectively, we recorded 55 cardiovascular deaths in men and 19 in women during a mean follow-up time of 7.6±3.6 and 8.1±3.4 years respectively. Overall, cardiovascular survival decreased with increasing CERT2 risk categories (figure 1). In Cox regression models, CERT2 significantly predicted the incidence of cardiovascular mortality in male patients with T2DM (unadj. HR 1.82 [1.39–2.37] per standard deviation; p We conclude that sex does not significantly impact the association of CERT2 with cardiovascular mortality in patients with T2DM. Funding Acknowledgement Type of funding sources: None. Figure 1

Diet is a major modifiable risk factor for cardiovascular disease (CVD). One explanation for this is its effect on specific lipids. However, knowledge on how the lipidome is affected is limited. We aimed to investigate if diet can change the new ceramide- and phospholipid-based CVD risk score CERT2 and the serum lipidome towards a more favorable CVD signature. In a crossover trial (ADIRA), 50 patients with rheumatoid arthritis (RA) had 10 weeks of a Mediterranean-style diet intervention or a Western-style control diet and then switched diets after a 4-month wash-out-period. Five hundred and thirty-eight individual lipids were measured in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipid risk scores were analyzed by Wilcoxon signed-rank test or mixed model and lipidomic data with multivariate statistical methods. In the main analysis, including the 46 participants completing ≥1 diet period, there was no significant difference in CERT2 after the intervention compared with the control, although several CERT2 components were changed within periods. In addition, triacylglycerols, cholesteryl esters, phosphatidylcholines, alkylphosphatidylcholines and alkenylphosphatidylcholines had a healthier composition after the intervention compared to after the control diet. This trial indicates that certain dietary changes can improve the serum lipid signature towards a less atherogenic profile in patients with RA.

Aims Trimethyllysine (TML) is involved in carnitine synthesis, serves as a precursor of trimethylamine N-oxide (TMAO) and is associated with cardiovascular events in patients with established coronary heart disease (CHD). We prospectively examined circulating TML as a predictor of all-cause and cardiovascular mortality in community-dwelling adults and patients with CHD. Methods and results By Cox regression modelling, risk associations were examined in 6393 subjects in the community-based Hordaland Health Study (HUSK). A replication study was conducted among 4117 patients with suspected stable angina pectoris in the Western Norway Coronary Angiography Cohort (WECAC). During a mean follow-up of 10.5 years in the HUSK-cohort, 884 (13.8%) subjects died, of whom 287 from cardiovascular causes. After multivariable adjustments for traditional cardiovascular risk factors, the hazard ratio (HR) [95% confidence interval (95% CI)] for all-cause mortality comparing the 4th vs. 1st TML-quartile was 1.66 (1.31–2.10, P Conclusions Elevated TML-levels were associated with increased risk of all-cause and cardiovascular mortality both in subjects with and without established CHD.

The recently introduced Coronary Event Risk Test version 2 (CERT2) is a validated cardiovascular risk predictor score that uses circulating ceramide and phosphatidylcholine concentrations. We here aimed at investigating the power of CERT2 to predict cardiovascular mortality in 280 male and 121 female patients with type 2 diabetes (T2DM). Prospectively, we recorded 55 cardiovascular deaths in men and 19 in women during a mean follow-up time of 7.6±3.6 and 8.1±3.4 years respectively. Overall, cardiovascular survival decreased with increasing CERT2 risk category (Figure), and cardiovascular mortality was higher in men than in women (20.1% vs. 15.8%; p=0.321). In Cox regression models, CERT2 significantly predicted the incidence of cardiovascular mortality in male patients with T2DM (unadj. HR 1.82 [1.39-2.37] per standard deviation; p Disclosure A. Leiherer: None. M. Laaperi: None. A. Jylha: None. P. Fraunberger: None. H. Drexel: None. A. Muendlein: None. C. H. Saely: None. B. Larcher: None. A. Mader: None. M. Maechler: None. L. Sprenger: None. B. Mutschlechner: None. R. Laaksonen: None.

Background and purpose Fenretinide, a synthetic retinoid derivative first investigated for cancer prevention and treatment, has been shown to ameliorate glucose tolerance, improve plasma lipid profile and reduce body fat mass. These effects, together with its ability to inhibit ceramide synthesis, suggest that fenretinide may have an anti-atherosclerotic action. Experimental approach To this aim, nine-week-old apoE-knockout (EKO) female mice were fed for twelve weeks a Western diet, without (control) or with (0.1% w/w) fenretinide. As a reference, wild-type (WT) mice were treated similarly. Growth and metabolic parameters were monitored throughout the study. Atherosclerosis development was evaluated in the aorta and at the aortic sinus. Blood and lymphoid organs were further characterized with thorough cytological/histological and immunocytofluorimetric analyses. Key results Fenretinide treatment significantly lowered body weight, glucose levels and plasma levels of total cholesterol, triglycerides, and phospholipids. In the liver, fenretinide remarkably reduced hepatic glycogenosis and steatosis driven by the Western diet. Treated spleens were abnormally enlarged, with severe follicular atrophy and massive extramedullary haematopoiesis. Severe renal hemosiderin deposition was observed in treated EKO mice. Treatment resulted in a threefold increase of total leukocytes (WT and EKO) and raised the activated/resting monocyte ratio in EKO mice. Finally, atherosclerosis development was markedly increased at the aortic arch, thoracic and abdominal aorta of fenretinide-treated mice. Conclusions and implications We provide the first evidence that, despite beneficial metabolic effects, fenretinide treatment may enhance the development of atherosclerosis.

BACKGROUND AND AIMS: High concentrations of low density lipoprotein (LDL) triglycerides have been associated with prevalent angiographic coronary artery disease. The present analysis was designed to investigate the association of LDL triglycerides with cardiovascular mortality and to explore possible mechanisms that may link LDL triglycerides to cardiovascular risk. METHODS: LDL triglycerides were measured in 3,140 participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. They were prospectively followed for cardiovascular mortality (median duration 9.9 years). Genome wide association data for LDL triglycerides were available for 2,900 LURIC participants. Genetic data and measurements of hepatic lipase activity were available for 478 participants of the HERITAGE Family study. Genome wide association data for cardiovascular disease were available for 184,305 participants of the CARDIoGRAMplusC4D consortium. RESULTS: There was a continuous positive association between LDL triglycerides and cardiovascular mortality (hazard ratio for 5(th) vs. 1(st) quintile = 2.53, p < 0.001) and this association was similar in males and females. Genome wide association analysis in LURIC revealed that LDL triglycerides were strongly associated with variation in the hepatic lipase region (p < 10(−15) for rs1800588 and rs10468017). The LDL triglyceride raising alleles in rs1800588 and rs10468017 were associated with low hepatic lipase activity in HERITAGE and increased cardiovascular risk in CARDIoGRAMplusC4D. Two-sample Mendelian randomization analysis (HERITAGE and CARDIoGRAMplusC4D) using rs1800588 and rs10468017 as instrumental variable suggested that low hepatic lipase activity may cause increased cardiovascular risk (p = 0.013). CONCLUSIONS: Low hepatic lipase activity may link high LDL triglycerides to increased cardiovascular risk.

Objective— It is known that specific plasma ceramides are associated with stress-induced reversible myocardial perfusion defects in patients with established or suspected coronary artery disease undergoing myocardial perfusion scintigraphy (MPS). However, it is currently uncertain whether plasma ceramides are also associated with reduced poststress myocardial perfusion in these patients. Approach and Results— We measured 6 previously identified high-risk plasma ceramide species (ceramide [d18:1/16:0], ceramide [d18:1/18:0], ceramide [d18:1/20:0], ceramide [d18:1/22:0], ceramide [d18:1/24:0], and ceramide [d18:1/24:1]) in 167 consecutive patients with established or suspected coronary artery disease undergoing stress MPS for clinical indications. Plasma ceramides were measured by a targeted liquid chromatography–tandem mass spectrometry assay both at baseline and after MPS. Multivariable linear regression analysis was undertaken to examine the associations (standardized B coefficients) between plasma ceramides and the percentage of poststress myocardial perfusion after adjustment for multiple cardiovascular risk factors. Seventy-eight patients had stress-induced myocardial ischemia on MPS (mainly located in the anteroapical wall). Of the 6 measured plasma ceramides, higher levels of basal ceramide (d18:1/18:0; B=−0.182; P =0.019), ceramide (d18:1/20:0; B=−0.224; P =0.004), ceramide (d18:1/22:0; B=−0.163; P =0.035), and ceramide (d18:1/24:1; B=−0.20; P =0.010) were associated with lower poststress anteroapical wall perfusion. Notably, these significant associations persisted even after adjustment for conventional cardiovascular risk factors, previous coronary artery disease, electrocardiographic left bundle branch block, left ventricular ejection fraction and type of stress testing. Similar results were observed for poststress plasma ceramides. Conclusions— Higher circulating levels of specific ceramides, both at baseline and after stress, were independently associated with lower poststress anteroapical wall perfusion in patients with suspected or established coronary artery disease referred for clinically indicated MPS.

Background The Global Registry of Acute Coronary Events (GRACE) score is an established clinical risk stratification tool for patients with acute coronary syndromes (ACS). We developed and internally validated a model for 1-year all-cause mortality prediction in ACS patients. Methods Between 2009 and 2012, 2’168 ACS patients were enrolled into the Swiss SPUM-ACS Cohort. Biomarkers were determined in 1’892 patients and follow-up was achieved in 95.8% of patients. 1-year all-cause mortality was 4.3% (n = 80). In our analysis we consider all linear models using combinations of 8 out of 56 variables to predict 1-year all-cause mortality and to derive a variable ranking. Results 1.3% of 1’420’494’075 models outperformed the GRACE 2.0 Score. The SPUM-ACS Score includes age, plasma glucose, NT-proBNP, left ventricular ejection fraction (LVEF), Killip class, history of peripheral artery disease (PAD), malignancy, and cardio-pulmonary resuscitation. For predicting 1-year mortality after ACS, the SPUM-ACS Score outperformed the GRACE 2.0 Score which achieves a 5-fold cross-validated AUC of 0.81 (95% CI 0.78–0.84). Ranking individual features according to their importance across all multivariate models revealed age, trimethylamine N-oxide, creatinine, history of PAD or malignancy, LVEF, and haemoglobin as the most relevant variables for predicting 1-year mortality. Conclusions The variable ranking and the selection for the SPUM-ACS Score highlight the relevance of age, markers of heart failure, and comorbidities for prediction of all-cause death. Before application, this score needs to be externally validated and refined in larger cohorts. Clinical Trial Registration NCT01000701.

Ceramides are sphingolipids that play roles as structural lipids and as second messengers in biological processes. Circulating ceramides are influenced by diet/food and predict major cardiovascular (CV) events, such as atrial fibrillation (AF). In 1227 patients with symptomatic chronic heart failure (HF), an association between diet and ceramides was found for coffee consumption of ≥3 cups and Cer(d18:1/24:0). Increased Cer(d18:1/24:0) was associated with lower incident AF (24.3% vs 15.4% tertile 1 vs 3, P = 0.016) and lower CV mortality (28.4% vs 12.0% tertile 1 vs 3, P < 0.0001). For coffee consumption, only an association with incident AF was found (24.5% never, 5.2% ≥3 cups). These inverse associations with AF were confirmed in survival analyses corrected for biomarkers (Cer(d18:1/24:0) HR: 0.79, P = 0.018; coffee consumption HR: 0.22, P = 0.001). In conclusion, higher coffee intake was associated with a lower risk of incident AF and with higher concentrations of Cer(d18:1/24:0). Cer(d18:1/24:0) was inversely associated to risk of AF.

Background: Osteoporosis and atherosclerosis are complex multifactorial diseases sharing common risk factors and pathophysiological mechanisms suggesting that these are comorbidities. Omics studies identifying joint molecular markers associated with these diseases are sparse. Subjects and methods: Using liquid chromatography-tandem mass spectrometry, we quantified 437 molecular lipid species from the Young Finns Study cohort (aged 30–45 years and 57% women) and performed lipidome-wide multivariate analysis of variance (MANOVA) with early markers for both diseases. Carotid intima-media thickness for atherosclerosis measured with ultrasound and bone mineral density from distal radius and tibia for osteoporosis measured with peripheral quantitative computed tomography were used as early markers of the diseases. Results: MANOVA adjusted with age, sex and body mass index, identified eight statistically significant (adjusted p-value (padj) < 0.05) and 15 suggestively significant (padj < 0.25) molecular lipid species associated with the studied markers. Similar analysis adjusted additionally for smoking habit, physical activity and alcohol consumption identified four significant and six suggestively significant molecular lipid species. These most significant lipid classes/species jointly associated with the studied markers were glycerolipid/TAG(18:0/18:0/18:1), glycerophospholipid/PC(40:3), sphingolipid/Gb3(d18:1/22:0), and sphingolipid/Gb3(d18:1/24:0). Conclusion: Our results support the osteoporosis-atherosclerosis comorbidity hypothesis and present potential new joint lipid biomarkers for these diseases. publishedVersion

Introduction: The Coronary Event Risk Test (CERT) is a validated cardiovascular risk predictor that uses circulating ceramide concentrations to allocate patients into one of four risk categories. This test has recently been updated (CERT-2), now additionally including phosphatidylcholine concentrations. Hypothesis: We hypothesize that CERT and CERT-2 have the power to predict cardiovascular mortality in patients with cardiovascular disease (CVD). Methods: We investigated a patient cohort of 999 patients with established CVD. Results: Overall, comparing survival curves (figure) for over 12 years of follow up and the predictive power of survival models using net reclassification improvement (NRI), CERT-2 was the best predictor of cardiovascular mortality, surpassing CERT (NRI=0.456; p=0.01) and also the 2019 ESC-SCORE (NRI=0.163; p=0.04). Patients in the highest risk category of CERT as compared to the lowest category had a HR of 3.63 [2.09-6.30] for cardiovascular death; for CERT-2 the corresponding HR was 6.02 [2.47-14.64]. Among patients with T2DM (n=322), the HR for cardiovascular death was 3.00 [1.44-6.23] using CERT and 7.06 [1.64-30.50] using CERT-2; the corresponding HRs among non-diabetic subjects were 2.99 [1.20-7.46] and 3.43 [1.03-11.43], respectively. Conclusions: We conclude that both, CERT and CERT-2 scores are powerful predictors of cardiovascular mortality in CVD patients, especially in those patients with T2D, performance is even higher with CERT-2.

The Coronary Event Risk Test (CERT) is a validated cardiovascular risk predictor that uses circulating ceramide concentrations to allocate patients into one of four risk categories. This test has recently been updated (CERT-2), now additionally including phosphatidylcholine concentrations. The purpose of this study was to investigate the power of CERT and CERT-2 to predict cardiovascular mortality in patients with cardiovascular disease (CVD). We investigated a cohort of 999 patients with established CVD. Overall, comparing survival curves (figure) for over 12 years of follow up and the predictive power of survival models using net reclassification improvement (NRI), CERT-2 was the best predictor of cardiovascular mortality, surpassing CERT (NRI=0.456; p=0.01) and also the 2019 ESC-SCORE (NRI=0.163; p=0.04). Patients in the highest risk category of CERT as compared to the lowest category had a HR of 3.63 [2.09–6.30] for cardiovascular death; for CERT-2 the corresponding HR was 6.02 [2.47–14.64]. Among patients with T2DM (n=322), the HR for cardiovascular death was 3.00 [1.44–6.23] using CERT and 7.06 [1.64–30.50] using CERT-2; the corresponding HRs among non-diabetic subjects were 2.99 [1.20–7.46] and 3.43 [1.03–11.43], respectively. We conclude that both, CERT and CERT-2 scores are powerful predictors of cardiovascular mortality in CVD patients, especially in those patients with T2D. Performance is even higher with CERT-2. Funding Acknowledgement Type of funding source: None

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the halflife, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than longchain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions. publishedVersion