Plasma ceramide and phospholipid-based risk score and the risk of cardiovascular death in patients after acute coronary syndrome.

AIMS: Ceramide (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 RESULTS: Prespecified 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. CONCLUSIONS: A 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.

Different rates of flux through the biosynthetic pathway for long-chain versus very-long-chain sphingolipids.

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 half-life, 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 long-chain (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.

Prediction of Residual Risk by Ceramide-Phospholipid Score in Patients With Stable Coronary Heart Disease on Optimal Medical Therapy.

Background Identification of patients with stable coronary heart disease who are at significant residual risk could be helpful for targeted prevention. Our aim was to determine the prognostic value of the recently introduced ceramide- and phospholipid-based risk score, the Cardiovascular Event Risk Test (CERT2), in patients with stable coronary heart disease on optimal medical therapy and to identify biological processes that contribute to the CERT2 score. Methods and Results Plasma samples (n=11 222) obtained from the STABILITY (Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy) trial were analyzed using a tandem liquid chromatography-mass spectrometry method. STABILITY was a trial in patients with stable coronary heart disease randomized to the lipoprotein-associated phospholipase A2 inhibitor darapladib or placebo on optimized medical therapy at baseline, with a median follow-up of 3.7 years. Hazard ratios per SD for the CERT2 risk score were 1.32 (95% CI, 1.25-1.39) for major adverse cardiovascular event, 1.47 (95% CI, 1.35-1.59) for cardiovascular death, 1.32 (95% CI, 1.16-1.49) for stroke, 1.23 (95% CI, 1.14-1.33) for myocardial infarction, and 1.56 (95% CI, 1.39-1.76) for hospitalization due to heart failure, when adjusted for traditional cardiovascular risk factors. CERT2 showed correlation (P<0.001, r>0.2) with inflammatory markers high-sensitivity C-reactive protein, interleukin 6, the heart failure marker N-terminal pro-B-type natriuretic peptide, and low-density lipoprotein cholesterol. After also adjusting for levels of other prognostic biomarkers, the CERT2 score was still independently related to the risk of cardiovascular death but not to nonfatal events. Conclusions The CERT2 risk score can detect residual risk in patients with stable coronary heart disease and is associated with biomarkers indicating inflammation, myocardial necrosis, myocardial dysfunction, renal dysfunction, and dyslipidemia. REGISTRATION URL: https://www.clini​caltr​ials.gov. Unique identifier: NCT00799903.

Development and validation of a ceramide- and phospholipid-based cardiovascular risk estimation score for coronary artery disease patients.

AIMS: Distinct ceramide lipids have been shown to predict the risk for cardiovascular disease (CVD) events, especially cardiovascular death. As phospholipids have also been linked with CVD risk, we investigated whether the combination of ceramides with phosphatidylcholines (PCs) would be synergistic in the prediction of CVD events in patients with atherosclerotic coronary heart disease in three independent cohort studies. METHODS AND RESULTS: Ceramides and PCs were analysed using liquid chromatography-mass spectrometry (LC-MS) in three studies: WECAC (The Western Norway Coronary Angiography Cohort) (N = 3789), LIPID (Long-Term Intervention with Pravastatin in Ischaemic Disease) trial (N = 5991), and KAROLA (Langzeiterfolge der KARdiOLogischen Anschlussheilbehandlung) (N = 1023). A simple risk score, based on the ceramides and PCs showing the best prognostic features, was developed in the WECAC study and validated in the two other cohorts. This score was highly significant in predicting CVD mortality [multiadjusted hazard ratios (HRs; 95% confidence interval) per standard deviation were 1.44 (1.28-1.63) in WECAC, 1.47 (1.34-1.61) in the LIPID trial, and 1.69 (1.31-2.17) in KAROLA]. In addition, a combination of the risk score with high-sensitivity troponin T increased the HRs to 1.63 (1.44-1.85) and 2.04 (1.57-2.64) in WECAC and KAROLA cohorts, respectively. The C-statistics in WECAC for the risk score combined with sex and age was 0.76 for CVD death. The ceramide-phospholipid risk score showed comparable and synergistic predictive performance with previously published CVD risk models for secondary prevention. CONCLUSION: A simple ceramide- and phospholipid-based risk score can efficiently predict residual CVD event risk in patients with coronary artery disease.

New evidence from plasma ceramides links apoE polymorphism to greater risk of coronary artery disease in Finnish adults.

apoE, a key regulator of plasma lipids, mediates altered functionalities in lipoprotein metabolism and thus affects the risk of coronary artery disease (CAD). The significance of different apoE polymorphisms remains unclear; although the ε4 allele is clearly associated with increased cholesterol levels (which inform CAD risk), direct studies about apoE polymorphisms on CAD risk and development have yielded controversial results. Furthermore, certain species of ceramides-complex lipids abundant in plasma LDL-are markers of increased risk of myocardial infarction and cardiovascular death. Using a high-throughput MS approach, we quantified 30 molecular plasma ceramide species from a cohort of 2,160 apoE-genotyped (rs7412, rs429358) young adults enrolled in the population-based Cardiovascular Risk in Young Finns Study. We then searched this lipidome data set to identify new indications of pathways influenced by apoE polymorphisms and possibly related to CAD risk. This approach revealed a previously unreported association between apoE polymorphism and a consistently documented high-risk CAD marker, Cer(d18:1/16:0). Compared with the apoE ε3/3 reference group, plasma levels of apoE ε4 were elevated and those of apoE ε2 were lowered in all subjects without evidence of apoE-by-sex interactions. apoE associated with seven ceramides that are connected to atherogenically potent macrophages and/or lipoprotein particles; these associations could indicate a plausible linkage between apoE polymorphism and ceramide metabolism, leading to adverse plasma LDL metabolism and atherogenesis. In conclusion, new evidence from plasma ceramides links apoE polymorphism with an increased risk of CAD and extends our understanding of the role of apoE in health and disease.

hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver.

Hepatocyte-like cells (HLCs) differentiated from human-induced pluripotent stem cells offer an alternative platform to primary human hepatocytes (PHHs) for studying the lipid metabolism of the liver. However, despite their great potential, the lipid profile of HLCs has not yet been characterized. Here, we comprehensively studied the lipid profile and fatty acid (FA) metabolism of HLCs and compared them with the current standard hepatocyte models: HepG2 cells and PHHs. We differentiated HLCs by five commonly used methods from three cell lines and thoroughly characterized them by gene and protein expression. HLCs generated by each method were assessed for their functionality and the ability to synthesize, elongate, and desaturate FAs. In addition, lipid and FA profiles of HLCs were investigated by both mass spectrometry and gas chromatography and then compared with the profiles of PHHs and HepG2 cells. HLCs resembled PHHs by expressing hepatic markers: secreting albumin, lipoprotein particles, and urea, and demonstrating similarities in their lipid and FA profile. Unlike HepG2 cells, HLCs contained low levels of lysophospholipids similar to the content of PHHs. Furthermore, HLCs were able to efficiently use the exogenous FAs available in their medium and simultaneously modify simple lipids into more complex ones to fulfill their needs. In addition, we propose that increasing the polyunsaturated FA supply of the culture medium may positively affect the lipid profile and functionality of HLCs. In conclusion, our data showed that HLCs provide a functional and relevant model to investigate human lipid homeostasis at both molecular and cellular levels.

Plasma concentrations of molecular lipid species predict long-term clinical outcome in coronary artery disease patients.

We investigated the associations of ten previously identified high risk molecular lipid species and three ceramide ratios with the occurrence of major adverse cardiac events (MACEs) during a median follow-up of 4.7 years in patients with coronary artery disease (CAD). Between 2008 and 2011, 581 patients underwent diagnostic coronary angiography or percutaneous coronary intervention for stable angina pectoris (SAP) or acute coronary syndrome (ACS). Blood was drawn prior to the index procedure and lipid species were determined. The primary endpoint was the occurrence of a MACE, comprising all-cause mortality, nonfatal ACS, or unplanned coronary revascularization. The secondary endpoint comprised all-cause mortality or nonfatal ACS. During a median follow-up of 4.7 [IQR: 4.2-5.6] years, 155 patients (27%) had MACEs. In multivariable analyses, Cer(d18:1/16:0) concentration was associated with MACEs {hazard ratio 2.32; 95% CI [1.09-4.96] per natural logarithm (ln) (pmol/ml) P = 0.030} after adjustment for cardiac risk factors, clinical presentation, statin use at baseline, and admission nonHDL cholesterol level. Furthermore, after multivariable adjustment, concentrations of Cer(d18:1/16:0), Cer(d18:1/20:0), Cer(d18:1/24:1), and their ratios to Cer(d18:1/24:0) were associated with the composite endpoint death or nonfatal ACS. The data together show the circulating ceramide lipids we investigated here are associated with adverse cardiac outcome during long-term follow-up independent of clinical risk factors.

Ceramide stearic to palmitic acid ratio predicts incident diabetes.

AIMS/HYPOTHESIS: Ceramide lipids have a role in the development of insulin resistance, diabetes and risk of cardiovascular disease. Here we investigated four ceramides and their ratios to find the best predictors of incident diabetes. METHODS: A validated mass-spectrometric method was applied to measure Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1) from serum or plasma samples. These ceramides were analysed in a population-based risk factor study (FINRISK 2002, n = 8045), in a cohort of participants undergoing elective coronary angiography for suspected stable angina pectoris (Western Norway Coronary Angiography Cohort [WECAC], n = 3344) and in an intervention trial investigating improved methods of lifestyle modification for individuals at high risk of the metabolic syndrome (Prevent Metabolic Syndrome [PrevMetSyn], n = 371). Diabetes risk score models were developed to estimate the 10 year risk of incident diabetes. RESULTS: Analysis in FINRISK 2002 showed that the Cer(d18:1/18:0)/Cer(d18:1/16:0) ceramide ratio was predictive of incident diabetes (HR per SD 2.23, 95% CI 2.05, 2.42), and remained significant after adjustment for several risk factors, including BMI, fasting glucose and HbA1c (HR 1.34, 95% CI 1.14, 1.57). The finding was validated in the WECAC study (unadjusted HR 1.81, 95% CI 1.53, 2.14; adjusted HR 1.39, 95% CI 1.16, 1.66). In the intervention trial, the ceramide ratio and diabetes risk scores significantly decreased in individuals who had 5% or more weight loss. CONCLUSIONS/INTERPRETATION: The Cer(d18:1/18:0)/Cer(d18:1/16:0) ratio is an independent predictive biomarker for incident diabetes, and may be modulated by lifestyle intervention.

Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers.

BACKGROUND: Exosomes have recently appeared as a novel source of noninvasive cancer biomarkers, since these nanovesicles contain molecules from cancer cells and can be detected in biofluids. We have here investigated the potential use of lipids in urinary exosomes as prostate cancer biomarkers. METHODS: A high-throughput mass spectrometry quantitative lipidomic analysis was performed to reveal the lipid composition of urinary exosomes in prostate cancer patients and healthy controls. RESULTS: Control samples were first analysed to characterise the lipidome of urinary exosomes and test the reproducibility of the method. In total, 107 lipid species were quantified in urinary exosomes. Several differences, for example, in cholesterol and phosphatidylcholine, were found between urinary exosomes and exosomes derived from cell lines, thus showing the importance of in vivo studies for biomarker analysis. The 36 most abundant lipid species in urinary exosomes were then quantified in 15 prostate cancer patients and 13 healthy controls. Interestingly, the levels of nine lipids species were found to be significantly different when the two groups were compared. The highest significance was shown for phosphatidylserine (PS) 18:1/18:1 and lactosylceramide (d18:1/16:0), the latter also showed the highest patient-to-control ratio. Furthermore, combinations of these lipid species and PS 18:0-18:2 distinguished the two groups with 93% sensitivity and 100% specificity. Finally, in agreement with the reported dysregulation of sphingolipid metabolism in cancer cells, alteration in specific sphingolipid lipid classes were observed. CONCLUSION: This study shows for the first time the potential use of exosomal lipid species in urine as prostate cancer biomarkers.

Data including GROMACS input files for atomistic molecular dynamics simulations of mixed, asymmetric bilayers including molecular topologies, equilibrated structures, and force field for lipids compatible with OPLS-AA parameters.

In this Data in Brief article we provide a data package of GROMACS input files for atomistic molecular dynamics simulations of multicomponent, asymmetric lipid bilayers using the OPLS-AA force field. These data include 14 model bilayers composed of 8 different lipid molecules. The lipids present in these models are: cholesterol (CHOL), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidyl-ethanolamine (SOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (SOPS), N-palmitoyl-D-erythro-sphingosyl-phosphatidylcholine (SM16), and N-lignoceroyl-D-erythro-sphingosyl-phosphatidylcholine (SM24). The bilayers׳ compositions are based on lipidomic studies of PC-3 prostate cancer cells and exosomes discussed in Llorente et al. (2013) [1], showing an increase in the section of long-tail lipid species (SOPS, SOPE, and SM24) in the exosomes. Former knowledge about lipid asymmetry in cell membranes was accounted for in the models, meaning that the model of the inner leaflet is composed of a mixture of PC, PS, PE, and cholesterol, while the extracellular leaflet is composed of SM, PC and cholesterol discussed in Van Meer et al. (2008) [2]. The provided data include lipids׳ topologies, equilibrated structures of asymmetric bilayers, all force field parameters, and input files with parameters describing simulation conditions (md.mdp). The data is associated with the research article "Interdigitation of Long-Chain Sphingomyelin Induces Coupling of Membrane Leaflets in a Cholesterol Dependent Manner" (Róg et al., 2016) [3].

Circulating Ceramides Predict Cardiovascular Outcomes in the Population-Based FINRISK 2002 Cohort.

OBJECTIVE: Ceramides are molecular lipids implicated in apoptosis, inflammation, obesity, and insulin resistance. An earlier study reported that ceramides were associated with fatal outcome among patients with coronary heart disease. Here, we examined whether ceramides are associated with major adverse cardiovascular events (MACEs) among apparently healthy individuals. APPROACH AND RESULTS: FINRISK 2002 is a population-based risk factor survey, which recruited men and women aged 25 to 74 years. The cohort was followed up until the end of 2014. We quantified 4 circulating ceramides, Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1), in 8101 serum samples by a targeted liquid chromatography-tandem mass spectrometry assay. Primary outcome of interest was incident MACE (n=813). Secondary analyses were performed for MACE death (n=116) without previous nonfatal MACE and for recurrent MACE (n=226) among survivors of a previous incident MACE. We used Cox proportional hazard models adjusted for the Framingham covariates to determine the association of ceramides with the outcomes. Of the ceramide species, Cer(d18:1/18:0) had the strongest association with incident MACE and the highest unadjusted hazard ratio of 1.31 (95% confidence interval, 1.21-1.41), which remained significant at 1.21 (95% confidence interval, 1.11-1.33) after Framingham risk factor adjustments. The hazard ratios were generally stronger for recurrent and fatal events than for first events. Clinical net reclassification improvement was 7.5% (P=6.9×10-5) for Cer(d18:1/18:0). CONCLUSIONS: Distinct serum ceramides are associated with the risk of incident MACE in apparently healthy individuals. These results should encourage more detailed analyses of ceramides in cardiovascular pathobiology and suggest new biomarkers of MACE risk.

Determining the Turnover of Glycosphingolipid Species by Stable-Isotope Tracer Lipidomics.

In this study, we used water-soluble stable mass isotope precursors to measure the turnover of endogenous ceramide (Cer) and glycosphingolipids (GSLs) in HEp-2 cells. Cells incubated in the presence of [13C-U]glucose showed rapid incorporation of hexose residues with an increased mass of 6Da into GSLs. Different turnover rates of GSL classes and their molecular species were observed. Approximately 30% of the glucosylceramide, 50% of the lactosylceramide, and 50% of the globotriaosylceramide species showed a much slower turnover than the rest. This demonstrates the existence of different lipid pools, where a certain fraction of species survived for a long time in the cells. The species with the shortest N-amidated fatty acyl groups (C16:0 and C18:0) showed a more rapid turnover than those with the longest N-amidated fatty acids (C24:0 and C24:1). Experiments with addition of [13C-U]serine were performed to study de novo synthesis of Cer from serine and palmitoyl-CoA. These experiments revealed that de novo synthesis contributes to a minor extent to the total synthesis of new sphingolipids and showed that there is a more rapid formation of the longest Cer species (C24:0 and C24:1) than of the shortest species (C16:0), that is, the opposite as observed for the GSLs in the experiments with [13C-U]glucose. In conclusion, this FLUX lipidomics experimental approach with the addition of [13C-U]glucose to cells allows us to not only study the total turnover but also permit observations of lipid intermediates and metabolic flow of endogenous GSL species at the molecular lipid level.

Gender, Contraceptives and Individual Metabolic Predisposition Shape a Healthy Plasma Lipidome.

Lipidomics of human blood plasma is an emerging biomarker discovery approach that compares lipid profiles under pathological and physiologically normal conditions, but how a healthy lipidome varies within the population is poorly understood. By quantifying 281 molecular species from 27 major lipid classes in the plasma of 71 healthy young Caucasians whose 35 clinical blood test and anthropometric indices matched the medical norm, we provided a comprehensive, expandable and clinically relevant resource of reference molar concentrations of individual lipids. We established that gender is a major lipidomic factor, whose impact is strongly enhanced by hormonal contraceptives and mediated by sex hormone-binding globulin. In lipidomics epidemiological studies should avoid mixed-gender cohorts and females taking hormonal contraceptives should be considered as a separate sub-cohort. Within a gender-restricted cohort lipidomics revealed a compositional signature that indicates the predisposition towards an early development of metabolic syndrome in ca. 25% of healthy male individuals suggesting a healthy plasma lipidome as resource for early biomarker discovery.

Plasma ceramides predict cardiovascular death in patients with stable coronary artery disease and acute coronary syndromes beyond LDL-cholesterol.

AIMS: The aim was to study the prognostic value of plasma ceramides (Cer) as cardiovascular death (CV death) markers in three independent coronary artery disease (CAD) cohorts. METHODS AND RESULTS: Corogene study is a prospective Finnish cohort including stable CAD patients (n = 160). Multiple lipid biomarkers and C-reactive protein were measured in addition to plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1). Subsequently, the association between high-risk ceramides and CV mortality was investigated in the prospective Special Program University Medicine-Inflammation in Acute Coronary Syndromes (SPUM-ACS) cohort (n = 1637), conducted in four Swiss university hospitals. Finally, the results were validated in Bergen Coronary Angiography Cohort (BECAC), a prospective Norwegian cohort study of stable CAD patients. Ceramides, especially when used in ratios, were significantly associated with CV death in all studies, independent of other lipid markers and C-reactive protein. Adjusted odds ratios per standard deviation for the Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio were 4.49 (95% CI, 2.24-8.98), 1.64 (1.29-2.08), and 1.77 (1.41-2.23) in the Corogene, SPUM-ACS, and BECAC studies, respectively. The Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio improved the predictive value of the GRACE score (net reclassification improvement, NRI = 0.17 and ΔAUC = 0.09) in ACS and the predictive value of the Marschner score in stable CAD (NRI = 0.15 and ΔAUC = 0.02). CONCLUSIONS: Distinct plasma ceramide ratios are significant predictors of CV death both in patients with stable CAD and ACS, over and above currently used lipid markers. This may improve the identification of high-risk patients in need of more aggressive therapeutic interventions.

Development and validation of a high-throughput LC-MS/MS assay for routine measurement of molecular ceramides.

Monitoring the levels of the ceramides (Cer) d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1 and ratios thereof in human plasma empowers the prediction of fatal outcome of coronary artery disease (CAD). We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for clinical-scaled measurement of the four distinct ceramides. Rapid plasma precipitation was accomplished in 96-well format. Excellent extraction recoveries in the range of 98-109% were achieved for each ceramide. Addition of corresponding D7-labeled ceramide standards facilitated precise quantification of each plasma ceramide species utilizing a novel short 5-min LC-MS/MS method. Neither matrix interference nor carryover was observed. Robust intra- and inter-assay accuracy and precision <15% at five different concentrations were obtained. Linear calibration lines with regressions, R(2) > 0.99, were achieved for all analytes. Short-term bench top, long-term plasma, and extract stability demonstrated that the distinct ceramides were stable in the conditions evaluated. The validity of the methodology was demonstrated by determining the precise ceramide concentrations in a small CAD case-control study. Thus, our LC-MS/MS methodology features simple sample preparation and short analysis time for accurate quantification of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1, designed for routine analysis.

Interdigitation of long-chain sphingomyelin induces coupling of membrane leaflets in a cholesterol dependent manner.

It has been a long-standing question how the two leaflets in a lipid bilayer modulate each others' physical properties. In this paper, we discuss how this interaction may take place through interdigitation. We use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on the lipidomics data determined for exosomes released by PC-3 prostate cancer cells. The simulations show interdigitation to be exceptionally strong for long-chain sphingomyelin (SM) molecules. In asymmetric membranes the amide-linked chain of SM is observed to extend deep into the opposing membrane leaflet. Interestingly, we find that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. Analysis of this finding reveals that the amide-linked SM chain interacts favorably with the lipid chains in the opposite leaflet, and that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet.

Molecular lipids identify cardiovascular risk and are efficiently lowered by simvastatin and PCSK9 deficiency.

CONTEXT: Coronary artery disease (CAD) is among the leading causes of mortality and morbidity worldwide. Traditional risk markers explain only a proportion of total cardiovascular risk. Thus, development and improvement of early diagnostic strategies and targeted initiation of preventive measures would be of great benefit. OBJECTIVE: We aimed to identify molecular lipids that are associated with fatal outcome of CAD patients. Furthermore, the effect of different lipid-lowering drugs on novel risk lipids was evaluated. METHODS: Serum samples of 445 CAD subjects participating in a long-term follow-up of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study were analyzed. In addition, samples obtained from a separate randomized parallel three-group study of subjects treated with simvastatin (n=24), ezetimibe (n=24), or their combination (n=24) were studied. Furthermore, samples from the LURIC participants with a loss-of-function mutation (R46L) in the PCSK9 gene (n=19) were analyzed and compared with major allele carriers (n=868). RESULTS: Distinct ceramide species were significantly associated with the fatal outcome of CAD patients. Simvastatin lowered plasma ceramides broadly by about 25%, but no changes in ceramides were observed in the ezetimibe group. PCSK9 deficiency was significantly associated (-13%) with lowered low-density lipoprotein cholesterol accompanied by a significant 20% reduction in CAD outcome risk-related ceramides. CONCLUSIONS: These data suggest that distinct ceramides associate significantly with CAD outcome independently of traditional risk factors and that the mechanism of lipid lowering is important.

Molecular lipidomics of exosomes released by PC-3 prostate cancer cells.

The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating an extraordinary discrimination of lipids sorted into these microvesicles. In particular, exosomes are highly enriched in glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (mol% of total lipids). Furthermore, lipid species, even of classes not enriched in exosomes, were selectively included in exosomes. Finally, it was found that there is an 8.4-fold enrichment of lipids per mg of protein in exosomes. The detailed lipid composition provided in this study may be useful to understand the mechanism of exosome formation, release and function. Several of the lipids enriched in exosomes could potentially be used as cancer biomarkers.

Shotgun Lipidomics by Sequential Precursor Ion Fragmentation on a Hybrid Quadrupole Time-of-Flight Mass Spectrometer.

Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.

High throughput quantitative molecular lipidomics.

Applications in biomedical research increasingly demand detailed lipid molecule information acquired at high throughput. Although the recent advances in lipidomics offer to delineate the lipidomes in detail, the challenge remains in performing such analyses at the requested quality and to maintain the quality also in a high throughput setting. In this review we describe a high throughput molecular lipidomic solution based on robotic assisted sample preparation and lipid extraction and multiple lipidomic platforms integrated with a sophisticated bioinformatics system. As demonstrated, the virtue of this lipidomic toolkit lies in its high throughput delivery of comprehensive quantitative lipidomic outputs at the molecular lipid level, its ease of scalability and its capability to serve in a regulatory setting. We anticipate that this toolkit will contribute to basic research, nutritional research and promote the discovery of new disease biomarkers, disease related mechanisms of actions and drug targets.