the ludwigshafen risk and cardiovascular health ( luric ) study is an ongoing prospective study of currently more than 3000 individuals of german ancestry in whom the cardiovascular and metabolic phenotypes cad , myocardial infarction ( mi ) , dyslipidemia , hypertension , metabolic syndrome , and diabetes mellitus have been defined or ruled out using standardized methodologies ( 8) . all the patients were in secondary prevention , ie , had known cad . patients with german ancestry were recruited at the cardiac center in ludwigshafen from 1997 to 2002 . after obtaining a written informed consent , baseline examination was done consisting of a standardized individual and family history questionnaire and extensive sampling of fasted venous blood in the early morning . we first selected male cad patients ( n = 258 ) from the luric cohort who died due to cardiovascular disease ( cvd ) reasons within the first 3 years of follow - up . the control group consisted of male cad patients who did not die during the follow - up . frequency matching was used for matching case and control groups for age , body mass index ( bmi ) , statin use , and smoking . the total number of controls ( n = 187 ) remained smaller than the total number of cases . this was due to the exclusion of numerous stable diabetic patients who had had events indicative of plaque vulnerability such as mi and/or stroke prior to the study entry . coronary angiography was used to verify cad ( stenosis > 20% in one or more coronary arteries ) in both study groups . cardiovascular deaths were defined as sudden cardiac death , fatal mi , death due to congestive heart failure , death immediately after intervention to treat cad , fatal stroke , and other causes of deaths due to cardiac disease . two experienced clinicians blinded of the study data independently went through death certificates to classify deaths to cvd and non - cvd causes ( 9 ) . patient characteristics abbreviation : apo , apolipoprotein ; crp , c - reactive protein ; dm2 , diabetes mellitus type 2 ; n.s . , not significant ; tc , total cholesterol ; tg , triglycerides . framingham score of coronary heart disease ( 10 y risk ) ( 17 ) . plasma samples of healthy males from a single - center , randomized , parallel , three - group study performed at the university of cologne ( 10 ) were analyzed to evaluate the potential of ezetimibe ( 10 mg ; n = 24 ) , simvastatin ( 40 mg ; n = 24 ) , or their combination ( n = 24 ) to reduce plasma concentrations of molecular lipids species that had been identified to be related to cardiovascular death in the luric cohort . furthermore , we used the luric study genome - wide association studies database and identified subjects who were carrying the previously described loss - of - function mutation r46l ( rs11591147 ) ( 11 ) of the pcsk9 gene to reveal the possible lipidomic effect of pcsk9 deficiency in men . altogether 19 heterozygous male mutation carriers and 868 male homozygous major allele carriers were identified from our database with both genetic information and serum lipidomic data available . known amounts of internal standards were added to the samples before extraction and the final lipid extracts were dried under nitrogen . sphingolipids were analyzed as described elsewhere ( 13 ) on a 4000 qtrap mass spectrometer ( applied biosystems / mds analytical technologies ) equipped with an ultrahigh pressure liquid chromatography system , ctc pal autosampler ( leap technologies ) , and rheos allegro ultrahigh pressure liquid chromatography ( flux instruments ) using multiple reaction monitoring . shotgun lipidomics was performed by multiple precursor ion and neutral loss scanning as described elsewhere ( 14 ) on a qtrap 5500 mass spectrometer ( applied biosystems / mds analytical technologies ) equipped with a robotic nanoflow ion source nanomate hd ( advion ) . mass spectrometry data files were processed using multiquant 1.1.0.26 or lipid profiler ( 15 ) ( applied biosystems / mds analytical technologies ) . identified lipids were quantified by normalizing against their respective internal standard and volume for plasma or serum . quality control ( qc ) samples were used to monitor the overall quality of the lipid extraction and mass spectrometry analyses ( 16 ) . the qc samples were mainly used to remove technical outliers and lipid species that were detected below the lipid class - based lower limit of quantification . in total the average coefficient of variation of all the lipids detected in the study samples was 20% . lipid class concentrations were calculated by summing up the concentrations of corresponding molecular lipids . for analysis of cad mortality and pcsk9 mutation data , an unpaired student t test was performed on log - transformed concentrations and lipid to lipid ratios because variables were approximately log normal . equality of variance was tested , and pooled t test was used in case of equal variance and satterthwaite t test was used in case of unequal variance . cad mortality case and controls groups were frequency matched ; therefore , an unpaired t test was used . the t test results are presented as volcano plots and heat maps ( figures 13 ) . in volcano plots , the magnitude of relative difference between groups ( horizontal axis ) is plotted against the statistical significance ( vertical axis ) . odds ratios were calculated using logistic regression model with and without adjustment for age , bmi , fasting glucose , hdl - c , ldl - c , c - reactive protein , and triglycerides . a paired t test was conducted for comparing baseline and after - treatment lipid concentrations in subjects receiving ezetemibe , simvastatin , or their combination . the false discovery rate q values were calculated to correct the multiple hypotheses testing results . volcano heat map indicating molecular lipid species difference ( percentage ) between stable and high - risk cad patients . right panel ( red color ) indicates lipids that associate with cvd outcome risk in cad patients . lipid concentrations in controls are taken as a reference ; thus , positive values correspond to higher values in cases vs controls . b , heat map of sphingolipid differences . volcano heat map indicating molecular lipids and ceramide to ceramide ratio differences between all studied stable cad patients and high - risk cad patients ( a ) , differences between stable cad patients and vulnerable cad patients with diabetes ( b ) , and patients without diabetes ( c ) . lipid concentrations in controls are taken as a reference ; thus , positive values correspond to higher values in cases vs controls . the ludwigshafen risk and cardiovascular health ( luric ) study is an ongoing prospective study of currently more than 3000 individuals of german ancestry in whom the cardiovascular and metabolic phenotypes cad , myocardial infarction ( mi ) , dyslipidemia , hypertension , metabolic syndrome , and diabetes mellitus have been defined or ruled out using standardized methodologies ( 8) . all the patients were in secondary prevention , ie , had known cad . patients with german ancestry were recruited at the cardiac center in ludwigshafen from 1997 to 2002 . after obtaining a written informed consent , baseline examination was done consisting of a standardized individual and family history questionnaire and extensive sampling of fasted venous blood in the early morning . we first selected male cad patients ( n = 258 ) from the luric cohort who died due to cardiovascular disease ( cvd ) reasons within the first 3 years of follow - up . the control group consisted of male cad patients who did not die during the follow - up . frequency matching was used for matching case and control groups for age , body mass index ( bmi ) , statin use , and smoking . the total number of controls ( n = 187 ) remained smaller than the total number of cases . this was due to the exclusion of numerous stable diabetic patients who had had events indicative of plaque vulnerability such as mi and/or stroke prior to the study entry . coronary angiography was used to verify cad ( stenosis > 20% in one or more coronary arteries ) in both study groups . cardiovascular deaths were defined as sudden cardiac death , fatal mi , death due to congestive heart failure , death immediately after intervention to treat cad , fatal stroke , and other causes of deaths due to cardiac disease . two experienced clinicians blinded of the study data independently went through death certificates to classify deaths to cvd and non - cvd causes ( 9 ) . patient characteristics abbreviation : apo , apolipoprotein ; crp , c - reactive protein ; dm2 , diabetes mellitus type 2 ; n.s . , not significant ; tc , total cholesterol ; tg , triglycerides . framingham score of coronary heart disease ( 10 y risk ) ( 17 ) . plasma samples of healthy males from a single - center , randomized , parallel , three - group study performed at the university of cologne ( 10 ) were analyzed to evaluate the potential of ezetimibe ( 10 mg ; n = 24 ) , simvastatin ( 40 mg ; n = 24 ) , or their combination ( n = 24 ) to reduce plasma concentrations of molecular lipids species that had been identified to be related to cardiovascular death in the luric cohort . furthermore , we used the luric study genome - wide association studies database and identified subjects who were carrying the previously described loss - of - function mutation r46l ( rs11591147 ) ( 11 ) of the pcsk9 gene to reveal the possible lipidomic effect of pcsk9 deficiency in men . altogether 19 heterozygous male mutation carriers and 868 male homozygous major allele carriers were identified from our database with both genetic information and serum lipidomic data available . amounts of internal standards were added to the samples before extraction and the final lipid extracts were dried under nitrogen . sphingolipids were analyzed as described elsewhere ( 13 ) on a 4000 qtrap mass spectrometer ( applied biosystems / mds analytical technologies ) equipped with an ultrahigh pressure liquid chromatography system , ctc pal autosampler ( leap technologies ) , and rheos allegro ultrahigh pressure liquid chromatography ( flux instruments ) using multiple reaction monitoring . shotgun lipidomics was performed by multiple precursor ion and neutral loss scanning as described elsewhere ( 14 ) on a qtrap 5500 mass spectrometer ( applied biosystems / mds analytical technologies ) equipped with a robotic nanoflow ion source nanomate hd ( advion ) . mass spectrometry data files were processed using multiquant 1.1.0.26 or lipid profiler ( 15 ) ( applied biosystems / mds analytical technologies ) . identified lipids were quantified by normalizing against their respective internal standard and volume for plasma or serum . quality control ( qc ) samples were used to monitor the overall quality of the lipid extraction and mass spectrometry analyses ( 16 ) . the qc samples were mainly used to remove technical outliers and lipid species that were detected below the lipid class - based lower limit of quantification . in total , 14 qc samples evenly distributed along analytical runs of the study were analyzed . the average coefficient of variation of all the lipids detected in the study samples was 20% . lipid class concentrations were calculated by summing up the concentrations of corresponding molecular lipids . for analysis of cad mortality and pcsk9 mutation data , an unpaired student t test was performed on log - transformed concentrations and lipid to lipid ratios because variables were approximately log normal . equality of variance was tested , and pooled t test was used in case of equal variance and satterthwaite t test was used in case of unequal variance . cad mortality case and controls groups were frequency matched ; therefore , an unpaired t test was used . the t test results are presented as volcano plots and heat maps ( figures 13 ) . in volcano plots , the magnitude of relative difference between groups ( horizontal axis ) is plotted against the statistical significance ( vertical axis ) . odds ratios were calculated using logistic regression model with and without adjustment for age , bmi , fasting glucose , hdl - c , ldl - c , c - reactive protein , and triglycerides . a paired t test was conducted for comparing baseline and after - treatment lipid concentrations in subjects receiving ezetemibe , simvastatin , or their combination . the false discovery rate q values were calculated to correct the multiple hypotheses testing results . volcano heat map indicating molecular lipid species difference ( percentage ) between stable and high - risk cad patients . right panel ( red color ) indicates lipids that associate with cvd outcome risk in cad patients . lipid concentrations in controls are taken as a reference ; thus , positive values correspond to higher values in cases vs controls . b , heat map of sphingolipid differences . volcano heat map indicating molecular lipids and ceramide to ceramide ratio differences between all studied stable cad patients and high - risk cad patients ( a ) , differences between stable cad patients and vulnerable cad patients with diabetes ( b ) , and patients without diabetes ( c ) . lipid concentrations in controls are taken as a reference ; thus , positive values correspond to higher values in cases vs controls . the classical risk markers were unable to distinguish cad patients who died during the follow - up from stable cad patients who had structural coronary disease at baseline . modestly higher total cholesterol concentrations ( 6% , p = .002 ) were recorded in the case groups and hdl - c and apoa1 were slightly lower in cases compared with controls ( 7.5% , p = .001 , and 7.5% , p = .001 , respectively ) ( table 1 ) . both case and control groups had median framingham 10-year risk of coronary heart disease ( 17 ) of 20% . the estimation of the 10-year risk of fatal cardiovascular disease in europe ( systematic coronary risk evaluation , score ) ( 18 ) was 7.1% in cases and 6.7% in controls , and the slight difference was not statistically significant . more pronounced differences between groups were observed through lipidomic analyses ( figure 1 and supplemental table 1 , published on the endocrine society 's journals online web site at http://jcem.endojournals.org ) . lactosylceramides laccer ( 18:1/18:0 ) and laccer ( 18:1/20:0 ) were among the risk - associated lipids together with cer ( 18:1/16:0 ) and cer ( 18:1/18:0 ) . several molecular lipid species such as ce20:5 ( 17.4% , p = .0001 ) and ce18:3 ( 15.1% , p = .000037 ) displayed reduced concentrations in cases as compared with the control group . abundance of several ceramide species containing specific fatty acids was higher along the axis of the glycosphingolipid pathway ( figure 2 ) in which ceramide is converted to galactosylceramide and glucosylceramide , glucosylceramide is converted to lactosylceramide , and lactosylceramide is metabolized to globotriaosylceramide ( gb3 ) . for example , d18:1/22:0-containing species were higher in ceramide , lactosylceramide , and globotriaosylceramide classes . a similar pattern is demonstrated by other long - chain containing ceramides ( d18:1/16:0 and d18:1/18:0 ) . however , cer(d18:1/24:0 ) lipid species concentration was lower in cases reflected in lower concentration of sphingomyelin containing the same fatty acid sm(d18:1/24:0 ) ( figure 2b ) . to account for lipid concentration differences due to metabolome influencing factors interestingly , when these patients were studied separately , very distinct lipidomic profiles were recorded for the two groups . in nondiabetic cad patients , the risk of fatal outcome was associated with elevated sphingolipids , whereas in diabetic cad patients , the risk was mainly associated with reduced cholesteryl ester species ( figure 3 ) . because we noted that factors like diabetes , smoking , and lipid - lowering treatment among others , could influence the expression of the risk - associated lipids , we decided to calculate ceramide and cerebroside ratios to check whether these would be less dependent on different patient characteristics while still indicative of risk . it turned out that the ratios of cer(18:1/16:0)/cer(18:1/24:0 ) and cer(18:1/22:0)/cer(18:1/24:0 ) were significantly related to increased risk of cvd death in all subjects and subgroups . cer(18:1/24:0)/cer(18:1/24:1 ) was indicative of a reduced risk of cvd death , regardless of diabetes status ( figure 3 ) . importantly , odds ratios for cvd death of these ratios remained significant after adjustment for traditional cvd death risk factors , such as ldl - c ( table 2 ) . unadjusted and adjusted odds ratios for cvd death of ceramide ratios abbreviation : or , odds ratio . models adjusted for age , bmi , fasting glucose , hdl - c , ldl - c , c - reactive protein , triglycerides , and systolic and diastolic blood pressure . predictive potential for cad mortality was tested by receiver - operating characteristic analysis for the ceramide ratios and compared with ldl - c . the area under curve and 95% confidence limits were 0.67 ( range 0.620.72 ) for cer(d18:1/16:0)/cer(d18:1/24:0 ) , 0.65 ( range 0.600.71 ) for cer(d18:1/20:0)/cer(d18:1/24:0 ) , and 0.68 ( range 0.630.73 ) for cer(d18:1/24:0)/cer(d18:1/24:1 ) , whereas for ldl - c , these values were 0.55 ( range 0.500.61 ) . finally , the effect of lipid - lowering treatments on ceramides and cerebrosides associated with increased risk of cvd death in statin - nave subjects as well as on ldl - c was evaluated ( figure 4 ) . as a comparison basis , we used the most significant lipids that separate the high - risk cad patients from stable cad patients ( figure 4a ) . the effect of different ldl - c - lowering methods on cad outcome - related lipidomic markers . results shown for top ranked cad risk lipids , ldl - c , and ceramide ( 18:1/24:0 ) . effect of ezetimibe ( 10 mg ) ( b ) , simvastatin ( 40 mg ) ( c ) , simvastatin + ezetimibe ( 40 mg+10 mg ) , ( d ) and pcsk9 loss - of - function mutation ( r46l ) ( e ) on cad mortality risk lipids . for evaluation of ezetimibe and simvastatin effects on these putative risk lipids we used plasma samples from a randomized clinical trial comparing simvastatin 40 mg , ezetimibe 10 mg , and their combination ( supplemental table 2 ) . as expected , simvastatin 40 mg lowered ldl - c significantly by 40% , and this reduction was accompanied by a significant reduction ( 25% ) also in all ceramides and cerebrosides recorded in this study ( figure 4c ) . ezetimibe treatment ( 10 mg ) in turn lowered ldl - c by 21% but affected only very modestly the plasma levels of cvd death - related lipid species ( figure 4b ) . in fact , cer18:1/18:0 and cer18:1/20:0 concentrations were slightly elevated in ezetimibe - treated subjects by 7.6% and 8.8% , respectively , whereas cer18:1/16:0 levels remained unchanged during this treatment . there was no statistically significant difference between treatment effects of simvastatin alone or in combination with ezetimibe on molecular lipids . however , combined treatment resulted in significantly lower ldl - c levels than simvastatin or ezetimibe alone ( figure 4d ) . drugs that inhibit pcsk9 are being developed as an alternative to statin lipid - lowering therapy , and we studied the effects of pcsk9 inhibition on the risk lipids ( supplemental table 3 ) . the effect was investigated by using subjects with the pcsk9 allelic variants from the luric patient population ( figure 4e ) . the pcsk9 loss - of - function mutation ( r46l ) resulted in a significant reduction of ceramides indicative of cad outcome risk comparable with that observed in simvastatin - treated subjects . interestingly , the mutation carriers had only modestly lower ldl - c levels ( 12.9% ) compared with major allele carriers while being still efficient in risk lipid reduction . furthermore , pcsk9 deficiency lowered the reduced cad outcome risk - associated cer(18:1/24:0 ) nonsignificantly by 6.4% , whereas simvastatin and simvastatin - ezetimibe combination both resulted in significant ( > 25% ) reductions . the classical risk markers were unable to distinguish cad patients who died during the follow - up from stable cad patients who had structural coronary disease at baseline . modestly higher total cholesterol concentrations ( 6% , p = .002 ) were recorded in the case groups and hdl - c and apoa1 were slightly lower in cases compared with controls ( 7.5% , p = .001 , and 7.5% , p = .001 , respectively ) ( table 1 ) . both case and control groups had median framingham 10-year risk of coronary heart disease ( 17 ) of 20% . the estimation of the 10-year risk of fatal cardiovascular disease in europe ( systematic coronary risk evaluation , score ) ( 18 ) was 7.1% in cases and 6.7% in controls , and the slight difference was not statistically significant . more pronounced differences between groups were observed through lipidomic analyses ( figure 1 and supplemental table 1 , published on the endocrine society 's journals online web site at http://jcem.endojournals.org ) . lactosylceramides laccer ( 18:1/18:0 ) and laccer ( 18:1/20:0 ) were among the risk - associated lipids together with cer ( 18:1/16:0 ) and cer ( 18:1/18:0 ) . several molecular lipid species such as ce20:5 ( 17.4% , p = .0001 ) and ce18:3 ( 15.1% , p = .000037 ) displayed reduced concentrations in cases as compared with the control group . abundance of several ceramide species containing specific fatty acids was higher along the axis of the glycosphingolipid pathway ( figure 2 ) in which ceramide is converted to galactosylceramide and glucosylceramide , glucosylceramide is converted to lactosylceramide , and lactosylceramide is metabolized to globotriaosylceramide ( gb3 ) . for example , d18:1/22:0-containing species were higher in ceramide , lactosylceramide , and globotriaosylceramide classes . a similar pattern is demonstrated by other long - chain containing ceramides ( d18:1/16:0 and d18:1/18:0 ) . however , cer(d18:1/24:0 ) lipid species concentration was lower in cases reflected in lower concentration of sphingomyelin containing the same fatty acid sm(d18:1/24:0 ) ( figure 2b ) . to account for lipid concentration differences due to metabolome influencing factors interestingly , when these patients were studied separately , very distinct lipidomic profiles were recorded for the two groups . in nondiabetic cad patients , the risk of fatal outcome was associated with elevated sphingolipids , whereas in diabetic cad patients , the risk was mainly associated with reduced cholesteryl ester species ( figure 3 ) . because we noted that factors like diabetes , smoking , and lipid - lowering treatment among others , could influence the expression of the risk - associated lipids , we decided to calculate ceramide and cerebroside ratios to check whether these would be less dependent on different patient characteristics while still indicative of risk . it turned out that the ratios of cer(18:1/16:0)/cer(18:1/24:0 ) and cer(18:1/22:0)/cer(18:1/24:0 ) were significantly related to increased risk of cvd death in all subjects and subgroups . cer(18:1/24:0)/cer(18:1/24:1 ) was indicative of a reduced risk of cvd death , regardless of diabetes status ( figure 3 ) . importantly , odds ratios for cvd death of these ratios remained significant after adjustment for traditional cvd death risk factors , such as ldl - c ( table 2 ) . unadjusted and adjusted odds ratios for cvd death of ceramide ratios abbreviation : or , odds ratio . models adjusted for age , bmi , fasting glucose , hdl - c , ldl - c , c - reactive protein , triglycerides , and systolic and diastolic blood pressure . predictive potential for cad mortality was tested by receiver - operating characteristic analysis for the ceramide ratios and compared with ldl - c . the area under curve and 95% confidence limits were 0.67 ( range 0.620.72 ) for cer(d18:1/16:0)/cer(d18:1/24:0 ) , 0.65 ( range 0.600.71 ) for cer(d18:1/20:0)/cer(d18:1/24:0 ) , and 0.68 ( range 0.630.73 ) for cer(d18:1/24:0)/cer(d18:1/24:1 ) , whereas for ldl - c , these values were 0.55 ( range 0.500.61 ) . finally , the effect of lipid - lowering treatments on ceramides and cerebrosides associated with increased risk of cvd death in statin - nave subjects as well as on ldl - c was evaluated ( figure 4 ) . as a comparison basis , we used the most significant lipids that separate the high - risk cad patients from stable cad patients ( figure 4a ) . the effect of different ldl - c - lowering methods on cad outcome - related lipidomic markers . results shown for top ranked cad risk lipids , ldl - c , and ceramide ( 18:1/24:0 ) . effect of ezetimibe ( 10 mg ) ( b ) , simvastatin ( 40 mg ) ( c ) , simvastatin + ezetimibe ( 40 mg+10 mg ) , ( d ) and pcsk9 loss - of - function mutation ( r46l ) ( e ) on cad mortality risk lipids . for evaluation of ezetimibe and simvastatin effects on these putative risk lipids we used plasma samples from a randomized clinical trial comparing simvastatin 40 mg , ezetimibe 10 mg , and their combination ( supplemental table 2 ) . as expected , simvastatin 40 mg lowered ldl - c significantly by 40% , and this reduction was accompanied by a significant reduction ( 25% ) also in all ceramides and cerebrosides recorded in this study ( figure 4c ) . ezetimibe treatment ( 10 mg ) in turn lowered ldl - c by 21% but affected only very modestly the plasma levels of cvd death - related lipid species ( figure 4b ) . in fact , cer18:1/18:0 and cer18:1/20:0 concentrations were slightly elevated in ezetimibe - treated subjects by 7.6% and 8.8% , respectively , whereas cer18:1/16:0 levels remained unchanged during this treatment . there was no statistically significant difference between treatment effects of simvastatin alone or in combination with ezetimibe on molecular lipids . however , combined treatment resulted in significantly lower ldl - c levels than simvastatin or ezetimibe alone ( figure 4d ) . drugs that inhibit pcsk9 are being developed as an alternative to statin lipid - lowering therapy , and we studied the effects of pcsk9 inhibition on the risk lipids ( supplemental table 3 ) . the effect was investigated by using subjects with the pcsk9 allelic variants from the luric patient population ( figure 4e ) . the pcsk9 loss - of - function mutation ( r46l ) resulted in a significant reduction of ceramides indicative of cad outcome risk comparable with that observed in simvastatin - treated subjects . interestingly , the mutation carriers had only modestly lower ldl - c levels ( 12.9% ) compared with major allele carriers while being still efficient in risk lipid reduction . furthermore , pcsk9 deficiency lowered the reduced cad outcome risk - associated cer(18:1/24:0 ) nonsignificantly by 6.4% , whereas simvastatin and simvastatin - ezetimibe combination both resulted in significant ( > 25% ) reductions . the present study demonstrates that specific molecular lipid species are significantly associated with mortality in cad . importantly , the predictive potential of distinct ceramides was superior to the currently used standard ldl - c measurement , underscoring the value of molecular lipidomic analyses . moreover , we show that these molecular lipid species are highly influenced by the choice of lipid - lowering treatment and that simple numerical ldl - c lowering per se is not necessarily translated to modulation of all risk - associated lipid species . in earlier experimental studies , ceramides and other sphingolipids have been associated with the development of atherosclerosis , and several enzymes in the ceramide synthetic pathway have been tested as potential drug targets in animal models ( 1921 ) . the present study suggests that different molecular ceramides associate with cad outcome risk , and thus , they may play a significant role also in plaque vulnerability in addition to development of atherosclerotic disease . notably , for some ceramide species containing specific fatty acids , higher concentrations were observed along the axis of glycosphingolipid pathway ( figure 2 ) with no significant differences observed in corresponding sphingomyelin species , although lower concentrations of other ceramide species with longer - chain fatty acids were reflected in lower concentrations of the corresponding sphingomyelin . these findings together with the fact that total sphingolipid levels remained unchanged indicate that alteration in sphingolipid balance is species dependent . a role of the glycosphingolipid pathway in atherosclerosis was previously suspected based on the following observations : lactosylceramide and glucosylceramide accumulate in the atherosclerotic plaque ( 22 ) , and both lactosylceramide and glucosylceramide suppress production of macrophage apoe and lead to an accumulation of cholesterol in macrophage foam cells ( 23 ) . importantly , inhibition of the glycosphingolipid pathway was previously shown to decrease atherosclerosis in mice ( 24 ) . to the best of our knowledge , our data for the first time link specific molecular lipid species from the glucosphingolipid pathway measured in human serum to cad mortality . on the other hand , a reverse association of long - chain ceramide and sphingomyelin d18:1/24:0 with cad mortality demonstrates the complexity of this disease , which is not seen by analyzing the lipid classes alone . further studies are needed to evaluate the potential connections between the ceramide species and thrombogenesis , fibrinolysis , and plaque instability that may explain the observed linkage between death from cvd reasons and molecular ceramides . another main finding of this study is that two different lipid - lowering drugs display distinct effects on the cvd risk - associated plasma ceramides , beyond their ldl - c - lowering effects . simvastatin 40 mg resulted in a broad lowering of all recorded ceramide and cerebroside species ; however , the magnitude of reduction was clearly lower than that for ldl - c . on the other hand , we demonstrated in this study that a very modest ldl - c effect due to pcsk9 deficiency resulted in a larger proportional reduction in cad outcome - related plasma ceramides . this suggests that there is an interaction between the means of ldl receptor ( ldlr ) up - regulation and the levels of circulating ceramides . both statins and pcsk9 inhibition increase ldlr - mediated hepatic lipid uptake , and it could thus be expected that they result in similar lipid - lowering profiles . however , statins are acting indirectly via inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase and inhibition of this rate - limiting enzyme of the mevalonate pathway has multiple effects on a number of genes related to lipid metabolism in hepatocytes . this is due to subsequent activation of the sterol regulatory element binding protein ( srebp)-1- and -2-mediated gene transcription ( 25 , 26 ) . pcsk9 inhibition , in turn , may act more precisely on ldlr in hepatocytes and thus may lead to more targeted lipid lowering without more systemic effects on lipid metabolism . therefore , pcsk9 inhibition seems a promising lipid - lowering method of choice from the plasma lipidomic point of view . in particular , this is the case if this inhibition leads to similar plasma lipidomic composition as the well - characterized loss - of - function mutation . however , these data are based on a 2-week intervention , and thus , the long - term effect must await confirmation in longer studies . on the other hand , taylor et al demonstrated recently that in the arterial biology for the investigation of the treatment effects of reducing cholesterol-6 trial , ezetimibe treatment resulted in a paradoxical progression of carotid intima - media thickness in association with both greater ldl - c reduction and cumulative drug exposure ( 27 ) . the authors concluded that their findings may suggest the presence of off - target actions of ezetimibe . based on our current results , one could hypothesize that the paradoxical effect on carotid intima - media thickness is due to the minimal effects on ceramides despite the substantial ldl - c - lowering effects . this study suggests that ldl - c and traditional risk factors provide only limited predictive information on fatal cvd complications in patients with established cad . however , molecular lipid assays can be used to improve the identification of cad patients at high risk , and such assay results may also serve as better efficacy indicators for lipid - modifying drugs . it is important to note that although traditional risk scores are developed for the general population , for the purpose of this study , we selected only diseased patients and investigated association of lipids with stable or unstable disease . as a result , both patient groups were at high risk from the start of the follow - up as indicated by framingham and european risk score values . a limitation of our study is that only male subjects from only one cohort were evaluated . we selected men for reducing variability in the data because atherosclerotic development is gender specific ( 28 ) , and traditionally risk scores are evaluated separately for men and women ( 17 , 18 ) . thus , our results should be validated in an independent large cohort and extended to women . in the present work , longer exposure time , greater subject numbers , and possibly study in patients with cad will be needed to further understand the dynamics of modulation of the risk lipids .
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 .