# Patent

## Title
Lipidomic biomarkers for identification of high-risk coronary artery disease patients

## Abstract

The present invention inter alia provides a method, and use thereof, of predicting severe CVD complications such as AMI or CVD death by detecting the lipid concentrations or lipid ratios of a biological sample and comparing it to a control and has identified specific lipid markers that are more specific and sensitive in predicting these CVD complications than currently utilized clinical markers. Also provided is an antibodies towards said lipids, and the use thereof for predicting, diagnosing, preventing and/or treating CVD complications. The invention additionally relates to kits comprising lipids and/or an antibody thereto, for use in the prediction and/or diagnosis of CVD complications.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/805,319, filed 18 Dec. 2012 (now U.S. Pat. No. 9,201,080), which is a U.S. National Stage application of PCT/EP2011/060253 filed 20 Jun. 2011, which claims priority to European patent application 10006399.9 filed 20 Jun. 2010 and U.S. Provisional Patent Application 61/356,675 filed 21 Jun. 2010, the entire disclosures of which are herein incorporated by reference in their entireties.


DESCRIPTION
Field of the Invention
This invention relates to methods and uses involving lipid levels to predict and prevent severe cardiovascular disease-associated fatal complications. The invention thus provides a means to identify and treat high-risk coronary artery disease patients. The methods include analyzing lipid levels of a biological sample, and comparing it to a control.
Background of the Invention
Worldwide, cardiovascular diseases (CVD) are among the leading causes of mortality and morbidity with ever-increasing prevalence. CVD is used to classify numerous conditions that affect the heart, heart valves, blood, and vasculature of the body. One of these conditions is coronary artery disease (CAD). Early targeted initiation of preventive measures of CVD-related fatal complications, such as acute myocardial infarction (AMI) and death, would be of great benefit and can provide a major opportunity in reducing mortality and morbidity in patients suffering from CVD. To this end, accurate identification of individuals who are at risk of developing CVD complications is essential. However, traditional risk assessment fails to recognize a substantial proportion of patients at high risk while a large proportion of individuals are classified as having intermediate risk, leaving patient management uncertain. Additional strategies to further refine risk assessment of high-risk CVD are therefore highly needed. To this end, the inventors have evaluated the role of novel lipidomic biomarkers as a prognostic tool for fatal cardiovascular events in CVD patients.
Plasma or serum total cholesterol, LDL-cholesterol or HDL-cholesterol concentrations have been used as gold standard biomarkers for CVD/CAD risk prediction. However, a number of coronary artery disease (CAD) or acute myocardial infarction (AMI) patients have LDL-C levels within the recommended range suggesting the need for additional diagnostic measures of the residual risk. It is evident from earlier large scale population studies that these measurements associate with the CAD risk and CAD endpoints such as AMI or cardiovascular death. Therefore, preventive treatment strategies have so far been addressed to lower LDL-C concentrations (mainly by statin treatment) and more recently also attempts to raise HDL-C have been made (e.g., by CETP-inhibitors). On the other hand, it has also been observed that one half of the AMI patients actually do have normal LDL cholesterol levels and that there is a substantial residual risk in statin treated patients despite a LDL-C lowering. Furthermore, recent publications have demonstrated that plasma levels of apolipoprotein B (apoB), the main surface protein on LDL particles, and LDL-C, the amount of cholesterol in those particles, are correlated and, considered separately, as positive risk factors. Plasma levels of apolipoprotein A1, the main surface protein on HDL particles, and HDL-C, the amount of cholesterol in those particles, are also correlated with each other and, considered separately, as negative risk factors. Importantly, for a given usual apoB, lower LDL-C has been observed to associate with a higher risk of AMI supporting the view that, on average, LDL particles with low cholesterol content per particle (small, dense LDL particles) are particularly hazardous. Thus, it seems possible that LDL-C associates directly with the more dangerous molecules carried by LDL-particle and that LDL-C is only an indirect measurement of the risk. Therefore, it is of importance to search for molecules e.g., certain lipid species that are directly related with hazardous (i.e., fatal) cardiovascular events.
Lipid metabolite imbalance is a probable cause of dyslipidemia and the ensuing atherosclerosis manifested in its gravest form as the vulnerable atherosclerotic plaque. Atherosclerotic plaques are complex molecular formations that contain numerous lipids. However, there are other factors than lipid rich plaques or LDL cholesterol that make lipids an attractive group of molecules for CVD studies. Lipids are tightly regulated which makes Lipidomic data robust and informative on the current state of the studied organism. Also, lipids are one of the culmination points of a biological system, more the true outcome than the predictor. Combining Lipidomic data with appropriate biobanked clinical material presents a good opportunity for biomarker discovery. Moreover, lipidomics can be used as a gauge of efficacy and safety in drug development and evolving theragnostics. Lipidomic biomarkers are prime candidates for true companion diagnostics in the CVD area and present many opportunities for improved translational medicine as well.
The plaque building blocks and lipoprotein components that are thought to traffic lipids to the site of lesion formation can now be resolved with Lipidomic studies correlating lipid structure and composition to function and thereby disease pathogenesis. While the number of lipid mediators in the human body is overwhelming, their identification and quantification is facilitated by the advances in mass spectrometry and lipid biochemistry, which today enable the simultaneous high throughput identification and quantification of hundreds of molecular lipid species in several lipid classes (Ejsing C S, et al: Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc Natl Acad Sci USA 2009, 106:2136-2141; Stahlman M, et al: High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009 Hiukka A, et al: ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan. Diabetes 2009, 58:2018-2026; Linden D, et al: Liver-directed overexpression of mitochondrial glycerol-3-phosphate acyltransferase results in hepatic steatosis, increased triacylglycerol secretion and reduced fatty acid oxidation. FASEB J 2006, 20:434-443.) collectively referred to as the lipidome. Lipidomic studies identify lipid cellular distribution and describe their biochemical mechanisms, interactions and dynamics. Importantly, lipidomics quantifies the exact chemical composition of lipidomes (Han X, Gross R W: Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 2003, 44:1071-1079).
Due to both high sensitivity and selectivity of lipidomics, even the smallest sample amounts can be analyzed today. The bulk of the lipid data in the art today presents lipids in a sum composition format, i.e. phosphatidylcholine (PC) 34:1 (Brugger B, et al: Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci USA 1997, 94:2339-2344) where the molecular lipid and the attached fatty acid tails remain unidentified. The identification of molecular lipid species, e.g., PC 16:0/18:1 (Ekroos K, et al: Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation. J Lipid Res 2003, 44:2181-2192) is the main feature of advanced lipidomics, which delivers highly resolved molecular lipid species rather than summed fatty acid information. For example, the information of the type of fatty acids and their positions of attachment to the glycerol backbone making up the particular PC molecule is revealed. There are conventional techniques such as thin-layer chromatography combined with gas chromatography but they not only require considerably larger sample amounts and laborious sample preparation, but they do not deliver the molecular lipid species. Despite multiple mass spectrometry techniques capable of characterizing lipid entities, most of them are still unable to deliver reliable high-quality quantitative data in terms of absolute or close-to absolute concentrations. In the context of the present invention, electrospray ionization mass spectrometry-based lipidomics is the preferred technology and can utilize both shotgun and targeted lipidomics for exhaustive deciphering and precise quantification of molecular lipidomes. The superior quality and specificity of shotgun and targeted lipidomics will meet stringent regulatory standards, such as good laboratory practice guidelines (GLP) when set-up in the proper environment. Using these technologies quantification of up to two thousand molecular lipids is possible even in a high throughput format.
Lipidomics is a tool for differentiating patients based on their molecular lipid profiles. Personalized medicine and diagnostics enabled by lipidomics will facilitate the mission of the right individual receiving the right drug at the right time and dose. Several works employing analytes consisting of lipids, proteins and hydrophilic molecules among many others have been conducted to meet the needs of personalized medicine. Recently, non-hypothesis-driven metabolomic screenings have been used to identify novel CVD biomarkers.
For example, WO2004/038381 discloses a method for metabolomically facilitating the diagnosis of a disease state of a subject, or for predicting whether a subject is predisposed to having a disease state wherein the small molecule profile from a subject is obtained and compared to a standard small molecule profile.
WO2008/148857 discloses a method to assess the risk of cardiovascular disease in a patient (including atherosclerosis) by isolating the HDL fraction and sub-fraction from a blood sample of the patient. The components of the HDL fraction or sub-fraction to be measured were Sphingosine-1-Phosphate (S1P), sphingomyelin (SM) and Apolipoprotein A-I (apoA-1).
WO2008/11943 further discloses markers for detecting coronary artery disease that can indicate a patient at risk of having or developing coronary artery disease. These include 15 “first-choice” molecules which were: C18:3 Cholesterol ester, C32:1 Phosphatidylcholine, Alanine, Lipid (mainly VLDL), Lysine, Hexadecanoic acid, C36:2 Phosphatidylcholine, Formate, C32:2 Phosphatidylcholine, C18:2 (Linoleic Acid), Cholesterol, C 18:2 Lyso-phosphatidylcholine, C36:3 Phosphatidylcholine, C34:4 Phosphatidylcholine and C34:3 Phosphatidylcholine.
Furthermore, US2007/0099242 describes a method to determine if a subject is at risk to develop, or is suffering from cardiovascular disease. The method involves determining a change in the amount of a biomarker in the biological sample or HDL sub-fraction thereof, compared to a control sample, wherein the biomarker is at least one of Apolipoprotein C-IV (“ApoC-IV”), Paraoxonase 1 (“PON-1”), Complement Factor 3 (“C3”), Apolipoprotein A-IV (“ApoA-IV”), Apolipoprotein E (“ApoE”), Apolipoprotein LI (“ApoL1”), Complement Factor C4 (“C4”), Complement Factor C4B1 (“C4B1”), Histone H2A, Apolipoprotein C-II (“ApoC-II”), Apolipoprotein M (“ApoM”), Vitronectin, Haptoglobin-related Protein and Clusterin. The document also discloses a method for detecting the presence of one or more atherosclerotic lesions wherein a change in the amount of a biomarker in the biological sample or HDL sub-fraction thereof is detected, compared to a control sample and wherein the biomarker is selected from PON-1, C3, C4, ApoE, ApoM and C4B1. All biomarkers mentioned in this document are protein or lipoprotein biomarkers.
From previous work it cannot be extrapolated that lipid analysis will yield by default a CVD biomarker predictive to the fatal outcomes associated with CVD/CAD. The present invention identifies biomarkers of high risk CVD by absolute, or close to absolute, quantification of defined molecular lipid species instead of profiling multiple analytes. Importantly, while many of the existing biomarker candidates are composite fingerprints of multiple factors, the lipidomics approach herein shows value already at a level of single species or ratios thereof.
In the present invention herein, lipid biomarker concentrations have been measured and quantified in patients with documented CAD who did not show fatal outcomes during the follow-up period (3 years) and in high-risk CAD patients who died due to cardiovascular events during the follow-up period. This invention thus enables accurate usage of the lipid-based biomarkers to identify high risk CVD/CAD patients. Another layer of accuracy was reached through a careful patient selection since it is important to control for factors which may affect the lipid concentration read-outs. Unlike the previous efforts described above, we used specific targeted platforms on a singular technology set-up to analyze lipid species in particular.
The technology and the way it was applied in the context of the inventive teaching presented herein is set apart from similar efforts in the field inter alia due to the following criteria. In sample preparation, samples are strictly controlled and treated identically to avoid potential artifacts that could arise from improper handling. In connection with the present invention, samples were carefully thawed slowly on ice and directly thereafter subjected to a custom-made automated lipid extraction which possesses currently the highest precision in liquid handling, therefore minimizing potential errors. Furthermore, sample freeze-thaw cycles were strictly controlled since this can dramatically affect the lipid stabilities. The automated lipid extraction is based on the method by Folch and colleagues (Folch J, et al: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957, 226(1):497-509) which uses chloroform and methanol. This method is preferred when a wide range, from polar to non-polar, of lipid classes are to be extracted with optimal recoveries thus preventing the loss of lipid species. Lipid class specific non-endogenous lipids, when applicable, were used as internal standards to gain highest precision in identification (minimizing false positives) and quantification of monitored molecular lipid species. In this way absolute or semi-absolute amounts of endogenous molecular lipids were determined with the highest precision that can be achieved with today's technologies. The endogenous lipids and respective standards were monitored at the molecular lipid level. In this way, not only false positive identifications were minimized, but molecular lipids could be precisely determined and quantified. Analysis quality was strictly controlled using a novel quality control system. This was mainly controlled by multiple internal standards (IS), external standards (ES), IS/ES ratios, and instrument control samples. By stringently controlling these components, technical and biological outliers were readily identified and rejected from further analysis. To obtain best precision in sensitivity, selectivity and quantification for each molecular lipid different targeted platforms were used. Some lipids are best analyzed using high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC) combined with mass spectrometry based multiple reaction monitoring (MRM) whereas others are best analyzed by direct infusion in combination with mass spectrometry-based precursor ion scanning and neutral loss scanning techniques.
SUMMARY OF THE INVENTION
The present invention provides novel lipidomic markers for predicting and preventing severe CVD/CAD-associated complications, including AMI and death. These markers thus provide a means to identify and treat high-risk coronary artery disease patients. Specifically, it has been found that the lipid molecules, lipid-lipid ratios and lipid-clinical concentration ratios provided herein, when displaying an increased or decreased level—as the case may be—in samples from CAD patients, are useful lipidomic markers for the methods and uses in accordance with the present invention. These sensitive and specific markers were specifically tested to display superior diagnostic and prognostic value compared to the current clinically-used markers predictive for CVD/CAD outcomes. In fact, the currently available biomarkers such as LDL-C or HDL-C have only very limited or no value in predicting the CVD death risk in CAD patients. The present invention therefore represents a significant advantage to other markers which are currently used to diagnose and/or predict CVD and CVD complications, which include LDL-C, total plasma/serum cholesterol and Apolipoprotein B and A1. Thus, the lipidomic markers provided herein allow better diagnosis of or assessment of the risk to develop major CVD complications such as AMI or CVD death.
In accordance with the present invention, methods are inter alia disclosed herein for determining the risk of a patient to develop CVD complications, determining warning signs of CVD risks, (including death, myocardial infarction (MI), angina pectoris, transischemic attack (TIA) and stroke) in said patient.
Methods according to the invention typically comprise the steps of: a) providing a biological sample from a CAD subject; b) determining a lipid concentration, lipid-lipid ratio, or lipid-clinical concentration ratio or (a) corresponding profile(s) from said sample (i.e., determining information on a lipidomic marker in accordance with the invention); and c) comparing said determined lipid concentration, lipid-lipid ratio, or lipid-clinical concentration ratio or said corresponding profile(s) to the corresponding lipid concentration, lipid-lipid ratio, or lipid-clinical concentration ratio or the corresponding profile(s) in a control.
The control may be a sample from (a) CAD patient(s) with no history of major CVD events. It may also be a sample that represents a combination of samples from a CAD patient population with no history of major CVD events. Alternatively, the control may be a set of data concerning a lipidomic marker in accordance with the present invention, e.g., information on the concentration of (a) lipid(s), lipid-lipid ratio(s), or lipid-clinical concentration ratio(s) in accordance with the present invention in a sample when taken from (a) CAD patient(s) with no history of major CVD events, or in a combination of samples taken from a CAD patient population with no history of major CVD events. Said information, and thus the corresponding set of data, may have been previously determined, calculated or extrapolated, or may have yet to be determined, calculated or extrapolated, or may also be taken from the literature.
As mentioned above, the lipidomic marker to be compared between the subject sample and the control (or control sample) may be one or more of the lipid concentration(s), lipid-lipid ratio(s), or lipid-clinical concentration ratio(s) or combinations thereof, i.e., the corresponding profile(s), as described and claimed herein. In this regard, the control or control sample allows establishment of the lipidomic marker baseline or starting point.
In connection with all aspects and embodiments of the invention described and claimed herein, the determination of the lipid concentration(s), the lipid-lipid ratio(s) or the lipid-clinical concentration ratio(s) is typically performed using an assay. Collecting information on a lipidomic marker (i.e., the concentration(s) of (a) lipid(s), lipid-lipid ratio(s), or lipid-clinical concentration ratio(s) or combinations thereof, i.e., corresponding profile(s)) from the sample of a patient and, where appropriate, a corresponding control sample, can be performed with various chemical and high-resolution analytical techniques. Suitable analytical techniques include, but are not limited to, mass spectrometry and nuclear resonance spectroscopy. Any high-resolution technique capable of resolving individual lipids or lipid classes and providing structural information of the same can be used to collect the information on the lipidomic marker in question, e.g., lipid profile from the biological sample. Collecting the information on the lipidomic marker with mass spectrometry (MS) is one of the preferred embodiments of the current invention. The MS instrument can be coupled to a direct sample infusion method, such as a robotic nanoflow ion source device, or to a high performance separation method such as high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC).
Again in accordance with all aspects and embodiments described and claimed herein, both the sample from the subject and the control sample is preferably a blood sample, more preferably a blood plasma sample, or also preferably a blood serum sample. It may also be a fraction of blood, blood plasma or blood serum, e.g., a lipoprotein fraction. A blood sample can be prepared and plasma or serum, or fractions thereof, can be separated therefrom with techniques well known to the person skilled in the art. Alternatively, both the sample from the subject and the control sample may also be a tissue sample, e.g., artery tissue, such as carotid artery tissue, or artery plaque material, such as carotid artery plaque material.
The lipidomic markers of the present invention allow for prediction and prevention of fatal CVD complications. This will facilitate earlier intervention, less symptom development and suffering and decreased morbidity/mortality associated with CVD. Thus, the lipidomic markers described and claimed herein allow for individual tailoring of drug intervention for patients being at risk to develop major CVD complications.
In other words, the present invention discloses diagnostic and/or predictive lipid markers and lipid-lipid or lipid-clinical concentration ratios for use in predicting CVD complications such as AMI or CVD death. The invention uses the measurement of lipid concentrations, lipid-lipid and/or lipid-clinical concentration ratios to determine the risk of said subject to develop CVD complications such as AMI and/or CVD death. The subject may have previously suffered from a cardiovascular disease event such as angina pectoris, myocardial infarction or stroke. The CVD may or may not be a result of atherosclerosis.
Accordingly, in one aspect of the invention, a method is provided for determining whether a subject is at risk to develop one or more CVD complications, such as AMI or CVD death, said method comprising determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications, such as AMI or CVD death, wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Tables 4a and 7a):
Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/24:1), GlcCer(d18:1/18:0), LacCer(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), PC O-32:0 (KDdiA-PC), PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl), PS O-18:2/16:0-alkenyl and Total LacCer; and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Tables 4a and 7a):

CE 14:0, CE 16:0, CE 17:1, CE 20:3, Cer(d18:0/22:0), Cer(d18:0/24:0), LPC 18:1, PC 16:0/18:2, PC 16:0/20:3, PC 16:0/20:4, PC 16:0/22:6, PC 18:0/18:1, PC 18:0/20:3, PC 18:0/20:4, PC 18:1/18:2, SM (d18:1/14:0) (d18:1/13:1-OH), SM (d18:1/23:0) (d18:1/22:1-OH), SM (d18:1/24:0) (d18:1/23:1-OH), Total CE, Total LPC and Total PC.

In a particular embodiment, a method is provided for determining whether a subject is at risk to develop one or more CVD complications, such as AMI or CVD death, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications, such as AMI or CVD death, and wherein the one or more lipid(s) whose increase in concentration is (are) compared to the control is (are) selected from (Tables 4b and 7b):
Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:1), Cer(d18:1/26:1), GlcCer(d18:1/18:0), GlcCer(d18:1/20:0), GlcCer(d18:1/24:1), GlcCer(d18:1/26:1), LacCer(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), LacCer(d18:1/24:1), PC O-32:0 (KDdiA-PC), PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:2/16:0-alkenyl, Total Cer, Total DAG and Total LacCer;

and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Tables 4b and 7b):

CE 14:0, CE 17:1, CE 20:3, Cer(d18:0/24:0), LPC 18:1, PC 16:0/20:3, PC 16:0/20:4, PC 18:0/20:4, PC O-40:3, SM (d18:1/14:0) (d18:1/13:1-OH), Total LPC and Total PC.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Table 8):
Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), PS O-18:2/16:0-alkenyl, PS O-16:1/18:2-alkyl, Total Cer, Total LacCer, GlcCer(d18:1/24:1), LacCer(d18:1/22:0) and Cer(d18:1/18:0).
In another preferred embodiment, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Table 8):
Total PC, PC 16:0/20:4, Cer(d18:0/24:0), Total LPC, CE 14:0, CE 20:3, CE 17:1, PC 16:0/20:3, LPC 18:1, PC 18:0/20:3, PC 18:0/18:1 and Cer(d18:0/22:0).
In a particularly preferred embodiment, the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), LacCer(d18:1/22:0) and Cer(d18:1/18:0).
In another particularly preferred embodiment, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Table 11):
PC 16:0/20:4 and Cer(d18:0/24:0).
In an alternative embodiment, the present invention relates to a method for determining whether a subject is at risk to develop one or more complications such as AMI or CVD death, comprising determining in a sample from said subject one or more lipid-lipid ratio(s), wherein (an) increased or decreased lipid-lipid ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications, such as AMI or CVD death, wherein the one or more lipid-lipid ratio(s) whose increase(s) is (are) compared to the control is (are) selected from (Tables 5a and 8a):
CE 16:0/CE 18:3, CE 18:2/CE 18:3, CE 19:1/Cer(d18:0/22:0), Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/16:0)/Total PC, Cer(d18:1/18:0)/LPC 16:0, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/18:0)/Total PC, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/20:0)/Total PC, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/22:0)/Total PC, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:1/24:1)/Total CE, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/24:1)/Total PC, Cer(d18:1/26:0)/PC O-40:0, GlcCer(d18:1/20:0)/PC 16:0/20:4, GlcCer(d18:1/20:0)/Total PC, GlcCer(d18:1/26:0)/Total CE, LacCer(d18:1/16:0)/Total LPC, LacCer(d18:1/18:0)/PC 16:0/18:1, LacCer(d18:1/18:0)/PC 16:0/20:3, LacCer(d18:1/18:0)/PC 18:0/18:1, LacCer(d18:1/18:0)/PC 18:0/20:3, LacCer(d18:1/18:0)/PC 18:1/18:1, LacCer(d18:1/18:0)/PC 18:1/18:2, LacCer(d18:1/18:0)/Total LPC, LacCer(d18:1/18:0)/Total PC, LacCer(d18:1/20:0)/PC 16:0/18:1, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/20:3 LacCer(d18:1/20:0)/PC 18:1/18:1, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/PC 18:2/18:2, LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0), LacCer(d18:1/20:0)/Total LPC, LacCer(d18:1/20:0)/Total PC, LacCer(d18:1/20:0)/Total SM, LacCer(d18:1/22:0)/PC 16:0/20:3, LacCer(d18:1/22:0)/PC 16:0/20:4, LacCer(d18:1/22:0)/PC 18:0/20:3, LacCer(d18:1/22:0)/SM (d18:1/14:0) (d18:1/13:1-OH), LacCer(d18:1/22:0)/Total LPC, LacCer(d18:1/22:0)/Total PC, LacCer(d18:1/24:0)/PC 16:0/20:3, LacCer(d18:1/24:0)/Total LPC, LacCer(d18:1/24:1)/Total LPC, LacCer(d18:1/24:1)/Total PC, LacCer(d18:1/24:1)/Total PC O, PC 16:0/16:0/PC 16:0/20:4, PC 16:0/16:0/Total PC, PC 16:0/18:2/Total PC, PC O-18:0/18:2-alkyl/PC O-36:5, PC O-32:0 (KDdiA-PC)/PC O-38:5, PS O-16:0/18:2-alkenyl/Total PS O, PS O-16:1/18:2-alkyl/Total PS O, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl)/Total PS O, PS O-18:2/16:0-alkenyl/Total PS O, Total Cer/Total PC, Total LacCer/Total PC and Total LacCer/Total PC O;

and wherein the one or more lipid-lipid ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from (Tables 5a and 8a):

CE 14:0/Cer(d18:1/24:1), CE 16:0/Cer(d18:1/24:1), CE 16:1/CE 19:1, CE 16:1/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/20:0), CE 16:1/Cer(d18:1/24:1), CE 16:1/GlcCer(d18:1/18:0), CE 16:1/GlcCer(d18:1/20:0), CE 16:1/LacCer(d18:1/16:0), CE 16:1/LacCer(d18:1/18:0), CE 16:1/LacCer(d18:1/20:0), CE 16:1/LacCer(d18:1/22:0), CE 16:1/LacCer(d18:1/24:0), CE 16:1/PC 16:0/16:0, CE 16:1/Total LacCer, CE 17:1/Cer(d18:1/24:1), CE 17:1/GlcCer(d18:1/24:1), CE 17:1/LacCer(d18:1/18:0), CE 18:1/Total LacCer, CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 18:3/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/22:0), CE 18:3/Cer(d18:1/24:0), CE 18:3/Cer(d18:1/24:1), CE 18:3/GlcCer(d18:1/18:0), CE 18:3/GlcCer(d18:1/20:0), CE 18:3/LacCer(d18:1/18:0), CE 18:3/LacCer(d18:1/20:0), CE 18:3/LacCer(d18:1/22:0), CE 18:3/LacCer(d18:1/24:0), CE 18:3/PC 16:0/16:0, CE 18:3/PC O-34:1, CE 18:3/PS O-16:0/18:1-alkenyl (PS O-16:1/18:1-alkyl), CE 18:3/PS O-16:0/18:2-alkenyl, CE 18:3/PS O-16:1/18:2-alkyl, CE 18:3/Total CE, CE 18:3/Total Cer, CE 18:3/Total LacCer, CE 20:3/Cer(d18:1/24:1), CE 20:3/LacCer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/LacCer(d18:1/24:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/22:0)/Total CE, Cer(d18:0/24:0)/Cer(d18:1/16:0), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/GlcCer(d18:1/20:0), Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/SM (d18:1/17:0) (d18:1/16:1-OH), Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Total Cer, Cer(d18:0/24:1)/Total CE, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/24:1), LPC 16:0/Total LacCer, LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), LPC 18:2/PS O-16:0/18:2-alkenyl, LPC 18:2/PS O-16:1/18:2-alkyl, PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, SM (d18:1/24:0) (d18:1/23:1-OH)/Total Cer and Total LPC/Total LacCer.

In an alternative embodiment, the present invention relates to a method for determining whether a subject is at risk to develop one or more complications such as AMI or CVD death, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject one or more lipid-lipid ratio(s), wherein (an) increased or decreased lipid-lipid ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications, such as AMI or CVD death, wherein the one or more lipid-lipid ratio(s) whose increase(s) is (are) compared to the control is (are) selected from (Tables 5b and 8b):
CE 16:0/CE 18:3, CE 18:0/CE 18:3, CE 18:2/CE 18:3, Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/16:0)/Total PC, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/18:1, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/18:0)/PC 18:1/18:1, Cer(d18:1/18:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/18:0)/SM (d18:1/17:2-OH), Cer(d18:1/18:0)/SM (d18:1/18:1), Cer(d18:1/18:0)/Total CE, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/18:0)/Total PC, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 18:0/18:1, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/20:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/20:0)/Total LPC, Cer(d18:1/20:0)/Total PC, Cer(d18:1/20:0)/Total PC O, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/22:0)/PC 18:0/20:4, Cer(d18:1/22:0)/Total PC, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/18:2, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/18:2, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/PC 18:1/18:1, Cer(d18:1/24:1)/PC 18:1/18:2, Cer(d18:1/24:1)/PC O-40:3, Cer(d18:1/24:1)/SM (d18:1/17:1-OH), Cer(d18:1/24:1)/SM (d18:1/18:0), Cer(d18:1/24:1)/SM (d18:1/23:0) (d18:1/22:1-OH), Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:1/24:1)/Total CE, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/24:1)/Total PC, GlcCer(d18:1/26:0)/Total CE, GlcCer(d18:1/26:1)/Total CE, LacCer(d18:1/18:0)/Total PC, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/20:3, LacCer(d18:1/20:0)/PC 18:0/20:4, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0), LacCer(d18:1/20:0)/Total CE, LacCer(d18:1/20:0)/Total LPC, LacCer(d18:1/20:0)/Total PC, LacCer(d18:1/20:0)/Total SM, LacCer(d18:1/24:0)/Total LPC, PC 16:0/16:0/PC 16:0/20:4, PC 16:0/16:0/Total PC, PC O-32:0 (KDdiA-PC)/Total PC O, PS O-16:0/18:2-alkenyl/Total PC, PS O-16:0/18:2-alkenyl/Total PC O, PS O-16:0/18:2-alkenyl/Total PS O, PS O-16:1/18:2-alkyl/Total PC, PS O-16:1/18:2-alkyl/Total PC O, PS O-16:1/18:2-alkyl/Total PS O, PS O-18:2/16:0-alkenyl/Total PC O, PS O-18:2/16:0-alkenyl/Total PS O, SM (d18:1/17:0) (d18:1/16:1-OH)/Total PC O, Total Cer/Total PC, Total DAG/Total LPC, Total DAG/Total PC, Total DAG/Total PC O and Total LacCer/Total PC;

and wherein the one or more lipid-lipid ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from (Tables 5b and 8b):

CE 14:0/Cer(d18:1/18:0), CE 14:0/Cer(d18:1/24:1), CE 14:0/Total DAG, CE 15:0/Cer(d18:1/20:0), CE 16:0/Cer(d18:1/18:0), CE 16:0/Cer(d18:1/24:1), CE 16:1/CE 19:1, CE 16:1/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/20:0), CE 16:1/Cer(d18:1/24:1), CE 16:1/GlcCer(d18:1/24:1), CE 16:1/LacCer(d18:1/18:0), CE 16:1/LacCer(d18:1/24:0), CE 16:1/Total LacCer, CE 17:1/Cer(d18:1/18:0), CE 17:1/Cer(d18:1/24:1), CE 18:2/Cer(d18:1/20:0), CE 18:2/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 18:3/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/22:0), CE 18:3/Cer(d18:1/24:0), CE 18:3/Cer(d18:1/24:1), CE 18:3/GlcCer(d18:1/20:0), CE 18:3/LacCer(d18:1/20:0), CE 18:3/LacCer(d18:1/22:0), CE 18:3/LacCer(d18:1/24:0), CE 18:3/PC 16:0/16:0, CE 18:3/PC O-34:1, CE 18:3/PS O-16:0/18:2-alkenyl, CE 18:3/PS O-16:1/18:2-alkyl, CE 18:3/Total CE, CE 18:3/Total Cer, CE 18:3/Total DAG, CE 18:3/Total LacCer, CE 20:3/Cer(d18:1/24:1), CE 20:3/LacCer(d18:1/20:0), CE 20:4/Cer(d18:1/18:0), CE 20:4/Cer(d18:1/24:1), CE 20:4/GlcCer(d18:1/20:0), CE 20:4/GlcCer(d18:1/24:1), CE 20:4/LacCer(d18:1/20:0), CE 20:5/LacCer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/22:0)/Total CE, Cer(d18:0/22:0)/Total DAG, Cer(d18:0/22:0)/Total GlcCer, Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Total Cer, DAG 16:0/18:1/Total DAG, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/24:0), LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 16:0/20:4/PS O-16:0/18:2-alkenyl, PC 16:0/20:4/PS O-16:1/18:2-alkyl, PC 16:0/20:4/Total DAG, PC 18:0/20:3/PS O-16:0/18:2-alkenyl, PC 18:0/20:3/PS O-16:1/18:2-alkyl, PC 18:0/20:3/PS O-18:2/16:0-alkenyl, PC 18:0/20:4/PS O-16:0/18:2-alkenyl, PC 18:0/20:4/PS O-16:1/18:2-alkyl, PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, PC 18:1/18:2/Total Cer, PC O-40:3/PS O-18:2/16:0-alkenyl, SM (d18:1/23:0) (d18:1/22:1-OH)/Total DAG, SM (d18:1/24:0) (d18:1/23:1-OH)/Total Cer, Total CE/Total DAG and Total LPC/Total LacCer.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 9):
GlcCer(d18:1/26:1)/Total CE, Cer(d18:1/24:1)/Total PC, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, Total Cer/Total PC, Total LacCer/Total PC, LacCer(d18:1/20:0)/PC 18:1/18:2, PS O-16:0/18:2-alkenyl/Total PS O, Cer(d18:1/18:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/Total LPC and LacCer(d18:1/20:0)/PC 16:0/20:4;
In another preferred embodiment, the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 9):
Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/Cer(d18:1/24:1), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Total Cer, Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), Total LPC/Total LacCer and GlcCer(d18:1/26:0)/LacCer(d18:1/22:0).
In a particularly preferred embodiment, the one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/24:1)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, PS O-16:0/18:2-alkenyl/Total PS 0 and Cer(d18:1/18:0)/PC 16:0/20:4;
and the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 11):
GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Total Cer, Total LPC/Total LacCer, GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl and Cer(d18:0/24:0)/LacCer(d18:1/24:0);
In yet another alternative embodiment the present invention relates to a method for determining whether a subject is at risk to develop one or more CVD complications, such as AMI or CVD death, comprising determining in a sample from said subject one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications, such as AMI or CVD death, wherein the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Tables 6a and 9a):
Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/HDL cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/triglycerides, GlcCer(d18:1/24:0)/total cholesterol, LacCer(d18:1/16:0)/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/HDL cholesterol, LacCer(d18:1/18:0)/LDL cholesterol, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/18:0)/total-c/HDL-c, LacCer(d18:1/18:0)/triglycerides, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/22:0)/total-c/HDL-c, LacCer(d18:1/22:0)/triglycerides, LacCer(d18:1/24:0)/apoA1/apoB, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, PC O-32:0 (KDdiA-PC)/apolipoprotein A-I, PC O-32:0 (KDdiA-PC)/triglycerides, PC O-34:1/triglycerides, PS O-16:0/18:1-alkenyl (PS O-16:1/18:1-alkyl)/triglycerides, PS O-16:0/18:2-alkenyl/triglycerides, PS O-16:1/18:2-alkyl/triglycerides, PS O-18:2/16:0-alkenyl/HDL cholesterol, PS O-18:2/16:0-alkenyl/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/total cholesterol, Total Cer/triglycerides, Total GlcCer/apolipoprotein B, Total GlcCer/total cholesterol, Total LacCer/apolipoprotein A-I, Total LacCer/apolipoprotein B, Total LacCer/total cholesterol and Total LacCer/triglycerides;

and wherein the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 6a and 9a):

CE 14:0/apolipoprotein B, CE 14:0/LDL cholesterol, CE 14:0/LDL-c/HDL-c, CE 14:0/total cholesterol, CE 14:0/total-c/HDL-c, CE 16:1/apolipoprotein B, CE 16:1/HDL cholesterol, CE 16:1/LDL cholesterol, CE 16:1/total cholesterol, CE 17:1/LDL-c/HDL-c, CE 18:3/apoA1/apoB, CE 18:3/apolipoprotein A-I, CE 18:3/apolipoprotein B, CE 18:3/HDL cholesterol, CE 18:3/LDL cholesterol, CE 18:3/LDL-c/HDL-c, CE 18:3/total cholesterol, CE 18:3/total-c/HDL-c, CE 20:3/apolipoprotein B, CE 20:3/LDL-c/HDL-c, CE 20:3/total-c/HDL-c, CE 20:5/apolipoprotein B, CE 20:5/HDL cholesterol, CE 20:5/LDL cholesterol, Cer(d18:0/22:0)/apolipoprotein B, Cer(d18:0/22:0)/LDL-c/HDL-c, Cer(d18:0/22:0)/total-c/HDL-c, Cer(d18:0/24:0)/apolipoprotein A-I, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/HDL cholesterol, Cer(d18:0/24:0)/LDL cholesterol, Cer(d18:0/24:0)/LDL-c/HDL-c, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/total-c/HDL-c, LPC 18:2/apoA1/apoB, LPC 18:2/apolipoprotein B, LPC 18:2/HDL cholesterol, LPC 18:2/LDL cholesterol, LPC 18:2/LDL-c/HDL-c, LPC 18:2/total cholesterol, PC 16:0/20:3/apolipoprotein B, PC 16:0/20:3/HDL cholesterol, PC 16:0/20:3/LDL-c/HDL-c, PC 16:0/20:3/total-c/HDL-c, PC 16:0/20:4/apolipoprotein A-I, PC 16:0/20:4/apolipoprotein B, PC 16:0/20:4/LDL cholesterol, PC 16:0/20:4/LDL-c/HDL-c, PC 16:0/20:4/total cholesterol, PC 16:0/20:4/total-c/HDL-c, PC 18:0/18:1/LDL-c/HDL-c, PC 18:0/20:3/LDL-c/HDL-c, PC 18:0/20:3/total-c/HDL-c, PC 18:0/20:4/apoA1/apoB, Total LPC/LDL-c/HDL-c, Total LPC/total-c/HDL-c, Total PC/apolipoprotein B, Total PC/LDL-c/HDL-c, Total PC/total cholesterol and Total PC/total-c/HDL-c.

In yet another alternative embodiment the present invention relates to a method for determining whether a subject is at risk to develop one or more CVD complications, such as AMI or CVD death, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more of CVD complications, such as AMI or CVD death, wherein the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Tables 6b and 9b):
Cer(d18:1/16:0)/apolipoprotein A-I, Cer(d18:1/16:0)/LDL cholesterol, Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/HDL cholesterol, Cer(d18:1/20:0)/LDL cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/total-c/HDL-c, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/22:0)/apolipoprotein A-I, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/total cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/24:0)/apolipoprotein A-I, Cer(d18:1/24:0)/apolipoprotein B, Cer(d18:1/24:0)/LDL cholesterol, Cer(d18:1/24:0)/total cholesterol, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/HDL cholesterol, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/LDL-c/HDL-c, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/triglycerides, GlcCer(d18:1/20:0)/apolipoprotein B, GlcCer(d18:1/20:0)/total cholesterol, GlcCer(d18:1/24:1)/apolipoprotein B, GlcCer(d18:1/26:1)/apolipoprotein A-I, LacCer(d18:1/16:0)/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, PC O-34:1/apolipoprotein B, PS O-16:0/18:2-alkenyl/triglycerides, PS O-16:1/18:2-alkyl/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/apolipoprotein B, Total Cer/LDL cholesterol, Total Cer/total cholesterol, Total DAG/apolipoprotein A-I, Total DAG/triglycerides, Total GlcCer/apolipoprotein B, Total LacCer/apolipoprotein A-I, Total LacCer/apolipoprotein B and Total LacCer/total cholesterol.

and wherein the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 6b and 9b):

CE 14:0/apoA1/apoB, CE 14:0/apolipoprotein B, CE 14:0/LDL-c/HDL-c, CE 14:0/total-c/HDL-c, CE 16:1/apoA1/apoB, CE 18:3/apoA1/apoB, CE 18:3/apolipoprotein A-I, CE 18:3/apolipoprotein B, CE 18:3/HDL cholesterol, CE 18:3/LDL cholesterol, CE 18:3/LDL-c/HDL-c, CE 18:3/total cholesterol, CE 18:3/total-c/HDL-c, CE 20:5/triglycerides, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/total-c/HDL-c, PC 18:0/20:4/apoA1/apoB, PC O-38:6/apolipoprotein A-I, Total LPC/apoA1/apoB and Total PC/apolipoprotein A-I.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 10):
Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total Cer/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/24:1)/apolipoprotein B.
In another preferred embodiment, the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 10):
Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/apolipoprotein B, PC 16:0/20:4/apolipoprotein B and Cer(d18:0/24:0)/apolipoprotein A-I.
In a particularly preferred embodiment, the lipid-clinical concentration ratio whose increase is compared to the control is (are) selected from (Table 11):
Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/18:0)/apolipoprotein A-I.
For the purposes of the invention, and particularly for lipid-clinical concentration ratios, an Apolipoprotein A-I measurement may alternatively be an Apolipoprotein A-II measurement.
In another aspect the present invention relates to a method for evaluating the effectiveness of a treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, comprising determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said treatment, wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Table 2a and 5a):
Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/24:1), GlcCer(d18:1/18:0), LacCer(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), PC 0-32:0 (KDdiA-PC), PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl), PS O-18:2/16:0-alkenyl and Total LacCer;

and wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Table 2a and 5a):

CE 14:0, CE 16:0, CE 17:1, CE 20:3, Cer(d18:0/22:0), Cer(d18:0/24:0), LPC 18:1, PC 16:0/18:2, PC 16:0/20:3, PC 16:0/20:4, PC 16:0/22:6, PC 18:0/18:1, PC 18:0/20:3, PC 18:0/20:4, PC 18:1/18:2, SM (d18:1/14:0) (d18:1/13:1-OH), SM (d18:1/23:0) (d18:1/22:1-OH), SM (d18:1/24:0) (d18:1/23:1-OH), Total CE, Total LPC and Total PC.

In a particular embodiment, the present invention relates to a method for evaluating the effectiveness of a treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, wherein the subject is not undergoing statin treatment and wherein said method comprises, determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said treatment, wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Table 2b and 5b):
Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:1), Cer(d18:1/26:1), GlcCer(d18:1/18:0), GlcCer(d18:1/20:0), GlcCer(d18:1/24:1), GlcCer(d18:1/26:1), LacCer(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), LacCer(d18:1/24:1), PC O-32:0 (KDdiA-PC), PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:2/16:0-alkenyl, Total Cer, Total DAG and Total LacCer;

and wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Table 2b and 5b):

CE 14:0, CE 17:1, CE 20:3, Cer(d18:0/24:0), LPC 18:1, PC 16:0/20:3, PC 16:0/20:4, PC 18:0/20:4, PC O-40:3, SM (d18:1/14:0) (d18:1/13:1-OH), Total LPC and Total PC.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid(s) whose decrease in concentration is (are) compared to the control is (are) selected from (Table 8):
Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), PS O-18:2/16:0-alkenyl, PS O-16:1/18:2-alkyl, Total Cer, Total LacCer, GlcCer(d18:1/24:1), LacCer(d18:1/22:0) and Cer(d18:1/18:0).
In another preferred embodiment, the one or more lipid(s) whose increase in concentration is (are) compared to the control is (are) selected from (Table 8):
Total PC, PC 16:0/20:4, Cer(d18:0/24:0), Total LPC, CE 14:0, CE 20:3, CE 17:1, PC 16:0/20:3, LPC 18:1, PC 18:0/20:3, PC 18:0/18:1 and Cer(d18:0/22:0).
In a particularly preferred embodiment, the one or more lipid(s) whose decrease in concentration is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), LacCer(d18:1/22:0) and Cer(d18:1/18:0);
and the one or more lipid(s) whose increase in concentration is (are) compared to the control is (are) selected from (Table 11): PC 16:0/20:4 and Cer(d18:0/24:0).
In another alternative embodiment the invention relates to a method for evaluating the effectiveness of a treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, comprising determining in a sample from said subject one or more lipid-lipid ratio(s), wherein (an) increased or decreased lipid-lipid ratio(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said treatment, wherein the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 5a and 8a):
CE 16:0/CE 18:3, CE 18:2/CE 18:3, CE 19:1/Cer(d18:0/22:0), Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/16:0)/Total PC, Cer(d18:1/18:0)/LPC 16:0, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/18:0)/Total PC, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/20:0)/Total PC, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/22:0)/Total PC, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:1/24:1)/Total CE, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/24:1)/Total PC, Cer(d18:1/26:0)/PC O-40:0, GlcCer(d18:1/20:0)/PC 16:0/20:4, GlcCer(d18:1/20:0)/Total PC, GlcCer(d18:1/26:0)/Total CE, LacCer(d18:1/16:0)/Total LPC, LacCer(d18:1/18:0)/PC 16:0/18:1, LacCer(d18:1/18:0)/PC 16:0/20:3, LacCer(d18:1/18:0)/PC 18:0/18:1, LacCer(d18:1/18:0)/PC 18:0/20:3, LacCer(d18:1/18:0)/PC 18:1/18:1, LacCer(d18:1/18:0)/PC 18:1/18:2, LacCer(d18:1/18:0)/Total LPC, LacCer(d18:1/18:0)/Total PC, LacCer(d18:1/20:0)/PC 16:0/18:1, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/20:3 LacCer(d18:1/20:0)/PC 18:1/18:1, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/PC 18:2/18:2, LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0), LacCer(d18:1/20:0)/Total LPC, LacCer(d18:1/20:0)/Total PC, LacCer(d18:1/20:0)/Total SM, LacCer(d18:1/22:0)/PC 16:0/20:3, LacCer(d18:1/22:0)/PC 16:0/20:4, LacCer(d18:1/22:0)/PC 18:0/20:3, LacCer(d18:1/22:0)/SM (d18:1/14:0) (d18:1/13:1-OH), LacCer(d18:1/22:0)/Total LPC, LacCer(d18:1/22:0)/Total PC, LacCer(d18:1/24:0)/PC 16:0/20:3, LacCer(d18:1/24:0)/Total LPC, LacCer(d18:1/24:1)/Total LPC, LacCer(d18:1/24:1)/Total PC, LacCer(d18:1/24:1)/Total PC O, PC 16:0/16:0/PC 16:0/20:4, PC 16:0/16:0/Total PC, PC 16:0/18:2/Total PC, PC O-18:0/18:2-alkyl/PC O-36:5, PC O-32:0 (KDdiA-PC)/PC O-38:5, PS O-16:0/18:2-alkenyl/Total PS O, PS O-16:1/18:2-alkyl/Total PS O, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl)/Total PS O, PS O-18:2/16:0-alkenyl/Total PS O, Total Cer/Total PC, Total LacCer/Total PC and Total LacCer/Total PC O;

and wherein one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Tables 5a and 8a):

CE 14:0/Cer(d18:1/24:1), CE 16:0/Cer(d18:1/24:1), CE 16:1/CE 19:1, CE 16:1/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/20:0), CE 16:1/Cer(d18:1/24:1), CE 16:1/GlcCer(d18:1/18:0), CE 16:1/GlcCer(d18:1/20:0), CE 16:1/LacCer(d18:1/16:0), CE 16:1/LacCer(d18:1/18:0), CE 16:1/LacCer(d18:1/20:0), CE 16:1/LacCer(d18:1/22:0), CE 16:1/LacCer(d18:1/24:0), CE 16:1/PC 16:0/16:0, CE 16:1/Total LacCer, CE 17:1/Cer(d18:1/24:1), CE 17:1/GlcCer(d18:1/24:1), CE 17:1/LacCer(d18:1/18:0), CE 18:1/Total LacCer, CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 18:3/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/22:0), CE 18:3/Cer(d18:1/24:0), CE 18:3/Cer(d18:1/24:1), CE 18:3/GlcCer(d18:1/18:0), CE 18:3/GlcCer(d18:1/20:0), CE 18:3/LacCer(d18:1/18:0), CE 18:3/LacCer(d18:1/20:0), CE 18:3/LacCer(d18:1/22:0), CE 18:3/LacCer(d18:1/24:0), CE 18:3/PC 16:0/16:0, CE 18:3/PC O-34:1, CE 18:3/PS O-16:0/18:1-alkenyl (PS O-16:1/18:1-alkyl), CE 18:3/PS O-16:0/18:2-alkenyl, CE 18:3/PS O-16:1/18:2-alkyl, CE 18:3/Total CE, CE 18:3/Total Cer, CE 18:3/Total LacCer, CE 20:3/Cer(d18:1/24:1), CE 20:3/LacCer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/LacCer(d18:1/24:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/22:0)/Total CE, Cer(d18:0/24:0)/Cer(d18:1/16:0), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/GlcCer(d18:1/20:0), Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/SM (d18:1/17:0) (d18:1/16:1-OH), Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Total Cer, Cer(d18:0/24:1)/Total CE, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/24:1), LPC 16:0/Total LacCer, LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), LPC 18:2/PS O-16:0/18:2-alkenyl, LPC 18:2/PS O-16:1/18:2-alkyl, PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, SM (d18:1/24:0) (d18:1/23:1-OH)/Total Cer and Total LPC/Total LacCer.

In another alternative embodiment the invention relates to a method for evaluating the effectiveness of a treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, wherein the subject is not undergoing statin treatment and wherein said method comprises comprising determining in a sample from said subject one or more lipid-lipid ratio(s), wherein (an) increased or decreased lipid-lipid ratio(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said treatment, wherein the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 5b and 8b):
CE 16:0/CE 18:3, CE 18:0/CE 18:3, CE 18:2/CE 18:3, Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/16:0)/Total PC, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/18:1, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/18:0)/PC 18:1/18:1, Cer(d18:1/18:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/18:0)/SM (d18:1/17:2-OH), Cer(d18:1/18:0)/SM (d18:1/18:1), Cer(d18:1/18:0)/Total CE, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/18:0)/Total PC, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 18:0/18:1, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/20:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/20:0)/Total LPC, Cer(d18:1/20:0)/Total PC, Cer(d18:1/20:0)/Total PC O, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/22:0)/PC 18:0/20:4, Cer(d18:1/22:0)/Total PC, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/18:2, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/18:2, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/PC 18:1/18:1, Cer(d18:1/24:1)/PC 18:1/18:2, Cer(d18:1/24:1)/PC O-40:3, Cer(d18:1/24:1)/SM (d18:1/17:1-OH), Cer(d18:1/24:1)/SM (d18:1/18:0), Cer(d18:1/24:1)/SM (d18:1/23:0) (d18:1/22:1-OH), Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:1/24:1)/Total CE, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/24:1)/Total PC, GlcCer(d18:1/26:0)/Total CE, GlcCer(d18:1/26:1)/Total CE, LacCer(d18:1/18:0)/Total PC, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/20:3, LacCer(d18:1/20:0)/PC 18:0/20:4, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0), LacCer(d18:1/20:0)/Total CE, LacCer(d18:1/20:0)/Total LPC, LacCer(d18:1/20:0)/Total PC, LacCer(d18:1/20:0)/Total SM, LacCer(d18:1/24:0)/Total LPC, PC 16:0/16:0/PC 16:0/20:4, PC 16:0/16:0/Total PC, PC O-32:0 (KDdiA-PC)/Total PC O, PS O-16:0/18:2-alkenyl/Total PC, PS O-16:0/18:2-alkenyl/Total PC O, PS O-16:0/18:2-alkenyl/Total PS O, PS O-16:1/18:2-alkyl/Total PC, PS O-16:1/18:2-alkyl/Total PC O, PS O-16:1/18:2-alkyl/Total PS O, PS O-18:2/16:0-alkenyl/Total PC O, PS O-18:2/16:0-alkenyl/Total PS O, SM (d18:1/17:0) (d18:1/16:1-OH)/Total PC O, Total Cer/Total PC, Total DAG/Total LPC, Total DAG/Total PC, Total DAG/Total PC O and Total LacCer/Total PC;

and wherein the one or more lipid-lipid ratio(s) whose increase(s) is (are) compared to the control is (are) selected from (Tables 5b and 8b):

CE 14:0/Cer(d18:1/18:0), CE 14:0/Cer(d18:1/24:1), CE 14:0/Total DAG, CE 15:0/Cer(d18:1/20:0), CE 16:0/Cer(d18:1/18:0), CE 16:0/Cer(d18:1/24:1), CE 16:1/CE 19:1, CE 16:1/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/20:0), CE 16:1/Cer(d18:1/24:1), CE 16:1/GlcCer(d18:1/24:1), CE 16:1/LacCer(d18:1/18:0), CE 16:1/LacCer(d18:1/24:0), CE 16:1/Total LacCer, CE 17:1/Cer(d18:1/18:0), CE 17:1/Cer(d18:1/24:1), CE 18:2/Cer(d18:1/20:0), CE 18:2/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 18:3/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/22:0), CE 18:3/Cer(d18:1/24:0), CE 18:3/Cer(d18:1/24:1), CE 18:3/GlcCer(d18:1/20:0), CE 18:3/LacCer(d18:1/20:0), CE 18:3/LacCer(d18:1/22:0), CE 18:3/LacCer(d18:1/24:0), CE 18:3/PC 16:0/16:0, CE 18:3/PC O-34:1, CE 18:3/PS O-16:0/18:2-alkenyl, CE 18:3/PS O-16:1/18:2-alkyl, CE 18:3/Total CE, CE 18:3/Total Cer, CE 18:3/Total DAG, CE 18:3/Total LacCer, CE 20:3/Cer(d18:1/24:1), CE 20:3/LacCer(d18:1/20:0), CE 20:4/Cer(d18:1/18:0), CE 20:4/Cer(d18:1/24:1), CE 20:4/GlcCer(d18:1/20:0), CE 20:4/GlcCer(d18:1/24:1), CE 20:4/LacCer(d18:1/20:0), CE 20:5/LacCer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/22:0)/Total CE, Cer(d18:0/22:0)/Total DAG, Cer(d18:0/22:0)/Total GlcCer, Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Total Cer, DAG 16:0/18:1/Total DAG, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/24:0), LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 16:0/20:4/PS O-16:0/18:2-alkenyl, PC 16:0/20:4/PS O-16:1/18:2-alkyl, PC 16:0/20:4/Total DAG, PC 18:0/20:3/PS O-16:0/18:2-alkenyl, PC 18:0/20:3/PS O-16:1/18:2-alkyl, PC 18:0/20:3/PS O-18:2/16:0-alkenyl, PC 18:0/20:4/PS O-16:0/18:2-alkenyl, PC 18:0/20:4/PS O-16:1/18:2-alkyl, PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, PC 18:1/18:2/Total Cer, PC O-40:3/PS O-18:2/16:0-alkenyl, SM (d18:1/23:0) (d18:1/22:1-OH)/Total DAG, SM (d18:1/24:0) (d18:1/23:1-OH)/Total Cer, Total CE/Total DAG and Total LPC/Total LacCer.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 9):
GlcCer(d18:1/26:1)/Total CE, Cer(d18:1/24:1)/Total PC, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, Total Cer/Total PC, Total LacCer/Total PC, LacCer(d18:1/20:0)/PC 18:1/18:2, PS O-16:0/18:2-alkenyl/Total PS O, Cer(d18:1/18:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/Total LPC and LacCer(d18:1/20:0)/PC 16:0/20:4.
In another preferred embodiment, the one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 9):
Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/Cer(d18:1/24:1), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Total Cer, Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), Total LPC/Total LacCer and GlcCer(d18:1/26:0)/LacCer(d18:1/22:0).
In a particularly preferred embodiment, the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/24:1)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, PS O-16:0/18:2-alkenyl/Total PS 0 and Cer(d18:1/18:0)/PC 16:0/20:4;
and the one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 11):
GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Total Cer, Total LPC/Total LacCer, GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl and Cer(d18:0/24:0)/LacCer(d18:1/24:0).
In yet another alternative embodiment the invention relates to a method for evaluating the effectiveness of a treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, comprising determining in a sample from said subject one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said treatment, wherein the one or more lipid-clinical concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from (Tables 6a and 9a):
Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/HDL cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/triglycerides, GlcCer(d18:1/24:0)/total cholesterol, LacCer(d18:1/16:0)/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/HDL cholesterol, LacCer(d18:1/18:0)/LDL cholesterol, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/18:0)/total-c/HDL-c, LacCer(d18:1/18:0)/triglycerides, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/22:0)/total-c/HDL-c, LacCer(d18:1/22:0)/triglycerides, LacCer(d18:1/24:0)/apoA1/apoB, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, PC O-32:0 (KDdiA-PC)/apolipoprotein A-I, PC O-32:0 (KDdiA-PC)/triglycerides, PC O-34:1/triglycerides, PS O-16:0/18:1-alkenyl (PS O-16:1/18:1-alkyl)/triglycerides, PS O-16:0/18:2-alkenyl/triglycerides, PS O-16:1/18:2-alkyl/triglycerides, PS O-18:2/16:0-alkenyl/HDL cholesterol, PS O-18:2/16:0-alkenyl/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/total cholesterol, Total Cer/triglycerides, Total GlcCer/apolipoprotein B, Total GlcCer/total cholesterol, Total LacCer/apolipoprotein A-I, Total LacCer/apolipoprotein B, Total LacCer/total cholesterol and Total LacCer/triglycerides;

and wherein the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from Tables 6a and 9a:

CE 14:0/apolipoprotein B, CE 14:0/LDL cholesterol, CE 14:0/LDL-c/HDL-c, CE 14:0/total cholesterol, CE 14:0/total-c/HDL-c, CE 16:1/apolipoprotein B, CE 16:1/HDL cholesterol, CE 16:1/LDL cholesterol, CE 16:1/total cholesterol, CE 17:1/LDL-c/HDL-c, CE 18:3/apoA1/apoB, CE 18:3/apolipoprotein A-I, CE 18:3/apolipoprotein B, CE 18:3/HDL cholesterol, CE 18:3/LDL cholesterol, CE 18:3/LDL-c/HDL-c, CE 18:3/total cholesterol, CE 18:3/total-c/HDL-c, CE 20:3/apolipoprotein B, CE 20:3/LDL-c/HDL-c, CE 20:3/total-c/HDL-c, CE 20:5/apolipoprotein B, CE 20:5/HDL cholesterol, CE 20:5/LDL cholesterol, Cer(d18:0/22:0)/apolipoprotein B, Cer(d18:0/22:0)/LDL-c/HDL-c, Cer(d18:0/22:0)/total-c/HDL-c, Cer(d18:0/24:0)/apolipoprotein A-I, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/HDL cholesterol, Cer(d18:0/24:0)/LDL cholesterol, Cer(d18:0/24:0)/LDL-c/HDL-c, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/total-c/HDL-c, LPC 18:2/apoA1/apoB, LPC 18:2/apolipoprotein B, LPC 18:2/HDL cholesterol, LPC 18:2/LDL cholesterol, LPC 18:2/LDL-c/HDL-c, LPC 18:2/total cholesterol, PC 16:0/20:3/apolipoprotein B, PC 16:0/20:3/HDL cholesterol, PC 16:0/20:3/LDL-c/HDL-c, PC 16:0/20:3/total-c/HDL-c, PC 16:0/20:4/apolipoprotein A-I, PC 16:0/20:4/apolipoprotein B, PC 16:0/20:4/LDL cholesterol, PC 16:0/20:4/LDL-c/HDL-c, PC 16:0/20:4/total cholesterol, PC 16:0/20:4/total-c/HDL-c, PC 18:0/18:1/LDL-c/HDL-c, PC 18:0/20:3/LDL-c/HDL-c, PC 18:0/20:3/total-c/HDL-c, PC 18:0/20:4/apoA1/apoB, Total LPC/LDL-c/HDL-c, Total LPC/total-c/HDL-c, Total PC/apolipoprotein B, Total PC/LDL-c/HDL-c, Total PC/total cholesterol and Total PC/total-c/HDL-c.

In yet another alternative embodiment the invention relates to a method for evaluating the effectiveness of a treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said treatment, wherein the one or more lipid-clinical concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from (Tables 6b and 9b):
Cer(d18:1/16:0)/apolipoprotein A-I, Cer(d18:1/16:0)/LDL cholesterol, Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/triglyceride s, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/HDL cholesterol, Cer(d18:1/20:0)/LDL cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/total-c/HDL-c, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/22:0)/apolipoprotein A-I, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/total cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/24:0)/apolipoprotein A-I, Cer(d18:1/24:0)/apolipoprotein B, Cer(d18:1/24:0)/LDL cholesterol, Cer(d18:1/24:0)/total cholesterol, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/HDL cholesterol, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/LDL-c/HDL-c, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/triglycerides, GlcCer(d18:1/20:0)/apolipoprotein B, GlcCer(d18:1/20:0)/total cholesterol, GlcCer(d18:1/24:1)/apolipoprotein B, GlcCer(d18:1/26:1)/apolipoprotein A-I, LacCer(d18:1/16:0)/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, PC O-34:1/apolipoprotein B, PS O-16:0/18:2-alkenyl/triglycerides, PS O-16:1/18:2-alkyl/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/apolipoprotein B, Total Cer/LDL cholesterol, Total Cer/total cholesterol, Total DAG/apolipoprotein A-I, Total DAG/triglycerides, Total GlcCer/apolipoprotein B, Total LacCer/apolipoprotein A-I, Total LacCer/apolipoprotein B and Total LacCer/total cholesterol.

and wherein the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Tables 6b and 9b):

CE 14:0/apoA1/apoB, CE 14:0/apolipoprotein B, CE 14:0/LDL-c/HDL-c, CE 14:0/total-c/HDL-c, CE 16:1/apoA1/apoB, CE 18:3/apoA1/apoB, CE 18:3/apolipoprotein A-I, CE 18:3/apolipoprotein B, CE 18:3/HDL cholesterol, CE 18:3/LDL cholesterol, CE 18:3/LDL-c/HDL-c, CE 18:3/total cholesterol, CE 18:3/total-c/HDL-c, CE 20:5/triglycerides, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/total-c/HDL-c, PC 18:0/20:4/apoA1/apoB, PC O-38:6/apolipoprotein A-I, Total LPC/apoA1/apoB and Total PC/apolipoprotein A-I.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 10):
Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total Cer/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/24:1)/apolipoprotein B.
In another preferred embodiment, the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 10):
Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/apolipoprotein B, PC 16:0/20:4/apolipoprotein B and Cer(d18:0/24:0)/apolipoprotein A-I.
In a particularly preferred embodiment, the lipid-clinical concentration ratio(s) whose decrease(s) is (are) compared to the control are selected from (Table 11):
Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/18:0)/apolipoprotein A-I.
For the purposes of the invention, and particularly for lipid-clinical concentration ratios, an Apolipoprotein A-I measurement may alternatively be an Apolipoprotein A-II measurement.
In yet another aspect the invention relates to a method of choosing an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, comprising determining in a sample from said subject the concentration of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of treatment or a change in, or supplementation of, an already administered treatment, wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Tables 4a and 7a):
Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/24:1), GlcCer(d18:1/18:0), LacCer(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), PC 0-32:0 (KDdiA-PC), PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl), PS O-18:2/16:0-alkenyl and Total LacCer;

and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Tables 4a and 7a):

CE 14:0, CE 16:0, CE 17:1, CE 20:3, Cer(d18:0/22:0), Cer(d18:0/24:0), LPC 18:1, PC 16:0/18:2, PC 16:0/20:3, PC 16:0/20:4, PC 16:0/22:6, PC 18:0/18:1, PC 18:0/20:3, PC 18:0/20:4, PC 18:1/18:2, SM (d18:1/14:0) (d18:1/13:1-OH), SM (d18:1/23:0) (d18:1/22:1-OH), SM (d18:1/24:0) (d18:1/23:1-OH), Total CE, Total LPC and Total PC.

In yet another aspect the invention relates to a method of choosing an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject the concentration of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of treatment or a change in, or supplementation of, an already administered treatment, wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from (Tables 4b and 7b):
Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:1), Cer(d18:1/26:1), GlcCer(d18:1/18:0), GlcCer(d18:1/20:0), GlcCer(d18:1/24:1), GlcCer(d18:1/26:1), LacCer(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), LacCer(d18:1/24:1), PC O-32:0 (KDdiA-PC), PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:2/16:0-alkenyl, Total Cer, Total DAG and Total LacCer;

and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from (Tables 4b and 7b):

CE 14:0, CE 17:1, CE 20:3, Cer(d18:0/24:0), LPC 18:1, PC 16:0/20:3, PC 16:0/20:4, PC 18:0/20:4, PC O-40:3, SM (d18:1/14:0) (d18:1/13:1-OH), Total LPC and Total PC.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid(s) whose increase in concentration is (are) compared to the control is (are) selected from (Table 8):
Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), PS O-18:2/16:0-alkenyl, PS O-16:1/18:2-alkyl, Total Cer, Total LacCer, GlcCer(d18:1/24:1), LacCer(d18:1/22:0) and Cer(d18:1/18:0).
In another preferred embodiment, the one or more lipid(s) whose decrease in concentration is (are) compared to the control is (are) selected from (Table 8):
Total PC, PC 16:0/20:4, Cer(d18:0/24:0), Total LPC, CE 14:0, CE 20:3, CE 17:1, PC 16:0/20:3, LPC 18:1, PC 18:0/20:3, PC 18:0/18:1 and Cer(d18:0/22:0).
In a particularly preferred embodiment, the one or more lipid(s) whose increase in concentration is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), LacCer(d18:1/22:0) and Cer(d18:1/18:0);
and the one or more lipid(s) whose decrease in concentration is (are) compared to the control is (are) selected from (Table 11): PC 16:0/20:4 and Cer(d18:0/24:0).
In an alternative embodiment the invention relates to a method of choosing an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, comprising determining in a sample from said subject one or more lipid-lipid ratio(s), wherein (an) increased or decreased lipid-lipid ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of treatment or a change in, or supplementation of, an already administered treatment, wherein the one or more lipid-lipid ratio(s) whose increase(s) is (are) compared to the control is (are) selected from (Tables 5a and 8a):
CE 16:0/CE 18:3, CE 18:2/CE 18:3, CE 19:1/Cer(d18:0/22:0), Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/16:0)/Total PC, Cer(d18:1/18:0)/LPC 16:0, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/18:0)/Total PC, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/20:0)/Total PC, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/22:0)/Total PC, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:1/24:1)/Total CE, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/24:1)/Total PC, Cer(d18:1/26:0)/PC O-40:0, GlcCer(d18:1/20:0)/PC 16:0/20:4, GlcCer(d18:1/20:0)/Total PC, GlcCer(d18:1/26:0)/Total CE, LacCer(d18:1/16:0)/Total LPC, LacCer(d18:1/18:0)/PC 16:0/18:1, LacCer(d18:1/18:0)/PC 16:0/20:3, LacCer(d18:1/18:0)/PC 18:0/18:1, LacCer(d18:1/18:0)/PC 18:0/20:3, LacCer(d18:1/18:0)/PC 18:1/18:1, LacCer(d18:1/18:0)/PC 18:1/18:2, LacCer(d18:1/18:0)/Total LPC, LacCer(d18:1/18:0)/Total PC, LacCer(d18:1/20:0)/PC 16:0/18:1, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/20:3 LacCer(d18:1/20:0)/PC 18:1/18:1, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/PC 18:2/18:2, LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0), LacCer(d18:1/20:0)/Total LPC, LacCer(d18:1/20:0)/Total PC, LacCer(d18:1/20:0)/Total SM, LacCer(d18:1/22:0)/PC 16:0/20:3, LacCer(d18:1/22:0)/PC 16:0/20:4, LacCer(d18:1/22:0)/PC 18:0/20:3, LacCer(d18:1/22:0)/SM (d18:1/14:0) (d18:1/13:1-OH), LacCer(d18:1/22:0)/Total LPC, LacCer(d18:1/22:0)/Total PC, LacCer(d18:1/24:0)/PC 16:0/20:3, LacCer(d18:1/24:0)/Total LPC, LacCer(d18:1/24:1)/Total LPC, LacCer(d18:1/24:1)/Total PC, LacCer(d18:1/24:1)/Total PC O, PC 16:0/16:0/PC 16:0/20:4, PC 16:0/16:0/Total PC, PC 16:0/18:2/Total PC, PC O-18:0/18:2-alkyl/PC O-36:5, PC O-32:0 (KDdiA-PC)/PC O-38:5, PS O-16:0/18:2-alkenyl/Total PS O, PS O-16:1/18:2-alkyl/Total PS O, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl)/Total PS O, PS O-18:2/16:0-alkenyl/Total PS O, Total Cer/Total PC, Total LacCer/Total PC and Total LacCer/Total PC O;

and wherein one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 5a and 8a):

CE 14:0/Cer(d18:1/24:1), CE 16:0/Cer(d18:1/24:1), CE 16:1/CE 19:1, CE 16:1/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/20:0), CE 16:1/Cer(d18:1/24:1), CE 16:1/GlcCer(d18:1/18:0), CE 16:1/GlcCer(d18:1/20:0), CE 16:1/LacCer(d18:1/16:0), CE 16:1/LacCer(d18:1/18:0), CE 16:1/LacCer(d18:1/20:0), CE 16:1/LacCer(d18:1/22:0), CE 16:1/LacCer(d18:1/24:0), CE 16:1/PC 16:0/16:0, CE 16:1/Total LacCer, CE 17:1/Cer(d18:1/24:1), CE 17:1/GlcCer(d18:1/24:1), CE 17:1/LacCer(d18:1/18:0), CE 18:1/Total LacCer, CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 18:3/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/22:0), CE 18:3/Cer(d18:1/24:0), CE 18:3/Cer(d18:1/24:1), CE 18:3/GlcCer(d18:1/18:0), CE 18:3/GlcCer(d18:1/20:0), CE 18:3/LacCer(d18:1/18:0), CE 18:3/LacCer(d18:1/20:0), CE 18:3/LacCer(d18:1/22:0), CE 18:3/LacCer(d18:1/24:0), CE 18:3/PC 16:0/16:0, CE 18:3/PC O-34:1, CE 18:3/PS O-16:0/18:1-alkenyl (PS O-16:1/18:1-alkyl), CE 18:3/PS O-16:0/18:2-alkenyl, CE 18:3/PS O-16:1/18:2-alkyl, CE 18:3/Total CE, CE 18:3/Total Cer, CE 18:3/Total LacCer, CE 20:3/Cer(d18:1/24:1), CE 20:3/LacCer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/LacCer(d18:1/24:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/22:0)/Total CE, Cer(d18:0/24:0)/Cer(d18:1/16:0), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/GlcCer(d18:1/20:0), Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/SM (d18:1/17:0) (d18:1/16:1-OH), Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Total Cer, Cer(d18:0/24:1)/Total CE, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/24:1), LPC 16:0/Total LacCer, LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), LPC 18:2/PS O-16:0/18:2-alkenyl, LPC 18:2/PS O-16:1/18:2-alkyl, PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, SM (d18:1/24:0) (d18:1/23:1-OH)/Total Cer and Total LPC/Total LacCer.

In an alternative embodiment the invention relates to a method of choosing an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject one or more lipid-lipid ratio(s), wherein (an) increased or decreased lipid-lipid ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of treatment or a change in, or supplementation of, an already administered treatment, wherein the one or more lipid-lipid ratio(s) whose increase(s) is (are) compared to the control is (are) selected from (Tables 5b and 8b):
CE 16:0/CE 18:3, CE 18:0/CE 18:3, CE 18:2/CE 18:3, Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/16:0)/Total PC, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/18:1, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/18:0)/PC 18:1/18:1, Cer(d18:1/18:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/18:0)/SM (d18:1/17:2-OH), Cer(d18:1/18:0)/SM (d18:1/18:1), Cer(d18:1/18:0)/Total CE, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/18:0)/Total PC, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 18:0/18:1, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/20:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/20:0)/Total LPC, Cer(d18:1/20:0)/Total PC, Cer(d18:1/20:0)/Total PC O, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/22:0)/PC 18:0/20:4, Cer(d18:1/22:0)/Total PC, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/18:2, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/18:2, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/PC 18:1/18:1, Cer(d18:1/24:1)/PC 18:1/18:2, Cer(d18:1/24:1)/PC O-40:3, Cer(d18:1/24:1)/SM (d18:1/17:1-OH), Cer(d18:1/24:1)/SM (d18:1/18:0), Cer(d18:1/24:1)/SM (d18:1/23:0) (d18:1/22:1-OH), Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:1/24:1)/Total CE, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/24:1)/Total PC, GlcCer(d18:1/26:0)/Total CE, GlcCer(d18:1/26:1)/Total CE, LacCer(d18:1/18:0)/Total PC, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/20:3, LacCer(d18:1/20:0)/PC 18:0/20:4, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0), LacCer(d18:1/20:0)/Total CE, LacCer(d18:1/20:0)/Total LPC, LacCer(d18:1/20:0)/Total PC, LacCer(d18:1/20:0)/Total SM, LacCer(d18:1/24:0)/Total LPC, PC 16:0/16:0/PC 16:0/20:4, PC 16:0/16:0/Total PC, PC O-32:0 (KDdiA-PC)/Total PC O, PS O-16:0/18:2-alkenyl/Total PC, PS O-16:0/18:2-alkenyl/Total PC O, PS O-16:0/18:2-alkenyl/Total PS O, PS O-16:1/18:2-alkyl/Total PC, PS O-16:1/18:2-alkyl/Total PC O, PS O-16:1/18:2-alkyl/Total PS O, PS O-18:2/16:0-alkenyl/Total PC O, PS O-18:2/16:0-alkenyl/Total PS O, SM (d18:1/17:0) (d18:1/16:1-OH)/Total PC O, Total Cer/Total PC, Total DAG/Total LPC, Total DAG/Total PC, Total DAG/Total PC O and Total LacCer/Total PC;

and wherein the one or more lipid-lipid ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from (Tables 5b and 8b):

CE 14:0/Cer(d18:1/18:0), CE 14:0/Cer(d18:1/24:1), CE 14:0/Total DAG, CE 15:0/Cer(d18:1/20:0), CE 16:0/Cer(d18:1/18:0), CE 16:0/Cer(d18:1/24:1), CE 16:1/CE 19:1, CE 16:1/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/20:0), CE 16:1/Cer(d18:1/24:1), CE 16:1/GlcCer(d18:1/24:1), CE 16:1/LacCer(d18:1/18:0), CE 16:1/LacCer(d18:1/24:0), CE 16:1/Total LacCer, CE 17:1/Cer(d18:1/18:0), CE 17:1/Cer(d18:1/24:1), CE 18:2/Cer(d18:1/20:0), CE 18:2/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 18:3/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/22:0), CE 18:3/Cer(d18:1/24:0), CE 18:3/Cer(d18:1/24:1), CE 18:3/GlcCer(d18:1/20:0), CE 18:3/LacCer(d18:1/20:0), CE 18:3/LacCer(d18:1/22:0), CE 18:3/LacCer(d18:1/24:0), CE 18:3/PC 16:0/16:0, CE 18:3/PC O-34:1, CE 18:3/PS O-16:0/18:2-alkenyl, CE 18:3/PS O-16:1/18:2-alkyl, CE 18:3/Total CE, CE 18:3/Total Cer, CE 18:3/Total DAG, CE 18:3/Total LacCer, CE 20:3/Cer(d18:1/24:1), CE 20:3/LacCer(d18:1/20:0), CE 20:4/Cer(d18:1/18:0), CE 20:4/Cer(d18:1/24:1), CE 20:4/GlcCer(d18:1/20:0), CE 20:4/GlcCer(d18:1/24:1), CE 20:4/LacCer(d18:1/20:0), CE 20:5/LacCer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/22:0)/Total CE, Cer(d18:0/22:0)/Total DAG, Cer(d18:0/22:0)/Total GlcCer, Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Total Cer, DAG 16:0/18:1/Total DAG, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/24:0), LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 16:0/20:4/PS O-16:0/18:2-alkenyl, PC 16:0/20:4/PS O-16:1/18:2-alkyl, PC 16:0/20:4/Total DAG, PC 18:0/20:3/PS O-16:0/18:2-alkenyl, PC 18:0/20:3/PS O-16:1/18:2-alkyl, PC 18:0/20:3/PS O-18:2/16:0-alkenyl, PC 18:0/20:4/PS O-16:0/18:2-alkenyl, PC 18:0/20:4/PS O-16:1/18:2-alkyl, PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, PC 18:1/18:2/Total Cer, PC O-40:3/PS O-18:2/16:0-alkenyl, SM (d18:1/23:0) (d18:1/22:1-OH)/Total DAG, SM (d18:1/24:0) (d18:1/23:1-OH)/Total Cer, Total CE/Total DAG and Total LPC/Total LacCer.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 9):
GlcCer(d18:1/26:1)/Total CE, Cer(d18:1/24:1)/Total PC, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, Total Cer/Total PC, Total LacCer/Total PC, LacCer(d18:1/20:0)/PC 18:1/18:2, PS O-16:0/18:2-alkenyl/Total PS O, Cer(d18:1/18:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/Total LPC and LacCer(d18:1/20:0)/PC 16:0/20:4;
In another preferred embodiment, the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 9):
Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/22:0)/Cer(d18:1/24:1), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Total Cer, Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), Total LPC/Total LacCer and GlcCer(d18:1/26:0)/LacCer(d18:1/22:0).
In a particularly preferred embodiment, the one or more lipid-lipid ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/24:1)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, PS O-16:0/18:2-alkenyl/Total PS 0 and Cer(d18:1/18:0)/PC 16:0/20:4;
and the one or more lipid-lipid ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 11):
GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Total Cer, Total LPC/Total LacCer, GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl and Cer(d18:0/24:0)/LacCer(d18:1/24:0).
In yet another embodiment the invention relates to a method of choosing an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, comprising determining in a sample from said subject one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of treatment or a change in, or supplementation of, an already administered treatment, wherein the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Tables 6a and 9a):
Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/HDL cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/triglycerides, GlcCer(d18:1/24:0)/total cholesterol, LacCer(d18:1/16:0)/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/HDL cholesterol, LacCer(d18:1/18:0)/LDL cholesterol, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/18:0)/total-c/HDL-c, LacCer(d18:1/18:0)/triglycerides, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/22:0)/total-c/HDL-c, LacCer(d18:1/22:0)/triglycerides, LacCer(d18:1/24:0)/apoA1/apoB, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, PC O-32:0 (KDdiA-PC)/apolipoprotein A-I, PC O-32:0 (KDdiA-PC)/triglycerides, PC O-34:1/triglycerides, PS O-16:0/18:1-alkenyl (PS O-16:1/18:1-alkyl)/triglycerides, PS O-16:0/18:2-alkenyl/triglycerides, PS O-16:1/18:2-alkyl/triglycerides, PS O-18:2/16:0-alkenyl/HDL cholesterol, PS O-18:2/16:0-alkenyl/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/total cholesterol, Total Cer/triglycerides, Total GlcCer/apolipoprotein B, Total GlcCer/total cholesterol, Total LacCer/apolipoprotein A-I, Total LacCer/apolipoprotein B, Total LacCer/total cholesterol and Total LacCer/triglycerides;

and wherein the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 6a and 9a):

CE 14:0/apolipoprotein B, CE 14:0/LDL cholesterol, CE 14:0/LDL-c/HDL-c, CE 14:0/total cholesterol, CE 14:0/total-c/HDL-c, CE 16:1/apolipoprotein B, CE 16:1/HDL cholesterol, CE 16:1/LDL cholesterol, CE 16:1/total cholesterol, CE 17:1/LDL-c/HDL-c, CE 18:3/apoA1/apoB, CE 18:3/apolipoprotein A-I, CE 18:3/apolipoprotein B, CE 18:3/HDL cholesterol, CE 18:3/LDL cholesterol, CE 18:3/LDL-c/HDL-c, CE 18:3/total cholesterol, CE 18:3/total-c/HDL-c, CE 20:3/apolipoprotein B, CE 20:3/LDL-c/HDL-c, CE 20:3/total-c/HDL-c, CE 20:5/apolipoprotein B, CE 20:5/HDL cholesterol, CE 20:5/LDL cholesterol, Cer(d18:0/22:0)/apolipoprotein B, Cer(d18:0/22:0)/LDL-c/HDL-c, Cer(d18:0/22:0)/total-c/HDL-c, Cer(d18:0/24:0)/apolipoprotein A-I, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/HDL cholesterol, Cer(d18:0/24:0)/LDL cholesterol, Cer(d18:0/24:0)/LDL-c/HDL-c, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/total-c/HDL-c, LPC 18:2/apoA1/apoB, LPC 18:2/apolipoprotein B, LPC 18:2/HDL cholesterol, LPC 18:2/LDL cholesterol, LPC 18:2/LDL-c/HDL-c, LPC 18:2/total cholesterol, PC 16:0/20:3/apolipoprotein B, PC 16:0/20:3/HDL cholesterol, PC 16:0/20:3/LDL-c/HDL-c, PC 16:0/20:3/total-c/HDL-c, PC 16:0/20:4/apolipoprotein A-I, PC 16:0/20:4/apolipoprotein B, PC 16:0/20:4/LDL cholesterol, PC 16:0/20:4/LDL-c/HDL-c, PC 16:0/20:4/total cholesterol, PC 16:0/20:4/total-c/HDL-c, PC 18:0/18:1/LDL-c/HDL-c, PC 18:0/20:3/LDL-c/HDL-c, PC 18:0/20:3/total-c/HDL-c, PC 18:0/20:4/apoA1/apoB, Total LPC/LDL-c/HDL-c, Total LPC/total-c/HDL-c, Total PC/apolipoprotein B, Total PC/LDL-c/HDL-c, Total PC/total cholesterol and Total PC/total-c/HDL-c.

In yet another embodiment the invention relates to a method of choosing an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death, in a subject, wherein the subject is not undergoing statin treatment and wherein said method comprises determining in a sample from said subject one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of treatment or a change in, or supplementation of, an already administered treatment, wherein the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Tables 6b and 9b):
Cer(d18:1/16:0)/apolipoprotein A-I, Cer(d18:1/16:0)/LDL cholesterol, Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/HDL cholesterol, Cer(d18:1/20:0)/LDL cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/total-c/HDL-c, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/22:0)/apolipoprotein A-I, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/total cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/24:0)/apolipoprotein A-I, Cer(d18:1/24:0)/apolipoprotein B, Cer(d18:1/24:0)/LDL cholesterol, Cer(d18:1/24:0)/total cholesterol, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/HDL cholesterol, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/LDL-c/HDL-c, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/triglycerides, GlcCer(d18:1/20:0)/apolipoprotein B, GlcCer(d18:1/20:0)/total cholesterol, GlcCer(d18:1/24:1)/apolipoprotein B, GlcCer(d18:1/26:1)/apolipoprotein A-I, LacCer(d18:1/16:0)/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, PC O-34:1/apolipoprotein B, PS O-16:0/18:2-alkenyl/triglycerides, PS O-16:1/18:2-alkyl/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/apolipoprotein B, Total Cer/LDL cholesterol, Total Cer/total cholesterol, Total DAG/apolipoprotein A-I, Total DAG/triglycerides, Total GlcCer/apolipoprotein B, Total LacCer/apolipoprotein A-I, Total LacCer/apolipoprotein B and Total LacCer/total cholesterol.

and wherein the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Tables 6b and 9b):

CE 14:0/apoA1/apoB, CE 14:0/apolipoprotein B, CE 14:0/LDL-c/HDL-c, CE 14:0/total-c/HDL-c, CE 16:1/apoA1/apoB, CE 18:3/apoA1/apoB, CE 18:3/apolipoprotein A-I, CE 18:3/apolipoprotein B, CE 18:3/HDL cholesterol, CE 18:3/LDL cholesterol, CE 18:3/LDL-c/HDL-c, CE 18:3/total cholesterol, CE 18:3/total-c/HDL-c, CE 20:5/triglycerides, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/total-c/HDL-c, PC 18:0/20:4/apoA1/apoB, PC O-38:6/apolipoprotein A-I, Total LPC/apoA1/apoB and Total PC/apolipoprotein A-I.

In a preferred embodiment (for subjects that are either undergoing or not undergoing statin treatment), the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 10):
Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total Cer/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/24:1)/apolipoprotein B.
In another preferred embodiment, the one or more lipid-clinical concentration ratio(s) whose decrease is (are) compared to the control is (are) selected from (Table 10):
Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/apolipoprotein B, PC 16:0/20:4/apolipoprotein B and Cer(d18:0/24:0)/apolipoprotein A-I.
In a particularly preferred embodiment, the one or more lipid-clinical concentration ratio(s) whose increase is (are) compared to the control is (are) selected from (Table 11):
Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/18:0)/apolipoprotein A-I.
For the purposes of the invention, and particularly for lipid-clinical concentration ratios, an Apolipoprotein A-I measurement may alternatively be an Apolipoprotein A-II measurement.
In one embodiment of the invention, the treatment the effectiveness of which is to be evaluated or which is to be chosen as appropriate in accordance with the methods described and claimed herein, is a lipid modifying treatment.
For the purposes of the invention, at least one lipid concentration, lipid-lipid ratio or lipid-clinical concentration ratio from Tables 4-13, or combinations thereof, may be determined to assess whether the patient is at risk to develop one or more of CVD complications such as AMI or CVD death; to evaluate the effectiveness of the treatment of CVD and/or one or more of its complications, such as AMI or CVD death in a subject; or to choose an appropriate treatment of CVD and/or one or more of its complications, such as AMI or CVD death in a subject. However, it is also possible, and may be advantageous, to determine at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 lipid concentrations, lipid-lipid ratios or lipid-clinical concentration ratios from Tables 4-13, or combinations thereof, in this regard. Where more than one lipidomic markers are determined and used for the assessment, it may be advantageous that a specific lipid concentration, lipid-lipid ratio, lipid-clinical concentration ratio or combination thereof, is given greater weight than others in the above-mentioned assessment, evaluation or choice.
Preferred embodiments of the invention are methods wherein the one or more lipid(s) or lipid ratio(s), or combination thereof, comprise(s): Cer(d18:1/20:0), LacCer(d18:1/20:0), Cer(d18:1/24:1), LacCer(d18:1/24:1), LacCer(d18:1/22:0), Cer(d18:1/18:0), PC 16:0/20:4, Cer(d18:0/24:0), Cer(d18:1/24:1)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 16:0/20:3, PS O-16:0/18:2-alkenyl/Total PS O, Cer(d18:1/18:0)/PC 16:0/20:4, GlcCer(d18:1/26:0)/LacCer(d18:1/20:0), DAG 16:0/18:1/Total DAG, Cer(d18:0/24:0)/Total Cer, Total LPC/Total LacCer, GlcCer(d18:1/26:0)/LacCer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein A-I, Total LacCer/apolipoprotein A-I, LacCer(d18:1/20:0)/HDL cholesterol and Cer(d18:1/18:0)/apolipoprotein A-I.
In the context of the present invention, CVD is typically characterized by coronary artery disease, peripheral artery disease, a stroke and/or CVD death. The CVD in the subject whose sample is analyzed in accordance with the invention may be atherosclerosis-induced. However, the invention also embodies methods involving subjects who are at risk of developing CVD, but who may or may not have atherosclerosis.
In a further embodiment, the methods of the invention may further comprise determining the serum level of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Apolipoprotein B (ApoB) and/or Apolipoprotein C-III in the subject's sample. In one embodiment of the invention, the subject does not have elevated serum levels of one or more of total cholesterol, low-density lipoprotein cholesterol (LDL-C), Apolipoprotein C-III or Apolipoprotein B (ApoB), or a decreased serum level of HDL-cholesterol (HDL-C).
As mentioned above, for the purposes of the present invention, a control sample may be obtained from (a) CAD patient(s) or a group of CAD patients that has/have remained free of any major CVD complications e.g., by mixing a variety of samples from said population. If a group of CAD patients is used then several lipid profiles from a population are combined and the lipidomic marker is created from this combination. The levels or amounts of the individual lipids or the lipid-lipid ratios or lipid-clinical concentration ratios in the sample from a subject are compared to the levels or amounts of the lipids or lipid ratios in the control for determining the risk of one or more of CVD complications, such as AMI or CVD death, in said subject.
The invention encompasses the analysis of lipid concentrations, lipid-lipid ratios and/or lipid-clinical concentration ratios in samples from a subject that has been or is being treated with one or more statins and/or any other HMG-CoA reductase inhibitor.
Alternatively, the invention encompasses the analysis of lipid concentrations, lipid-lipid ratios and/or lipid-clinical concentration ratios in samples from a subject that has not yet undergone statin therapy or therapy with any other HMG-CoA reductase inhibitor.
In accordance with the aspects and embodiments of the invention described and claimed herein, the statin may be one selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, fluvastatin XL, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.
Collecting information on a lipidomic marker or a lipidomic profile from a subject's biological sample can be performed via various chemical and high resolution analytical techniques. Suitable analytical techniques include but are not limited to mass spectrometry and nuclear resonance spectroscopy. Any high resolution technique capable of resolving individual lipids or lipid classes and providing structural information of the same can be used to collect the lipid profile from the biological sample. For methods of the present invention the level of the lipid is determined by using mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy or dual polarisation interferometry, a high performance separation method such as HPLC or UPLC and/or an immunoassay such as an ELISA. According to an alternative or further embodiment an analyte in a sample can be detected and/or quantified by combining the analyte with a binding moiety capable of specifically binding the analyte. The binding moiety can include, for example, a member of a ligand-receptor pair, i.e., a pair of molecules capable of having a specific binding interaction. The binding moiety can also include, for example, a member of a specific binding pair, such as antibody-antigen, enzyme-substrate, nucleic acid-based ligands, other protein ligands, or other specific binding pairs known in the art. In a preferred embodiment, the lipidomic profile is collected with mass spectrometry (MS), wherein the MS instrument may be coupled to direct infusion methods and high performance separation methods such as HPLC or HPLC. The amount of the individual lipids or lipid classes in the collected lipidomic profile is used when comparing the collected lipid profile to a control.
The methods of the present invention may be used for determining a risk of said patient to develop CVD complications, particularly severe CVD complications such as death and myocardial infarction (MI), including acute myocardial infarction (AMI).
In one embodiment of the invention, a method for treating or preventing CVD complications, such as AMI or CVD death, in a subject in need thereof is provided. The method comprises administering a therapeutically effective dose of a drug capable of modulating one or more of the lipid concentration(s), lipid-lipid ratio(s) or lipid-clinical concentration ratio(s) described in Tables 4-13, wherein the dose is such that said one or more lipid concentration(s), lipid-lipid ratio(s) or lipid-clinical concentration ratio(s) in a sample of said subject does not significantly change when compared to (a) corresponding lipid concentration(s), (a) corresponding lipid-lipid ratio(s) or (a) corresponding lipid-clinical concentration ratio(s) in a control, e.g., a control sample. In a preferred embodiment, the drug is a statin or another HMG CoA reductase inhibitor. Particularly preferred statins in this regard are atorvastatin, cerivastatin, fluvastatin, fluvastatin XL, lovastatin, pitavastatin, pravastatin, rosuvastatin or simvastatin. In another preferred embodiment, the drug is niacin (nicotinic acid); a cholesterol absorption inhibitor, such as ezetimibe or SCH-48461; a cholesteryl ester transfer protein (CETP) inhibitor, such as torcetrapib, anacetrapib or JTT-705; a bile acids sequestrant, such as colesevelam, cholestyramine and colestipol; or a fibrate, such as fenofibrate, gemfibrozil, clofibrate, and bezafibrate. Alternatively, it may also be a phytosterol.
Also embodied by the present invention is a lipid as described herein, e.g. a lipid from any of Tables 4, 7, 10 or 13, for use in preventing or treating a subject at risk to develop CVD complications such as AMI or CVD death, wherein the said lipid is to be taken as a dietary supplement or a medicament. A corresponding method of treatment is likewise encompassed. Likewise, the invention also encompasses a modulator for use for modulating a lipid concentration, lipid-lipid ratio or lipid-clinical concentration ratio as described herein, e.g., in Tables 4-13, in a subject at risk to develop CVD and/or one or more of its complications such as AMI or CVD death. A corresponding method of treatment is likewise encompassed. In a further embodiment, the said modulator is a small molecule, an antisense RNA, a small interfering RNA (siRNA) or a natural or modified lipid.
In one embodiment of the present invention, an antibody against any one of the lipids in Tables 4-13 is used for predicting one or more CVD complications such as AMI or CVD death. In another embodiment of the invention, the antibody may be used for preventing or treating one or more of the above complications in a subject.
Any of the methods, drugs, lipids or antibodies of the present invention may be used for a subject which is at risk to develop or has suffered from one or more CVD complications such as acute myocardial infarction and/or a cardiovascular death. For the purposes of the invention, CVD complication(s) includes severe CVD complication(s), particularly death.
Also encompassed by the present invention is a kit for predicting CVD complications or for performing any of the methods or uses of the present invention, wherein the kit comprises a lipid standard chosen from the lipids in Tables 4, 7, 10 or 13, one or more control lipidomic markers, an antibody against one of the said lipids, and reagents for performing the method.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1.



This FIGURE demonstrates the importance of molecular lipid measurements. Two examples are given in this FIGURE to illustrate that two closely related molecular lipids may have a very different or even opposite effect on CVD complications. First quadrangles are used to indicate two Lactosylceramides (LacCer). LacCer (18:1/20:0) is a significant predictor for CVD Death, while closely related LacCer (18:1/16:0) has only limited or no value as a risk predictor. Second example proves that that two lipid species from the same lipid class may have even an opposite effect on CVD events. PC (18:0/20:4) proved to be a protective lipid against CVD death while PC (16:0/16:0) seems to increase CVD complications.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
Coronary vascular disease/cardiovascular disease (CVD) has its general meaning in the art and is used to classify numerous conditions that affect the heart, heart valves, blood, and vasculature of the body, including CAD. In the present invention the terms CVD and CAD may be used interchangeably. Cardiovascular diseases include endothelial dysfunction, coronary artery disease, angina pectoris, myocardial infarction, atherosclerosis, congestive heart failure, hypertension, cerebrovascular disease, stroke, transient ischemic attacks, deep vein thrombosis, peripheral artery disease, cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Such diseases frequently involve atherosclerosis. In a preferred embodiment of the invention, the cardiovascular disease is a cardiovascular disease associated with atherosclerosis.
CAD is coronary artery disease, AMI is acute myocardial infarction, ACS is acute coronary syndrome, CAC is coronary artery calcification, RCT is reverse cholesterol transport, LDL is low density lipoprotein, HDL is high density lipoprotein, LDL-C is low density lipoprotein cholesterol, HDL-C is high density lipoprotein cholesterol, ApoA is Apolipoprotein A, ApoB is Apolipoprotein B, ApoC is apolipoprotein C, MS is mass spectrometry, HPLC is high performance liquid chromatography, and UPLC is ultra performance liquid chromatography.
As used herein, “a subject” includes all mammals, including without limitation humans, but also non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.
A “sample” is defined as any biological sample obtained from a subject or a group or population of subjects. For the purposes of the present invention, the biological sample may be whole blood, blood serum, or blood plasma. It may also be a tissue sample. However, a preferred embodiment is wherein the biological sample is plasma or serum. Taking a blood sample of a patient is a part of normal clinical practice. The blood sample can be taken in connection with e.g. measuring the cholesterol levels in the patients. The collected blood sample can be prepared and serum or plasma can be separated with techniques well known to a person skilled in the art. Vena blood samples can be collected from patients using a needle and a BD Vacutainer® Plastic Tubes or Vacutainer® Plus Plastic Tubes (BD Vacutainer® SST′ Tubes contain spray-coated silia and a polymer gel for serum separation). Serum can be separated by centrifugation at 1300 RCF for 10 min at room temperature and stored in small plastic tubes at −80° C.
For the purposes of the present invention, lipids from the Lipidomic analysis were named according to the following nomenclature: CE is cholesteryl ester, Cer is ceramide, DAG is diacylglycerol, PC O is ether-linked PC, GD is disialogangliosides, GlcCer is galactosyl- and glucosylceramides, GM is monosialogangliosides, LacCer is lactosylceramides, LPC is lysophosphatidylcholine, PC is Phosphatidylcholine, PE is Phosphatidylethanolamine, PI is Phosphatidylinositol, SM is Sphingomyelin, S1P is sphingosine-1-phosphate.
The nomenclature X:Y indicates, X number of total carbon atoms in the fatty acid(s) portions of the molecule, and Y the total number of double bonds in the fatty acid portion(s) of the molecule.
The nomenclature AB indicates, for a molecule of DAG and PC, A and B types of fatty acid moieties attached to the glycerol backbone of the molecule.
The nomenclature (dC/A) indicates, for a molecule of Cer, GlcCer, LacCer and SM, C the type of long-chain base with an amide-linked, A, fatty acid moiety.
The wording “compared to a control sample” as used herein will be understood to include embodiments where control samples are actually analyzed in respect of a lipidomic marker of interest, i.e., in respect of the concentration of one or more of the lipid(s), the lipid-lipid ratios, or the lipid-clinical concentration ratios or combinations thereof as specifically described herein in connection with the various aspects and embodiments of the present invention. It will be appreciated, however, that the above wording also includes embodiments where the corresponding information on said lipidomic marker in said control sample is merely taken from the literature, or has been previously determined, calculated or extrapolated, or is yet to be determined, calculated or extrapolated.
As used herein, the term “antibody” includes monoclonal and polyclonal antibodies, whole antibodies, antibody fragments, and antibody sub-fragments that exhibit specific binding to a said lipid. Thus, suitable “antibodies” can be whole immunoglobulins of any class, e.g., IgG, IgM, IgA, IgD, IgE, chimeric antibodies or hybrid antibodies with dual or multiple antigen or epitope specificities, or fragments, e.g., F(ab′)2, Fab′, Fab and the like, including hybrid fragments, and additionally includes any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab′)2, a Fd fragment, a Fv fragment and dAb fragments) as well as complete antibodies. For example, Fab molecules can be expressed and assembled in a genetically transformed host like E. coli. A lambda vector system is available thus to express a population of Fab's with a potential diversity equal to or exceeding that of subject generating the predecessor antibody. See Huse W D, et al., Science 1989, 246:1275-81. Such Fab's are included in the definition of “antibody.” The ability of a given molecule, including an antibody fragment or sub-fragment, to act like an antibody and specifically bind to a specific antigen can be determined by binding assays known in the art, for example, using the antigen of interest as the binding partner.
Antibodies against lipids in accordance with the present invention may be prepared by methods well known to those skilled in the art. For example, mice may be immunized with a lipid with adjuvant. Splenocytes are harvested as a pool from the mice that were administered 3 immunizations at 2-week intervals with test bleeds performed on alternate weeks for serum antibody titers. Splenocytes are prepared as 3 aliquots that are either used immediately in fusion experiments or stored in liquid nitrogen for use in future fusions.
Fusion experiments are then performed according to the procedure of Stewart & Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants from wells with growing hybrids are screened by enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody (MAb) secretors on 96-well ELISA plates coated with the said lipid. ELISA positive cultures are cloned by limiting dilutions, typically resulting in hybridomas established from single colonies after 2 serial cloning experiments.
EXAMPLES
Example 1
Materials and Methods
This study is a sub-cohort of the LURIC study that is a large scale prospective study on cardiovascular epidemiology. LURIC database contains clinical information over 3000 patients including baseline coronary angiography, clinically used biomarker data and also e.g. CVD mortality data for the follow-up period (3 years). In this biomarker study the inventors compared CAD cases (n=62) that died during the follow-up due to CVD with patients (n=173) having a stable CAD. Subjects with a significant atherosclerosis level in the angiogram but no CVD related death during the follow-up were used as controls, while the case group had similarly a significant atherosclerosis based on the angiography at baseline and in addition they died during the follow-up due to acute cardiovascular events. A statistical analysis was performed separately also for cases (n=48) and controls (n=124) that were not treated with statins. The clinical characteristics are described in Table 1.







TABLE 1







Background characteristics for LURIC patients


analyzed with lipidomics









Variable
Controls (n = 173)
Cases (n = 62)












Age (average)
60
67


LDL-C
122
117.5


HDL-C
37.2
35.3


DM2 patients
62 (36%)
36 (58%)


Hypertensive patients
101 (58%) 
39 (63%)


Lipid lowering users
49 (28%)
14 (23%)


Smokers (active or quit less than 3
46 (27%)
 8 (13%)


years before sampling)









Definition of Cases: All cases had a significant vessel disease (>=20% stenosis) in coronary angiogram and they all died due to CVD during the follow-up.

Definition of Controls: All controls had a significant vessel disease (>=20% stenosis) in coronary angiogram, but they did not die due to CVD during the follow-up.

Example 2
Analytical Methods
Mass Spectrometry Driven Lipidomics
Direct infusion coupled to tandem mass spectrometry, i.e. shotgun lipidomics, and two liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches, i.e. ceramide and cerebroside lipidomics, were used to identify lipid biomarkers for coronary artery disease (CVD) risk by analyzing molecular lipid species in human serum, plasma, and carotid artery plaques. The applied methods were optimized especially for quantification of molecular cholesteryl esters (CE), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and other lysophospholipids (LPL), ether-linked phosphatidylcholines (PC O) and other ether-linked phospholipids (PL O), phosphatidylserines (PS), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidic acids (PA), diacylglycerols (DAG), ceramides (Cer), glucosylceramides (GlcCer) and lactosylceramides (LacCer).
The following materials were used according to the methods. HPLC or LC-MS grade of chloroform, methanol, water, acetonitrile, formic acid, methanol, isopropanol, ammonium acetate, acetic acid, potassium chloride and butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich (St. Louis, Mo., USA).
HPLC column (Acquity BEH C18, 2.1×50 mm id. 1.7 μm) was purchased from Waters (Milford, Mass., USA). HPLC pre-column (Widepore C18 4×2.0 mm) was purchased from Phenomenex (Torrance, Calif., USA). All labware used for the extraction were resistant to chloroform. Aerosol resistant filter tips (Molecular BioProducts) and Eppendorf 2 ml safe-lock tubes, 96-well twin.tec PCR plates, and Pierce-it-lite thermo-sealing foils were purchased from VWR International (West Chester, Pa., USA). CO-RE Filter Tips and 96-well 2 ml Whatman Uniplates were purchased from Hamilton Robotics (Bonaduz, Switzerland). Synthetic lipid standards were purchased from Avanti Polar Lipids (Alabaster, Ala., USA) and from Matreya (Pleasant Gap, Pa., USA).
Lipids were extracted in chloroform:methanol according to the following protocols. Samples were spiked with known amounts of non-endogenous synthetic internal standards for data normalization and endogenous lipid quantification. In shotgun lipidomics analysis; LPC 17:0, PC 17:0/17:0, PA 17:0/17:0, PE 17:0/17:0, PG 17:0/17:0, PS 17:0/17:0, DAG 17:0/17:0, D6-CE 18:0, in ceramide and cerebroside lipidomics; Cer d18:1/17:0, D3-LacCer d18:1/16:0, and D3-GlcCer d18:1/16:0, were used as internal standards. Post-extract spiked non-endogenous synthetic external standards were used for quality controlling. Stock solutions of standards were prepared by dissolving appropriately weighed amounts of each standard in chloroform:methanol (2:1, V:V) to achieve a final concentration of 500 μM. An internal standard mixture containing each of the standard stock was created and used in lipid extraction.
Samples and quality control samples for each extraction batch were thawed on ice. The carotid artery plaque samples were weighed on ice by using a cryo-box and homogenized in ice-cold 70% methanol in water. The Mixer Mill 301 Teflon adapters were kept at −20° C. Homogenization was performed at 15-25 Hz for 2-15 minutes with Mixer Mill 301 (Retch GmbH, Germany).
Lipid extraction of human samples was carried out in automated fashion using a Hamilton MICROLAB STAR system (Hamilton Robotics, Switzerland). Well-mixed samples were aliquoted into a 96-well 2 ml Whatman Uniplate containing ice-cold methanol and 0.1% BHT. 5 μl of serum and plasma and 30 μl of carotid artery plaques were used for shotgun- and ceramide and cerebroside lipidomics and 100 μl of serum and plasma. The samples were mixed thoroughly after each step in the extraction protocol. The extraction proceeded at room temperature by adding an appropriate volume of internal standard mixture and chloroform. In shotgun and ceramide and cerebroside lipidomics, the organic phase separation was facilitated by adding 20 mM acetic acid and centrifuging the plate for 5 min at 500× g. The organic phase was transferred into a new 96-well 2 ml Whatman Uniplate. The remaining water-containing phase was washed by adding appropriate volume of chloroform followed by centrifugation. The two organic phases were pooled and evaporated under N2 until dryness. The lipid extracts were then re-dissolved in chloroform:methanol (1:2, v:v) including the addition of the synthetic external standard.
In shotgun lipidomics, lipid extracts were analyzed on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB Sciex) equipped with a robotic nanoflow ion source (NanoMate HD, Advion Biosciences). The instruments were operated in positive and negative ion modes. In positive ion the spray voltage was set to 1.0 to 1.4 kV and in negative ion mode to −1.0 to −1.4 kV. A gas pressure of 0.3-0.8 psi was used and the interface heater was set at 60° C. The collision energy (CE) and declustering potential (DP) was optimized for each lipid class using synthetic standards. The mass spectrometer was operated in unit resolution mode using a scan speed of 200 Da/s. Molecular lipids were analyzed in both positive and negative ion modes using multiple precursor ion scanning (MPIS) and neutral loss scanning (NLS) as described by Stahlman and colleagues (Stahlman M, et al. High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009).
In ceramide and cerebroside lipidomics, the high performance liquid chromatography (HPLC) analyses were conducted in the following way. Chromatographic apparatus consisted of a CTC HTC PAL autosampler (CTC Analytics AG, Switzerland), a Rheos Allegro UHPLC pump (Flux Instruments AG, Switzerland), an external column heater set to 60° C. for ceramide and cerebroside lipidomics. The extracted samples, 10 μl of each, were injected into the pre-column followed by the analytical column and delivered to the mass spectrometer at a flow rate of 500 μl/min. In ceramide and cerebroside lipidomics, A gradient was used for lipid analyte separation with solvent A comprising 10 mM ammonium acetate in HPLC grade water containing 0.1% formic acid and solvent B of 10 mM ammonium acetate in acetonitrile:isopropanol (4:3, V:V) containing 0.1% formic acid. The gradient was constructed in the following way: 0 min—65% B; 2 min-65% B; 2.5 min-75% B; 17.5 min-100% B; 22.5 min-100% B; 22.6 min-65% B; 25 min-65% B.
The lipid extracts were analyzed by HPLC-MS/MS. The MS analysis was performed on a hybrid triple quadrupole/linear ion trap mass spectrometer equipped with the Turbo VTM Ion Source (4000 QTRAP, AB Sciex). The instrument was operating in positive and negative ion modes. The ion source voltage was set to 5500V for ceramide and cerebroside lipidomics. The collision energy (CE) and declustering potential (DP) was optimized for each lipid class using synthetic standards. A 20 sec dwell time was applied for each scan.
The data processing was done in the following way. Initially the retention time (in LC mode) and identification of each peak was done using endogenous standards and by Information Dependent Acquisition (IDA) experiments where applicable. The raw data were processed according to peak detected and retention time (in LC mode) in automated fashion. A stringent cutoff was applied for separating background noise from actual lipid peaks. Each sample was controlled and only accepted when fulfilling the stringent acceptance criteria. Peak area counts (cps) of detected peaks were converted into a list of corresponding lipid names. Lipids were normalized to their respective internal standard and sample volume or tissue weight to retrieve their concentrations.
Several quality controls were used in the lipidomic analyses. A calibration line using synthetic or isolated standards was obtained prior to sample analysis. Synthetic standards were chosen based on application and had similar properties to the endogenous lipids or analyte(s) of interest. The calibration line consisted of a minimum of five standards points covering the expected quantification range. A sample extracted without standard and standards extracted with no matrix, were included with the calibration line.
The calibration line was used to determine the dynamic quantification range for each lipid class monitored, e.g., the linear quantification limits. As the internal standards used behave in the same way as endogenous lipids they were used for quantifying endogenous lipid species. The calibration lines were based on the same internal standards that were used for quantification of the endogenous lipids.
In each sample extracted for lipids, the ratio of synthetic internal standards (IS) to corresponding post-extract spiked external standard (ES) was determined. The peak area (cps) ratio of internal to external standard (IS/ES) was used for calculating the Coefficient of Variation (CV) across all samples. The IS/ES ratio enabled the calculation of lipid extraction recovery.
Instrument control (IC) was included at the start, middle and end of each run. IC sample analyzed was an extracted reference plasma sample and a set of standards to monitor the instrument's performance, i.e., the intra- and inter-assay variation.
For each platform, a stringent cutoff was applied for separating background noise from actual lipid peaks. Each sample was controlled and only accepted when fulfilling the stringent acceptance criteria. Masses and counts of detected peaks were converted into a list of corresponding lipid names. Lipids were normalized to their respective internal standard and sample volume to retrieve their concentrations.
Statistical Analyses
Percentage changes in lipid concentrations between control and case groups were calculated as follows:

100*(AVG[C] in case group−AVG[C] in control group)/AVG[C] in control group

Statistical significance was assigned based on standard t-test p-values.

In addition, ROC curves were used for finding lipid molecules and concentration cutoffs that separate the best cases from controls. Selectivity is calculated as a number of correctly identified cases divided by the total number of cases. Specificity is calculated as a number of correctly identified controls divided by the total number of controls. Selectivity and specificity was calculated for each lipid concentration, lipid to lipid ratio and ratio of lipid to clinical concentrations.
Example 3
Ethics
The LURIC study was approved by the ethics review committee at the “Landesärztekammer Rheinland-Pfalz” (Mainz, Germany). Written informed consent was obtained from each of the participants.
Results
In this LURIC study sub-cohort, the traditional biomarkers including LDL-cholesterol and HDL-cholesterol concentrations were practically identical in both groups and therefore were not predictive of CVD-related mortality in this study.
Multiple lipidomic markers appeared as significant predictors of CVD death (Tables 4-13). A total of 151 molecular lipids were quantified. The significant predictors were selected based on the top fifty candidates from each category, when available. The biomarker candidates based on molecular lipid concentrations are presented in Tables 4, 7, 10 and 13. The candidates were selected according to the following criteria: t-test p-value≦0.05 or sensitivity≧60% and specificity≧60%. From traditional clinical chemistry only apolipoprotein A1 and total cholesterol reached statistical significance with p-value lower than 0.05, but % change was less than 10% between controls and cases, other clinical values did not show any statistical significance. The predictive value of new lipidomic biomarkers was increased when their levels were expressed as distinct lipid-lipid ratios or lipid-clinical ratios (e.g. LDL-C or HDL-C). The top biomarker candidates are presented in Table 11. Top candidates from each category, when available, were selected based on the following selection criteria: t-test p-value≦0.05 and sensitivity≧60% and specificity≧60%.
Importance of Detailed Molecular Lipid Analyses
Recent evolvement of mass spectrometry driven lipid analysis approaches has made it possible to resolve complex lipidomes to their molecular lipid species level at high-throughput and quality required for analyses of clinical cohorts. As a result of the high sensitivity and selectivity of the methods, a lipidome-wide analysis of minute sample amounts has become feasible. Present technologies are capable of identifying lipids with different sum compositions, i.e. phosphatidylcholine (PC) 34:1, but more important is the identification of molecular lipid species, e.g. PC 16:0/18:1. In the latter analysis, information of the type of fatty acids and their positions attached to the glycerol backbone making up the particular PC molecule is retrieved.
The seminal work of Shinzawa-Itoh and colleagues showed by highly sophisticated experiments that the oxygen transfer mechanism in cytochrome c oxidase requires a specific phosphatidylglycerol molecular lipid with palmitate and vaccenate at the sn-1 and sn-2 positions respectively on the glycerol backbone (Shinzawa-Itoh K, Aoyama H, Muramoto K et al: Structures and physiological roles of 13 integral lipids of bovine heart cytochrome c oxidase. EMBO J 2007, 26:1713-1725). In line with other studies, this undoubtedly indicates that the lipid structure is an essential determinant of the biological effect. Therefore, molecular lipidomics is an essential for biomarker discovery. FIG. 1 illustrates the importance of molecular lipid data by comparing the biomarker value of two PC and LacCer molecules in predicting CVD mortality in the LURIC cohort. The data reveals that while LacCer(d18:1/20:0) is a significant CVD predictor, LacCer (d18:1/18:16:0) has low biomarker potential. In addition, two PC molecules PC (18:0/20:4) and PC (18:0/16:0) have even opposite effects on CVD complications. Thus, it is always necessary to identify and quantify all lipid species for lipid classes of interest including but not limited to cholesterol esters, different phospholipid classes, ceramides, cerebrosides (lactosylceramides, glycosylceramides), and gangliosides.







TABLE 2







Significant lipids in LURIC study sorted by p-value. Lipid names,


p-values and % change for negative correlation are presented.


Table 2a shows significant lipids from all study subjects. The


significant lipids from subjects not undergoing


statin treatment are listed in table 2b.









Lipid name
p-value
Percentage change










2a) Significant lipids in LURIC study sorted by p-value


from all study subjects.









Positive correlation




LacCer(d18:1/20:0)
0.00008
29.52421


LacCer(d18:1/22:0)
0.00046
22.75541


LacCer(d18:1/18:0)
0.00094
26.78692


Cer(d18:1/18:0)
0.00177
23.30373


Cer(d18:1/20:0)
0.00302
17.32385


LacCer(d18:1/24:1)
0.00361
24.72456


PS O-18:2/16:0-alkenyl
0.00571
49.89286


PS O-16:0/18:2-alkenyl
0.00670
49.99084


PS O-16:1/18:2-alkyl
0.00670
49.99084


Cer(d18:1/24:1)
0.01142
14.88898


Total LacCer
0.01669
13.83938


PS O-18:0/18:2-alkenyl
0.02899
40.93773


(PS O-18:1/18:2-alkyl)




LacCer(d18:1/24:0)
0.04425
15.80958


PC O-32:0 (KDdiA-PC)
0.04546
17.40815


GlcCer(d18:1/18:0)
0.04913
12.83156


Negative correlation




Total PC
0.00011
−16.07367


PC 16:0/20:4
0.00077
−18.06547


Total LPC
0.00126
−17.02070


CE 14:0
0.00181
−22.73309


PC 16:0/20:3
0.00191
−18.78110


Cer(d18:0/24:0)
0.00254
−27.27562


PC 18:0/20:4
0.00303
−16.02110


CE 20:3
0.00312
−19.02446


CE 17:1
0.00694
−16.90145


PC 18:0/20:3
0.00726
−17.20664


PC 18:0/18:1
0.00765
−18.18002


Cer(d18:0/22:0)
0.01158
−22.37263


PC 16:0/22:6
0.01180
−16.65050


LPC 18:1
0.01457
−14.45827


SM (d18:1/23:0) (d18:1/22:1-OH)
0.01920
−14.02360


CE 16:0
0.02427
−10.63490


Total CE
0.02745
−11.83333


SM (d18:1/24:0) (d18:1/23:1-OH)
0.02784
−13.83715


PC 18:1/18:2
0.03666
−10.83371







2b) Significant lipids in LURIC study sorted by p-value


from subjects not undergoing statin treatment.









Positive correlation




Cer(d18:1/20:0)
0.00004
28.00357


Cer(d18:1/18:0)
0.00009
34.32550


LacCer(d18:1/20:0)
0.00010
32.91154


Cer(d18:1/24:1)
0.00039
23.37606


LacCer(d18:1/22:0)
0.00048
25.61851


LacCer(d18:1/18:0)
0.00144
29.19850


LacCer(d18:1/24:1)
0.00199
29.83279


PS O-18:2/16:0-alkenyl
0.00432
33.81177


Cer(d18:1/22:0)
0.00473
18.32126


PS O-16:0/18:2-alkenyl
0.00590
32.17190


PS O-16:1/18:2-alkyl
0.00590
32.17190


Total DAG
0.00794
31.67365


Cer(d18:1/16:0)
0.00952
16.71359


Total Cer
0.00932
15.44601


Total LacCer
0.01105
16.00541


LacCer(d18:1/24:0)
0.01989
19.85622


GlcCer(d18:1/20:0)
0.02288
17.74772


PC O-32:0 (KDdiA-PC)
0.02467
22.23265


GlcCer(d18:1/18:0)
0.02584
16.12961


GlcCer(d18:1/24:1)
0.03290
18.89331


GlcCer(d18:1/26:1)
0.04702
17.52675


Cer(d18:1/26:1)
0.04802
13.59618


Negative correlation




Total PC
0.00921
−12.44220


CE 14:0
0.01090
−21.01258


CE 20:3
0.02157
−16.03606


CE 17:1
0.02204
−15.93952


PC 16:0/20:4
0.02256
−14.96966


PC 18:0/20:4
0.03376
−13.60917


Cer(d18:0/24:0)
0.03376
−21.87004


Total LPC
0.03443
−12.91576


PC 16:0/20:3
0.04337
−13.43056
















TABLE 3







Table of significant lipid to lipid ratios in LURIC study sorted by p-value.


Lipid names, p-values, % change both for positive and negative


correlation are presented. Table 3a shows significant lipids from


all study subjects. The significant lipids from subjects not


undergoing statin treatment are listed in table 3b.











Percentage


Lipid name/lipid name
p-value
change










3a) Significant lipid to lipid ratios in LURIC study sorted


by p-value from all study subjects.









Positive correlation




GlcCer(d18:1/26:0)/Total CE
0.0000000
12.8050228


GlcCer(d18:1/26:1)/Total CE
0.0000000
29.5576923


PS O-16:0/18:2-alkenyl/Total PS O
0.0000000
26.0723978


PS O-16:1/18:2-alkyl/Total PS O
0.0000000
26.0723978


PS O-18:2/16:0-alkenyl/Total PS O
0.0000000
28.7826715


Cer(d18:1/24:1)/Total PC
0.0000000
38.6612692


PC 16:0/16:0/Total PC
0.0000000
26.1328496


LacCer(d18:1/20:0)/Total PC
0.0000000
55.3076855


Cer(d18:1/24:1)/PC 16:0/20:4
0.0000000
52.6701270


Cer(d18:1/20:0)/Total PC
0.0000000
40.0940286


LacCer(d18:1/18:0)/Total PC
0.0000000
53.5855166


Cer(d18:1/18:0)/Total PC
0.0000000
48.2586905


Cer(d18:1/18:0)/PC 16:0/20:4
0.0000000
65.7060860


Cer(d18:1/24:1)/PC 16:0/18:1
0.0000000
34.2415459


PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl)/
0.0000000
18.7146206


Total PS O




Total LacCer/Total PC
0.0000001
40.0389043


Cer(d18:1/24:1)/PC 16:0/20:3
0.0000001
39.6033049


Cer(d18:1/22:0)/Total PC
0.0000001
31.3604924


Cer(d18:1/20:0)/PC 16:0/20:4
0.0000001
55.6619154


LacCer(d18:1/22:0)/Total PC
0.0000001
48.7385500


LacCer(d18:1/20:0)/PC 16:0/20:3
0.0000001
57.2475183


Cer(d18:1/18:0)/PC 16:0/20:3
0.0000002
53.4975004


LacCer(d18:1/20:0)/PC 18:0/20:3
0.0000002
56.3125345


LacCer(d18:1/18:0)/PC 18:0/20:3
0.0000002
58.4642532


Cer(d18:1/22:0)/PC 16:0/20:4
0.0000002
42.5755107


LacCer(d18:1/20:0)/PC 18:0/18:1
0.0000004
50.6042672


Cer(d18:1/24:1)/LPC 18:1
0.0000004
37.9852355


LacCer(d18:1/20:0)/PC 16:0/18:1
0.0000004
46.3528827


LacCer(d18:1/20:0)/PC 18:1/18:1
0.0000004
49.6378601


Cer(d18:1/18:0)/PC 16:0/18:1
0.0000004
42.1348344


Cer(d18:1/18:0)/PC 18:0/20:4
0.0000005
55.4207048


Cer(d18:1/24:1)/PC 18:0/20:4
0.0000005
40.7129933


Cer(d18:1/20:0)/PC 16:0/20:3
0.0000005
44.8451813


LacCer(d18:1/18:0)/PC 18:0/18:1
0.0000006
50.0860210


Cer(d18:1/20:0)/PC 16:0/18:1
0.0000006
35.2897482


LacCer(d18:1/20:0)/PC 18:1/18:2
0.0000007
47.4807020


Cer(d18:1/24:1)/Total LPC
0.0000007
37.5873387


Total Cer/Total PC
0.0000007
28.8638001


Cer(d18:1/20:0)/PC 18:0/20:4
0.0000007
46.2204972


LacCer(d18:1/18:0)/PC 18:1/18:1
0.0000007
47.3110220


LacCer(d18:1/18:0)/PC 16:0/20:3
0.0000007
55.5925567


LacCer(d18:1/24:1)/Total PC
0.0000008
56.5726916


Cer(d18:1/24:1)/PC 18:0/18:1
0.0000008
38.3563305


Cer(d18:1/24:1)/PC 18:0/20:3
0.0000013
39.6802706


LacCer(d18:1/22:0)/PC 16:0/20:4
0.0000013
57.8428560


LacCer(d18:1/18:0)/PC 16:0/18:1
0.0000014
45.2070855


LacCer(d18:1/22:0)/PC 18:0/20:3
0.0000014
50.7183795


Cer(d18:1/20:0)/PC 18:1/18:1
0.0000016
40.1833717


LacCer(d18:1/18:0)/PC 18:1/18:2
0.0000017
46.5169450


Cer(d18:1/22:0)/PC 16:0/20:3
0.0000018
33.7513783


Negative correlation




Cer(d18:0/22:0)/Total CE
0.0000000
−11.7767698


Cer(d18:0/24:0)/Total CE
0.0000000
−18.9531828


Cer(d18:0/24:1)/Total CE
0.0000000
−1.2444181


CE 18:3/Cer(d18:1/24:1)
0.0000000
−35.9591235


Cer(d18:0/24:0)/Cer(d18:1/24:1)
0.0000004
−36.1288066


CE 18:3/Total Cer
0.0000021
−30.3105704


CE 18:3/PC 16:0/16:0
0.0000028
−30.4716021


CE 18:3/LacCer(d18:1/20:0)
0.0000028
−44.3063424


CE 20:3/Cer(d18:1/24:1)
0.0000034
−28.1216690


CE 18:3/PS O-16:0/18:2-alkenyl
0.0000036
−38.5094276


CE 18:3/PS O-16:1/18:2-alkyl
0.0000036
−38.5094276


CE 18:3/Total LacCer
0.0000037
−34.1308012


CE 18:3/Cer(d18:1/20:0)
0.0000037
−33.6203392


LPC 18:2/LacCer(d18:1/20:0)
0.0000040
−41.6604394


Cer(d18:0/24:0)/Total Cer
0.0000049
−31.4531758


Cer(d18:0/24:0)/Cer(d18:1/18:0)
0.0000052
−40.2960344


Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl
0.0000059
−40.8329837


Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl
0.0000059
−40.8329837


Cer(d18:0/22:0)/Cer(d18:1/24:1)
0.0000064
−32.1886967


Cer(d18:0/24:0)/LacCer(d18:1/24:0)
0.0000067
−37.0287234


Cer(d18:0/22:0)/Cer(d18:1/18:0)
0.0000071
−36.5400374


CE 14:0/Cer(d18:1/24:1)
0.0000076
−29.9054883


CE 18:3/Cer(d18:1/16:0)
0.0000079
−32.5598715


CE 18:3/Cer(d18:1/18:0)
0.0000082
−38.1543108


Cer(d18:0/24:0)/Cer(d18:1/20:0)
0.0000087
−35.4631184


CE 18:3/GlcCer(d18:1/20:0)
0.0000087
−36.4665498


CE 18:3/Cer(d18:1/22:0)
0.0000101
−30.6595286


Cer(d18:0/24:0)/Cer(d18:1/22:0)
0.0000105
−32.4086711


Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl
0.0000114
−37.4226868


Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl
0.0000114
−37.4226868


CE 18:3/PC O-34:1
0.0000122
−33.4941413


CE 18:3/Total CE
0.0000151
−20.2882080


PC 16:0/20:3/PS O-16:0/18:2-alkenyl
0.0000154
−29.5280580


PC 16:0/20:3/PS O-16:1/18:2-alkyl
0.0000154
−29.5280580


PC 18:1/18:2/PS O-16:0/18:2-alkenyl
0.0000155
−23.7129392


PC 18:1/18:2/PS O-16:1/18:2-alkyl
0.0000155
−23.7129392


Cer(d18:0/22:0)/Cer(d18:1/20:0)
0.0000165
−31.5266149


CE 18:3/LacCer(d18:1/22:0)
0.0000174
−37.1525849


LPC 18:2/PS O-16:0/18:2-alkenyl
0.0000175
−30.3450779


LPC 18:2/PS O-16:1/18:2-alkyl
0.0000175
−30.3450779


CE 17:1/Cer(d18:1/24:1)
0.0000186
−24.8594478


CE 16:0/Cer(d18:1/24:1)
0.0000188
−19.2509390


CE 18:3/GlcCer(d18:1/18:0)
0.0000221
−35.5601491


CE 18:3/LacCer(d18:1/18:0)
0.0000326
−37.4416706


CE 18:3/PS O-16:0/18:1-alkenyl
0.0000326
−34.1672013


(PS O-16:1/18:1-alkyl)




CE 18:3/Cer(d18:1/24:0)
0.0000331
−27.4645341


Cer(d18:0/22:0)/Cer(d18:1/22:0)
0.0000377
−28.0899033


LPC 16:0/LacCer(d18:1/20:0)
0.0000391
−35.6188222


Cer(d18:0/24:0)/Cer(d18:1/16:0)
0.0000446
−34.6695918


Total LPC/Total LacCer
0.0000469
−26.0881987







3b) Significant lipid to lipid ratios in LURIC study sorted by


p-value from subjects not undergoing statin treatment









Positive correlation




GlcCer(d18:1/26:0)/Total CE
0.000000
13.329316


GlcCer(d18:1/26:1)/Total CE
0.000000
35.356592


PS O-16:0/18:2-alkenyl/Total PS O
0.000000
26.426208


PS O-16:1/18:2-alkyl/Total PS O
0.000000
26.426208


PS O-18:2/16:0-alkenyl/Total PS O
0.000000
29.590682


Cer(d18:1/24:1)/Total PC
0.000000
43.040800


Cer(d18:1/24:1)/PC 16:0/20:4
0.000000
59.876132


Cer(d18:1/24:1)/PC 18:0/20:4
0.000000
46.504629


Cer(d18:1/20:0)/Total PC
0.000000
46.216544


PC 16:0/16:0/Total PC
0.000000
28.139641


Total DAG/Total LPC
0.000000
57.426180


Cer(d18:1/18:0)/PC 16:0/20:4
0.000000
73.073704


Cer(d18:1/18:0)/Total PC
0.000000
53.769121


Cer(d18:1/22:0)/Total PC
0.000000
34.704834


Cer(d18:1/22:0)/PC 16:0/20:4
0.000000
47.860118


Cer(d18:1/20:0)/PC 16:0/20:4
0.000000
64.045788


LacCer(d18:1/20:0)/Total PC
0.000000
55.686130


Cer(d18:1/18:0)/PC 18:0/20:4
0.000000
61.943925


Cer(d18:1/20:0)/PC 18:0/20:4
0.000000
53.941480


Cer(d18:1/24:1)/PC 16:0/18:1
0.000000
35.646899


Cer(d18:1/24:1)/PC 16:0/20:3
0.000000
44.382440


Cer(d18:1/18:0)/PC 16:0/20:3
0.000001
61.224227


Cer(d18:1/20:0)/PC 16:0/20:3
0.000001
52.891307


Cer(d18:1/24:1)/PC 18:0/18:1
0.000001
42.115354


Cer(d18:1/22:0)/PC 18:0/20:4
0.000001
36.949978


Cer(d18:1/20:0)/PC 18:0/18:1
0.000004
47.396000


SM (d18:1/17:0) (d18:1/16:1-OH)/Total PC O
0.000004
39.660537


PC O-32:0 (KDdiA-PC)/Total PC O
0.000005
46.418572


Cer(d18:1/20:0)/PC 16:0/18:1
0.000005
37.704904


Cer(d18:1/24:1)/LPC 18:1
0.000005
40.092181


Cer(d18:1/24:1)/PC 18:1/18:1
0.000005
38.720001


Cer(d18:1/18:0)/PC 16:0/18:1
0.000006
44.307054


LacCer(d18:1/18:0)/Total PC
0.000006
50.539560


Cer(d18:1/20:0)/PC 18:1/18:1
0.000006
44.664936


Cer(d18:1/18:0)/PC 18:0/18:1
0.000006
54.563687


LacCer(d18:1/20:0)/PC 18:0/20:4
0.000007
66.072520


Cer(d18:1/24:1)/PC 18:0/20:3
0.000007
44.320399


LacCer(d18:1/20:0)/PC 16:0/20:3
0.000008
56.972652


Cer(d18:1/22:0)/PC 16:0/20:3
0.000008
37.291136


PC 16:0/16:0/PC 16:0/20:4
0.000009
42.512740


PS O-16:0/18:2-alkenyl/Total PC
0.000009
53.059092


PS O-16:1/18:2-alkyl/Total PC
0.000009
53.059092


LacCer(d18:1/20:0)/PC 18:0/20:3
0.000009
55.403274


Total Cer/Total PC
0.000011
29.949121


Total LacCer/Total PC
0.000012
37.815865


Total DAG/Total PC
0.000012
57.897424


Cer(d18:1/18:0)/PC 18:1/18:1
0.000012
49.427440


LacCer(d18:1/20:0)/PC 18:1/18:2
0.000013
48.432788


Total DAG/Total PC O
0.000014
52.258466


Cer(d18:1/24:1)/Total LPC
0.000014
38.684740


Negative correlation




Cer(d18:0/22:0)/Total CE
0.000000
−6.628640


Cer(d18:0/24:0)/Total CE
0.000000
−13.307278


DAG 16:0/18:1/Total DAG
0.000000
−17.305276


CE 18:3/Cer(d18:1/24:1)
0.000000
−37.975733


CE 16:0/Cer(d18:1/24:1)
0.000001
−23.257135


Cer(d18:0/24:0)/Cer(d18:1/24:1)
0.000003
−36.760563


CE 20:4/Cer(d18:1/24:1)
0.000004
−28.636879


CE 20:3/Cer(d18:1/24:1)
0.000007
−30.787067


CE 14:0/Cer(d18:1/24:1)
0.000008
−32.409429


Cer(d18:0/22:0)/Cer(d18:1/24:1)
0.000011
−34.086320


CE 20:4/Cer(d18:1/18:0)
0.000017
−31.313949


CE 18:3/LacCer(d18:1/20:0)
0.000017
−45.652383


CE 18:3/Cer(d18:1/20:0)
0.000019
−34.938575


CE 17:1/Cer(d18:1/24:1)
0.000020
−27.626744


CE 18:3/Cer(d18:1/18:0)
0.000022
−39.797182


CE 18:3/PS O-16:0/18:2-alkenyl
0.000023
−40.724368


CE 18:3/PS O-16:1/18:2-alkyl
0.000023
−40.724368


CE 18:3/PC O-34:1
0.000025
−36.379680


CE 18:3/Total DAG
0.000025
−40.454303


CE 18:3/PC 16:0/16:0
0.000026
−31.169278


CE 18:2/Cer(d18:1/24:1)
0.000027
−23.500781


Cer(d18:0/22:0)/Cer(d18:1/18:0)
0.000031
−38.447200


CE 18:3/Total Cer
0.000034
−30.424215


Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl
0.000045
−37.930990


Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl
0.000045
−37.930990


LPC 18:2/LacCer(d18:1/20:0)
0.000045
−40.925010


PC 18:0/20:4/PS O-16:0/18:2-alkenyl
0.000046
−31.467609


PC 18:0/20:4/PS O-16:1/18:2-alkyl
0.000046
−31.467609


PC 16:0/20:4/Total DAG
0.000046
−36.637600


CE 16:0/Cer(d18:1/18:0)
0.000047
−26.423623


Cer(d18:0/22:0)/Cer(d18:1/20:0)
0.000051
−32.923442


PC 16:0/20:3/PS O-16:0/18:2-alkenyl
0.000051
−30.612669


PC 16:0/20:3/PS O-16:1/18:2-alkyl
0.000051
−30.612669


Cer(d18:0/24:0)/Cer(d18:1/18:0)
0.000052
−41.966868


CE 17:1/Cer(d18:1/18:0)
0.000062
−30.441023


CE 20:4/LacCer(d18:1/20:0)
0.000062
−34.937892


Cer(d18:0/24:0)/Cer(d18:1/20:0)
0.000077
−36.424116


CE 18:3/GlcCer(d18:1/20:0)
0.000078
−38.775015


PC 16:0/20:4/PS O-16:0/18:2-alkenyl
0.000079
−31.649115


PC 16:0/20:4/PS O-16:1/18:2-alkyl
0.000079
−31.649115


Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl
0.000082
−40.069396


Cer(d18:0/24:0)/PS O-16:1/18:2-alkyl
0.000082
−40.069396


Cer(d18:0/24:0)/Total Cer
0.000091
−30.775984


Cer(d18:0/22:0)/Total DAG
0.000096
−39.183597


CE 18:3/Cer(d18:1/16:0)
0.000101
−32.942871


PC 18:1/18:2/PS O-16:0/18:2-alkenyl
0.000106
−24.667433


PC 18:1/18:2/PS O-16:1/18:2-alkyl
0.000106
−24.667433


CE 18:3/Cer(d18:1/22:0)
0.000109
−30.876034


CE 14:0/Cer(d18:1/18:0)
0.000117
−35.001327


PC 18:0/20:3/PS O-16:0/18:2-alkenyl
0.000119
−29.825217
















TABLE 4





Table of significant lipid to clinical ratios in LURIC study 


sorted by p-value. Lipid names and clinical measurement, p-values 


and percentage change both for positive and negative correlation 


are presented. Table 4a shows significant lipids from all study 


subjects. The significant lipids from subjects not undergoing statin 


treatment are listed in table 4b.







4a) Significant lipid to clinical ratios in LURIC study sorted 


by p-value from all study subjects.











Percentage 


Lipid name/clinical measurement
p-value
change





Positive correlation




LacCer(d18:1/20:0)/apolipoprotein A-I
0.00000
 43.04967


LacCer(d18:1/20:0)/total cholesterol
0.00000
 41.46919


LacCer(d18:1/22:0)/total cholesterol
0.00000
 32.24669


LacCer(d18:1/20:0)/triglycerides
0.00001
 54.05564


LacCer(d18:1/22:0)/apolipoprotein A-I
0.00001
 32.96039


Cer(d18:1/18:0)/triglycerides
0.00001
 40.06643


Cer(d18:1/18:0)/total cholesterol
0.00002
 31.56099


LacCer(d18:1/20:0)/apolipoprotein B
0.00002
 37.03057


Cer(d18:1/20:0)/triglycerides
0.00003
 34.46408


Cer(d18:1/20:0)/total cholesterol
0.00003
 25.23403


LacCer(d18:1/18:0)/triglycerides
0.00004
 52.60972


LacCer(d18:1/24:1)/triglycerides
0.00004
 60.79934


LacCer(d18:1/22:0)/apolipoprotein B
0.00004
 29.83861


Cer(d18:1/20:0)/apolipoprotein A-I
0.00004
 31.35691


LacCer(d18:1/18:0)/total cholesterol
0.00005
 38.87517


Cer(d18:1/24:1)/triglycerides
0.00005
 32.27486


LacCer(d18:1/20:0)/HDL cholesterol
0.00006
 39.32664


LacCer(d18:1/22:0)/triglycerides
0.00006
 49.54525


LacCer(d18:1/18:0)/apolipoprotein A-I
0.00007
 41.61371


LacCer(d18:1/24:1)/total cholesterol
0.00007
 36.35465


Total LacCer/total cholesterol
0.00008
 23.69633


Cer(d18:1/18:0)/apolipoprotein A-I
0.00008
 37.38237


Cer(d18:1/24:1)/total cholesterol
0.00011
 21.94547


Cer(d18:1/24:1)/apolipoprotein A-I
0.00011
 27.12684


Cer(d18:1/18:0)/apolipoprotein B
0.00017
 27.27914


LacCer(d18:1/20:0)/total-c/HDL-c
0.00018
 33.48550


LacCer(d18:1/24:1)/apolipoprotein A-I
0.00018
 36.33201


Total LacCer/triglycerides
0.00022
 38.52220


LacCer(d18:1/24:1)/apolipoprotein B
0.00022
 33.98045


LacCer(d18:1/18:0)/apolipoprotein B
0.00022
 33.69302


LacCer(d18:1/22:0)/HDL cholesterol
0.00024
 29.19905


Total LacCer/apolipoprotein A-I
0.00025
 25.87122


PC O-32:0 (KDdiA-PC)/triglycerides
0.00029
 48.31010


LacCer(d18:1/20:0)/LDL cholesterol
0.00031
 31.95659


Cer(d18:1/22:0)/triglycerides
0.00037
 28.23304


Total Cer/triglycerides
0.00043
 26.80115


LacCer(d18:1/22:0)/LDL cholesterol
0.00050
 24.24011


Cer(d18:1/20:0)/apolipoprotein B
0.00052
 20.79615


LacCer(d18:1/22:0)/total-c/HDL-c
0.00061
 27.84411


Total LacCer/apolipoprotein B
0.00065
 20.63257


PC O-34:1/triglycerides
0.00066
 38.80785


PC O-32:0 (KDdiA-PC)/apolipoprotein A-I
0.00066
 36.57664


Cer(d18:1/20:0)/HDL cholesterol
0.00067
 30.67863


LacCer(d18:1/18:0)/total-c/HDL-c
0.00073
 30.67436


PS O-18:2/16:0-alkenyl/HDL cholesterol
0.00073
 57.54661


LacCer(d18:1/18:0)/HDL cholesterol
0.00079
 38.04132


Cer(d18:1/16:0)/triglycerides
0.00083
 28.51323


Cer(d18:1/18:0)/HDL cholesterol
0.00085
 36.16483


Cer(d18:1/18:0)/total-c/HDL-c
0.00087
 21.75165


LacCer(d18:1/20:0)/apoA1/apoB
0.00090
 32.36387


Negative correlation




CE 18:3/apolipoprotein B
0.00016
−25.31907


CE 18:3/total cholesterol
0.00026
−23.67665


CE 18:3/LDL cholesterol
0.00052
−27.21759


CE 18:3/apolipoprotein A-I
0.00054
−23.09816


CE 18:3/LDL-c/HDL-c
0.00060
−31.33346


CE 18:3/apoA1/apoB
0.00068
−27.55825


CE 18:3/total-c/HDL-c
0.00070
−27.28733


CE 18:3/HDL cholesterol
0.00072
−24.04060


Cer(d18:0/24:0)/apolipoprotein B
0.00181
−26.25271


CE 14:0/LDL-c/HDL-c
0.00241
−27.07756


Cer(d18:0/24:0)/total cholesterol
0.00281
−24.73116


Cer(d18:0/24:0)/total-c/HDL-c
0.00347
−27.84469


CE 14:0/total-c/HDL-c
0.00356
−23.22809


PC 16:0/20:3/total-c/HDL-c
0.00364
−19.07121


CE 14:0/apolipoprotein B
0.00375
−19.76414


Total PC/LDL-c/HDL-c
0.00402
−21.24583


Cer(d18:0/24:0)/LDL-c/HDL-c
0.00502
−33.76759


Total PC/apolipoprotein B
0.00513
−12.91145


Total PC/total-c/HDL-c
0.00535
−16.13350


CE 20:3/LDL-c/HDL-c
0.00547
−21.16631


PC 16:0/20:4/apolipoprotein B
0.00576
−15.39247


PC 16:0/20:4/total-c/HDL-c
0.00682
−18.06323


Cer(d18:0/22:0)/LDL-c/HDL-c
0.00748
−29.05891


Total PC/total cholesterol
0.00760
−10.60231


Cer(d18:0/22:0)/total-c/HDL-c
0.00765
−23.72182


PC 16:0/20:3/LDL-c/HDL-c
0.00784
−22.56336


PC 18:0/20:3/total-c/HDL-c
0.00794
−17.83335


CE 14:0/total cholesterol
0.00816
−17.44260


CE 20:3/total-c/HDL-c
0.00819
−17.34055


PC 16:0/20:4/LDL-c/HDL-c
0.00828
−21.49898


CE 17:1/LDL-c/HDL-c
0.00832
−21.33179


CE 14:0/LDL cholesterol
0.00832
−20.55966


Cer(d18:0/24:0)/LDL cholesterol
0.00848
−30.08563


LPC 18:2/LDL-c/HDL-c
0.00873
−21.70580


Total LPC/LDL-c/HDL-c
0.00881
−20.39287


PC 18:0/20:3/LDL-c/HDL-c
0.00947
−22.19538


Cer(d18:0/24:0)/apolipoprotein A-I
0.01052
−22.39254


Cer(d18:0/22:0)/apolipoprotein B
0.01094
−21.02661


CE 20:3/apolipoprotein B
0.01105
−15.18746


PC 16:0/20:4/total cholesterol
0.01131
−13.37554


Total LPC/total-c/HDL-c
0.01183
−16.82334


LPC 18:2/apoA1/apoB
0.01189
−19.23316


PC 16:0/20:3/apolipoprotein B
0.01273
−15.11848


CE 16:1/apolipoprotein B
0.01456
−26.99397


Cer(d18:0/24:0)/HDL cholesterol
0.01507
−23.10073


CE 16:1/LDL cholesterol
0.01581
−29.68280


PC 18:0/18:1/LDL-c/HDL-c
0.01581
−25.72002


CE 20:5/HDL cholesterol
0.01584
−23.45029


CE 20:5/apolipoprotein B
0.01619
−27.03163


LPC 18:2/LDL cholesterol
0.01648
−17.26480










4b) Significant lipid to clinical ratios in LURIC study sorted by 


p-value from subjects not undergoing statin treatment.











Percentage 


Lipid name/clinical measurement
p-value
change





Positive correlation




Cer(d18:1/20:0)/total cholesterol
0.00000
 38.78716


Cer(d18:1/20:0)/apolipoprotein B
0.00000
 35.29473


Cer(d18:1/18:0)/total cholesterol
0.00000
 44.87416


Cer(d18:1/24:1)/total cholesterol
0.00000
 31.81230


Cer(d18:1/18:0)/apolipoprotein B
0.00000
 41.46939


Cer(d18:1/20:0)/apolipoprotein A-I
0.00000
 43.22923


Cer(d18:1/24:1)/apolipoprotein A-I
0.00000
 36.15491


LacCer(d18:1/20:0)/apolipoprotein A-I
0.00001
 47.52465


Cer(d18:1/18:0)/apolipoprotein A-I
0.00001
 49.43693


LacCer(d18:1/20:0)/total cholesterol
0.00001
 47.43498


Cer(d18:1/22:0)/total cholesterol
0.00001
 26.21569


LacCer(d18:1/22:0)/total cholesterol
0.00001
 36.57029


Cer(d18:1/24:1)/apolipoprotein B
0.00001
 29.03655


LacCer(d18:1/22:0)/apolipoprotein A-I
0.00001
 35.92744


Cer(d18:1/18:0)/triglycerides
0.00002
 45.88769


Cer(d18:1/20:0)/triglycerides
0.00002
 41.38147


LacCer(d18:1/20:0)/apolipoprotein B
0.00002
 43.58427


Total Cer/total cholesterol
0.00002
 22.69184


Total DAG/triglycerides
0.00003
 42.61436


LacCer(d18:1/20:0)/LDL cholesterol
0.00003
 42.53680


Cer(d18:1/18:0)/total-c/HDL-c
0.00003
 32.87649


Cer(d18:1/24:1)/triglycerides
0.00004
 37.38060


Cer(d18:1/20:0)/HDL cholesterol
0.00004
 44.64484


LacCer(d18:1/22:0)/apolipoprotein B
0.00004
 35.19942


Cer(d18:1/22:0)/apolipoprotein B
0.00005
 24.23877


LacCer(d18:1/24:1)/total cholesterol
0.00006
 43.51653


LacCer(d18:1/20:0)/HDL cholesterol
0.00006
 45.00931


Total LacCer/total cholesterol
0.00007
 27.38505


Cer(d18:1/18:0)/HDL cholesterol
0.00008
 50.71310


Cer(d18:1/24:1)/HDL cholesterol
0.00008
 36.69128


LacCer(d18:1/20:0)/triglycerides
0.00009
 53.20068


LacCer(d18:1/24:1)/apolipoprotein B
0.00009
 42.74199


Cer(d18:1/20:0)/total-c/HDL-c
0.00010
 26.38965


LacCer(d18:1/22:0)/LDL cholesterol
0.00010
 31.99739


Cer(d18:1/22:0)/apolipoprotein A-I
0.00011
 28.85911


LacCer(d18:1/24:1)/triglycerides
0.00013
 64.74061


Total Cer/apolipoprotein B
0.00015
 21.06506


Total Cer/apolipoprotein A-I
0.00016
 25.36811


Cer(d18:1/16:0)/total cholesterol
0.00016
 27.48182


LacCer(d18:1/24:1)/apolipoprotein A-I
0.00017
 41.35186


Cer(d18:1/20:0)/LDL cholesterol
0.00018
 35.64759


LacCer(d18:1/18:0)/total cholesterol
0.00019
 42.30922


LacCer(d18:1/22:0)/HDL cholesterol
0.00020
 33.63345


LacCer(d18:1/18:0)/apolipoprotein A-I
0.00022
 44.38352


Cer(d18:1/18:0)/LDL cholesterol
0.00023
 42.34532


Total LacCer/apolipoprotein A-I
0.00031
 28.05057


Total LacCer/apolipoprotein B
0.00035
 24.82894


Cer(d18:1/22:0)/triglycerides
0.00039
 32.11726


LacCer(d18:1/20:0)/total-c/HDL-c
0.00040
 37.19117


Cer(d18:1/16:0)/apolipoprotein A-I
0.00042
 30.73655


Negative correlation




CE 18:3/apoA1/apoB
0.00213
−27.06449


CE 18:3/apolipoprotein A-I
0.00385
−21.63982


CE 18:3/apolipoprotein B
0.00576
−21.66212


CE 18:3/total cholesterol
0.00593
−20.58905


CE 18:3/HDL cholesterol
0.00621
−21.44255


CE 18:3/total-c/HDL-c
0.01166
−23.94433


CE 18:3/LDL-c/HDL-c
0.01395
−25.96634


CE 18:3/LDL cholesterol
0.01410
−22.13225


CE 14:0/total-c/HDL-c
0.02379
−20.97683


CE 14:0/LDL-c/HDL-c
0.03038
−22.86773


Cer(d18:0/24:0)/apolipoprotein B
0.03428
−21.10483


CE 14:0/apolipoprotein B
0.03640
−16.53355


Cer(d18:0/24:0)/total-c/HDL-c
0.04122
−23.22174


Cer(d18:0/24:0)/total cholesterol
0.04309
−19.71324


CE 14:0/apoA1/apoB
0.04354
−18.62921


CE 16:1/apoA1/apoB
0.04511
−23.73367









The biomarker ability of measured lipids was assessed also by calculating the sensitivity and specificity values for each lipid and their ratios to other lipids or classical biomarkers such as LDL-C and apolipoproteins. This ROC curve analysis revealed a number of biomarker candidates that have equal of higher than 60% sensitivity and specificity for predicting CVD complications (Tables 7-9).







TABLE 5





Significant lipids in LURIC study sorted by top sensitivity 


and specificity. Table 5a shows significant lipids from all study 


subjects. The significant lipids from subjects not undergoing 


statin treatment are listed in table 5b.







5a) Significant lipids in LURIC study sorted by top sensitivity 


and specificity from all study subjects.













Percentage 


Lipid name
Sensitivity
Specificity
change





Positive correlation





LacCer(d18:1/20:0)
66.03774
63.70370
 29.52421


Total LacCer
63.15789
60.12270
 13.83938


Cer(d18:1/24:1)
61.40351
60.12270
 14.88898


Negative correlation





Total PC
71.92982
63.29114
−16.07367


PC 16:0/20:3
71.42857
60.75949
−18.78110


Total LPC
70.17544
60.75949
−17.02070


PC 18:0/20:3
66.03774
63.05732
−17.20664


LPC 18:1
64.91228
60.12658
−14.45827


SM (d18:1/14:0) 
63.79310
60.50955
−10.49341


(d18:1/13:1-OH)





Cer(d18:0/22:0)
61.40351
61.34969
−22.37263


PC 16:0/18:2
61.40351
60.12658
 −8.30551


PC 16:0/22:6
60.37736
61.14650
−16.65050










5b) Significant lipids in LURIC study sorted by top sensitivity 


and specificity from subjects not undergoing statin treatment.













Percentage 


Lipid name
Sensitivity
Specificity
change





Positive correlation





LacCer(d18:1/20:0)
70.45455
60.00000
 29.52421


Cer(d18:1/24:1)
69.56522
60.34483
 14.88898


Cer(d18:1/20:0)
65.21739
64.65517
 17.32385


Cer(d18:1/22:0)
60.86957
62.93103
 10.65433


GlcCer(d18:1/24:1)
60.86957
62.06897
 12.75066


LacCer(d18:1/22:0)
60.00000
61.76471
 22.75541


Negative correlation





Total PC
69.56522
61.60714
−16.07367


PC 16:0/20:3
64.44444
60.71429
−18.78110


SM (d18:1/14:0)
63.82979
60.71429
−10.49341


(d18:1/13:1-OH)





LPC 18:1
63.04348
60.71429
−14.45827


Total LPC
63.04348
60.71429
−17.02070


PC O-40:3
60.97561
60.43956
 −1.88354


PC 16:0/20:4
60.86957
60.71429
−18.06547
















TABLE 6





Table of significant lipid to lipid ratios in LURIC study sorted 


by top sensitivity and specificity. Table 6a shows significant lipids 


from all study subjects. The significant lipids from subjects not 


undergoing statin treatment are listed in table 6b.







6a) Table of significant lipid to lipid ratios in LURIC study 


sorted by top sensitivity and specificity from all study subjects.













Percentage


Lipid name/Lipid name
Sensitivity
Specificity
change





Positive correlation





PS O-16:0/18:2-alkenyl/Total PS O
98.24561
63.92405
 26.07240


PS O-16:1/18:2-alkyl/Total PS O
98.24561
63.92405
 26.07240


PS O-18:2/16:0-alkenyl/Total PS O
85.45455
70.66667
 28.78267


LacCer(d18:1/20:0)/PC 16:0/20:3
80.39216
65.11628
 57.24752


CE 18:2/CE 18:3
79.31034
60.37736
 26.34366


LacCer(d18:1/20:0)/Total LPC
78.84615
61.24031
 53.68111


LacCer(d18:1/20:0)/Total PC
78.84615
64.34109
 55.30769


LacCer(d18:1/20:0)/PC 18:0/20:3
77.08333
60.15625
 56.31253


Cer(d18:1/24:1)/LPC 18:2
75.92593
63.69427
 48.79017


CE 16:0/CE 18:3
75.86207
60.37736
 25.64615


LacCer(d18:1/20:0)/Total SM
75.47170
60.60606
 29.12486


Cer(d18:1/16:0)/Total PC
75.00000
61.14650
 32.91697


Cer(d18:1/18:0)/PC 16:0/20:4
75.00000
63.05732
 65.70609


Cer(d18:1/18:0)/Total LPC
75.00000
61.14650
 45.10211


Cer(d18:1/24:1)/SM (d18:1/24:0)
75.00000
62.42038
 36.77357


(d18:1/23:1-OH)





Cer(d18:1/24:1)/Total LPC
75.00000
63.05732
 37.58734


Cer(d18:1/24:1)/Total PC
75.00000
60.50955
 38.66127


LacCer(d18:1/18:0)/Total LPC
75.00000
60.13072
 55.13308


LacCer(d18:1/20:0)/SM
75.00000
62.60163
 36.30844


(d18:1/17:1-OH)





LacCer(d18:1/20:0)/SM
75.00000
62.60163
 36.30844


(d18:1/18:0)





Total Cer/Total PC
75.00000
63.92405
 28.86380


LacCer(d18:1/24:0)/Total LPC
74.54545
61.94030
 36.27249


LacCer(d18:1/20:0)/PC 18:2/18:2
74.46809
60.31746
 47.81762


PC 16:0/16:0/Total PC
73.68421
65.18987
 26.13285


Cer(d18:1/22:0)/Total PC
73.21429
61.78344
 31.36049


GlcCer(d18:1/20:0)/Total PC
73.21429
60.50955
 36.81380


LacCer(d18:1/16:0)/Total LPC
73.21429
60.50955
 26.35547


LacCer(d18:1/18:0)/Total PC
73.21429
61.43791
 53.58552


LacCer(d18:1/22:0)/SM (d18:1/14:0)
73.21429
60.00000
 30.07361


(d18:1/13:1-OH)





Total LacCer/Total PC
73.21429
60.50955
 40.03890


LacCer(d18:1/20:0)/PC 16:0/18:1
73.07692
62.01550
 46.35288


LacCer(d18:1/20:0)/PC 18:1/18:2
73.07692
60.46512
 47.48070


Cer(d18:1/26:0)/PC O-40:0
72.72727
62.22222
 9.97951


LacCer(d18:1/22:0)/Total LPC
72.72727
64.92537
 41.61463


LacCer(d18:1/24:1)/Total LPC
72.72727
60.44776
 45.29327


PC O-18:0/18:2-alkyl/PC O-36:5
72.72727
61.18421
 25.99402


PC O-32:0 (KDdiA-PC)/PC O-38:5
72.54902
61.36364
 34.00112


Cer(d18:1/22:0)/LPC 18:2
72.22222
61.78344
 41.72749


LacCer(d18:1/22:0)/PC 16:0/20:3
72.22222
66.41791
 51.38070


LacCer(d18:1/24:0)/PC 16:0/20:3
72.22222
61.19403
 39.48118


Cer(d18:1/24:1)/Total CE
71.92982
61.39241
 31.32927


PC 16:0/16:0/PC 16:0/20:4
71.92982
61.39241
 38.61464


PC 16:0/18:2/Total PC
71.92982
60.75949
 8.88941


Total LacCer/Total PC O
71.92982
60.24845
 17.49430


Cer(d18:1/16:0)/LPC 18:1
71.42857
63.69427
 30.55279


Cer(d18:1/18:0)/LPC 16:0
71.42857
61.14650
 40.41256


Cer(d18:1/18:0)/LPC 18:1
71.42857
61.78344
 45.33865


GlcCer(d18:1/20:0)/PC 16:0/20:4
71.42857
60.50955
 46.04915


LacCer(d18:1/24:1)/Total PC O
71.42857
60.14493
 21.09716


CE 19:1/Cer(d18:0/22:0)
71.15385
60.14493
 39.21301


Negative correlation





CE 18:3/LacCer(d18:1/20:0)
84.90566
62.87879
−44.30634


CE 18:3/Cer(d18:1/24:1)
80.70175
60.12658
−35.95912


Cer(d18:0/24:0)/Total Cer
80.70175
60.12270
−31.45318


LPC 18:2/LacCer(d18:1/20:0)
80.00000
64.34109
−41.66044


CE 18:3/Total CE
79.31034
61.00629
−20.28821


GlcCer(d18:1/26:0)/
79.16667
61.40351
−23.71130


LacCer(d18:1/20:0)





CE 16:1/Cer(d18:1/20:0)
78.94737
61.39241
−33.84979


CE 16:1/Cer(d18:1/24:1)
77.19298
61.39241
−36.17234


CE 16:1/LacCer(d18:1/18:0)
77.19298
60.38961
−37.93756


CE 18:3/Cer(d18:1/20:0)
77.19298
60.75949
−33.62034


LPC 18:1/LacCer(d18:1/20:0)
76.92308
63.56589
−34.40451


CE 18:3/LacCer(d18:1/22:0)
76.78571
61.02941
−37.15258


CE 16:1/CE 19:1
75.47170
60.86957
−66.77151


CE 16:1/Cer(d18:1/18:0)
75.43860
61.39241
−39.42716


CE 16:1/LacCer(d18:1/16:0)
75.43860
60.12658
−30.53235


CE 16:1/Total LacCer
75.43860
60.75949
−34.87060


CE 18:3/PC 16:0/16:0
75.43860
60.00000
−30.47160


CE 18:3/PS O-16:0/18:2-alkenyl
75.43860
60.64516
−38.50943


CE 18:3/PS O-16:1/18:2-alkyl
75.43860
60.64516
−38.50943


CE 18:3/Total LacCer
75.43860
63.29114
−34.13080


Cer(d18:0/24:0)/Cer(d18:1/22:0)
75.43860
66.25767
−32.40867


CE 16:1/LacCer(d18:1/22:0)
75.00000
61.02941
−37.29505


CE 16:1/LacCer(d18:1/24:0)
75.00000
60.29412
−36.84318


GlcCer(d18:1/26:0)/
75.00000
60.86957
−20.64622


LacCer(d18:1/22:0)





LPC 16:0/LacCer(d18:1/20:0)
75.00000
61.24031
−35.61882


Total LPC/Total LacCer
75.00000
61.78344
−26.08820


LPC 16:0/LacCer(d18:1/24:1)
74.54545
60.44776
−24.61533


CE 17:1/GlcCer(d18:1/24:1)
74.07407
60.25641
−24.94421


CE 16:1/GlcCer(d18:1/18:0)
73.68421
60.12658
−36.48698


CE 16:1/GlcCer(d18:1/20:0)
73.68421
60.75949
−38.30453


CE 16:1/PC 16:0/16:0
73.68421
60.64516
−29.10430


CE 18:1/Total LacCer
73.68421
60.12658
−20.87773


CE 18:3/Cer(d18:1/16:0)
73.68421
67.08861
−32.55987


CE 18:3/Cer(d18:1/22:0)
73.68421
61.39241
−30.65953


CE 20:3/Cer(d18:1/24:1)
73.68421
61.39241
−28.12167


Cer(d18:0/22:0)/Cer(d18:1/24:1)
73.68421
61.96319
−32.18870


Cer(d18:0/24:0)/Cer(d18:1/18:0)
73.68421
63.80368
−40.29603


Cer(d18:0/24:0)/Cer(d18:1/24:1)
73.68421
65.64417
−36.12881


Cer(d18:0/24:0)/GlcCer(d18:1/20:0)
73.68421
60.73620
−40.34621


CE 16:1/LacCer(d18:1/20:0)
73.58491
62.12121
−39.47654


CE 20:3/LacCer(d18:1/20:0)
73.58491
62.12121
−35.94789


CE 18:3/LacCer(d18:1/24:0)
73.21429
62.50000
−35.91095


CE 18:3/PS O-16:0/18:1-alkenyl 
73.21429
60.64516
−34.16720


(PS O-16:1/18:1-alkyl)





Cer(d18:0/22:0)/LacCer(d18:1/24:0)
73.21429
60.00000
−31.24173


Cer(d18:0/24:0)/LacCer(d18:1/24:0)
73.21429
62.85714
−37.02872


LPC 16:0/Total LacCer
73.21429
61.78344
−24.37341


SM(d18:1/24:0) 
73.21429
61.14650
−13.97590


(d18:1/23:1-OH)/Total Cer





Cer(d18:0/24:0)/SM (d18:1/17:0) 
72.91667
60.00000
−44.87505


(d18:1/16:1-OH)





LPC 16:0/LacCer(d18:1/22:0)
72.72727
67.16418
−27.47521


CE 17:1/LacCer(d18:1/18:0)
72.22222
60.52632
−27.90709










6b) Table of significant lipid to lipid ratios in LURIC study 


sorted by top sensitivity and specificity from subjects not undergoing 


statin treatment.













Percentage


Lipid name/Lipid name
Sensitivity
Specificity
change





Positive correlation





PS O-16:0/18:2-alkenyl/Total PS O
97.82609
62.50000
 26.42621


PS O-16:1/18:2-alkyl/Total PS O
97.82609
62.50000
 26.42621


PS O-18:2/16:0-alkenyl/Total PS O
88.63636
72.64151
 29.59068


Cer(d18:1/24:1)/LPC 18:2
81.39535
62.16216
 51.68188


CE 18:2/CE 18:3
80.85106
60.52632
 24.84398


Cer(d18:1/18:0)/PC 16:0/20:4
80.00000
62.16216
 73.07370


Cer(d18:1/24:1)/PC 16:0/18:2
80.00000
60.36036
 27.45405


Cer(d18:1/24:1)/Total LPC
80.00000
62.16216
 38.68474


Cer(d18:1/24:1)/Total PC
80.00000
62.16216
 43.04080


LacCer(d18:1/20:0)/PC 16:0/20:4
79.06977
68.42105
 82.44735


LacCer(d18:1/20:0)/PC 16:0/20:3
78.57143
64.21053
 56.97265


Cer(d18:1/16:0)/Total PC
77.77778
60.36036
 34.07762


Cer(d18:1/18:0)/LPC 18:1
77.77778
60.36036
 45.52166


Cer(d18:1/24:1)/SM (dl 8:1/24:0)
77.77778
60.17699
 32.27927


(d18:1/23:1-OH)





Cer(d18:1/24:1)/PC 0-40:3
77.50000
60.43956
 20.46959


LacCer(d18:1/20:0)/Total LPC
76.74419
60.00000
 49.56627


LacCer(d18:1/20:0)/Total PC
76.74419
63.15789
 55.68613


Cer(d18:1/18:0)/Total CE
76.08696
60.17699
 49.43112


Cer(d18:1/20:0)/SM (d18:1/14:0) 
76.08696
60.71429
 28.98221


(d18:1/13:1-OH)





Cer(d18:1/20:0)/Total PC O
76.08696
60.86957
 29.01076


Cer(d18:1/24:1)/Total CE
76.08696
60.17699
 39.34990


PS O-16:0/18:2-alkenyl/Total PC O
76.08696
61.60714
 20.69456


PS O-16:1/18:2-alkyl/Total PC O
76.08696
61.60714
 20.69456


Cer(d18:1/16:0)/LPC 18:1
75.55556
60.36036
 26.36925


Cer(d18:1/18:0)/Total LPC
75.55556
63.06306
 44.40414


Cer(d18:1/22:0)/Total PC
75.55556
63.06306
 34.70483


Total Cer/Total PC
75.55556
61.60714
 29.94912


LacCer(d18:1/20:0)/Total SM
75.00000
61.61616
 27.48798


LacCer(d18:1/24:0)/Total LPC
75.00000
67.01031
 34.46432


PS O-18:2/16:0-alkenyl/Total PC O
75.00000
60.37736
 21.65448


CE 16:0/CE 18:3
74.46809
61.40351
 25.51627


CE 18:0/CE 18:3
74.46809
65.13761
 29.77017


Cer(d18:1/22:0)/LPC 18:2
74.41860
60.36036
 44.40272


LacCer(d18:1/20:0)/PC 18:1/18:2
74.41860
64.21053
 48.43279


LacCer(d18:1/20:0)/SM 
74.41860
60.86957
 34.96305


(d18:1/17:1-OH)





LacCer(d18:1/20:0)/SM
74.41860
60.86957
 34.96305


(d18:1/18:0)





LacCer(d18:1/20:0)/PC 18:0/20:3
74.35897
62.76596
 55.40327


Cer(d18:1/18:0)/SM (d18:1/14:0)
73.91304
74.10714
 34.95249


(d18:1/13:1-OH)





Cer(d18:1/18:0)/SM (d18:1/17:2-OH)
73.91304
60.00000
 25.13954


Cer(d18:1/18:0)/SM (d18:1/18:1)
73.91304
60.00000
 25.13954


Cer(d18:1/24:1)/SM (d18:1/23:0)
73.91304
60.71429
 29.95139


(d18:1/22:1-OH)





PC 16:0/16:0/PC 16:0/20:4
73.91304
60.71429
 42.51274


Cer(d18:1/20:0)/Total LPC
73.33333
61.26126
 36.63159


Cer(d18:1/24:1)/PC 16:0/20:4
73.33333
62.16216
 59.87613


Cer(d18:1/24:1)/PC 18:0/18:2
73.33333
62.16216
 29.37710


Cer(d18:1/24:1)/PC 18:1/18:2
73.33333
61.26126
 36.88389


Cer(d18:1/24:1)/SM (d18:1/17:1-OH)
73.33333
64.42308
 23.41916


Cer(d18:1/24:1)/SM (d18:1/18:0)
73.33333
64.42308
 23.41916


LacCer(d18:1/20:0)/PC 18:0/18:1
73.17073
62.10526
 50.18761


LacCer(d18:1/20:0)/Total CE
72.72727
62.62626
 53.71776


Negative correlation





DAG 16:0/18:1/Total DAG
88.88889
61.16505
−17.30528


CE 18:3/LacCer(d18:1/20:0)
86.36364
66.66667
−45.65238


LPC 18:2/LacCer(d18:1/20:0)
82.92683
62.10526
−40.92501


CE 18:3/Cer(d18:1/24:1)
82.60870
61.06195
−37.97573


GlcCer(d18:1/26:0)/
80.48780
64.77273
−26.83109


LacCer(d18:1/20:0)





CE 14:0/Total DAG
80.00000
61.16505
−39.38279


CE 18:3/Total CE
78.72340
62.28070
−19.58872


PC 18:0/20:3/PS O-16:0/18:2-alkenyl
78.57143
60.36036
−29.82522


PC 18:0/20:3/PS O-16:1/18:2-alkyl
78.57143
60.36036
−29.82522


CE 16:1/Cer(d18:1/24:1)
78.26087
61.06195
−37.53854


CE 18:3/PS O-16:0/18:2-alkenyl
78.26087
60.90909
−40.72437


CE 18:3/PS O-16:1/18:2-alkyl
78.26087
60.90909
−40.72437


CE 18:3/Total LacCer
78.26087
60.17699
−32.64187


Cer(d18:0/24:0)/Total Cer
78.26087
62.06897
−30.77598


GlcCer(d18:1/26:0)/
78.04878
61.79775
−21.27116


LacCer(d18:1/22:0)





CE 18:3/LacCer(d18:1/22:0)
77.77778
63.63636
−34.76187


CE 20:3/LacCer(d18:1/20:0)
77.27273
60.60606
−36.58675


PC O-40:3/PS O-18:2/16:0-alkenyl
76.92308
62.79070
−28.59638


LPC 18:1/LacCer(d18:1/20:0)
76.74419
60.00000
−33.84809


CE 16:0/Cer(d18:1/24:1)
76.08696
61.06195
−23.25713


CE 16:1/Cer(d18:1/18:0)
76.08696
63.71681
−41.11023


CE 16:1/Cer(d18:1/20:0)
76.08696
66.37168
−34.98271


CE 16:1/GlcCer(d18:1/24:1)
76.08696
60.17699
−39.81218


CE 16:1/LacCer(d18:1/18:0)
76.08696
60.36036
−37.28535


CE 18:3/Cer(d18:1/20:0)
76.08696
63.71681
−34.93858


CE 18:3/Cer(d18:1/22:0)
76.08696
61.94690
−30.87603


Cer(d18:0/22:0)/Cer(d18:1/24:1)
76.08696
61.20690
−34.08632


Cer(d18:0/22:0)/Total GlcCer
76.08696
60.34483
−28.95910


Cer(d18:0/24:0)/Cer(d18:1/20:0)
76.08696
60.34483
−36.42412


PC 18:0/20:3/PS O-18:2/16:0-alkenyl
75.60976
60.95238
−29.83126


CE 16:1/LacCer(d18:1/24:0)
75.55556
60.60606
−36.83943


CE 18:3/LacCer(d18:1/24:0)
75.55556
60.60606
−37.84417


PC 18:1/18:2/Total Cer
75.55556
60.71429
−19.58862


SM (d18:1/23:0) 
75.55556
63.10680
−37.45206


(d18:1/22:1-OH)/Total DAG





SM (d18:1/24:0) 
75.55556
61.94690
−11.07113


(d18:1/23:1-OH)/Total Cer





Total CE/Total DA
75.55556
66.01942
−31.35698


Total LPC/Total LacCer
75.55556
69.36937
−23.44706


CE 16:1/CE 19:1
75.00000
60.82474
−56.18741


CE 20:5/LacCer(d18:1/20:0)
75.00000
61.61616
−37.54401


LPC 16:0/LacCer(d18:1/24:0)
75.00000
60.82474
−25.29458


CE 15:0/Cer(d18:1/20:0)
74.41860
60.57692
−21.94830


CE 16:0/Cer(d18:1/18:0)
73.91304
61.06195
−26.42362


CE 16:1/Total LacCer
73.91304
63.71681
−33.50629


CE 18:2/Cer(d18:1/20:0)
73.91304
61.94690
−19.85408


CE 18:3/Cer(d18:1/24:0)
73.91304
61.40351
−28.00256


CE 18:3/GlcCer(d18:1/20:0)
73.91304
65.48673
−38.77502


CE 18:3/PC 16:0/16:0
73.91304
63.63636
−31.16928


CE 20:3/Cer(d18:1/24:1)
73.91304
61.06195
−30.78707


CE 20:4/GlcCer(d18:1/20:0)
73.91304
62.83186
−30.95570


CE 20:4/GlcCer(d18:1/24:1)
73.91304
61.06195
−28.82844
















TABLE 7





Table of significant lipid to clinical ratios in LURIC study 


sorted by top sensitivity and specificity. Table 7a shows 


significant lipids from all study subjects. The significant 


lipids from subjects not undergoing statin treatment are listed 


in table 7b.







7a) Table of significant lipid to clinical ratios in LURIC study 


sorted by top sensitivity and specificity from all study subjects.













Percentage


Lipid name/Clinical measurement
Sensitivity
Specificity
change





Positive correlation





LacCer(d18:1/20:0)/
75.47170
61.48148
 43.04967


apolipoprotein A-I





Cer(d18:1/24:1)/total cholesterol
70.17544
60.73620
 21.94547


Cer(d18:1/24:1)/triglycerides
70.17544
60.12270
 32.27486


LacCer(d18:1/20:0)/HDL cholesterol
69.81132
62.22222
 39.32664


LacCer(d18:1/20:0)/apoA1/apoB
67.92453
61.48148
 32.36387


LacCer(d18:1/22:0)/HDL cholesterol
67.85714
60.00000
 29.19905


Cer(d18:1/16:0)/triglycerides
66.66667
61.34969
 28.51323


Cer(d18:1/22:0)/apolipoprotein B
66.66667
60.12270
 14.20773


Cer(d18:1/24:1)/apolipoprotein B
66.66667
62.57669
 18.05222


Total Cer/total cholesterol
66.66667
65.85366
 15.12131


LacCer(d18:1/20:0)/LDL-c/HDL-c
66.03774
60.74074
 23.54484


LacCer(d18:1/20:0)/total cholesterol
66.03774
62.96296
 41.46919


LacCer(d18:1/20:0)/total-c/HDL-c
66.03774
60.00000
 33.48550


Cer(d18:1/18:0)/triglycerides
64.91228
60.73620
 40.06643


Cer(d18:1/22:0)/total cholesterol
64.91228
61.34969
 17.20335


Cer(d18:1/24:1)/total-c/HDL-c
64.91228
61.34969
 13.34657


GlcCer(d18:1/24:0)/total cholesterol
64.91228
61.96319
 17.04608


LacCer(d18:1/18:0)/
64.91228
60.37736
 41.61371


apolipoprotein A-I





LacCer(d18:1/18:0)/total cholesterol
64.91228
61.00629
 38.87517


Total LacCer/total cholesterol
64.91228
65.03067
 23.69633


LacCer(d18:1/22:0)/apoA1/apoB
64.28571
62.85714
 24.17423


LacCer(d18:1/22:0)/
64.28571
62.85714
 32.96039


apolipoprotein A-I





LacCer(d18:1/22:0)/total cholesterol
64.28571
60.00000
 32.24669


LacCer(d18:1/24:0)/apoA1/apoB
64.28571
62.14286
 15.23336


LacCer(d18:1/20:0)/LDL cholesterol
64.15094
61.48148
 31.95659


LacCer(d18:1/20:0)/apolipoprotein B
64.15094
60.00000
 37.03057


PS O-18:2/16:0-alkenyl/triglycerides
63.63636
63.33333
 99.88127


Cer(d18:1/20:0)/apolipoprotein B
63.15789
60.12270
 20.79615


Cer(d18:1/20:0)/total cholesterol
63.15789
61.96319
 25.23403


Cer(d18:1/22:0)/LDL cholesterol
63.15789
60.12270
 11.30545


Cer(d18:1/24:1)/LDL cholesterol
63.15789
60.12270
 14.20759


PS O-16:0/18:2-alkenyl/triglycerides
63.15789
63.29114
 97.78843


PS O-16:1/18:2-alkyl/triglycerides
63.15789
63.29114
 97.78843


Total GlcCer/total cholesterol
63.15789
60.73620
 17.56924


Total LacCer/apolipoprotein A-I
63.15789
61.34969
 25.87122


Total LacCer/apolipoprotein B
63.15789
61.96319
 20.63257


LacCer(d18:1/24:1)/triglycerides
62.50000
62.14286
 60.79934


PS O-16:0/18:1-alkenyl (PS O-16:1/
62.50000
61.39241
 65.88734


18:1-alkyl)/triglycerides





Cer(d18:1/20:0)/triglycerides
61.40351
60.73620
 34.46408


Cer(d18:1/22:0)/triglycerides
61.40351
63.19018
 28.23304


Cer(d18:1/24:1)/apolipoprotein A-I
61.40351
65.03067
 27.12684


LacCer(d18:1/16:0)/triglycerides
61.40351
64.41718
 25.01312


LacCer(d18:1/18:0)/HDL cholesterol
61.40351
60.37736
 38.04132


LacCer(d18:1/18:0)/LDL cholesterol
61.40351
64.15094
 28.50180


LacCer(d18:1/18:0)/apolipoprotein B
61.40351
61.00629
 33.69302


LacCer(d18:1/18:0)/triglycerides
61.40351
62.26415
 52.60972


Total Cer/apolipoprotein A-I
61.40351
63.41463
 19.09944


Total GlcCer/apolipoprotein B
61.40351
60.12270
 14.92180


Total LacCer/triglycerid es
61.40351
62.57669
 38.52220


LacCer(d18:1/22:0)/apolipoprotein B
60.71429
65.00000
 29.83861


Negative correlation





CE 18:3/total-c/HDL-c
67.24138
62.89308
−27.28733


LPC 18:2/apolipoprotein B
65.45455
60.12658
−16.19596


Total PC/apolipoprotein B
64.91228
61.39241
−12.91145


Total PC/total cholesterol
64.91228
60.75949
−10.60231


CE 16:1/HDL cholesterol
63.79310
61.63522
−22.01197


CE 18:3/HDL cholesterol
63.79310
62.26415
−24.04060


CE 18:3/LDL cholesterol
63.79310
62.26415
−27.21759


LPC 18:2/LDL-c/HDL-c
63.63636
61.39241
−21.70580


Total PC/total-c/HDL-c
63.15789
60.12658
−16.13350


PC 16:0/20:3/HDL cholesterol
62.50000
62.65823
−11.45558


PC 16:0/20:3/apolipoprotein B
62.50000
60.12658
−15.11848


CE 16:1/apolipoprotein B
62.06897
62.26415
−26.99397


CE 16:1/total cholesterol
62.06897
60.37736
−24.28770


CE 18:3/LDL-c/HDL-c
62.06897
62.89308
−31.33346


CE 18:3/apolipoprotein A-I
62.06897
60.37736
−23.09816


LPC 18:2/HDL cholesterol
61.81818
60.75949
−16.07209


LPC 18:2/apoA1/apoB
61.81818
63.92405
−19.23316


PC 16:0/20:4/LDL cholesterol
61.40351
60.12658
−15.98292


PC 16:0/20:4/apolipoprotein A-I
61.40351
60.75949
−12.23661


PC 16:0/20:4/total cholesterol
61.40351
61.39241
−13.37554


Total PC/LDL-c/HDL-c
61.40351
62.65823
−21.24583


PC 16:0/20:3/total-c/HDL-c
60.71429
61.39241
−19.07121


PC 18:0/20:4/apoA1/apoB
60.71429
62.02532
−13.36540


CE 18:3/apoA1/apoB
60.34483
60.37736
−27.55825


CE 18:3/apolipoprotein B
60.34483
61.00629
−25.31907


CE 20:5/HDL cholesterol
60.34483
60.37736
−23.45029


CE 20:5/LDL cholesterol
60.34483
60.37736
−25.93288


LPC 18:2/LDL cholesterol
60.00000
62.02532
−17.26480


LPC 18:2/total cholesterol
60.00000
62.65823
−14.92954










7b) Table of significant lipid to clinical ratios in LURIC study


sorted by top sensitivity and specificity from subjects not undergoing


statin treatment.













Percentage


Lipid name/Clinical measurement
Sensitivity
Specificity
change





Positive correlation





Cer(d18:1/24:1)/apolipoprotein B
82.60870
62.06897
 18.05222


Cer(d18:1/24:1)/total cholesterol
80.43478
61.20690
 21.94547


LacCer(d18:1/20:0)/
77.27273
67.00000
 43.04967


apolipoprotein A-1





Cer(d18:1/22:0)/LDL cholesterol
73.91304
60.34483
 11.30545


Total Cer/LDL cholesterol
73.91304
62.39316
 8.25598


Total Cer/total cholesterol
73.91304
70.08547
 15.12131


LacCer(d18:1/20:0)/HDL cholesterol
72.72727
61.00000
 39.32664


LacCer(d18:1/20:0)/apoA1/apoB
72.72727
60.00000
 32.36387


Cer(d18:1/22:0)/total cholesterol
71.73913
61.20690
 17.20335


Cer(d18:1/24:1)/LDL cholesterol
71.73913
61.20690
 14.20759


Cer(d18:1/24:1)/total-c/HDL-c
71.73913
60.34483
 13.34657


Total Cer/apolipoprotein A-I
71.73913
61.53846
 19.09944


Total Cer/apolipoprotein B
71.73913
61.53846
 12.34421


LacCer(d18:1/20:0)/LDL cholesterol
70.45455
61.00000
 31.95659


LacCer(d18:1/20:0)/LDL-c/HDL-c
70.45455
61.00000
 23.54484


Cer(d18:1/20:0)/LDL cholesterol
69.56522
60.34483
 18.51094


Cer(d18:1/20:0)/apolipoprotein B
69.56522
68.10345
 20.79615


Cer(d18:1/20:0)/total cholesterol
69.56522
71.55172
 25.23403


Cer(d18:1/20:0)/total-c/HDL-c
69.56522
60.34483
 14.96816


Cer(d18:1/22:0)/apolipoprotein B
69.56522
65.51724
 14.20773


Cer(d18:1/24:0)/total cholesterol
69.56522
60.68376
 11.30123


Cer(d18:1/24:1)/LDL-c/HDL-c
69.56522
62.93103
 5.34922


Cer(d18:1/24:1)/apolipoprotein A-I
69.56522
60.34483
 27.12684


Cer(d18:1/24:1)/triglycerides
69.56522
63.79310
 32.27486


GlcCer(d18:1/24:1)/apolipoprotein B
69.56522
61.20690
 17.03707


Total DAG/apolipoprotein A-I
68.88889
60.19417
 15.46034


LacCer(d18:1/20:0)/total cholesterol
68.18182
61.00000
 41.46919


PC O-34:1/apolipoprotein B
68.18182
60.00000
 22.73127


Cer(d18:1/16:0)/LDL cholesterol
67.39130
62.06897
 10.42412


Cer(d18:1/18:0)/total-c/HDL-c
67.39130
61.20690
 21.75165


Cer(d18:1/18:0)/triglycerides
67.39130
64.65517
 40.06643


Cer(d18:1/24:0)/LDL cholesterol
67.39130
62.39316
 4.69599


Cer(d18:1/24:0)/apolipoprotein A-I
67.39130
61.53846
 14.67208


GlcCer(d18:1/20:0)/total cholesterol
67.39130
60.34483
 20.60005


Total GlcCer/apolipoprotein B
67.39130
60.34483
 14.92180


Total LacCer/total cholesterol
67.39130
62.93103
 23.69633


LacCer(d18:1/22:0)/apoA1/apoB
66.66667
61.76471
 24.17423


LacCer(d18:1/22:0)/
66.66667
60.78431
 32.96039


apolipoprotein A-I





LacCer(d18:1/20:0)/apolipoprotein B
65.90909
61.00000
 37.03057


LacCer(d18:1/20:0)/total-c/HDL-c
65.90909
62.00000
 33.48550


Cer(d18:1/16:0)/triglycerides
65.21739
62.06897
 28.51323


Cer(d18:1/22:0)/apolipoprotein A-I
65.21739
66.37931
 21.04730


Cer(d18:1/24:0)/apolipoprotein B
65.21739
62.39316
 9.26738


GlcCer(d18:1/20:0)/apolipoprotein B
65.21739
60.34483
 17.00627


GlcCer(d18:1/26:1)/
65.21739
61.60714
 21.90520


apolipoprotein A-I





LacCer(d18:1/16:0)/triglycerides
65.21739
60.34483
 25.01312


LacCer(d18:1/18:0)/apolipoprotein B
65.21739
61.40351
 33.69302


LacCer(d18:1/18:0)/total cholesterol
65.21739
62.28070
 38.87517


PS O-16:0/18:2-alkenyl/triglycerides
65.21739
61.60714
 97.78843


PS O-16:1/18:2-alkyl/triglycerides
65.21739
61.60714
 97.78843


Negative correlation





CE 18:3/total-c/HDL-c
68.08511
62.28070
−27.28733


CE 18:3/LDL-c/HDL-c
63.82979
60.52632
−31.33346


CE 18:3/apoA1/apoB
61.70213
64.03509
−27.55825


CE 20:5/triglycerides
61.70213
60.52632
−15.26342


PC O-38:6/apolipoprotein A-I
60.86957
62.03704
 −3.87776


Total LPC/apoA1/apoB
60.86957
60.71429
−14.81039


Total PC/apolipoprotein A-I
60.86957
62.50000
 −8.50761


PC 18:0/20:4/apoA1/apoB
60.00000
64.28571
−13.36540









The preferred lipid molecules of the invention were selected as follows: a) it was likely to be biologically meaningful, b) it preferably belongs to a family of lipids that are behaving similarly, c) it is expressed in meaningful & measurable concentrations, d) it has very significant p-value or good AUC-value (>0.65) and for most also the %-change is substantial (>20%), and e) it appeared significant in different tests. About 15 lipids or lipid ratios, each with either a positive or negative CVD correlation, were selected based on the highest p-values and best sensitivity and specificity subjectively ensuring the balanced representation of all lipid classes. Sensitivity and specificity thresholds were annotated in cases where the threshold of 60 and 70 were reached, respectively. The preferred embodiment lipids, lipid-lipid ratios and lipid-clinical ratios are presented in tables 8-11.







TABLE 8







The preferred embodiment lipids selected from significant lipids detected


from LURIC sample set.













Percentage 




Lipid name
p-value
change
Sensitivity
Specificity





Positive correlation






Cer(d18:1/20:0)
0.00004
 28.00357




LacCer(d18:1/20:0)
0.00010
 32.91154
70.45455
60.00000


Cer(d18:1/24:1)
0.00039
 23.37606
69.56522
60.34483


LacCer(d18:1/24:1)
0.00199
 29.83279




PS O-18:2/16:0-alkenyl
0.00432
 33.81177




PS O-16:1/18:2-alkyl
0.00590
 32.17190




Total Cer
0.00932
 15.44601




Total LacCer
0.01105
 16.00541




GlcCer(d18:1/24:1)

 12.75066
60.86957
62.06897


LacCer(d18:1/22:0)
0.00046
 22.75541




Cer(d18:1/18:0)
0.00009
 34.32550




Negative correlation






Total PC
0.00921
−12.44220




PC 16:0/20:4
0.02256
−14.96966




Cer(d18:0/24:0)
0.03376
−21.87004




Total LPC
0.03443
−12.91576




CE 14:0
0.01090
−21.01258




CE 20:3
0.02157
−16.03606




CE 17:1
0.02204
−15.93952




PC 16:0/20:3

−18.78110
64.44444
60.71429


LPC 18:1

−14.45827
63.04348
60.71429


PC 18:0/20:3
0.00726
−17.20664




PC 18:0/18:1
0.00765
−18.18002




Cer(d18:0/22:0)
0.01158
−22.37263
















TABLE 9







Preferred embodiments from significant lipid to lipid ratios detected 


from LURIC sample set.













Percentage




Lipid name/Lipid name
p-value
change
Sensitivity
Specificity














Positive correlation






GlcCer(d18:1/26:1)/Total CE
0.000000
35.356592




Cer(d18:1/24:1)/Total PC
0.000000
43.040800




Cer(d18:1/24:1)/PC 16:0/20:4
0.000000
59.876132




Cer(d18:1/20:0)/PC 16:0/20:4
0.000000
64.045788




LacCer(d18:1/20:0)/PC 16:0/20:3
0.000008
56.972652
80.39216
65.11628


Total Cer/Total PC
0.000011
29.949121




Total LacCer/Total PC
0.000012
37.815865




LacCer(d18:1/20:0)/PC 18:1/18:2
0.000013
48.432788




PS O-16:0/18:2-alkenyl/Total PS O

26.42621
97.82609
62.50000


Cer(d18:1/18:0)/PC 16:0/20:4

73.07370
80.00000
62.16216


LacCer(d18:1/20:0)/Total LPC

53.68111
78.84615
61.24031


LacCer(d18:1/20:0)/PC 16:0/20:4

82.44735
79.06977
68.42105


Negative correlation






Cer(d18:0/24:0)/Cer(d18:1/24:1)
0.000003
−36.760563




Cer(d18:0/22:0)/Cer(d18:1/24:1)
0.000011
−34.086320




DAG 16:0/18:1/Total DAG

−17.30528
88.88889
61.16505


Cer(d18:0/24:0)/Cer(d18:1/22:0)

−32.40867
75.43860
66.25767


Cer(d18:0/24:0)/Total CE
0.0000000
−18.9531828




Cer(d18:0/24:0)/Cer(d18:1/24:1)
0.000003
−36.760563




Cer(d18:0/24:0)/Total Cer
0.0000049
−31.4531758




Cer(d18:0/24:0)/Cer(d18:1/18:0)
0.0000052
−40.2960344




Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl
0.0000059
−40.8329837




Cer(d18:0/24:0)/LacCer(d18:1/24:0)
0.0000067
−37.0287234




Cer(d18:0/22:0)/Cer(d18:1/18:0)
0.0000071
−36.5400374




Cer(d18:0/24:0)/Cer(d18:1/22:0)
0.0000105
−32.4086711




Cer(d18:0/22:0)/Cer(d18:1/20:0)
0.0000165
−31.5266149




Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl
0.000045
−37.930990




Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl
0.000045
−37.930990




GlcCer(d18:1/26:0)/LacCer(d18:1/20:0)

−26.83109
80.48780
64.77273


Total LPC/Total LacCer
0.0000469
−26.08820




GlcCer(d18:1/26:0)/LacCer(d18:1/22:0)

−21.27116
78.04878
61.79775
















TABLE 10







Preferred embodiments from significant lipid to clinical ratios from LURIC


sample set.











Lipid name/Clinical measurement
p-value
Percentage change
Sensitivity
Specificity














Positive correlation






Cer(d18:1/20:0)/apolipoprotein A-I
0.00000
43.22923




Cer(d18:1/24:1)/apolipoprotein A-I
0.00000
36.15491
69.56522
60.34483


LacCer(d18:1/20:0)/apolipoprotein A-I
0.00001
47.52465
77.27273
67.00000


Total Cer/apolipoprotein A-I
0.00016
25.36811




Total LacCer/apolipoprotein A-I
0.00031
28.05057
71.73913
61.53846


Cer(d18:1/18:0)/apolipoprotein A-I
0.00001
49.43693




LacCer(d18:1/22:0)/apolipoprotein A-I
0.00001
35.92744




LacCer(d18:1/20:0)/HDL cholesterol

39.32664
72.72727
61.00000


Cer(d18:1/24:1)/apolipoprotein B

18.05222
82.60870
62.06897


Negative correlation






Cer(d18:0/24:0)/apolipoprotein B
0.03428
−21.10483




Cer(d18:0/24:0)/total cholesterol
0.04309
−19.71324




Cer(d18:0/24:0)/apolipoprotein B
0.00181
−26.25271




PC 16:0/20:4/apolipoprotein B
0.00576
−15.39247




Cer(d18:0/24:0)/apolipoprotein A-I
0.01052
−22.39254
















TABLE 11







Top candidates from each category, if available, are listed. The best


candidates were selected based on following criteria: t-test 


p-value ≦ 0.05 and sensitivity ≧ 60% and specificity ≧ 60%.











Percentage 


Measurement name
p-value
change












Positive correlation




Cer(d18:1/20:0)
0.00004
28.00357


LacCer(d18:1/20:0)
0.00010
32.91154


Cer(d18:1/24:1)
0.00039
23.37606


LacCer(d18:1/24:1)
0.00199
29.83279


LacCer(d18:1/22:0)
0.00046
22.75541


Cer(d18:1/18:0)
0.00009
34.32550


Cer(d18:1/24:1)/PC 16:0/20:4
0.000000
59.876132


LacCer(d18:1/20:0)/PC 16:0/20:3
0.000008
56.972652


PS O-16:0/18:2-alkenyl/Total PS O

26.42621


Cer(d18:1/18:0)/PC 16:0/20:4
0.000000
73.073704


Cer(d18:1/20:0)/apolipoprotein A-I
0.00000
43.22923


Cer(d18:1/24:1)/apolipoprotein A-I
0.00000
36.15491


LacCer(d18:1/20:0)/apolipoprotein A-I
0.00001
47.52465


Total LacCer/apolipoprotein A-I
0.00031
28.05057


LacCer(d18:1/20:0)/HDL cholesterol

39.32664


Cer(d18:1/18:0)/apolipoprotein A-I
0.00001
49.43693


Negative correlation




PC 16:0/20:4
0.02256
−14.96966


Cer(d18:0/24:0)
0.03376
−21.87004


GlcCer(d18:1/26:0)/LacCer(d18:1/20:0)

−26.83109


DAG 16:0/18:1/Total DAG

−17.30528


Cer(d18:0/24:0)/Total Cer

−31.45318


Total LPC/Total LacCer

−26.08820


GlcCer(d18:1/26:0)/LacCer(d18:1/22:0)

−21.27116


Cer(d18:0/24:0)/Cer(d18:1/24:1)
0.0000004
−36.1288066


Cer(d18:0/24:0)/Cer(d18:1/18:0)
0.0000052
−40.2960344


Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl
0.0000059
−40.8329837


Cer(d18:0/24:0)/Cer(d18:1/22:0)
0.0000105
−32.4086711


Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl
0.000045
−37.930990


Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl
0.000045
−37.930990


Cer(d18:0/24:0)/LacCer(d18:1/24:0)
0.0000067
−37.0287234









Lipidomic analysis proved to be efficient in identifying novel plasma biomarkers for CVD complications.
Molecular lipid to molecular lipid ratio could be an important indicator of cellular lipid metabolism including e.g., enzyme activities in the lipid metabolism pathways. Thus, these ratios may provide more information as the absolute plasma concentrations of the molecular lipids alone. As the absolute molecular lipid plasma concentration differences in general between healthy individuals and atherosclerotic patients seem to be between 30-70%, it might be reasonable to calculate and use different ratios instead of absolute concentrations only. As lipoprotein particles (e.g. LDL, HDL, and VLDL) are serving as carriers for most of the lipids in the blood stream it is appropriate to relate molecular lipid concentrations to lipoprotein data. Thus, the molecular lipid to HDL-cholesterol, LDL-cholesterol, apolipoprotein A-I and apolipoprotein B ratios were calculated. In fact, a number of ratios between the concentrations of different molecular lipids outperformed absolute plasma concentrations as disease biomarkers in CVD patients.
As the detected lipids are carried in the lipoprotein particles (LDL, VLDL and HDL) it is obvious that the corresponding lipoprotein fraction concentrations will even improve the prediction potential of molecular lipids from the results of the present study in total serum/plasma samples.
The lipid lowering drug efficiency measurements have so far been based on LDL-C and HDL-C assays. As the inventors have herein observed more potential biomarkers that predict the development of high-risk CVD complications better than these classical analyses, future drug efficiency profiling should be based on new sensitive and specific biomarkers that are more directly related to the risk of severe CVD-related complications rather than to LDL-C.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein both in the Examples in the body of the entire patent description. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

## Claims

1. A method for determining whether a subject is at risk to develop one or more Cardiovascular Disease (CVD) complications, comprising:
(a) assaying a sample from said subject to determine one or more lipid-lipid concentration ratio(s), wherein (an) increased or decreased lipid-lipid concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications,
wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0)/LPC 18:1,
Cer(d18:1/18:0)/LPC 16:0,
Cer(d18:1/18:0)/LPC 18:1,
Cer(d18:1/18:0)/Total LPC,
Cer(d18:1/20:0)/Total LPC,
Cer(d18:1/22:0)/LPC 18:2,
Cer(d18:1/24:1)/LPC 18:1,
Cer(d18:1/24:1)/LPC 18:2,
Cer(d18:1/24:1)/Total LPC,
Cer(d18:1/22:0)/PC 16:0/20:4,
Cer(d18:1/18:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/PC 18:0/20:4,
Cer(d18:1/20:0)/PC 18:0/20:4,
Cer(d18:1/22:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/Total CE,
Cer(d18:1/18:0)/Total CE, and
CE 19:1/Cer(d18:0/22:0),
and wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0)/Cer(d18:1/22:0),
Cer(d18:0/24:0)/Cer(d18:1/16:0),
Cer(d18:0/24:0)/Cer(d18:1/20:0),
Cer(d18:0/22:0)/Total CE,
Cer(d18:0/24:1)/Total CE,
CE 18:3/Cer(d18:1/24:1),
CE 20:3/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/20:0),
CE 14:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/16:0),
CE 18:3/Cer(d18:1/18:0),
CE 18:3/Cer(d18:1/22:0),
CE 17:1/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/24:0),
CE 20:4/Cer(d18:1/24:1),
CE 20:4/Cer(d18:1/18:0),
CE 18:2/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/18:0),
CE 17:1/Cer(d18:1/18:0),
CE 14:0/Cer(d18:1/18:0),
CE 16:1/Cer(d18:1/20:0),
CE 16:1/Cer(d18:1/24:1),
CE 16:1/Cer(d18:1/18:0),
CE 15:0/Cer(d18:1/20:0),
CE 18:2/Cer(d18:1/20:0), and
CE 18:3/Total Cer,

or
(b) assaying a sample from said subject to determine one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications,
wherein the one or more lipid-clinical concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0)/apolipoprotein A-I,
Cer(d18:1/16:0)/LDL cholesterol,
Cer(d18:1/16:0)/triglycerides,
Cer(d18:1/18:0)/apolipoprotein B,
Cer(d18:1/18:0)/HDL cholesterol,
Cer(d18:1/18:0)/total-c/HDL-c,
Cer(d18:1/18:0)/total cholesterol,
Cer(d18:1/18:0)/triglycerides,
Cer(d18:1/20:0)/apolipoprotein B,
Cer(d18:1/20:0)/HDL cholesterol,
Cer(d18:1/20:0)/total cholesterol,
Cer(d18:1/20:0)/triglycerides,
Cer(d18:1/20:0)/total-c/HDL-c,
Cer(d18:1/20:0)/LDL cholesterol,
Cer(d18:1/22:0)/apolipoprotein A-I,
Cer(d18:1/22:0)/apolipoprotein B,
Cer(d18:1/22:0)/LDL cholesterol,
Cer(d18:1/22:0)/triglycerides,
Cer(d18:1/22:0)/total cholesterol,
Cer(d18:1/24:0)/apolipoprotein A-I,
Cer(d18:1/24:0)/apolipoprotein B,
Cer(d18:1/24:0)/LDL cholesterol,
Cer(d18:1/24:0)/total cholesterol,
Cer(d18:1/24:1)/HDL cholesterol,
Cer(d18:1/24:1)/LDL-c/HDL-c,
Cer(d18:1/24:1)/total-c/HDL-c,
Cer(d18:1/24:1)/LDL cholesterol,
Cer(d18:1/24:1)/total cholesterol,
Cer(d18:1/24:1)/triglycerides,
Total Cer/apolipoprotein B,
Total Cer/LDL cholesterol,
Total Cer/total cholesterol, and
Total Cer/triglycerides,
and wherein the one or more lipid-clinical concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0)/apolipoprotein B,
Cer(d18:0/22:0)/LDL-c/HDL-c,
Cer(d18:0/22:0)/total-c/HDL-c,
Cer(d18:0/24:0)/HDL cholesterol,
Cer(d18:0/24:0)/LDL-c/HDL-c,
Cer(d18:0/24:0)/total-c/HDL-c, and
Cer(d18:0/24:0)/LDL cholesterol, wherein said CVD complications comprise myocardial infarction (MI) and/or death.

2. A method for evaluating the effectiveness of a lipid modifying treatment of CVD and/or one or more of its complications, in a subject, comprising:
a) assaying a sample from said subject to determine one or more lipid-lipid concentration ratio(s), wherein (an) increased or decreased lipid-lipid concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said lipid modifying treatment,
wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0)/LPC 18:1,
Cer(d18:1/18:0)/LPC 16:0,
Cer(d18:1/18:0)/LPC 18:1,
Cer(d18:1/18:0)/Total LPC,
Cer(d18:1/20:0)/Total LPC,
Cer(d18:1/22:0)/LPC 18:2,
Cer(d18:1/24:1)/LPC 18:1,
Cer(d18:1/24:1)/LPC 18:2,
Cer(d18:1/24:1)/Total LPC,
Cer(d18:1/22:0)/PC 16:0/20:4,
Cer(d18:1/18:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/PC 18:0/20:4,
Cer(d18:1/20:0)/PC 18:0/20:4,
Cer(d18:1/22:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/Total CE,
Cer(d18:1/18:0)/Total CE, and
CE 19:1/Cer(d18:0/22:0),
and wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0)/Cer(d18:1/22:0),
Cer(d18:0/24:0)/Cer(d18:1/16:0),
Cer(d18:0/24:0)/Cer(d18:1/20:0),
Cer(d18:0/22:0)/Total CE,
Cer(d18:0/24:1)/Total CE,
CE 18:3/Cer(d18:1/24:1),
CE 20:3/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/20:0),
CE 14:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/16:0),
CE 18:3/Cer(d18:1/18:0),
CE 18:3/Cer(d18:1/22:0),
CE 17:1/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/24:0),
CE 20:4/Cer(d18:1/24:1),
CE 20:4/Cer(d18:1/18:0),
CE 18:2/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/18:0),
CE 17:1/Cer(d18:1/18:0),
CE 14:0/Cer(d18:1/18:0),
CE 16:1/Cer(d18:1/20:0),
CE 16:1/Cer(d18:1/24:1),
CE 16:1/Cer(d18:1/18:0),
CE 15:0/Cer(d18:1/20:0),
CE 18:2/Cer(d18:1/20:0), and
CE 18:3/Total Cer,

or
b) assaying a sample from said subject to determine one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of effectiveness of said lipid modifying treatment,
wherein the one or more lipid-clinical concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0)/apolipoprotein A-I,
Cer(d18:1/16:0)/LDL cholesterol,
Cer(d18:1/16:0)/triglycerides,
Cer(d18:1/18:0)/apolipoprotein B,
Cer(d18:1/18:0)/HDL cholesterol,
Cer(d18:1/18:0)/total-c/HDL-c,
Cer(d18:1/18:0)/total cholesterol,
Cer(d18:1/18:0)/triglycerides,
Cer(d18:1/20:0)/apolipoprotein B,
Cer(d18:1/20:0)/HDL cholesterol,
Cer(d18:1/20:0)/total cholesterol,
Cer(d18:1/20:0)/triglycerides,
Cer(d18:1/20:0)/total-c/HDL-c,
Cer(d18:1/20:0)/LDL cholesterol,
Cer(d18:1/22:0)/apolipoprotein A-I,
Cer(d18:1/22:0)/apolipoprotein B,
Cer(d18:1/22:0)/LDL cholesterol,
Cer(d18:1/22:0)/triglycerides,
Cer(d18:1/22:0)/total cholesterol,
Cer(d18:1/24:0)/apolipoprotein A-I,
Cer(d18:1/24:0)/apolipoprotein B,
Cer(d18:1/24:0)/LDL cholesterol,
Cer(d18:1/24:0)/total cholesterol,
Cer(d18:1/24:1)/HDL cholesterol,
Cer(d18:1/24:1)/LDL-c/HDL-c,
Cer(d18:1/24:1)/total-c/HDL-c,
Cer(d18:1/24:1)/LDL cholesterol,
Cer(d18:1/24:1)/total cholesterol,
Cer(d18:1/24:1)/triglycerides,
Total Cer/apolipoprotein B,
Total Cer/LDL cholesterol,
Total Cer/total cholesterol, and
Total Cer/triglycerides,
and wherein the one or more lipid-clinical concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0)/apolipoprotein B,
Cer(d18:0/22:0)/LDL-c/HDL-c,
Cer(d18:0/22:0)/total-c/HDL-c,
Cer(d18:0/24:0)/HDL cholesterol,
Cer(d18:0/24:0)/LDL-c/HDL-c,
Cer(d18:0/24:0)/total-c/HDL-c, and
Cer(d18:0/24:0)/LDL cholesterol, wherein said CVD complications comprise myocardial infarction (MI) and/or death.

3. A method of choosing an appropriate lipid modifying treatment of CVD and/or one or more of its complications, in a subject, comprising:
(a) assaying a sample from said subject one or more lipid-lipid concentration ratio(s), wherein (an) increased or decreased lipid-lipid concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of a lipid modifying treatment or a change in, or supplementation of, an already administered lipid modifying treatment,
wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0)/LPC 18:1,
Cer(d18:1/18:0)/LPC 16:0,
Cer(d18:1/18:0)/LPC 18:1,
Cer(d18:1/18:0)/Total LPC,
Cer(d18:1/20:0)/Total LPC,
Cer(d18:1/22:0)/LPC 18:2,
Cer(d18:1/24:1)/LPC 18:1,
Cer(d18:1/24:1)/LPC 18:2,
Cer(d18:1/24:1)/Total LPC,
Cer(d18:1/22:0)/PC 16:0/20:4,
Cer(d18:1/18:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/PC 18:0/20:4,
Cer(d18:1/20:0)/PC 18:0/20:4,
Cer(d18:1/22:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/Total CE,
Cer(d18:1/18:0)/Total CE, and
CE 19:1/Cer(d18:0/22:0),
and wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0)/Cer(d18:1/22:0),
Cer(d18:0/24:0)/Cer(d18:1/16:0),
Cer(d18:0/24:0)/Cer(d18:1/20:0),
Cer(d18:0/22:0)/Total CE,
Cer(d18:0/24:1)/Total CE,
CE 18:3/Cer(d18:1/24:1),
CE 20:3/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/20:0),
CE 14:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/16:0),
CE 18:3/Cer(d18:1/18:0),
CE 18:3/Cer(d18:1/22:0),
CE 17:1/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/24:0),
CE 20:4/Cer(d18:1/24:1),
CE 20:4/Cer(d18:1/18:0),
CE 18:2/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/18:0),
CE 17:1/Cer(d18:1/18:0),
CE 14:0/Cer(d18:1/18:0),
CE 16:1/Cer(d18:1/20:0),
CE 16:1/Cer(d18:1/24:1),
CE 16:1/Cer(d18:1/18:0),
CE 15:0/Cer(d18:1/20:0),
CE 18:2/Cer(d18:1/20:0), and
CE 18:3/Total Cer,

or
(b) assaying a sample from said subject to determine one or more lipid-clinical concentration ratio(s), wherein (an) increased or decreased lipid-clinical concentration ratio(s) in said sample, when compared to a control sample, is (are) indicative of said subject being in need of a lipid modifying treatment or a change in, or supplementation of, an already administered lipid modifying treatment,
wherein the one or more lipid-clinical concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0)/apolipoprotein A-I,
Cer(d18:1/16:0)/LDL cholesterol,
Cer(d18:1/16:0)/triglycerides,
Cer(d18:1/18:0)/apolipoprotein B,
Cer(d18:1/18:0)/HDL cholesterol,
Cer(d18:1/18:0)/total-c/HDL-c,
Cer(d18:1/18:0)/total cholesterol,
Cer(d18:1/18:0)/triglycerides,
Cer(d18:1/20:0)/apolipoprotein B,
Cer(d18:1/20:0)/HDL cholesterol,
Cer(d18:1/20:0)/total cholesterol,
Cer(d18:1/20:0)/triglycerides,
Cer(d18:1/20:0)/total-c/HDL-c,
Cer(d18:1/20:0)/LDL cholesterol,
Cer(d18:1/22:0)/apolipoprotein A-I,
Cer(d18:1/22:0)/apolipoprotein B,
Cer(d18:1/22:0)/LDL cholesterol,
Cer(d18:1/22:0)/triglycerides,
Cer(d18:1/22:0)/total cholesterol,
Cer(d18:1/24:0)/apolipoprotein A-I,
Cer(d18:1/24:0)/apolipoprotein B,
Cer(d18:1/24:0)/LDL cholesterol,
Cer(d18:1/24:0)/total cholesterol,
Cer(d18:1/24:1)/HDL cholesterol,
Cer(d18:1/24:1)/LDL-c/HDL-c,
Cer(d18:1/24:1)/total-c/HDL-c,
Cer(d18:1/24:1)/LDL cholesterol,
Cer(d18:1/24:1)/total cholesterol,
Cer(d18:1/24:1)/triglycerides,
Total Cer/apolipoprotein B,
Total Cer/LDL cholesterol,
Total Cer/total cholesterol, and
Total Cer/triglycerides,
and wherein the one or more lipid-clinical concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0)/apolipoprotein B,
Cer(d18:0/22:0)/LDL-c/HDL-c,
Cer(d18:0/22:0)/total-c/HDL-c,
Cer(d18:0/24:0)/HDL cholesterol,
Cer(d18:0/24:0)/LDL-c/HDL-c,
Cer(d18:0/24:0)/total-c/HDL-c, and
Cer(d18:0/24:0)/LDL cholesterol, wherein said CVD complications comprise myocardial infarction (MI) and/or death.

4. The method of claims 1 or 3, wherein
(a) the one or more lipid-lipid concentration ratio(s) whose increase is compared to the control is selected from:
Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/18:0)/LPC 16:0, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/18:0)/PC 18:0/20:4 and Cer(d18:1/24:1)/PC 18:0/20:4,
(b) the one or more lipid-lipid concentration ratio(s) whose decrease is compared to the control is selected from:
Cer(d18:0/22:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/16:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/24:1), CE 20:3/Cer(d18:1/24:1), CE 14:0/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 16:0/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/24:0), CE 20:4/Cer(d18:1/24:1), CE 20:4/Cer(d18:1/18:0), CE 18:2/Cer(d18:1/24:1), CE 16:0/Cer(d18:1/18:0), CE 14:0/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/24:1) and CE 16:1/Cer(d18:1/18:0),
(c) the one or more lipid-clinical concentration ratio(s) whose increase is compared to the control is selected from:
Cer(d18:1/16:0)/apolipoprotein A-I, Cer(d18:1/16:0)/LDL cholesterol, Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/24:0)/apolipoprotein A-I, Cer(d18:1/24:0)/apolipoprotein B, Cer(d18:1/24:0)/LDL cholesterol, Cer(d18:1/24:0)/total cholesterol, Cer(d18:1/24:1)/HDL cholesterol, Cer(d18:1/24:1)/LDL-c/HDL-c, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/total cholesterol and Cer(d18:1/24:1)/triglycerides, and/or
(d) the one or more lipid-clinical concentration ratio(s) whose decrease is compared to the control is selected from:
Cer(d18:0/22:0)/apolipoprotein B, Cer(d18:0/24:0)/HDL cholesterol and Cer(d18:0/24:0)/LDL cholesterol.

5. The method of any one of claims 1-3, wherein the one or more lipid-lipid concentration ratios comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 lipid-lipid concentration ratios, and wherein the one or more lipid-clinical ratios comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 lipid-clinical concentration ratios, or combinations thereof.

6. The method of any one of claims 1-3, wherein
a) the CVD complication(s) is(are) atherosclerosis-induced; and/or
b) the subject has atherosclerosis; or
c) the subject does not have atherosclerosis.

7. The method of any one of claims 1-3, wherein
a) the method further comprises determining the serum level of total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Apolipoprotein B (ApoB) and/or Apolipoprotein C-III (ApoC-III) in said sample; and/or
b) the subject does not have elevated serum levels of one or more of total cholesterol, low-density lipoprotein cholesterol (LDL-C), Apolipoprotein C-III (ApoC-III) or Apolipoprotein B (ApoB), or a decreased serum level of HDL-cholesterol (HDL-C).

8. The method of any one of claims 1-3, wherein the subject
a) is being or has been treated with one or more statins and/or any other HMG-CoA reductase inhibitor; or
b) has not yet undergone statin therapy or therapy with any other HMG-CoA reductase inhibitor.

9. The method of any one of claims 1-3, wherein the lipid-lipid concentration ratio(s) or the lipid-clinical concentration ratio(s) is (are) assayed by using mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy or dual polarization interferometry, a high performance separation method, an immunoassay and/or with a binding moiety capable of specifically binding the analyte.

10. The method of claim 8, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, fluvastatin XL, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.

11. The method of any one of claims 1-3, wherein the subject is at risk to develop or has suffered from one or more CVD complications.

12. The method of any one of claims 1-3, wherein the control sample is from (a) CAD patient(s) or a group of CAD patients that has/have remained free of any major CVD complications, wherein the sample is a blood sample, or a serum sample.

13. The method of any one of claims 1-3, wherein the sample is blood, serum or plasma and wherein the lipid-lipid concentration ratio(s) or the lipid-clinical concentration ratio(s) is (are) determined by using mass spectrometry.

14. The method of claim 2, wherein:
(a) the one or more lipid-lipid concentration ratio(s) whose decrease is compared to the control is selected from:
Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/18:0)/LPC 16:0, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:2, Cer(d18:1/18:0)/PC 18:0/20:4 and Cer(d18:1/24:1)/PC 18:0/20:4,
(b) the one or more lipid-lipid concentration ratio(s) whose increase is compared to the control is selected from:
Cer(d18:0/22:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/16:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), CE 18:3/Cer(d18:1/24:1), CE 20:3/Cer(d18:1/24:1), CE 14:0/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/16:0), CE 18:3/Cer(d18:1/18:0), CE 16:0/Cer(d18:1/24:1), CE 18:3/Cer(d18:1/24:0), CE 20:4/Cer(d18:1/24:1), CE 20:4/Cer(d18:1/18:0), CE 18:2/Cer(d18:1/24:1), CE 16:0/Cer(d18:1/18:0), CE 14:0/Cer(d18:1/18:0), CE 16:1/Cer(d18:1/24:1) and CE 16:1/Cer(d18:1/18:0),
(c) the one or more lipid-clinical concentration ratio(s) whose decrease is compared to the control is selected from:
Cer(d18:1/16:0)/apolipoprotein Cer(d18:1/16:0)/LDL cholesterol, Cer(d18:1/16:0)/triglycerides, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/24:0)/apolipoprotein Cer(d18:1/24:0)/apolipoprotein B, Cer(d18:1/24:0)/LDL cholesterol, Cer(d18:1/24:0)/total cholesterol, Cer(d18:1/24:1)/HDL cholesterol, Cer(d18:1/24:1)/LDL-c/HDL-c, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/total cholesterol and Cer(d18:1/24:1)/triglycerides, and/or
(d) the one or more lipid-clinical concentration ratio(s) whose increase is compared to the control is selected from:
Cer(d18:0/22:0)/apolipoprotein B, Cer(d18:0/24:0)/HDL cholesterol and Cer(d18:0/24:0)/LDL cholesterol.

15. The method of claim 2, further comprising adjusting the treatment based on the determining step of a), b), and/or c.

16. The method of claim 3, further comprising treating the subject or changing or supplementing an already administered treatment.

17. A method of treating or preventing cardiovascular disease (CVD) complications in a subject, the method comprising administering to the subject a therapeutically effective dose of a drug capable of modulating one or more of the lipid-lipid concentration ratio(s) or lipid-clinical concentration ratio(s) according to claim 1, wherein prior to administering the drug, the subject is determined to be at risk to develop the one or more CVD complications by the method according to claim 1.

18. The method of claim 17, wherein the drug is a statin or any other HMG-CoA reductase inhibitor, niacin (nicotinic acid); a cholesterol absorption inhibitor; a cholesteryl ester transfer protein (CETP) inhibitor; a bile acids sequestrant; a fibrate; or a phytosterol.

19. The method of claim 18, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, fluvastatin XL, lovastatin, pitavastatin, pravastatin, rosuvastatin and/or simvastatin.

20. A method for detecting a lipid concentration(s), a lipid-lipid concentration ratio(s) and/or a lipid-clinical concentration ratio(s) in a sample from a coronary artery disease subject, comprising:
(a) assaying the sample from the coronary artery disease subject to detect the concentration(s) of one or more lipid(s), wherein the one or more lipid(s) is (are) selected from:
Cer(d18:1/16:0),
Cer(d18:1/18:0),
Cer(d18:1/20:0),
Cer(d18:1/22:0),
Cer(d18:1/26:1),
Cer(d18:0/22:0), and
Cer(d18:0/24:0);
(b) assaying the sample from the coronary artery disease subject to detect the concentration(s) of one or more lipid-lipid concentration ratio(s), wherein the one or more lipid-lipid concentration ratio(s) is (are) selected from:
Cer(d18:1/16:0)/LPC 18:1,
Cer(d18:1/18:0)/LPC 16:0,
Cer(d18:1/18:0)/LPC 18:1,
Cer(d18:1/18:0)/Total LPC,
Cer(d18:1/20:0)/Total LPC,
Cer(d18:1/22:0)/LPC 18:2,
Cer(d18:1/24:1)/LPC 18:1,
Cer(d18:1/24:1)/LPC 18:2,
Cer(d18:1/24:1)/Total LPC,
Cer(d18:1/22:0)/PC 16:0/20:4,
Cer(d18:1/18:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/PC 18:0/20:4,
Cer(d18:1/20:0)/PC 18:0/20:4,
Cer(d18:1/22:0)/PC 18:0/20:4,
Cer(d18:1/24:1)/Total CE,
Cer(d18:1/18:0)/Total CE,
CE 19:1/Cer(d18:0/22:0),
Cer(d18:0/22:0)/Cer(d18:1/22:0),
Cer(d18:0/24:0)/Cer(d18:1/16:0),
Cer(d18:0/24:0)/Cer(d18:1/20:0),
Cer(d18:0/22:0)/Total CE,
Cer(d18:0/24:1)/Total CE,
CE 18:3/Cer(d18:1/24:1),
CE 20:3/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/20:0),
CE 14:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/16:0),
CE 18:3/Cer(d18:1/18:0),
CE 18:3/Cer(d18:1/22:0),
CE 17:1/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/24:1),
CE 18:3/Cer(d18:1/24:0),
CE 20:4/Cer(d18:1/24:1),
CE 20:4/Cer(d18:1/18:0),
CE 18:2/Cer(d18:1/24:1),
CE 16:0/Cer(d18:1/18:0),
CE 17:1/Cer(d18:1/18:0),
CE 14:0/Cer(d18:1/18:0),
CE 16:1/Cer(d18:1/20:0),
CE 16:1/Cer(d18:1/24:1),
CE 16:1/Cer(d18:1/18:0),
CE 15:0/Cer(d18:1/20:0),
CE 18:2/Cer(d18:1/20:0), and
CE 18:3/Total Cer,

or
(c) assaying the sample from the coronary artery disease subject to detect the concentration(s) of one or more lipid-clinical concentration ratio(s) wherein the one or more lipid-clinical concentration ratio(s) is (are) selected from:
Cer(d18:1/16:0)/apolipoprotein A-I,
Cer(d18:1/16:0)/LDL cholesterol,
Cer(d18:1/16:0)/triglycerides,
Cer(d18:1/18:0)/apolipoprotein B,
Cer(d18:1/18:0)/HDL cholesterol,
Cer(d18:1/18:0)/total-c/HDL-c,
Cer(d18:1/18:0)/total cholesterol,
Cer(d18:1/18:0)/triglycerides,
Cer(d18:1/20:0)/apolipoprotein B,
Cer(d18:1/20:0)/HDL cholesterol,
Cer(d18:1/20:0)/total cholesterol,
Cer(d18:1/20:0)/triglycerides,
Cer(d18:1/20:0)/total-c/HDL-c,
Cer(d18:1/20:0)/LDL cholesterol,
Cer(d18:1/22:0)/apolipoprotein A-I,
Cer(d18:1/22:0)/apolipoprotein B,
Cer(d18:1/22:0)/LDL cholesterol,
Cer(d18:1/22:0)/triglycerides,
Cer(d18:1/22:0)/total cholesterol,
Cer(d18:1/24:0)/apolipoprotein A-I,
Cer(d18:1/24:0)/apolipoprotein B,
Cer(d18:1/24:0)/LDL cholesterol,
Cer(d18:1/24:0)/total cholesterol,
Cer(d18:1/24:1)/HDL cholesterol,
Cer(d18:1/24:1)/LDL-c/HDL-c,
Cer(d18:1/24:1)/total-c/HDL-c,
Cer(d18:1/24:1)/LDL cholesterol,
Cer(d18:1/24:1)/total cholesterol,
Cer(d18:1/24:1)/triglycerides,
Total Cer/apolipoprotein B,
Total Cer/LDL cholesterol,
Total Cer/total cholesterol, and
Total Cer/triglycerides,
Cer(d18:0/22:0)/apolipoprotein B,
Cer(d18:0/22:0)/LDL-c/HDL-c,
Cer(d18:0/22:0)/total-c/HDL-c,
Cer(d18:0/24:0)/HDL cholesterol,
Cer(d18:0/24:0)/LDL-c/HDL-c,
Cer(d18:0/24:0)/total-c/HDL-c, and
Cer(d18:0/24:0)/LDL cholesterol.

21. A method for determining whether a subject is at risk to develop one or more Cardiovascular Disease (CVD) complications, comprising:
a) assaying a sample from said subject to determine the concentration(s) of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications,
wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0),
Cer(d18:1/18:0),
Cer(d18:1/20:0),
Cer(d18:1/22:0),
Cer(d18:1/24:1),
Cer(d18:1/26:1), and
and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0), and
Cer(d18:0/24:0);
b) treating the subject indicated as being at risk of developing one or more CVD complications based on one or more of the determining steps with a lipid modifying treatment, wherein said CVD complications comprise myocardial infarction (MI) and/or death, wherein the lipid modifying treatment is a statin or any other HMG-CoA reductase inhibitor, niacin (nicotinic acid); a cholesterol absorption inhibitor; a cholesteryl ester transfer protein (CETP) inhibitor; a bile acids sequestrant; a fibrate; or a phytosterol.

22. The method of claim 1, further comprising assaying a sample from said subject to determine the concentration(s) of one or more lipid(s), wherein (an) increased or decreased concentration(s) in said sample, when compared to a control sample, is (are) indicative of said subject having an increased risk of developing one or more CVD complications,
wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from:
Cer(d18:1/16:0),
Cer(d18:1/18:0),
Cer(d18:1/20:0),
Cer(d18:1/22:0),
Cer(d18:1/24:1),
Cer(d18:1/26:1), and
Total Cer,
and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from:
Cer(d18:0/22:0), and
Cer(d18:0/24:0).