Abstract:
The present invention inter alia provides a method, and uses thereof, to measure drug efficacy and specificity of treatment with an inhibitor of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) by detecting the concentrations of lipids and/or lipid-lipid concentration ratios of a biological sample and comparing it to a control. The invention is applicable, inter alia, to determining whether a PCSK9 inhibiting drug is functioning efficiently in lowering serum low-density lipoprotein (LDL) concentration and whether a PCSK9 inhibiting drug displays any adverse side-effects, such as liver toxicity. Provided are lipid markers that are more specific and sensitive in detecting drug efficacy and possible adverse drug-induced side-effects than the currently utilized clinical markers. Also provided is an antibody towards said lipids, and the use thereof for predicting and diagnosing of PCSK9 inhibiting drug-induced adverse reactions. The invention additionally relates to kits comprising lipids and/or an antibody thereto, for the determination of PCSK9 inhibiting drug efficacy and drug-induced adverse reactions.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a U.S. National Stage application of PCT/EP2013/060816 filed 24 May 2013, which claims priority to European patent application 12169517.5 filed 25 May 2012 and U.S. Provisional Patent Application 61/651,569 filed 25 May 2012, the entire disclosures of which are hereby incorporated herein by reference in their entireties. 
     
    
     FIELD OF THE INVENTION 
       [0002]    This invention relates to methods involving measuring levels of lipids and lipid-lipid concentration ratios to measure drug efficacy and specificity of treatments that target Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9). The invention is applicable, inter alia, to determining whether a PCSK9 targeting treatment is functioning efficiently and whether a PCSK9 targeting compound displays off-target effects. The invention is also useful for evaluating the compliance of patients to PCSK9 targeting treatments. The methods include analyzing lipid biomarker levels of a biological sample, and comparing it to a control. 
       BACKGROUND OF THE INVENTION 
       [0003]    Plasma low-density lipoprotein (LDL) cholesterol is an established risk factor for coronary vascular diseases. Generally, high blood cholesterol levels are treated with statins. However, even high dosages of statins might not be efficient enough to decrease cholesterol levels satisfactorily if patients have high initial plasma cholesterol values or exhibit resistance to statin treatment. Also, high statin dosages might increase the risk of side-effects. Consequently, new cholesterol-lowering interventions are needed to prevent and treat coronary vascular disease (CVD). 
         [0004]    Human genetic studies indicate that PCSK9 plays a central role in the regulation of plasma LDL levels (Abifadel, M. et al. 2003. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.  Nat Genet  34: 154-156). PCSK9 belongs to a family of serine proteases, the proprotein convertases (Seidah, N. G. et al. 2003. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.  Proc Natl Acad Sci USA  100: 928-933). However, independently of its enzymatic activity, it decreases the number of LDL-receptors (LDL-R) expressed on the hepatocyte surface by binding to LDL-R extracellularly and facilitating its lysosomal degradation, thus inhibiting the recycling of LDL-R back to the cell surface (Horton, J. D. et al. 2009. PCSK9: a convertase that coordinates LDL catabolism.  J Lipid Res  50 Suppl: S172-177). Individuals with loss-of-function mutations in PCSK9 gene have reduced plasma LDL cholesterol levels and are protected from CVD (Cohen, J. et al. 2005. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9 . Nat Genet  37: 161-165; Abifadel, M. et al. 2003. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.  Nat Genet  34: 154-156). In contrast, gain-of-function mutations in the PCSK9 gene have been shown to be associated with elevated plasma LDL levels and premature CVD (Abifadel, M. et al. 2003). Moreover, by expressing human D374Y gain-of-function PCSK9 in mice, it has been shown that reduced LDL-R activity might not be the sole cause of hypercholesterolemia as D374Y mice were shown to secrete more triacylglycerol-rich lipoproteins into the circulation as compared to wild-type mice (Herbert, B. et al. 2010. Increased secretion of lipoproteins in transgenic mice expressing human D374Y PCSK9 under physiological genetic control.  Arterioscler Thromb Vasc Biol  30: 1333-1339). These observations position PCSK9 as a potential target in the treatment of hypercholesterolemia. 
         [0005]    In mouse, PCSK9 is expressed predominantly in liver, small intestine, and kidney (Zaid, A. et al. 2008. Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.  Hepatology  48: 646-654). Complete knockout of PCSK9 in mouse has been shown to result in approximately 40% reduction in circulating LDL cholesterol levels (Rashid, S. et al. 2005. Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9 . Proc Natl Acad Sci USA  102: 5374-5379). Administration of statins, the HMG-coenzyme A reductase inhibitors, was shown to decrease the LDL levels even more indicating the potential of simultaneous use of statins and PCSK9 inhibition in the treatment of CVD. However, PCSK9 has been shown to play a role in mouse liver regeneration (Zaid, A. et al. 2008. Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.  Hepatology  48: 646-654). Therefore, complete inhibition of PCSK9 function might increase the risk of complications or even death upon hepatic damage. On the other hand, in the same study, it was demonstrated in PCSK9 heterozygote knockout mice, that 50% decrease in PCSK9 activity is not deleterious and does not result in impairment in liver regeneration. 
         [0006]    Recently, in humans, statins and other lipid-lowering drugs have been shown to elevate serum PCSK9 protein levels through activation of sterol regulatory element-binding protein-2 (SREBP-2) (Careskey, H. E. et al. 2008. Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9.  J Lipid Res  49: 394-398 and Konrad, R. J. et al. 2011. Effects of currently prescribed LDL-C-lowering drugs on PCSK9 and implications for the next generation of LDL-C-lowering agents.  Lipids Health Dis  10: 38). These observations suggest that PCSK9 inhibition in combination with statins or other LDL-lowering medications would be required to achieve the most efficient treatment outcome. 
         [0007]    As PCSK9 is a potential target for the treatment of dyslipidemia, the inventor investigated the effect of PCSK9 deficiency on plasma lipidomes in heterozygote and homozygote PCSK9 knockout mice using mass spectrometric applications aiming to identify changes in lipid homeostasis at the level of molecular lipids. Such changes were investigated also in humans by taking advantage of existing knowledge on functionality of know genetic variants of the PCSK9 gene. In short, the lipidomic profile in subjects carrying a known loss-of-function mutation was compared to that in subjects carrying the major allele. Such a specific efficacy read-out will be useful while developing and selecting new compounds acting on PCSK9 and monitoring clinical efficacy of PCSK9 inhibitors. A typical and precise efficacy read-out can also be used to monitor unwanted off-target effects as deviations from the predetermined efficacy profile may be indicative of such unspecific potentially harmful drug effects. 
         [0008]    According to the present invention, the lipids may be analyzed by a variety of techniques. In the context of the present invention, electrospray ionization mass spectrometry-based lipidomics is the preferred technology. The superior quality and specificity of shotgun and targeted analysis methods will meet stringent regulatory standards, such as good laboratory practice guidelines (GLP) when set-up in the proper environment. 
         [0009]    Compared to the state of the art, the biomarkers identified herein can be analyzed from even the smallest sample amounts, due to both high sensitivity and specificity of the technology. 
         [0010]    The present invention identifies biomarkers indicative of efficacy and specificity of PCSK9 inhibiting drugs. The biomarkers will facilitate the mission of making sure the individual receives the right PCSK9 inhibiting drug at the right time and dose, thereby opening this therapeutic area towards personalizing otherwise more generally applied medicines and/or treatment regimes. Identified biomarkers can also be used by developers of new drug agents against PCSK9 to select specific lead compounds among agent candidates. 
       SUMMARY OF THE INVENTION 
       [0011]    The present invention inter alia provides lipids and lipid ratios that are indicative of PCSK9 inhibition. This is based on knock-out animal data and human translation data on loss-of-function mutations which display equivalent lipid composition as analyzed with lipidomic platforms. The identified lipids can be used to monitor the extent of PCSK9 inhibition and its specificity. This offers a novel and improved way to look at PCSK9 inhibition, which normally relies on LDL cholesterol read-outs. However, LDL-cholesterol measurement does not provide any information whether the PCSK9 inhibition also lowers beneficial and essential lipids, which may cause unwanted side effects. The challenge is also that if the level of LDL-cholesterol is reduced too much, it may cause harmful effects to the patient. The identified lipid markers herein provide improved insight on the efficacy of PCSK9 inhibition and its specificity. 
         [0012]    As PCSK9 is a potential target for the treatment of dyslipidemia, the inventor investigated the effect of PCSK9 deficiency on plasma lipidomes in heterozygote and homozygote PCSK9 knockout mice using mass spectrometric applications, with the aim of identifying changes in lipid homeostasis at the level of molecular lipids. Lipid profiles were generated from wildtype, heterozygote and homozygote PCSK9 knock-out mice which were given either regular chow or Western chow, in order to investigate the effect of PCSK9 deficiency and the difference in lipid biomarkers caused by a high-fat diet in mice lacking one or both PCSK9 allele. Such changes were investigated also in humans by taking advantage of existing knowledge on functionality of known genetic variants of the PCSK9 gene. In short, the lipidomic profiles of samples from subjects carrying a known loss-of-function mutation were compared to lipidomic profiles of samples from subjects carrying the major allele. Such a specific efficacy read-out will be useful while developing and selecting new compounds acting on PCSK9 and monitoring clinical efficacy of PCSK9 inhibitors. A typical and precise efficacy read-out can also be used to monitor unwanted off-target effects as deviations from the predetermined efficacy profile may be indicative of such unspecific potentially harmful drug effects. 
         [0013]    Natural PCSK9 inhibition due to a genetic loss-of-function mutation results in a favorable molecular lipid change in plasma that may at least partly explain why carriers of this mutation have lower coronary artery disease (CAD) risk. Thus, the identified biomarkers can be used as specificity indicators for any therapy targeting PCSK9 since deviations from the changes that result from the loss of PCSK9 function could be due to undesired non-specific off-target effects.  FIG. 1  shows that PCSK9 inhibition lowers lipids which increase CAD risk. Additionally, PCSK9 inhibition was compared to lipid lowering effects of the statin treatment. Statin class drugs lower plasma lipids by upregulating hepatic LDL-receptor and thus increasing LDL-receptor mediated lipid removal from circulation. PCSK9 inhibition also increases the expression of hepatic LDL-receptors and causes lipid lowering due to increased LDL-receptor mediated lipid removal from circulation. Therefore, in theory these two treatment modalities should result in similar plasma lipid changes.  FIG. 1  shows that specific lipid changes that result from PCSK9 loss-of-function can be used as efficacy and specificity read-outs for therapies targeting PCSK9. In this regard,  FIG. 2  shows that statins affects the level of many lipid biomarkers differently than loss of PCSK9 function, indicating that they have a broader, less specific effect on lowering plasma lipids than a specific PCSK9 inhibitor/silencer. 
         [0014]    This invention relates to methods involving lipid levels to measure drug efficacy and specificity of treatments that target PCSK9. The invention is applicable, inter alia, to determining whether a PCSK9 targeting treatment is functioning efficiently and whether a PCSK9 targeting compound has off-target effects. Also, it can be used to evaluate the compliance of patients on PCSK9 targeting treatments. The methods include analyzing lipid levels of a biological sample, and comparing it to a control. 
         [0015]    In one aspect of the present invention, methods, lipidomic markers, agents such as antibodies and kits are disclosed and/or claimed herein for analyzing functions of new drug compounds as well as for detecting drug efficacy and specificity in patients on treatments targeting PCSK9. 
         [0016]    Methods according to the invention may, e.g., comprise the steps of: a) providing a biological sample from a subject or a test animal being treated, to be treated, or having been treated with a PCSK9 inhibiting drug; b) determining the concentration(s) of one or more lipid(s) and/or one or more lipid ratio(s) identified herein as useful lipidomic markers in accordance with the invention in said sample; and c) comparing said determined lipid concentration(s) and/or lipid ratio(s) to the corresponding lipid concentration(s) in a control. 
         [0017]    The lipidomic markers of the present invention allow for sensitive and specific detection of efficacy and specificity of PCSK9 inhibiting drugs. This will facilitate improving patient care and treatment outcome achievement, lessening toxicity symptom development and suffering, and achieving decreased morbidity/mortality associated with drug-induced off-target effects. Thus, the lipidomic markers described and claimed herein allow for individual tailoring of drug intervention regarding patients treated, or to be treated with PCSK9 inhibiting drugs. Also, the invention is applicable to animal experiments where PCSK9 inhibiting compounds are tested. The invention will allow a better specificity assessment of novel lipid lowering medications to be made. 
         [0018]    Accordingly, in one aspect of the invention, a method is provided for determining the efficacy of a treatment with a lipid-lowering drug in a subject, said method comprising determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (a) decreased or increased concentration(s) in said sample, when compared to a control, is (are) indicative of high efficacy 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 the decreased lipids in Tables 2 to 5; and wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 2 to 5. 
         [0019]    In a preferred embodiment, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids in Tables 4 to 5; and the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 4 to 5. 
         [0020]    In an alternative embodiment, the present invention relates to a method for determining the efficacy of a treatment with a lipid-lowering drug in a subject, said method comprising determining in a sample from said subject one or more lipid-lipid concentration ratio(s), wherein (a) decreased or increased lipid-lipid concentration ratio(s) in said sample, when compared to a control, is (are) indicative of high efficacy of said treatment, wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 2 to 5; and wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 2 to 5. 
         [0021]    In a preferred embodiment, the one or more lipid-lipid concentration ratios whose decrease(s) is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 4 to 5; and the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 4 to 5. 
         [0022]    In another aspect of the invention, a method is provided for predicting the efficacy of a treatment with a lipid-lowering drug in a subject (i.e., in a subject that has yet to receive a lipid-lowering treatment), said method comprising determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (a) increased or decreased concentration(s) in said sample, when compared to a control, is (are) indicative that said treatment will be efficacious, wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids in Tables 2 to 5; and wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 2 to 5. 
         [0023]    In a preferred embodiment, the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids in Tables 4 to 5; and the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 4 to 5. 
         [0024]    In an alternative embodiment, the present invention relates to a method for predicting the efficacy of a treatment with a lipid-lowering drug in a subject (i.e., in a subject that has yet to receive a lipid-lowering treatment), said method comprising determining in a sample from said subject one or more lipid-lipid concentration ratio(s), wherein (a) increased or decreased lipid-lipid concentration ratio in said sample, when compared to a control, is (are) indicative said treatment will be efficacious, wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 2 to 5; and wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 2 to 5. 
         [0025]    In a preferred embodiment, the one or more lipid-lipid concentration ratios whose increase(s) is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 4 to 5; and the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 4 to 5. 
         [0026]    The above embodiment is useful for subjects who have not yet received said treatment with a lipid-lowering drug. 
         [0027]    In yet another embodiment, the present invention relates to a method for determining the compliance of a subject with a lipid-lowering drug treatment, said method comprising determining in a sample from said subject the concentration(s) of one or more lipid(s), wherein (a) decreased or increased concentration(s) in said sample, when compared to a control, is (are) indicative of good treatment compliance, wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to a control is (are) selected from the decreased lipids in Tables 2 to 5; and wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to a control is (are) selected from the increased lipids in Tables 2 to 5. 
         [0028]    In a preferred embodiment, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids in Tables 4 to 5; and the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 4 to 5. 
         [0029]    In an alternative embodiment, the present invention relates to a method for determining the compliance of a subject with a lipid-lowering drug treatment, said method comprising determining in a sample from said subject one or more lipid-lipid concentration ratio(s), wherein (a) decreased or increased lipid-lipid concentration ratio(s) in said sample, when compared to a control, is (are) indicative of good treatment compliance, wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) in concentration is (are) compared to a control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 2 to 5; and wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to a control is (are) selected from the increased lipid-lipid concentration ratios in Tables 2 to 5. 
         [0030]    In a preferred embodiment, the one or more lipid-lipid concentration ratio(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 4 to 5; and the one or more lipid-lipid concentration ratio(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 4 to 5. 
         [0031]    In a further embodiment, the present invention relates to a method for identifying compounds that are useful as lipid-lowering drugs or for treating cardiovascular disease and its complications, said method comprising determining in a sample from said subject undergoing treatment with said compound, the concentration(s) of one or more lipid(s), wherein (a) decreased or increased concentration(s) in said sample, when compared to a control, is (are) indicative of usefulness as lipid-lowering drug, wherein the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids in Tables 2 to 5; and wherein the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 2 to 5. 
         [0032]    In a preferred embodiment, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids in Tables 4 to 5; and the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipids in Tables 4 to 5. 
         [0033]    In an alternative embodiment, the present invention relates to a method for identifying compounds that are useful as lipid-lowering drugs or for treating cardiovascular disease and its complications, said method comprising determining in a sample from said subject one or more lipid-lipid concentration ratio(s), wherein (a) decreased or increased concentration(s) in said sample, when compared to a control, is (are) indicative of usefulness as lipid-lowering drug, wherein the one or more lipid-lipid concentration ratio(s) whose decrease(s) is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 2 to 5; and wherein the one or more lipid-lipid concentration ratio(s) whose increase(s) is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 2 to 5. 
         [0034]    In a preferred embodiment, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipid-lipid concentration ratios in Tables 4 to 5; and the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipid-lipid concentration ratios in Tables 4 to 5. 
         [0035]    In a preferred embodiment of any of the methods or other embodiments of the present invention, the said lipid-lowering drug is a PCSK9 inhibitor/silencer. 
         [0036]    In methods of the invention may further comprise measuring the level of circulating LDL. If the level of LDL increases after administration, this indicates that the compound is not useful as a lipid-lowering drug. Conversely, if the level of LDL decreases after administration, this indicates that the compound is useful as a lipid-lowering drug. 
         [0037]    Alternatively, the methods of the invention may further comprise measuring the ability of the compound to prevent PCSK9 from binding LDL receptors. If the compound does not prevent PCSK9 from binding LDL receptors after administration, this indicates that the compound is not useful as a lipid-lowering drug. If the compound prevents PCSK9 from binding LDL receptors after administration, this indicates that the compound is useful as a lipid-lowering drug. 
         [0038]    In a further embodiment, the present invention relates to a method for determining specificity of a PCSK9 inhibitor/silencer, said method comprising comparing the concentration(s) of one or more lipids(s) or lipid-lipid concentration ratio(s) in a sample from a subject to a control, wherein the said one or more lipid(s) or lipid-lipid concentration ratio(s) is (are) selected from the lipids and lipid-lipid concentration ratios in Tables 2 to 5; and wherein the control is a sample or value, derived from one or more subject(s) having a PCSK9 loss-of-function mutation. 
         [0039]    In an alternative embodiment of this method, the control is a sample or value derived from one or more subject(s) having a PCSK9 loss-of-function-type lipid profile. A PCSK9 loss-of-function-type lipid profile may be generated by determining the concentration(s) of one or more lipids or lipid-lipid concentrations ratios from a control. 
         [0040]    In yet another alternative embodiment of this method, the control is a sample or value, derived from one or more subject(s) treated with a known specific PCSK9 inhibitor/silencer. 
         [0041]    The absence of a difference between the said one or more lipid(s) or lipid-lipid concentration ratio(s) in the sample and the control according to this method is indicative of the specificity of the treatment with said PCSK9 inhibitor/silencer. Conversely, the presence of a difference between the said one or more lipid(s) or lipid-lipid concentration ratio(s) in the sample and the control is indicative of non-specific effects caused by said PCSK9 inhibitor/silencer or compound, such as one or more adverse side-effects. 
         [0042]    In a preferred embodiment of this method, the one or more lipid(s) whose decrease(s) in concentration is (are) compared to the control is (are) selected from the decreased lipids and lipid-lipid concentration ratios in Tables 4 to 5; and the one or more lipid(s) whose increase(s) in concentration is (are) compared to the control is (are) selected from the increased lipids and lipid-lipid concentration ratios in Tables 4 to 5. 
         [0043]    In connection with all aspects and embodiments of the invention described and claimed herein, the said subject in respect of which comparison is made may be (a) a patient undergoing treatment with a PCSK9 inhibitor/silencer or another compound targeting PCSK9; (b) a test animal undergoing treatment with a PCSK9 inhibitor/silencer or another compound targeting PCSK9; (c) a patient or test animal undergoing treatment with a lipid-lowering drug other than a PCSK9 inhibitor/silencer; or (d) a patient or test animal who/which has not undergone and is not undergoing treatment with a PCSK9 inhibitor/silencer, another compound targeting PCSK9 or a lipid-lowering drug other than a PCSK9 inhibitor/silencer. 
         [0044]    For the methods disclosed herein for determining the efficacy or predicting the efficacy of a treatment with a lipid-lowering drug (e.g., a PCSK9 inhibitor/silencer), for determining the compliance of a subject with a lipid-lowering drug treatment (e.g., a PCSK9 inhibitor/silencer), or for identifying compounds that are useful as lipid-lowering drugs (e.g., a PCSK9 inhibitor/silencer), the control to which a comparison is made may be a control sample obtained from the same subject prior to treatment with a PCSK9 inhibitor/silencer or during discontinuation of said treatment. 
         [0045]    For the purposes of the present invention, a control sample may also be obtained from (a) a group of patients e.g., by mixing a variety of samples from a population. If a group of 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 concentration ratios in the sample from a subject are compared to the levels or amounts of the lipids or lipid-lipid concentration ratios in the control, for the purposes of the methods herein described and/or claimed. 
         [0046]    In one embodiment, the control to which a comparison is made may be a control value established from one or more healthy subject(s) not previously treated with a PCSK9 inhibitor/silencer. It may also be a sample that represents a combination of samples from a patient population. Alternatively, the control may be a control value established from one or more healthy subject(s) not undergoing treatment with a PCSK9 inhibitor/silencer. As a further alternative, 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) or lipid-lipid concentration ratio(s), in accordance with the present invention, in a sample when taken from a control sample from one or more subjects who carry any loss-of-function mutation in the PCSK9 gene, such as R46L (rs11591147). As another alternative, the control may be a control value established from one or more subject(s) on treatment with a PCSK9 inhibitor/silencer and with no signs or history of drug-induced off-target effects. 
         [0047]    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. 
         [0048]    Preferably, the control sample is blood, plasma, serum, urine or tissue, or a lipoprotein fraction thereof 
         [0049]    In connection with all aspects and embodiments of the invention, any of the methods herein claimed and/or described may further comprise determining or evaluating the level of LDL cholesterol in said subject or in a sample from said subject. In one embodiment, the subject has reduced LDL cholesterol levels. 
         [0050]    In accordance with the methods of the invention, the sample may be blood, blood plasma, blood serum, or urine. The sample may also be a fraction of blood, blood plasma, blood serum or urine, e.g., a lipoprotein fraction. A blood sample can be prepared and plasma or serum, or fractions thereof, can be separated there from 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., a tissue biopsy) or a lipoprotein fraction thereof. In a further alternative, the sample can be any mammalian cells (e.g., erythrocytes). 
         [0051]    Collecting information on a lipidomic marker (i.e., a lipid, lipid concentration, or lipidomic marker combination as described and claimed herein) according to the methods of the present invention from the subject&#39;s sample, and also from the control sample, can be performed via various chemical and high resolution analytical techniques. Particularly suitable analytical techniques include, but are not limited to, mass spectrometry and nuclear magnetic resonance spectroscopy. Indeed, any high resolution technique capable of resolving individual lipids or lipid classes and providing structural information of the same can be used to determine the lipidomic markers according to the invention from the subject&#39;s sample, and also from the control sample. For the purposes of the methods of the present invention the lipid concentration(s) or lipid ratio(s) are thus preferably determined by using mass spectrometry. However, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy or dual polarisation interferometry, high performance separation methods such as HPLC or UPLC, an immunoassay such as an ELISA and/or the use of a binding moiety capable of specifically binding the lipid analyte are also useful in this regard. 
         [0052]    As indicated above, according to an alternative or further embodiment of the methods of the invention, a lipid 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. 
         [0053]    In a particularly preferred embodiment, the lipidomic markers of the present invention are determined with mass spectrometry (MS), wherein the MS instrument is optionally coupled to direct infusion methods and high performance separation methods such as HPLC or UPLC. The amount of the individual lipids or lipid classes in the collected lipidomic markers is used when comparing the collected lipid profile to a control. 
         [0054]    The present invention also includes a PCSK9 inhibitor/silencer for use in therapy. Preferably such therapy is treatment of hypercholesteremia. Likewise preferably, such therapy is reducing the risk of, or treating a cardiovascular disease, such as atherosclerosis, coronary artery disease, acute myocardial infarction and/or stroke. In the context of this aspect of the invention, the said PCSK9 inhibitor/silencer decreases the concentration in a subject of one or more lipid(s) selected from the decreased lipids in Tables 2 to 5. A corresponding method of treatment is likewise encompassed. In a preferred embodiment, the said PCSK9 inhibitor/silencer decreases in a subject one or more lipid(s) selected from the decreased lipids in Tables 4 to 5. 
         [0055]    In an alternative embodiment, the said PCSK9 inhibitor/silencer increases in a subject one or more lipid(s) selected from the increased lipids in Tables 2 to 5. In a preferred embodiment, the said PCSK9 inhibitor/silencer increases in a subject one or more lipid(s) selected from the increased lipids in Tables 4 to 5. 
         [0056]    In a further alternative embodiment the said PCSK9 inhibitor/silencer decreases one or more lipid-lipid concentration ratio(s) selected from the decreased lipid-lipid concentration ratios in Tables 2 to 5. In a preferred embodiment, the said PCSK9 inhibitor/silencer decreases one or more lipid-lipid concentration ratio(s) selected from the decreased lipid-lipid concentration ratios in Tables 4 to 5. 
         [0057]    In a yet a further alternative embodiment, the said PCSK9 inhibitor/silencer increases one or more lipid-lipid concentration ratio(s) selected from the increased lipid-lipid concentration ratios in Tables 2 to 5. In a preferred embodiment, the PCSK9 inhibitor/silencer increases one or more lipid-lipid concentration ratio(s) selected from the increased lipid-lipid concentration ratios in Tables 4 to 5. 
         [0058]    For the purposes of the present invention, a PCSK9 inhibitor/silencer may be selected from (a) one or more antibodies against PCSK9, (b) a drug inhibitor of the PCSK9, (c) a small molecule that inhibits the interaction of the LDL-receptor with PCSK9; (d) a peptide that mimics the interaction domain of the LDL-receptor with PCSK9, (e) one or more siRNAs specific for PCSK9 and/or (e) one or more antisense oligonucleotides specific for PCSK9. An antibody against PCSK9 is particularly preferred as lipid lowering drug, or PCSK9 inhibitor/silencer, for the purposes of the present invention. 
         [0059]    Also encompassed by the present invention is using an antibody against any one of the lipids or against any one of the lipids in the lipid-lipid concentration ratios defined in Tables 2 to 5 for predicting or determining the efficacy of a treatment with a lipid-lowering drug. In a preferred embodiment, the antibody is one against any one of the lipids or against any one of the lipids in the lipid-lipid concentration ratios defined in Tables 4 to 5. A corresponding method of treatment is likewise encompassed. In a preferred embodiment, the lipid-lowering drug is a PCSK9 inhibitor/silencer. 
         [0060]    Also part of the present invention is using an antibody against any one of the lipids or against any one of the lipids in the lipid-lipid concentration ratios defined in Tables 2 to 5 for preventing or treating one or more adverse side-effects due to treatment with a PCSK9 inhibitor/silencer in a subject. In a preferred embodiment, the antibody is against any one of the lipids or against any one of the lipids in the lipid-lipid concentration ratios defined in Table 4 to 5. A corresponding method of treatment is likewise encompassed. 
         [0061]    For the purposes of the present invention, an adverse side-effect due to or caused by treatment with a PCSK9 inhibitor/silencer may be liver toxicity. 
         [0062]    Also encompassed by the present invention is a kit for performing the methods and uses described and/or claimed herein, wherein the kit comprises reagents and reference compounds. The reference compounds may be one or more of the following, but are not limited to: (a) (a) lipid standard(s) chosen from the lipids in Tables 2 to 5, (b) one or more control markers (for example, a lipid or lipids, preferably a lipid corresponding to any of the lipidomic markers described and/or claimed herein, or (an)other lipid(s), e.g., total PC, or another molecule, e.g., a protein; c) positive and/or negative controls; d) internal and/or external standards; e) calibration line controls; (f) an antibody or other binding moiety capable of binding any one of the lipids in Tables 2 to 5. The reagents are solution(s), solvent(s), and/or buffer(s) useful for performing said methods or uses. 
         [0063]    In one embodiment of the invention, a kit is provided for performing the methods described and/or claimed herein, wherein the kit comprises reagents and reference compounds. The reference compounds may be one or more of the following, but are not limited to: (a) (a) lipid standard(s) chosen from the lipids defined in Tables 2 to 5; and optionally one or more further reference compounds selected from: (b) one or more control markers (for example, a lipid or lipids, preferably a lipid corresponding to any of the lipidomic markers described and/or claimed herein, or another lipid(s), e.g., total PC, or another molecule, e.g., a protein); (c) positive and/or negative controls; (d) internal and/or external standards, which may or may not be chemically modified, tagged or non-endogenously occurring molecules in human; (e) calibration line controls; and (f) an agent, optionally an antibody, capable of binding any one of the lipids in Tables 2 to 5 and (g) (a) reagent(s) for performing said methods or uses. 
         [0064]    Preferred kits according to the invention comprise, for example, the following combinations of the above listed constituents: (a) and (b), and optionally (g); (a) and (c), and optionally (g); (a) and (d), and optionally (g); (a) and (e), and optionally (g); (a) and (f), and optionally (g); (a), (b) and (c), and optionally (g); (a), (b) and (d), and optionally (g); (a), (b) and (e), and optionally (g); (a), (b) and (f), and optionally (g); (a), (c) and (d), and optionally (g); (a), (c) and (e), and optionally (g); (a), (c) and (f), and optionally (g); (a), (d) and (e), and optionally (g); (a), (d) and (f), and optionally (g); or (a), (e) and (f), and optionally (g). 
         [0065]    In one preferred embodiment, the one or more control marker(s) of the claimed kit is/are (a) molecule(s) that is/are regularly measured in a clinical setting. For example, preferred are embodiments wherein the one or more said control marker(s) is apoA, apoB, albumin or total PC, or a combination thereof. 
         [0066]    Preferably, the kit is used for any of the purposes of the present invention, wherein the lipid concentration(s), lipid ratio(s) or (a) lipid combination(s) thereof in a sample from a subject is (are) determined by using mass spectrometry. The sample may be subjected to purification and/or other sample pre-preparation step(s) before mass spectrometry analysis. The purification step may be, but is not limited to chromatography, for example, high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) and/or ultra high performance liquid chromatography (UHPLC). The sample pre-preparation step may be, but is not limited to solid-phase extraction (SPE), derivatization, liquid-liquid extraction and/or lipoprotein fractionation. The said mass spectrometry determination may be done by tandem mass spectrometry. 
         [0067]    In an alternative preferred embodiment, the kit is used, wherein the lipid concentration(s), lipid ratio(s) or (a) lipid combination(s) thereof in a sample from a subject is (are) determined by using an enzyme-linked immunosorbent assay (ELISA). 
         [0068]    In another embodiment of the invention, a kit is provided that can be use in an immunoassay for performing the methods of the invention. An exemplary kit comprises, but is not limited to: (a) (an) antibody(ies) capable of binding any one of the lipids in Tables 2 to 5; and optionally one or more of the following: 
         [0069]    (b) a substrate specific for said enzyme; 
         [0070]    (c) a stop solution; 
         [0071]    (d) an assay plate coated with (an) antibody(ies) capable of binding any of the lipids in Tables 2 to 5; 
         [0072]    (e) (a) standard(s) and/or (a) calibration line standard(s); and 
         [0073]    (f) necessary buffers and/or reagents required to perform the assay. 
         [0074]    Another exemplary kit comprises, but is not limited to: 
         [0075]    (a) (an) antibody(ies) capable of binding any one of the lipids in Tables 2 to 5, conjugated to an enzyme; and optionally one or more of the following: 
         [0076]    (b) a substrate specific for said enzyme; 
         [0077]    (c) a stop solution; 
         [0078]    (d) an assay plate coated with (an) antibody(ies) capable of binding any of the lipids in Tables 2 to 5; 
         [0079]    (e) (a) standard(s) and/or (a) calibration line standard(s); and 
         [0080]    (f) necessary buffers and/or reagents required to perform the assay. 
         [0081]    A further exemplary kit comprises, but is not limited to: 
         [0082]    (a) any one of the lipid(s) in Tables 2 to 5 conjugated to an enzyme; and optionally one or more of the following: 
         [0083]    (b) a substrate specific for said enzyme; 
         [0084]    (c) a stop solution; 
         [0085]    (d) an assay plate coated with (an) antibody(ies) capable of binding any of the lipids in Tables 2 to 5; 
         [0086]    (e) (a) standard(s) and/or (a) calibration line standard(s); and 
         [0087]    (f) necessary buffers and/or reagents required to perform the assay. 
         [0088]    A further exemplary kit comprises, but is not limited to: 
         [0089]    (a) (an) antibody(ies) capable of binding any one of the lipids in Tables 2 to 5, conjugated to a detectable label, e.g., biotin; and optionally one or more of the following: 
         [0090]    (b) a substrate specific for said enzyme; 
         [0091]    (c) a stop solution; 
         [0092]    (d) an assay plate coated with (an) antibody(ies) capable of binding any of the lipids in Tables 2 to 5; 
         [0093]    (e) (a) standard(s) and/or (a) calibration line standard(s); and 
         [0094]    (f) necessary buffers and/or reagents required to perform the assay. 
         [0095]    Yet a further exemplary kit comprises, but is not limited to: 
         [0096]    (a) any one of the lipid(s) in Tables 2 to 5 conjugated to a detectable label; and optionally one or more of the following: 
         [0097]    (b) a substrate specific for said enzyme; 
         [0098]    (c) a stop solution; 
         [0099]    (d) an assay plate coated with (an) antibody(ies) capable of binding any of the lipids in Tables 2 to 5; 
         [0100]    (e) (a) standard(s) and/or (a) calibration line standard(s); and 
         [0101]    (f) necessary buffers and/or reagents required to perform the assay. 
         [0102]    Preferred kits according to the above embodiments comprise, for example, the following combinations of the above listed constituents: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f), (a), (e) and (f), (a), (b), (c) and (d); (a) (c), (d) and (e); (a), (d), (e) and (f); (a), (b), (c), (d) and (e); or (a), (c), (d), (e) and (f). 
         [0103]    In another preferred example of a kit of the above embodiment, the kit further comprises an antibody which is conjugated to an enzyme and which is capable of binding to the antibody in (a) of the above embodiments. 
         [0104]    In a further preferred example of a kit of the above embodiment, the kit further comprises an antibody which is conjugated to a detectable label; e.g., biotin or a fluorescent label, and which is capable of binding to the antibody in (a) of the above embodiments. 
         [0105]    In a further preferred example of a kit of the above embodiments, the kit further comprises an enzyme conjugated to a protein, which is specific to the detectable label on the antibody in (a) of the above embodiments, e.g., alkaline phosphatase conjugated to streptavidin. 
         [0106]    Also comprised in the invention is the use of the kits of the invention for performing the methods described and claimed herein. The kit used in this regard may be a competitive ELISA, and comprise 
         [0107]    (a) (an) antibody(ies) or antiserum against any one of the lipids in Tables 2 to 5; 
         [0108]    (b) any one of the lipid(s) in any one of Tables 2 to 5 conjugated to an enzyme; 
         [0109]    (c) (a) standard(s) and/or (a) calibration line standard(s); 
         [0110]    (d) an assay plate coated with an antibody capable of binding an antibody, e.g., an antibody capable of binding a rabbit Ig, a goat Ig, a mouse Ig, a guinea pig Ig, a rat Ig, or a sheep Ig; 
         [0111]    (e) a substrate specific for said enzyme; 
         [0112]    (f) a stop solution; and 
         [0113]    (g) reagent(s) for performing said methods or uses. 
         [0114]    In one example, the standard(s) and/or calibration line standard(s) is (are) lipid standard(s) chosen from the lipids defined in any one of the lipids in Tables 2 to 5. 
         [0115]    An exemplary use of this competitive ELISA kit will be as follows: 
         [0116]    1. Wells of an assay plate are coated with an antibody capable of binding a rabbit antibody. 
         [0117]    2. Samples are added to wells, and optionally (a) calibration line standard(s) is (are) added to other wells. 
         [0118]    3. A solution of any one of the lipids in Tables 2 to 5, conjugated to alkaline phosphatase, is added followed by a rabbit polyclonal antibody capable of binding any one of the lipids in Tables 2 to 5. 
         [0119]    4. During sample incubation the rabbit polyclonal antibody binds in a competitive manner to any one of the lipids in Tables 2 to 5, in the samples or conjugate. 
         [0120]    5. The plate is washed leaving only the bound lipid in the samples or conjugate. 
         [0121]    6. A specific substrate solution is added, which results in a color reaction when catalyzed by the alkaline phosphatase on the lipid conjugate. 
         [0122]    7. The reaction is stopped by a specific stop solution and the color development in each well is read at a proper light length, e.g., at 405 nm. 
         [0123]    8. In a competitive ELISA, the color intensity is indirectly proportional to the amount of lipid, in the sample. 
         [0124]    All kits of the present invention may be accompanied by instructions to use them (i) for determining the efficacy of a treatment with a lipid-lowering drug in a subject, (ii) for predicting the efficacy of a treatment with a lipid-lowering drug in a subject, (iii) for determining the compliance of a subject with a lipid-lowering drug treatment, (iv) for identifying compounds that are useful as lipid-lowering drugs or for treating cardiovascular disease and its complications, or (v) for determining the specificity of a PCSK9 inhibitor/silencer, all as defined herein. 
         [0125]    In the context of all aspects and embodiments of the invention described and claimed herein, the determination of the lipid concentration(s) or the lipid ratio(s) is typically performed using an assay. 
         [0126]    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&#39;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), ultra performance liquid chromatography (UPLC) 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. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0127]      FIG. 1 . 
           [0128]    The mean percentage changes in lipid concentrations between patients who died due to CVD complications vs. patients with stable CAD (A), carriers of R46L loss-of-function mutation vs. control (B), patients after treatment with atorvastatin vs. same patients before treatment (C), and patients after treatment with simvastatin vs. same patients before treatment (D). Cer, ceramide; LacCer, lactosylceramide. 
           [0129]      FIG. 2 . 
           [0130]    The lipid changes caused by specific PSCK9 inhibition (patients having one PCSK9 loss-of-function gene). Also shown are the lipid changes caused by the lipid lowering drugs atorvastatin and simvastatin. 
           [0131]      FIG. 3 . 
           [0132]    Percentage differences in molecular lipid mean concentrations in plasma of PCSK9 −/−  (A) and PCSK9 +/−  (B) mice on regular chow or on Western diet (C and D, respectively) as compared to wildtype (WT). Significance is based on Student&#39;s t-test. Each symbol corresponds to a lipid molecule of a certain category measured by MS application. 
           [0133]      FIG. 4 . 
       
    
    
       [0134]    The percentage differences in molecular lipid mean concentrations in plasma of human male CAD patients who carry the R46L variant in PCSK9 gene in comparison to the CAD patients without the R46L mutation. Significance in based on Student&#39;s t-test. Each point corresponds to a lipid molecule measured by MS application or by a clinical kit. 
       DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
       [0135]    Some abbreviations used herein have the following meaning: ADR is adverse drug reaction, MS is mass spectrometry, HPLC is high performance liquid chromatography, and UPLC is ultra high performance liquid chromatography. 
         [0136]    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. 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. 
         [0137]    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. 
         [0138]    For the purposes of the present invention, a lipid lowering drug or medication is preferably a PCSK9 inhibitor or silencer. 
         [0139]    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 (e.g., mice and rats). A particularly preferred subject in accordance with the present invention is a human. 
         [0140]    As used herein a high risk subject is typically a subject, particularly a human, on high drug dose and/or on multiple medications (causing a risk for drug interactions), having a disease that may affect the drug efficacy or increase the risk of adverse events (e.g., hypothyroidism, renal insufficiency or a liver disease). 
         [0141]    A sample as used herein 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, with blood serum and blood plasma being preferred. 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. Venous 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. The sample may also be a fraction of whole blood, blood plasma or blood serum, e.g., a lipoprotein fraction. In another preferred embodiment, the sample may also be a tissue sample, e.g., muscle biopsy tissue, or urine, or a fraction thereof (e.g., a lipoprotein fraction). 
         [0142]    The lipids or other molecules in the control to which the comparison is made in accordance with the present invention are referred to herein also as control markers. 
         [0143]    As used herein, a control may be a control sample or merely a control value. In case it is a control value, it will be appreciated that it may have already been determined, calculated or extrapolated prior to initiating the methods of the invention. Alternatively, the control value may be determined, calculated or extrapolated after conducting the determination of the concentration(s) of said one or more lipid(s) or said one or more lipid ratio(s) in accordance with the methods of the present invention. Thus, it will be appreciated that a suitable control value in accordance with the present invention may well be one that is taken from the literature. 
         [0144]    As used herein, the reference to a control sample from the same subject or from a(nother) subject may mean that the control sample has been directly obtained from said subject. Alternatively, however, it may also mean that it has been obtained as the result of a physical or chemical treatment of a sample directly obtained or taken from said subject, such as centrifugation, fractionation, enzymatic digestion, precipitation, and the like. The same applies to any reference herein to a control sample from one or more subjects, from from a group of subjects or from a population of subjects. 
         [0145]    The terms control sample from one or more subjects, or control sample from a group of subjects or control sample from a population of subjects as used herein furthermore preferably entail that the control sample is representative of said more than one subjects, group of subjects or population of subjects. In this context, representative shall mean that the concentration(s) of the one or more lipids in said control sample to which a comparison is made in the context of the present invention corresponds to the average concentration(s) of said lipid(s) in corresponding individual samples from the subjects of said group or population. Preferably, the concentrations of all lipids in said control sample correspond to the average concentrations of said lipids in corresponding individual samples from the subjects of said group or population. Likewise, where a comparison is made in the context of the present invention to one or more other molecules, e.g., other lipids or proteins, such as total PC, or apoA, apoB, or albumin, respectively, a representative control sample is one where the concentration(s) of this (these) molecule(s) corresponds to the average concentration(s) of said molecule(s) in corresponding individual samples from the subjects of said group or population. In a preferred embodiment, a control sample from one or more subjects, a control sample from a group of subjects or a control sample from a population of subjects in the sense of the present invention is obtained by mixing equal amounts of samples directly obtained or taken from the subjects of said more than one subjects, group or population, or by mixing equal amounts of fractions, constituents or reaction products (e.g., enzymatic reaction products or precipitates) thereof. 
         [0146]    As used herein a control sample corresponds to the subject&#39;s sample if it has been obtained from the same type of biological tissue or source in the same, or essentially the same, manner. For example, if the subject&#39;s sample is a whole blood, blood plasma or blood serum sample, or a fraction thereof, a corresponding control sample will likewise be a whole blood, blood plasma or blood serum sample, or a fraction thereof, respectively. It will be appreciated that such corresponding control sample would include whole blood, blood plasma or blood serum samples, or fractions thereof, obtained by mixing the whole blood, blood plasma or blood serum samples, or certain fractions thereof, from a group or population of subjects (see also the further explanations herein and the claims regarding suitable control samples in accordance with the invention). The same applies mutatis mutandis to, e.g., tissue and urine samples. 
         [0147]    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 concentration ratios, or the lipid-clinical concentration ratios or combinations thereof as specifically described and/or claimed 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. 
         [0148]    A lipid as used herein is defined as hydrophobic or amphiphilic small molecule. 
         [0149]    For the purposes of the present invention, lipids are referred to according to the following nomenclature: CE is cholesteryl ester, DAG is diacylglycerol, TAG is triacylglycerol, PC is phosphatidylcholine, PC O is alkyl-linked PC, PC P is alkenyl-linked PC, LPC is lysophosphatidylcholine, PE is phosphatidylethanolamine, PE O is alkyl-linked PE, PE P is alkenyl-linked PE, PI is phosphatidylinositol, Cer is ceramide, Glc/GalCer is galactosyl- or glucosylceramide, LacCer is lactosylceramide, Gb3 is Globotriaosylceramide, SM is sphingomyelin, S1P is sphingosine-1-phosphate, SPH is sphingosine, SA1P is sphinganine-1-phosphate, SPA is sphinganine. 
         [0150]    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. 
         [0151]    The nomenclature A/B indicates, for a molecule of DAG and PC, A and B types of fatty acid moieties attached to the glycerol backbone of the molecule. 
         [0152]    The nomenclature (dC/A) indicates, for a molecule of Cer, Gb, GlcCer, LacCer and SM, C the type of long-chain base with an amide-linked, A, fatty acid moiety. 
         [0153]    An “increase”, “decrease”, or “difference” compared to a control, according to the invention, is one that is (i) indicative of efficacy of a treatment with a lipid-lowering drug in a subject, (ii) predictive of efficacy of a treatment with a lipid-lowering drug in a subject, (iii) indicative of compliance of a subject with a lipid-lowering drug treatment, (iv) indicative of compounds that are useful as lipid-lowering drugs or for treating cardiovascular disease and its complications, or (v) indicative of specificity of a PCSK9 inhibitor/silencer, respectively. Preferably, it is an increase, decrease or difference of at least 5%. More preferably, it is an increase, decrease or difference of at least 10%. Other preferred increases, decreases or differences compared to the control in accordance with the invention are increases, decreases or differences of at least 15%, more preferably at least 20%, and even more preferably of at least 25%, 50%, 75% or 100%. Increases, decreases or differences of more than 100% are likewise particularly preferred. 
         [0154]    For the purposes of the present invention, a PCSK9 inhibitor/silencer is a molecule that prevents PCSK9 from binding to the LDL receptor, particularly to the LDL receptors present in the liver. As noted earlier herein, the PCSK9 inhibitor/silencer is preferably (a) an antibody against PCSK9; (b) a drug inhibitor of PCSK9; (c) a small molecule that inhibits the interaction of the LDL-receptor with PCSK9; (d) a peptide that mimics the interaction domain of the LDL-receptor with PCSK9, (e) an siRNA specific for PCSK9, particularly PCSK9 mRNA; or (0 an antisense oligonucleotide specific for PCSK9, particularly PCSK9 mRNA. 
         [0155]    In a preferred embodiment, the PCSK9 inhibitor/silencer is an antibody that prevents PCSK9 from binding to the LDL receptor. Such antibodies are well known in the art (see, e.g., WO2009/055783, WO2008/063382, WO2009/100297, WO2008/125623 or WO2009/026558, all incorporated herein by reference) and may be suitably used in the context of the present invention. 
         [0156]    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, singe chain variable domain 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&#39;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&#39;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. 
         [0157]    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. 
         [0158]    Fusion experiments are then performed according to the procedure of Stewart &amp; 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. 
         [0159]    As used herein the term small molecule refers to a low molecular weight organic compound. Preferably, the upper molecular weight limit for a small molecule in accordance with the present invention is 2,500 Daltons, more preferably 1,500 Daltons, and particularly preferred 800 Daltons. The size and charge of the small molecules of the present invention will preferably be such that they allow for the possibility to rapidly diffuse across cell membranes so that the small molecules can reach intracellular sites of action. 
         [0160]    A drug inhibitor of PCSK9 as used herein may be a small molecule. It may, however, also be a polymer, e.g., a polypeptide other than an antibody, a glycoprotein, a proteoglycan, a nucleic acid, e.g., an aptamer, a carbohydrate, or a lipid. 
       EXAMPLES 
     Example 1 
     Materials and Methods 
       [0161]    Plasma samples from wild-type (Wt), PCSK9 homozygote knock-out (Pcsk9−/−), and PCSK9 heterozygote knock-out (Pcsk9+/−) animals were used for lipidomic analyses. Each group had 18 male mice aged 3 months. Up to 3 months&#39; age the mice were on the same regular chow diet (day 0). Thereafter, mice were first on regular chow-diet (2018 Teklad Global, Harlan Laboratories) for two weeks after which 3 mice from each group were sacrificed for tissue sampling (day 15). The remaining mice were switched to standard Western diet (TD.88137 Harlan Teklad) for a period of two weeks after which all remaining mice were sacrificed (day 30). The Western diet contained 34%, 21%, and 0.2% of sugar, fat, and cholesterol, respectively, whereas the regular chow diet contained 5%, 6%, and 0% of these ingredients, respectively. 
         [0162]    Mice were kept fasted for 4 h before bleeding. Cheek bleeds of about 250 μl were drawn using the 500 μl microcontainers (BD) containing EDTA. The blood samples were centrifuged at 3000 rpm for 15 min at 4° C. The supernatants (50 to 100 μl) were transferred to clean Eppendorf tubes. The samples were frozen immediately upon sampling and stored at −80° C. prior to lipidomic analyses. 
         [0163]    For Shotgun lipidomic analyses, 10 μl of mouse plasma were used for lipid extraction. For quantification of ceramides and cerebrosides, 50 μl of mouse plasma were used for lipid extraction. This study design allowed inventors to determine a typical lipidomic profile induced by total (−/−) or partial (+/−) PCSK9 inhibition both on regular chow and Western diet. 
       Example 2 
     Materials and Methods 
       [0164]    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 and routine clinical laboratory data. In this study, the inventors compared lipidomic profile in subjects carrying a know loss-of-function mutation (R46L, rs11591147, Abifadel, M. et al. 2003. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.  Nat Genet  34: 154-156) with the lipidomic profile in subjects carrying the major allele with normal PCSK9 function. This comparison allowed inventors to determine a typical lipidomic profile induced by PCSK9 partial deficiency. The clinical characteristics are described in Table 1. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Background characteristics for LURIC patients analyzed with lipidomics 
               
             
          
           
               
                 Variable 
                 Controls (n = 541) 
                 Cases (n = 12) 
               
               
                   
               
             
          
           
               
                 Age (average) 
                 65.1 
                 66.3 
               
               
                 LDL-C (mg/dL) 
                 116.8 
                 108.3 
               
               
                 HDL-C (mg/dL) 
                 36.8 
                 38.2 
               
               
                 Lipid lowering users 
                 248 
                 2 
               
               
                   
               
             
          
         
       
     
         [0165]    Analytical Methods 
         [0166]    Mass Spectrometry Driven Lipidomics 
         [0167]    Direct infusion coupled to tandem mass spectrometry, i.e. shotgun lipidomics, triacylglycerol lipidomics, and a liquid chromatography tandem mass spectrometry (LC-MS/MS) approach, i.e. ceramide and cerebroside and spingosine lipidomics, were used to identify the effect of diminished PCSK9 concentration by analyzing molecular lipid species in human and mouse serum. The applied methods were optimized especially for quantification of molecular cholesteryl esters (CE), free cholesterol (FC), phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and other lysophospholipids (LPL), alkyl- and alkenyl-linked phosphatidylcholines (PC O and PC P, respectively) and other alkyl- and alkenyl-linked phospholipids (PL O and PL P, respectively), phosphatidylserines (PS), phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI), phosphatidic acids (PA), diacylglycerols (DAG), triacylglycerols (TAG), ceramides (Cer), glucosyl/galactosylceramides (Glc/GalCer), lactosylceramides (LacCer), globotriaosylceramides (Gb3), sphingosines (SPH), sphingosine-1-phosphates (S1P), sphinganines (SPA), and sphinganine-1-phosphates (SA1P). 
         [0168]    The following materials were used according to the methods. High performance liquid chromatography (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). 
         [0169]    In ceramide and cerebroside lipidomics, the ultra high performance liquid chromatography (UHPLC) 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). In sphingosine and sphingosine-1-phophate lipidomics, the hydrophilic interaction liquid chromatography (HILIC) column (Atlantis HILIC 3 mm 2.1×50 mm) and the HPLC guard-column (Atlantis HILIC 3 mm 2 1×10 mm) were purchased from Waters (Milford, Mass., 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), Matreya (Pleasant Gap, Pa., USA), and Cayman Chemical (Ann Arbor, Mich., USA). 
         [0170]    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. 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 or 1000 μM. An internal standard mixture containing each of the standard stock was created and used in lipid extraction. 
         [0171]    10 μl of human and mouse plasma were used for shotgun and/or triacylglycerol lipidomics and/or quantification of free cholesterol and 10 μl and 50 μl of human and mouse plasma, respectively, for ceramide and cerebroside and/or sphingosine and sphingosine-1-phophate lipidomics. Human samples were not analyzed for triacylglycerols, free cholesterol, and sphingoid bases. Lipid extractions were 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. 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 and methanol. In shotgun, triacylglycerol, ceramide and cerebroside, and sphingosine and sphingosine-1-phophate 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 N 2  until dryness. The lipid extracts were then re-dissolved in chloroform:methanol (1:2, v/v) including the addition of the synthetic external standard. The extracts were stored in 2 ml safe-lock Eppendorf tubes at −20° C. prior to MS analysis. Required volumes of lipid extracts were aliquoted into an Eppendorf 96-well twin.tec PCR plate and the plate was heat-sealed with aluminum foil to avoid evaporation. 
         [0172]    In shotgun and triacylglycerol lipidomics as well as when quantifying free cholesterol, 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). Triacylglycerols were analyzed in positive ion mode using neutral loss scanning. Free cholesterol was derivatized into CE 2:0 by acetyl chloride before analysis (Liebisch, G., et al.,  High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry  ( ESI - MS/MS ).  Biochim Biophys Acta,  2006. 1761(1): p. 121-8). 
         [0173]    In ceramide and cerebroside lipidomics, the LC-MS/MS 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, and the Acquity BEH C18 column with an in-line pre-column. 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. 
         [0174]    In sphingosine and sphingosine-1-phosphate lipidomics, the LC-MS/MS 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 50° C., and the Atlantis HILIC column with an in-line guard-column. The extracted samples, 10 μl of each, were injected into the guard-column followed by the analytical column and delivered to the mass spectrometer at a flow rate of 500 μl/min. In sphingosine and sphingosine-1-phosphate lipidomics, a gradient was used for lipid analyte separation with solvent A comprising 50 mmol/1 ammonium formate in ultra pure water with 0.2% formic acid and solvent B of acetonitrile with 0.2% formic acid. The gradient was constructed in the following way: 0 min-95% B; 0.70 min-95% B; 1.50 min-75% B; 1.51 min-50% B; 1.70 min-50% B; 1.71 min-85% B; 3.00 min-85% B; 3.10 min-95% B; 4.00 min-95% B. 
         [0175]    In both ceramide and cerebroside lipidomics and sphingosine and sphingosine-1-phosphate lipidomics, the lipid extracts were analyzed by LC-MS/MS. The MS analysis was performed on a hybrid triple quadrupole/linear ion trap mass spectrometer equipped with the Turbo V™ Ion Source (4000 QTRAP, AB Sciex). The instrument was operating in positive ion mode. The ion source voltage was set to 5500V and source temperature at 400° C. The collision energy (CE) and declustering potential (DP) was optimized for each lipid class using synthetic standards. A 20/25 sec dwell time was applied for each scan. Multiple reaction monitoring (MRM) scan mode was applied and based on the description by Sullards and colleagues (Sullards M C, et al:  Structure - specific, quantitative methods for analysis of sphingolipids by liquid chromatography - tandem mass spectrometry: “inside - out” sphingolipidomics. Methods Enzymol  2007). 
         [0176]    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 to retrieve their concentrations. 
         [0177]    The ratio of synthetic Internal Standards (IS) to corresponding post-extract spiked External Standards (ES), and MS analysis of extracted matrix and solvents served as quality controls (QC) of the analysis. In addition, extracted reference plasma samples were analyzed for monitoring the instruments&#39; performance, i.e., the intra- and inter-assay variation. 
         [0178]    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. 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. 
         [0179]    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 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. 
         [0180]    Statistical Analyses 
         [0181]    Percentage changes in lipid concentrations between control and case groups were calculated as follows: 
         [0000]      100*(AVG[ C ] in case group−AVG[ C ] in control group)/AVG[C] in control group.
 
         [0182]    Statistical significance was assigned based on t-test. 
         [0183]    Ethics 
         [0184]    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. The mouse study was approved by the IRCM bioethics committee for animal care. 
         [0185]    Results 
         [0186]    The lipidomic biomarkers appeared as significant biomarkers of the diminished PCSK9 activity. A loss of one Pcsk9 allele was sufficient to induce significant changes in sphingolipid concentrations, comparable to those seen in Pcsk9 −/−  mice. The fatty acid 16:0 containing sphingomyelin [SM(d18:1/16:0)], ceramide [Cer(d18:1/16:0)], glucosyl/galactosylceramide [Glc/GalCer(d18:1/16:0)], and lactosylceramide [LacCer(d18:1/16:0)] species appeared as the most affected lipid species in both Pcsk9 −/−  and Pcsk9 +/−  mice when the animals were on chow diet ( FIGS. 1A and 1B , and Table 2a and 2b, respectively). On the contrary, triacylglycerols (TAG) were shown to be significantly more concentrated in the plasma of Pcsk9 −/−  and Pcsk9 +/−  mice than in wild-type (Wt) mice. 
         [0187]    As compared to the regular chow diet condition, clearly less significant changes were recorded for Pcsk9 −/−  mice on Western diet ( FIG. 1C , Table 2c). Interestingly, on Western diet, Pcsk9 +/−  mice demonstrated more significant changes in lipid concentrations than Pcsk9 −/−  mice ( FIG. 1D , Table 2d). A typical change in the plasma of PCSK9-deficient mice on Western diet appeared to be decreased Cer, Glc/GalCer, and LacCer species with long fatty acyl chains, such as the Glc/GalCer(d18:1/24:0), the Cer(d18:1/24:0) and the LacCer(d18:1/24:0). 
         [0188]    A similar lipidomic change was observed in humans carrying a PCSK9 loss-of-function mutation. The most significantly reduced lipid species in human carriers of the known loss-of-function variant in the PCSK9 gene (R46L) included the same Glc/GalCer, LacCer and SM species, as already observed in the Pcsk9 −/−  and Pcsk9 +/−  mouse models ( FIG. 2 , Table 3). However, due to the limited number of human samples most of the observed changes did not reach the level of statistical significance. Furthermore, in fasting human plasma samples, no separation of lipid species based on fatty acyl chain length could be observed. In contrast to mouse plasma, concentrations of two cholesteryl ester species, the CE 20:3 and the CE 20:4 were significantly reduced in human plasma due to the PCSK9-deficiency. 
         [0189]    As a total 109 molecular lipids and 257 TAG fatty acids were quantified in this study as described above. Out of those 63 molecular lipids and 105 TAG fatty acids were significant biomarkers based on set criteria. The significant biomarker candidates based on molecular lipid concentrations are presented in Tables 2 and 3. In human, the selected biomarkers had improved performance over traditionally used biomarkers such as LDL-cholesterol. The individual lipids having improved performance over traditional markers are listed in Table 3a. Table 3b lists lipid-lipid concentration ratios having improved performance over individual lipids. 
         [0190]    The preferred embodiments selected among the identified biomarker candidates are listed in Tables 4 and 5. 
         [0191]    Table 6 presents the brutto TAG species and the possible fatty acid combinations contributing to each brutto species. Brutto TAG presents the sum of the three fatty acids and the number of double bonds of a TAG molecule. Table 6 presents few examples for each brutto TAG species but there might be other combinations as well. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2a 
               
               
                   
               
               
                 Significant biomarkers based on individual lipid or lipid-lipid concentration ratio 
               
               
                 measurement detected in PCSK9 −/−  mice on regular chow diet in comparison to wildtype. Species 
               
               
                 names, p-values, and percentage changes are presented. The different fatty acid compositions are 
               
               
                 described in Table 6. 
               
               
                 PCSK9 −/−  vs. wildtype mice on regular chow diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
                   
               
               
                 Decreased 
                 change 
                 p-value 
                 Increased 
                 change 
                 p-value 
               
               
                   
               
               
                 TAG 60:12 FA 20:4 
                 −64.990 
                 2.508E−04 
                 TAG 50:5 
                 156.815 
                 1.316E−02 
               
               
                 TAG 60:12 
                 −64.990 
                 2.508E−04 
                 TAG 55:3 
                 152.265 
                 2.968E−02 
               
               
                 TAG 58:10 FA 20:4 
                 −64.073 
                 1.341E−03 
                 TAG 51:4 FA 15:0 
                 86.98016 
                 1.780E−02 
               
               
                 TAG 58:10 
                 −22.734 
                 1.905E−01 
                 TAG 51:4 
                 145.866 
                 9.870E−03 
               
               
                 LacCer(d18:1/24:0) 
                 −61.734 
                 1.802E−06 
                 TAG 50:2 FA 18:2 
                 76.248 
                 7.679E−02 
               
               
                 Glc/GalCer(d18:1/20:0) 
                 −61.304 
                 5.021E−07 
                 TAG 50:2 
                 123.880 
                 1.296E−01 
               
               
                 Glc/GalCer(d18:1/18:0) 
                 −60.966 
                 7.032E−14 
                 TAG 50:4 FA 14:0 
                 77.875 
                 2.216E−02 
               
               
                 Glc/GalCer(d18:1/22:0) 
                 −58.750 
                 2.897E−06 
                 TAG 50:4 
                 128.492 
                 7.890E−02 
               
               
                 TAG 56:9 FA 20:4 
                 −57.499 
                 4.513E−04 
                 TAG 54:3 FA 16:0 
                 62.577 
                 3.325E−02 
               
               
                 TAG 56:9 FA 18:3 
                 −37.299 
                 1.072E−02 
                 TAG 54:3 FA 18:2 
                 99.394 
                 1.796E−02 
               
               
                 TAG 56:9 
                 −28.883 
                 1.264E−01 
                 TAG 54:3 
                 106.898 
                 3.787E−02 
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −56.593 
                 4.968E−12 
                 TAG 53:4 FA 18:2 
                 88.635 
                 8.278E−03 
               
               
                 Total Glc/GalCer 
                 −55.866 
                 6.261E−07 
                 TAG 53:4 
                 106.150 
                 6.500E−03 
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH) 
                 −54.947 
                 2.058E−16 
                 TAG 52:6 
                 101.188 
                 2.561E−02 
               
               
                 LacCer(d18:1/16:0) 
                 −54.523 
                 8.517E−10 
                 TAG 54:4 FA 18:2 
                 84.418 
                 2.500E−02 
               
               
                 TAG 58:9 FA 20:4 
                 −53.838 
                 6.457E−04 
                 TAG 54:4 FA 18:1 
                 78.619 
                 4.889E−02 
               
               
                 TAG 58:9 
                 −1.979 
                 8.979E−01 
                 TAG 54:4 FA 18:0 
                 77.628 
                 9.456E−03 
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −53.028 
                 6.967E−05 
                 TAG 54:4 
                 83.059 
                 3.721E−02 
               
               
                 Total LacCer 
                 −51.984 
                 4.137E−08 
                 TAG 52:4 FA 18:2 
                 70.069 
                 2.305E−02 
               
               
                 CE 16:1 
                 −51.509 
                 3.221E−05 
                 TAG 52:4 FA 16:0 
                 64.013 
                 2.820E−02 
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −51.294 
                 5.712E−05 
                 TAG 52:4 
                 73.643 
                 2.485E−02 
               
               
                 Glc/GalCer(d18:1/26:0) 
                 −50.524 
                 2.374E−04 
                 TAG 56:4 FA 18:1 
                 91.940 
                 4.847E−02 
               
               
                 Cer(d18:1/22:0) 
                 −49.683 
                 9.847E−08 
                 TAG 56:4 
                 89.544 
                 5.546E−02 
               
               
                 Cer(d18:1/20:0) 
                 −48.392 
                 2.989E−09 
                 TAG 54:5 FA 18:1 
                 75.955 
                 1.000E−02 
               
               
                 SM (d18:1/16:1) (d18:1/15:2-OH) 
                 −47.581 
                 6.693E−07 
                 TAG 54:5 FA 18:2 
                 74.494 
                 4.962E−03 
               
               
                 Cer(d18:1/18:0) 
                 −46.286 
                 3.817E−06 
                 TAG 54:5 
                 70.166 
                 1.239E−02 
               
               
                 CE 14:0 
                 −45.541 
                 3.299E−05 
                 TAG 52:5 FA 18:2 
                 71.092 
                 4.892E−02 
               
               
                 CE 16:0 
                 −45.218 
                 1.010E−12 
                 TAG 52:5 FA 18:3 
                 61.858 
                 3.526E−02 
               
               
                 LacCer(d18:1/24:1) 
                 −44.119 
                 1.054E−05 
                 TAG 52:5 
                 69.317 
                 4.417E−02 
               
               
                 CE 18:1 
                 −42.857 
                 1.547E−10 
                 TAG 54:6 FA 18:2 
                 78.005 
                 2.522E−03 
               
               
                 CE 18:2 
                 −41.802 
                 6.076E−11 
                 TAG 54:6 FA 18:1 
                 65.617 
                 1.854E−02 
               
               
                 FC 
                 −41.774 
                 1.139E−10 
                 TAG 54:6 FA 18:3 
                 57.726 
                 3.365E−02 
               
               
                 CE 18:3 
                 −41.356 
                 2.182E−10 
                 TAG 54:6 
                 52.606 
                 1.530E−02 
               
               
                 LacCer(d18:1/22:0) 
                 −41.134 
                 1.239E−03 
                 TAG 56:5 FA 18:2 
                 71.286 
                 2.510E−02 
               
               
                 TAG 56:8 FA 20:4 
                 −40.099 
                 9.167E−03 
                 TAG 56:5 FA 20:1 
                 68.247 
                 2.564E−02 
               
               
                 TAG 56:8 
                 8.278 
                 6.507E−01 
                 TAG 56:5 
                 71.820 
                 4.739E−02 
               
               
                 CE 22:5 
                 −39.993 
                 1.070E−02 
                 TAG 56:6 FA 18:2 
                 58.754 
                 3.922E−02 
               
               
                 Total CE 
                 −39.638 
                 1.073E−09 
                 TAG 56:6 
                 30.327 
                 2.893E−01 
               
               
                 PC 16:0/16:0 
                 −39.452 
                 4.758E−06 
                 TAG 54:7 FA 18:2 
                 52.396 
                 2.256E−02 
               
               
                 CE 22:6 
                 −39.271 
                 2.050E−08 
                 TAG 54:7 FA 18:3 
                 50.258 
                 4.750E−02 
               
               
                 PC 16:0/18:1 
                 −39.038 
                 8.272E−06 
                 TAG 54:7 
                 40.748 
                 4.048E−02 
               
               
                 Total Cer 
                 −38.285 
                 5.399E−06 
                 Total TAG 
                 47.255 
                 4.230E−02 
               
               
                 Total LPE 
                 −38.219 
                 3.272E−02 
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/26:1) 
                 −37.439 
                 1.426E−02 
                   
                   
                   
               
               
                 TAG 56:7 FA 20:4 
                 −3 6.094 
                 2.192E−03 
                   
                   
                   
               
               
                 TAG 56:7 
                 4.516 
                 8.231E−01 
                   
                   
                   
               
               
                 PC 18:0/18:1 
                 −35.534 
                 2.324E−05 
                   
                   
                   
               
               
                 CE 20:4 
                 −34.208 
                 1.783E−05 
                   
                   
                   
               
               
                 CE 20:3 
                 −33.966 
                 3.821E−02 
                   
                   
                   
               
               
                 SM (d18:1/24:1) (d18:1/23:2-OH) 
                 −33.856 
                 7.152E−04 
                   
                   
                   
               
               
                 Cer(d18:1/24:1) 
                 −33.646 
                 9.245E−05 
                   
                   
                   
               
               
                 CE 17:1 
                 −33.524 
                 4.173E−03 
                   
                   
                   
               
               
                 PC 18:1/18:1 
                 −33.350 
                 1.936E−02 
                   
                   
                   
               
               
                 Cer(d18:1/24:0) 
                 −33.095 
                 1.606E−03 
                   
                   
                   
               
               
                 CE 20:5 
                 −32.845 
                 8.999E−03 
                   
                   
                   
               
               
                 Total PC 
                 −32.432 
                 1.481E−07 
                   
                   
                   
               
               
                 PC 16:0/20:3 
                 −32.324 
                 1.542E−02 
                   
                   
                   
               
               
                 PC 16:0/18:2 
                 −32.309 
                 2.042E−07 
                   
                   
                   
               
               
                 PC 18:0/18:2 
                 −32.095 
                 1.116E−05 
                   
                   
                   
               
               
                 PC 18:0/22:6 
                 −30.973 
                 2.463E−04 
                   
                   
                   
               
               
                 PC 18:1/20:4 
                 −30.513 
                 1.352E−02 
                   
                   
                   
               
               
                 PC 16:0/22:6 
                 −30.321 
                 1.159E−05 
                   
                   
                   
               
               
                 PI 18:0/20:4 
                 −29.759 
                 3.149E−06 
                   
                   
                   
               
               
                 CE 15:0 
                 −27.608 
                 1.578E−02 
                   
                   
                   
               
               
                 Cer(d18:1/16:0) 
                 −26.897 
                 1.773E−03 
                   
                   
                   
               
               
                 Cer(d18:1/26:1) 
                 −30.721 
                 7.786E−03 
                   
                   
                   
               
               
                 LPC 16:0 
                 −20.017 
                 4.196E−04 
                   
                   
                   
               
               
                 Total LPC 
                 −17.349 
                 9.137E−04 
               
               
                   
               
               
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 change 
                 p-value 
                 Increased 
                 change 
                 p-value 
               
               
                   
               
               
                 Glc/GalCer(d18:1/16:0)/LPC 18:2 
                 −50.893 
                 9.485E−10 
                 Cer(d18:0/24:0)/ 
                 185.871 
                 3.695E−06 
               
               
                   
                   
                   
                 Glc/GalCer(d18:1/18:0) 
                   
                   
               
               
                 Cer(d18:1/18:0)/Sphingosine  
                 −51.057 
                 1.878E−06 
                 Cer(d18:0/24:0)/ 
                 141.343 
                 2.425E−05 
               
               
                 d16:1 
                   
                   
                 LacCer(d18:1/16:0) 
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0)/PC  
                 −51.694 
                 1.060E−06 
                 TAG 58:10/TAG 60:12 
                 139.279 
                 6.521E−07 
               
               
                 18:2/18:2 
                   
                   
                   
                   
                   
               
               
                 CE 16:1/LPC 20:4 
                 −52.821 
                 7.039E−06 
                 Cer(d18:0/24:0)/SM 
                 138.225 
                 1.317E−06 
               
               
                   
                   
                   
                 (d18:1/16:0) (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0)/ 
                 −55.633 
                 1.568E−07 
                 Cer(d18:0/22:0)/SM 
                 119.864 
                 1.274E−07 
               
               
                 Sphinganine-1-phosphate d18:0 
                   
                   
                 (d18:1/16:0) (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0)/LPC 18:2 
                 −55.805 
                 2.189E−09 
                 LPC 18:2/SM (d18:1/16:0) 
                 98.384 
                 7.626E−12 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 LacCer(d18:1/16:0)/Sphingosine  
                 −57.265 
                 5.627E−06 
                 LPC 18:2/LacCer(d18:1/16:0) 
                 94.112 
                 3.915E−10 
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH)/ 
                 −58.151 
                 3.565E−08 
                 Cer(d18:0/24:0)/FC 
                 89.233 
                 0.000E+00 
               
               
                 Sphingosine d16:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0)/ 
                 −61.257 
                 1.345E−07 
                 Cer(d18:1/26:1)/ 
                 89.153 
                 3.214E−05 
               
               
                 Sphingosine d16:1 
                   
                   
                 Glc/GalCer(d18:1/18:0) 
                   
                   
               
               
                 CE 18:1/TAG 54:7 
                 −62.395 
                 8.602E−05 
                 Cer(d18:0/22:0)/ 
                 82.249 
                 1.926E−07 
               
               
                   
                   
                   
                 Cer(d18:1/22:0) 
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0)/ 
                 −66.767 
                 4.538E−06 
                 Cer(d18:1/26:1)/ 
                 81.958 
                 5.201E−05 
               
               
                 Sphingosine d18:1 
                   
                   
                 Glc/GalCer(d18:1/16:0) 
                   
                   
               
               
                 Cer(d18:1/18:0)/TAG 52:5 
                 −68.662 
                 9.244E−05 
                 LPC 16:0/SM (d18:1/16:0) 
                 79.420 
                 2.663E−10 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH)/ 
                 −72.138 
                 7.999E−05 
                 PC 18:0/20:4/SM 
                 70.011 
                 5.229E−08 
               
               
                 TAG 54:6 
                   
                   
                 (d18:1/16:0) (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/20:0)/TAG 52:5 
                 −79.327 
                 9.153E−05 
                 TAG 52:5/TAG 56:7 
                 66.808 
                 4.896E−07 
               
               
                 Glc/GalCer(d18:1/18:0)/TAG 54:6 
                 −79.363 
                 3.995E−05 
                 Cer(d18:1/16:0)/Glc/GalCer  
                 63.539 
                 2.374E−09 
               
               
                   
                   
                   
                 (d18:1/16:0) 
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0)/TAG 52:3 
                 −79.470 
                 6.181E−05 
                 CE 20:4/Glc/ 
                 62.767 
                 2.045E−08 
               
               
                   
                   
                   
                 GalCer(d18:1/18:0) 
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0)/TAG 54:3 
                 −79.758 
                 5.002E−05 
                 Cer(d18:0/24:0)/PC 16:0/18:2  
                 59.693 
                 0.000E+00 
               
               
                 Glc/GalCer(d18:1/16:0)/TAG 52:4 
                 −79.855 
                 3.166E−05 
                 CE 22:6/Glc/ 
                 50.609 
                 1.465E−07 
               
               
                   
                   
                   
                 GalCer(d18:1/18:0) 
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0)/TAG 52:4 
                 −82.312 
                 4.937E−05 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2b 
               
               
                   
               
               
                 Significant biomarkers based on individual lipid or lipid-lipid concentration ratio 
               
               
                 measurement detected in PCSK9 +/−  mice on regular chow diet in comparison to wildtype. Species 
               
               
                 names, p-values, and percentage changes are presented. 
               
               
                 PCSK9 +/−  vs. wildtype mice on regular chow diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
               
               
                 Decreased 
                 change 
                 p-value 
                 Increased 
                 change 
               
               
                   
               
               
                 TAG 60:12 FA 20:4 
                 −44.777 
                   
                 TAG 53:3 FA 17:0 
                 135.356 
               
               
                 TAG 60:12 
                 −44.777 
                   
                 TAG 53:3 FA 18:2 
                 110.794 
               
               
                 Glc/GalCer(d18:1/22:0) 
                 −38.116 
                 1.072E−03 
                 TAG 53:3 FA 18:1 
                 63.497 
               
               
                 TAG 58:7 FA 22:5 
                 −36.296 
                   
                 TAG 53:3 
                 98.444 
               
               
                 TAG 58:7 
                 −16.719 
                   
                 TAG 49:2 FA 18:2 
                 128.655 
               
               
                 Glc/GalCer(d18:1/20:0) 
                 −36.166 
                 1.126E−03 
                 TAG 49:2 FA 15:0 
                 81.288 
               
               
                 TAG 58:9 FA 20:4 
                 −35.280 
                   
                 TAG 49:2 FA 16:0 
                 46.357 
               
               
                 TAG 58:9 
                 −12.061 
                   
                 TAG 49:2 
                 81.946 
               
               
                 Total Glc/GalCer 
                 −34.200 
                 8.552E−04 
                 TAG 54:8 FA 18:3 
                 72.857 
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −34.067 
                 4.625E−03 
                 TAG 54:8 
                 45.668 
               
               
                 TAG 54:7 FA 16:1 
                 −31.485 
                   
                 TAG 50:4 FA 16:2 
                 71.219 
               
               
                 TAG 54:7 FA 20:4 
                 −19.366 
                   
                 TAG 50:4 FA 14:0 
                 65.347 
               
               
                 TAG 54:7 
                 25.776 
                   
                 TAG 50:4 FA 18:2 
                 52.949 
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −30.734 
                 1.378E−02 
                 TAG 50:4 FA 16:0 
                 31.487 
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH) 
                 −30.205 
                 1.297E−08 
                 TAG 50:4 
                 46.374 
               
               
                 TAG 58:10 FA 20:4 
                 −29.587 
                   
                 TAG 56:3 FA 20:1 
                 69.839 
               
               
                 TAG 58:10 
                 −6.838 
                   
                 TAG 56:3 FA 18:1 
                 23.266 
               
               
                 Glc/GalCer(d18:1/18:0) 
                 −29.219 
                 7.332E−06 
                 TAG 56:3 
                 41.617 
               
               
                 TAG 56:9 FA 20:4 
                 −28.188 
                   
                 TAG 54:3 FA 18:2 
                 67.212 
               
               
                 TAG 56:9 
                 −25.833 
                   
                 TAG 54:3 FA 20:1 
                 59.887 
               
               
                 TAG 56:5 FA 20:3 
                 −27.595 
                   
                 TAG 54:3 FA 16:0 
                 40.549 
               
               
                 TAG 56:5 
                 9.946 
                   
                 TAG 54:3 
                 57.889 
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −27.063 
                 6.051E−05 
                 TAG 54:4 FA 18:0 
                 63.987 
               
               
                 LacCer(d18:1/24:0) 
                 −35.343 
                 2.402E−03 
                 TAG 54:4 FA 18:2 
                 45.810 
               
               
                 TAG 54:4 FA 20:3 
                 −25.768 
                   
                 TAG 54:4 FA 20:2 
                 43.761 
               
               
                 TAG 54:4 
                 35.266 
                   
                 TAG 54:4 FA 18:1 
                 28.831 
               
               
                 TAG 58:8 FA 18:2 
                 −24.677 
                   
                 TAG 54:4 FA 16:0 
                 16.648 
               
               
                 TAG 58:8 
                 −3.510 
                   
                 TAG 54:4 
                 35.266 
               
               
                 Total LacCer 
                 −24.058 
                 3.203E−03 
                 TAG 50:2 FA 18:2 
                 62.114 
               
               
                 LacCer(d18:1/22:0) 
                 −23.147 
                 4.961E−02 
                 TAG 50:2 FA 16:0 
                 27.001 
               
               
                 TAG 56:7 FA 20:4 
                 −22.839 
                   
                 TAG 50:2 FA 14:0 
                 25.767 
               
               
                 TAG 56: 
                 −10.897 
                   
                 TAG 50:2 
                 35.859 
               
               
                 Glc/GalCer(d18:1/26:0) 
                 −22.797 
                 6.959E−02 
                 TAG 53:4 FA 18:2 
                 61.260 
               
               
                 Cer(d18:1/22:0) 
                 −22.584 
                 5.909E−03 
                 TAG 53:4 
                 43.469 
               
               
                 TAG 56:8 FA 20:4 
                 −21.569 
                   
                 TAG 54:6 FA 18:2 
                 61.238 
               
               
                 TAG 56:8 
                 −6.916 
                   
                 TAG 54:6 FA 18:1 
                 32.069 
               
               
                 LacCer(d18:1/16:0) 
                 −20.192 
                 4.711E−03 
                 TAG 54:6 FA 18:3 
                 16.262 
               
               
                 LacCer(d18:1/24:1) 
                 −19.792 
                 2.750E−02 
                 TAG 54:6 
                 32.549 
               
               
                 CE 18:3 
                 −19.265 
                 4.349E−04 
                 TAG 54:5 FA 18:2 
                 61.074 
               
               
                 Cer(d18:1/18:0) 
                 −18.329 
                 4.180E−02 
                 TAG 54:5 FA 18:1 
                 56.979 
               
               
                 Cer(d18:0/22:0) 
                 −17.737 
                 1.486E−01 
                 TAG 54:5 
                 48.831 
               
               
                 PC 18:0/22:6 
                 −17.696 
                 2.093E−02 
                 TAG 50:3 FA 18:3 
                 56.000 
               
               
                 FC 
                 −16.776 
                 1.655E−03 
                 TAG 50:3 FA 18:2 
                 32.037 
               
               
                 Glc/GalCer(d18:1/26:1) 
                 −16.753 
                 1.901E−01 
                 TAG 50:3 
                 14.123 
               
               
                 Cer(d18:0/24:1) 
                 −16.574 
                 1.908E−01 
                 TAG 56:5 FA 18:2 
                 52.163 
               
               
                 PC 16:0/16:0 
                 −16.026 
                 3.590E−02 
                 TAG 56:5 FA 20:1 
                 44.606 
               
               
                 TAG 56:6 FA 20:4 
                 −16.001 
                   
                 TAG 56:5 FA 20:2 
                 28.250 
               
               
                 TAG 56:6 
                 −5.937 
                   
                 TAG 56:5 
                 9.946 
               
               
                 Total Cer 
                 −15.497 
                 4.131E−02 
                   
                   
               
               
                   
               
             
          
           
               
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 change 
                 p-value 
                 Increased 
                 change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/22:0)/PC 16:0/18:2 
                 −12.964 
                 0.000E+00 
                 CE 20:5/Glc/GalCer(d18:1/24:0) 
                 82.189 
                 7.377E−03 
               
               
                 FC/PC 18:0/20:3 
                 −25.581 
                 3.816E−03 
                 CE 20:5/Glc/GalCer(d18:1/22:0) 
                 82.061 
                 2.129E−03 
               
               
                 CE 18:3/CE 20:5 
                 −26.155 
                 3.455E−03 
                 PC 18:2/18:2/SM (d18:1/16:0) 
                 63.587 
                 9.679E−06 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 FC/LPC 16:1 
                 −30.754 
                 5.212E−03 
                 CE 20:5/SM (d18:1/16:0) 
                 62.662 
                 2.087E−04 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0)/PC 
                 −31.147 
                 2.324E−05 
                 Cer(d18:0/24:0)/ 
                 61.005 
                 1.146E−02 
               
               
                 18:0/20:4 
                   
                   
                 LacCer(d18:1/24: 0) 
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0)/LPC 
                 −31.822 
                 1.556E−05 
                 LPC 16:1/SM (d18:1/16:0) 
                 57.606 
                 2.495E−03 
               
               
                 18:2 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0)/PC 
                 −33.636 
                 1.669E−05 
                 PC 18:1/20:4/SM (d18:1/16:0) 
                 55.007 
                 3.384E−04 
               
               
                 16:0/20:4 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 CE 18:3/LPC 16:1 
                 −34.274 
                 5.376E−03 
                 LPC 20:4/SM (d18:1/16:0) 
                 53.454 
                 5.406E−04 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 SM (d18:1/16:0) (d18:1/15: 
                 −35.255 
                 1.603E−04 
                 PC 18:1/18:2/SM (d18:1/16:0) 
                 52.347 
                 6.620E−05 
               
               
                 1-OH)/Sphingosine d16:1 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Cer(d18:1/22:0)/LPC 20:4 
                 −35.825 
                 3.493E−03 
                 CE 20:5/LacCer(d18:1/16:0) 
                 51.636 
                 9.063E−03 
               
               
                 Glc/GalCer(d18:1/22:0)/ 
                 −43.303 
                 1.522E−02 
                 CE 20:4/SM (d18:1/16:0) 
                 42.452 
                 1.598E−05 
               
               
                 Sphingosine d16:1 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/22:0)/PC 
                 −44.396 
                 9.622E−04 
                 CE 20:5/FC 
                 41.327 
                 2.339E−03 
               
               
                 18:1/18:2 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/20:0)/LPC 
                 −49.264 
                 1.000E−02 
                 LPC 16:0/SM (d18:1/16:0) 
                 40.008 
                 1.817E−04 
               
               
                 16:1 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/20:0)/LPC 
                 −49.948 
                 6.554E−04 
                 PI 18:0/20:4/SM (d18:1/16:0) 
                 33.148 
                 7.497E−04 
               
               
                 20:4 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/22:0)/LPC 
                 −50.721 
                 8.458E−03 
                 CE 20:5/PC 16:0/16:0 
                 31.095 
                 2.629E−02 
               
               
                 16:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/22:0)/LPC 
                 −51.506 
                 8.433E−04 
                 LPC 18:2/LacCer(d18:1/16:0) 
                 30.712 
                 9.605E−03 
               
               
                 20:4 
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2c 
               
               
                   
               
               
                 Significant biomarkers based on individual lipid or lipid-lipid concentration ratio 
               
               
                 measurement detected in PCSK9 −/−  mice on Western diet in comparison to wildtype. Species names, 
               
               
                 p-values, and percentage changes are presented. 
               
               
                 PCSK9 −/−  vs. wildtype mice on Western diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
                   
               
               
                 Decreased 
                 change 
                 p-value 
                 Increased 
                 change 
                 p-value 
               
               
                   
               
               
                 CE 18:0 
                 −53.952 
                 3.305E−03 
                 Sphingosine (SPH) d16:1 
                 64.323 
                 1.533E−03 
               
               
                 TAG 56:5 FA 18:2 
                 −69.447 
                   
                 TAG 50:2 FA 18:1 
                 55.940 
                 4.029E−02 
               
               
                 TAG 56:5 FA 18:1 
                 −40.184 
                 3.929E−01 
                 TAG 50:2 FA 16:0 
                 47.004 
                 4.740E−02 
               
               
                 TAG 56:5 FA 20:2 
                 −64.835 
                   
                 TAG 50:2 FA 14:0 
                 21.986 
                 3.838E−01 
               
               
                 TAG 56:5 FA 20:3 
                 −36.442 
                 4.788E−01 
                 TAG 50:2 FA 18:2 
                 17.037 
                 4.435E−01 
               
               
                 TAG 56:5 
                 −49.388 
                 3.306E−01 
                 TAG 50:2 
                 47.040 
                 5.626E−02 
               
               
                 TAG 58:8 FA 18:1 
                 −65.029 
                   
                   
                   
                   
               
               
                 TAG 58:8 FA 22:6 
                 −56.332 
                   
                 Total SPH 
                 50.856 
                 5.789E−03 
               
               
                 TAG 58:8 
                 −60.483 
                   
                 TAG 50:3 FA 16:1 
                 50.410 
                 2.678E−02 
               
               
                 TAG 58:7 FA 18:1 
                 −63.977 
                   
                 TAG 50:3 FA 18:3 
                 37.040 
                   
               
               
                 TAG 58:7 FA 22:5 
                 −48.248 
                   
                 TAG 50:3 FA 18:2 
                 33.534 
                 1.033E−01 
               
               
                 TAG 58:7 
                 −55.754 
                   
                 TAG 50:3 FA 16:0 
                 19.397 
                 3.852E−01 
               
               
                 TAG 56:8 FA 22:6 
                 −48.846 
                   
                 TAG 50:3 
                 39.099 
                 5.636E−02 
               
               
                 TAG 56:8 FA 16:0 
                 −35.490 
                   
                 Sphingosine d18:1 
                 49.840 
                 7.175E−03 
               
               
                 TAG 56:8 FA 18:2 
                 −33.612 
                   
                 TAG 50:4 FA 16:1 
                 47.909 
                   
               
               
                 TAG 56:8 
                 −46.650 
                   
                 TAG 50:4 
                 16.664 
                   
               
               
                 TAG 56:6 FA 18:1 
                 −47.995 
                   
                 TAG 52:3 FA 16:1 
                 43.537 
                 6.661E−02 
               
               
                 TAG 56:6 FA 20:4 
                 −43.581 
                   
                 TAG 52:3 FA 18:0 
                 35.461 
                   
               
               
                 TAG 56:6 FA 20:3 
                 −35.657 
                   
                 TAG 52:3 FA 18:1 
                 26.714 
                 2.263E−01 
               
               
                 TAG 56:6 FA 18:2 
                 −24.983 
                   
                 TAG 52:3 
                 18.534 
                 3.544E−01 
               
               
                 TAG 56:6 total 
                 −29.092 
                   
                 TAG 49:2 FA 16:0 
                 42.140 
                   
               
               
                 Cer(d18:0/24:0) 
                 −36.791 
                 2.958E−02 
                 TAG 49:2 FA 15:0 
                 15.169 
                   
               
               
                 Cer(d18:1/26:1) 
                 −32.004 
                 2.558E−04 
                 TAG 49:2 total 
                 22.766 
                   
               
               
                 Cer(d18:0/22:0) 
                 −28.800 
                 3.316E−02 
                 Sphinganine d18:0 
                 41.602 
                 8.815E−03 
               
               
                 Glc/GalCer(d18:1/26:1) 
                 −27.701 
                 4.231E−03 
                 TAG 53:3 FA 18:1 
                 36.680 
                   
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −26.867 
                 2.746E−02 
                 TAG 53:3 
                 23.391 
                   
               
               
                 Cer(d18:1/26:0) 
                 −26.304 
                 7.863E−03 
                 TAG 52:2 FA 16:0 
                 31.623 
                 1.379E−01 
               
               
                 Cer(d18:1/24:0) 
                 −25.281 
                 1.231E−03 
                 TAG 52:2 FA 18:1 
                 30.549 
                 2.502E−01 
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −25.124 
                 1.452E−02 
                   
                   
                   
               
               
                 TAG 52:2 
                 30.933 
                 1.966E−01 
                   
                   
                   
               
               
                 CE 16:0 
                 −22.506 
                 1.299E−04 
                 TAG 54:7 FA 18:2 
                 29.707 
                   
               
               
                 Total Glc/GalCer 
                 −22.165 
                 1.058E−01 
                 TAG 54:7 FA 18:3 
                 16.926 
                   
               
               
                 Cer(d18:1/22:0) 
                 −22.045 
                 1.310E−01 
                 TAG 54:7 
                 2.091 
                   
               
               
                 Glc/GalCer(d18:1/20:0) 
                 −20.419 
                 2.949E−01 
                 LPC 16:1 
                 27.750 
                 1.120E−01 
               
               
                 LacCer(d18:1/24:0) 
                 −20.357 
                 1.060E−01 
                 Sphingosine-1-phosphate (S1P) d18:1 
                 21.375 
                 5.453E−02 
               
               
                 CE 18:1 
                 −19.571 
                 3.429E−02 
                 Total S1P 
                 21.375 
                 5.453E−02 
               
               
                 Cer(d18:1/20:0) 
                 −19.501 
                 3.481E−01 
                 Total TAG 
                 20.052 
                 3.701E−01 
               
               
                 LacCer(d18:1/22:0) 
                 −19.021 
                 7.362E−02 
                 TAG 56:6 FA 22:4 
                 17.596 
                   
               
               
                 Total Cer 
                 −18.476 
                 5.787E−02 
                 TAG 56:6 
                 −29.092 
                   
               
               
                 PC 16:0/16:0 
                 −18.244 
                 1.262E−01 
                 Sphinganine-1-phosphate d18:0 
                 16.834 
                 2.617E−01 
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −16.527 
                 2.436E−01 
                   
                   
                   
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH) 
                 −15.898 
                 1.986E−01 
               
               
                   
               
               
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 change 
                 p-value 
                 Increased 
                 change 
                 p-value 
               
               
                   
               
               
                 CE 16:0/CE 20:3 
                 −48.833 
                 1.67E−04 
                 LPC 16:1/TAG 56:5 
                 117.120 
                 4.74E−03 
               
               
                 Cer(d18:0/24:0)/Cer(d18:1/18:0) 
                 −49.008 
                 8.69E−04 
                 Cer(d18:1/18:0)/Cer(d18:1/26:1) 
                 88.778 
                 1.09E−02 
               
               
                 Cer(d18:1/24:0)/LPC 16:1 
                 −49.150 
                 3.63E−04 
                 TAG 50:2/TAG 54:5 
                 86.223 
                 3.35E−02 
               
               
                 CE 18:1/Sphingosine d16:1 
                 −49.803 
                 1.16E−05 
                 CE 20:3/Glc/GalCer(d18:1/16:0) 
                 84.705 
                 1.31E−02 
               
               
                 PC 16:0/16:0/Sphingosine 
                 −50.221 
                 9.85E−06 
                 CE 20:3/TAG 54:4 
                 82.091 
                 2.72E−02 
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:0/22:0)/Sphingosine 
                 −51.551 
                 3.75E−05 
                 PC 18:2/18:2/TAG 56:5 
                 76.494 
                 4.27E−02 
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:1/24:0)/Sphingosine 
                 −51.685 
                 4.14E−06 
                 CE 20:3/PC 16:0/16:0 
                 70.816 
                 7.47E−03 
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:0/24:0)/PC 18:2/18:2 
                 −52.891 
                 8.28E−03 
                 Cer(d18:1/18:0)/Glc/ 
                 64.951 
                 7.32E−03 
               
               
                   
                   
                   
                 GalCer(d18:1/24:0) 
                   
                   
               
               
                 Cer(d18:0/24:0)/PC 16:0/20:4 
                 −53.440 
                 1.63E−02 
                 CE 20:3/SM (d18:1/16:0) 
                 58.191 
                 1.14E−02 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Cer(d18:0/24:0)/Sphinganine 
                 −54.294 
                 9.41E−03 
                 Cer(d18:1/18:0)/Cer(d18:1/22:0) 
                 54.941 
                 1.35E−02 
               
               
                 d18:0 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:1/26:1)/LPC 16:1 
                 −54.395 
                 1.43E−03 
                 LPC 16:1/PC 16:0/16:0 
                 53.424 
                 3.69E−03 
               
               
                 Cer(d18:0/24:0)/PC 16:0/20:3 
                 −54.574 
                 2.28E−02 
                 CE 20:3/FC 
                 52.675 
                 2.54E−02 
               
               
                 Glc/GalCer(d18:1/16:0)/ 
                 −54.630 
                 1.22E−04 
                 LPC 16:1/SM (d18:1/16:0) 
                 47.406 
                 6.67E−03 
               
               
                 Sphingosine d16:1 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Cer(d18:0/24:0)/Sphingosine 
                 −58.674 
                 4.36E−04 
                 Cer(d18:1/24:1)/Cer(d18:1/26:1) 
                 46.396 
                 2.48E−03 
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:0/24:0)/LPC 16:1 
                 −61.145 
                 1.34E−03 
                 Sphingosine d16:1/Sphingosine-1- 
                 37.012 
                 2.81E−03 
               
               
                   
                   
                   
                 phosphate d18:1 
                   
                   
               
               
                   
                   
                   
                 PI 18:0/20:4/SM (d18:1/16:0) 
                 36.227 
                 9.39E−03 
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2d 
               
               
                   
               
               
                 Significant biomarkers based on individual lipid or lipid-lipid concentration ratio 
               
               
                 measurement detected in PCSK9 +/−  mice on Western diet in comparison to wildtype. Species 
               
               
                 names, p-values, and percentage changes are presented. 
               
               
                 PCSK9 +/−  vs. wildtype mice on Western diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Measurement Name 
                 Percentage 
                   
                 Measurement Name 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/22:0) 
                 −58.820 
                 3.240E−05 
                 Sphingosine (SPH) d16:1 
                 107.934 
                 4.509E−07 
               
               
                 Cer(d18:0/24:0) 
                 −57.003 
                 5.688E−04 
                 Total SPH 
                 75.244 
                 4.162E−05 
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −54.076 
                 9.882E−07 
                 Sphingosine d18:1 
                 72.779 
                 7.344E−05 
               
               
                 Glc/GalCer(d18:1/26:1) 
                 −51.762 
                 5.315E−07 
                 Sphinganine-1-phosphate d18:0 
                 70.319 
                 1.192E−05 
               
               
                 Cer(d18:0/24:1) 
                 −51.729 
                 4.012E−03 
                 Sphinganine d18:0 
                 62.423 
                 7.539E−05 
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −48.741 
                 6.131E−04 
                 Sphingosine-1-phosphate d18:1 
                 53.308 
                 6.250E−06 
               
               
                 Cer(d18:1/24:0) 
                 −47.795 
                 2.927E−08 
                 CE 20:4 
                 50.933 
                 1.436E−02 
               
               
                 Cer(d18:1/26:1) 
                 −47.142 
                 2.122E−07 
                 SM (d18:1/24:0) (d18:1/23:1-OH) 
                 49.321 
                 4.971E−02 
               
               
                 LacCer(d18:1/24:0) 
                 −45.674 
                 2.048E−04 
                 PC 18:1/22:6 
                 41.215 
                 4.282E−02 
               
               
                 Glc/GalCer(d18:1/26:0) 
                 −43.777 
                 3.426E−04 
                 PC 18:1/20:4 
                 39.998 
                 4.105E−02 
               
               
                 Total Glc/GalCer 
                 −43.704 
                 1.188E−03 
                 LPC 16:1 
                 30.783 
                 5.904E−02 
               
               
                 Glc/GalCer(d18:1/22:0) 
                 −43.038 
                 9.953E−03 
                 PC 16:0/20:3 
                 30.331 
                 1.023E−01 
               
               
                 CE 18:0 
                 −38.988 
                 3.123E−02 
                 PC 16:0/22:6 
                 26.848 
                 7.083E−02 
               
               
                 LacCer(d18:1/22:0) 
                 −38.829 
                 2.682E−04 
                 CE 22:6 
                 26.051 
                 1.431E−01 
               
               
                 Total Cer 
                 −36.509 
                 1.904E−04 
                 CE 18:2 
                 25.895 
                 5.265E−02 
               
               
                 Cer(d18:1/26:0) 
                 −35.073 
                 2.640E−04 
                 Total CE 
                 25.389 
                 4.560E−02 
               
               
                 LacCer(d18:1/24:1) 
                 −33.935 
                 2.435E−04 
                 PC 16:0/20:4 
                 22.578 
                 9.245E−02 
               
               
                 Cer(d18:1/22:0) 
                 −32.533 
                 1.857E−02 
                 CE 16:1 
                 21.981 
                 1.514E−01 
               
               
                 Cer(d18:1/24:1) 
                 −29.447 
                 1.722E−02 
                 CE 18:3 
                 21.230 
                 8.476E−02 
               
               
                 Total LacCer 
                 −29.179 
                 2.627E−04 
                 Cer(d18:1/18:0) 
                 20.846 
                 2.314E−01 
               
               
                 Glc/GalCer(d18:1/20:0) 
                 −28.509 
                 1.167E−01 
                 PI 18:0/20:4 
                 19.870 
                 1.635E−01 
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −24.831 
                 2.714E−02 
                 PC 18:2/18:2 
                 19.299 
                 1.191E−01 
               
               
                 CE 16:0 
                 −19.411 
                 3.007E−04 
                 LPC 20:4 
                 16.370 
                 2.785E−01 
               
               
                   
                   
                   
                 PC 18:1/18:2 
                 15.307 
                 1.773E−01 
               
               
                   
               
               
                 Lipid-lipid 
                   
                   
                 Lipid-lipid 
                   
                   
               
               
                 concentration ratio 
                 Percentage 
                   
                 concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Measurement Name 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/24:1)/FC 
                 −56.408 
                 0.000E+00 
                 CE 20:3/Glc/GalCer(d18:1/26:1) 
                 360.547 
                 1.498E−03 
               
               
                 LacCer(d18:1/16:0)/Sphing 
                 −56.691 
                 9.211E−10 
                 CE 20:3/Cer(d18:0/24:0) 
                 305.964 
                 1.938E−04 
               
               
                 osine d16:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0)/PC 
                 −61.038 
                 2.913E−10 
                 CE 20:3/Cer(d18:1/24:0) 
                 300.319 
                 4.185E−04 
               
               
                 16:0/20:4 
                   
                   
                   
                   
                   
               
               
                 CE 16:0/Sphingosine d16:1 
                 −64.159 
                 1.949E−08 
                 CE 20:3/Cer(d18:0/22:0) 
                 299.250 
                 1.003E−04 
               
               
                 Glc/GalCer(d18:1/24:1)/Sp 
                 −68.880 
                 7.661E−06 
                 CE 20:3/Cer(d18:1/26:1) 
                 282.779 
                 9.631E−05 
               
               
                 hingosine d18:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:1/24:0)/Sphingosi 
                 −69.354 
                 7.730E−09 
                 CE 20:4/Cer(d18:0/22:0) 
                 268.566 
                 1.737E−06 
               
               
                 ne d18:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0)/Sp 
                 −69.806 
                 1.083E−08 
                 CE 20:5/Cer(d18:0/22:0) 
                 255.932 
                 6.261E−06 
               
               
                 hingosine-1-phosphate 
                   
                   
                   
                   
                   
               
               
                 d18:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0)/Sp 
                 −72.584 
                 4.488E−08 
                 CE 20:4/Cer(d18:0/24:0) 
                 241.592 
                 2.033E−05 
               
               
                 hingosine d18:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0)/Sp 
                 −72.647 
                 3.066E−08 
                 CE 20:4/Glc/GalCer(d18:1/24:0) 
                 219.850 
                 4.296E−08 
               
               
                 hinganine-1-phosphate 
                   
                   
                   
                   
                   
               
               
                 d18:0 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:0/24:0)/PC 
                 −73.450 
                 7.227E−04 
                 CE 20:5/Cer(d18:0/24:1) 
                 210.249 
                 2.136E−05 
               
               
                 18:1/20:4 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:1/24:0)/Sphingosi 
                 −75.303 
                 5.327E−10 
                 CE 22:6/Cer(d18:0/22:0) 
                 210.101 
                 7.122E−06 
               
               
                 ne d16:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0)/Sp 
                 −78.076 
                 1.085E−10 
                 CE 16:1/Cer(d18:0/22:0) 
                 197.486 
                 8.513E−06 
               
               
                 hingosine d16:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:0/24:0)/Sphingosi 
                 −79.943 
                 1.970E−06 
                 Cer(d18:1/18:0)/Glc/ 
                 176.575 
                 6.844E−04 
               
               
                 ne d16:1 
                   
                   
                 GalCer(d18:1/26:1) 
                   
                   
               
               
                   
                   
                   
                 CE 18:2/Glc/GalCer(d18:1/24:0) 
                 171.724 
                 5.882E−09 
               
               
                   
                   
                   
                 CE 18:3/Glc/GalCer(d18:1/24:0) 
                 157.031 
                 4.734E−09 
               
               
                   
                   
                   
                 Cer(d18:1/18:0)/Glc/ 
                 155.600 
                 1.811E−08 
               
               
                   
                   
                   
                 GalCer(d18:1/24:0) 
                   
                   
               
               
                   
                   
                   
                 LPC 16:1/LacCer(d18:1/24:0) 
                 129.430 
                 4.599E−03 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 3a 
               
             
             
               
                   
               
               
                 Significant biomarkers based on individual lipid measurement in  
               
               
                 human samples. Species names, p-values, and percentage changes are  
               
               
                 presented. Traditionally used biomarkers and their percentage change  
               
               
                 and p-value is also listed. The listed individual lipid biomarkers has  
               
               
                 improved separation of PCSK9 inhibition compared to the traditionally 
               
               
                 used markers, e.g. LDL-cholesterol. 
               
               
                 Human R46L vs. control 
               
             
          
           
               
                   
                 Percentage 
                   
               
               
                   
                 Change 
                 p-value 
               
               
                   
               
               
                 Measurement Name 
                   
                   
               
               
                 Decreased 
                   
                   
               
               
                 CE 20:3 
                 −29.391 
                 1.547E−02 
               
               
                 CE 20:5 
                 −27.746 
                 1.640E−01 
               
               
                 CE 17:1 
                 −27.380 
                 1.629E−02 
               
               
                 SM (d18:1/17:0) (d18:1/16:1-OH) 
                 −25.747 
                 1.721E−01 
               
               
                 CE 20:4 
                 −25.511 
                 3.724E−02 
               
               
                 CE 16:1 
                 −25.210 
                 1.962E−01 
               
               
                 LPC 16:0 
                 −22.167 
                 3.356E−02 
               
               
                 LacCer(d18:1/18:0) 
                 −21.812 
                  3.98E−04 
               
               
                 CE 18:1 
                 −21.788 
                 4.631E−02 
               
               
                 SM (d18:1/23:0) (d18:1/22:1-OH) 
                 −19.747 
                 1.492E−01 
               
               
                 SM (d18:1/24:1) (d18:1/23:2-OH) 
                 −19.672 
                 1.301E−01 
               
               
                 GlcCer(d18:1/18:0) 
                 −19.105 
                 2.016E−01 
               
               
                 Cer(d18:1/18:0) 
                 −19.012 
                 1.895E−01 
               
               
                 SM (d18:1/16:1) (d18:1/15:2-OH) 
                 −18.823 
                 4.836E−02 
               
               
                 CE 14:0 
                 −18.283 
                 1.695E−01 
               
               
                 Cer(d18:1/16:0) 
                 −17.722 
                 1.212E−01 
               
               
                 Cer(d18:0/22:0) 
                 −17.713 
                  6.35E−02 
               
               
                 CE 18:3 
                 −17.559 
                 2.100E−01 
               
               
                 LacCer(d18:1/16:0) 
                 −16.866 
                 6.083E−02 
               
               
                 SM (d18:1/18:0) 
                 −16.804 
                 1.568E−01 
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH) 
                 −16.758 
                 8.385E−02 
               
               
                 CE 16:0 
                 −15.966 
                 5.712E−02 
               
               
                 CE 15:0 
                 −15.068 
                 2.266E−01 
               
               
                 GlcCer(d18:1/16:0) 
                 −14.968 
                 1.989E−01 
               
               
                 CE 18:0 
                 −14.770 
                 2.346E−01 
               
               
                 Cer(d18:1/20:0) 
                 −14.495 
                  5.55E−03 
               
               
                 GlcCer(d18:1/24:0) 
                 −13.668 
                  7.13E−02 
               
               
                 PC 18:0/20:3 
                 −11.595 
                  6.83E−02 
               
               
                 PC 16:0/16:0 
                 −11403 
                  5.19E−02 
               
               
                 Traditional markers 
                   
                   
               
               
                 LDL-cholesterol (clinical) 
                 −10.105 
                  9.77E−02 
               
               
                 Total cholesterol (clinical) 
                 −8.417 
                  4.94E−02 
               
               
                 Triglycerides (clinical) 
                 −4.372 
                  5.98E−01 
               
               
                 HDL-cholesterol (clinical) 
                 −0.330 
                  9.55E−01 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3b 
               
             
             
               
                   
               
               
                 Significant biomarkers based on lipid or lipid-lipid 
               
               
                 concentration ratios in human samples. Species names, 
               
               
                 p-values, and percentage changes are presented. 
               
             
          
           
               
                   
                 Percentage  
                   
               
               
                 Lipid-lipid ratio 
                 Change 
                 P-value 
               
               
                   
               
             
          
           
               
                 Decreased 
                   
                   
               
               
                 CE 22:2/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −50.105 
                 5.44E−04 
               
               
                 LacCer(d18:1/18:0)/supersensitive C-reactive  
                 −49.645 
                 3.60E−04 
               
               
                 protein(mg/L) 
                   
                   
               
               
                 CE 20:0/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −47.596 
                 5.03E−03 
               
               
                 CE 19:1/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −41.469 
                 8.90E−04 
               
               
                 LacCer(d18:1/24:1)/supersensitive C-reactive  
                 −41.057 
                 1.50E−02 
               
               
                 protein(mg/L) 
                   
                   
               
               
                 Cer(d18:1/16:0)/SM (d18:1/23:1) (d18:1/ 
                 −39.696 
                 1.28E−04 
               
               
                 22:2-OH) 
                   
                   
               
               
                 DAG 16:0/18:1/lipoprotein(a) (EDTA) (mg/dL) 
                 −38.988 
                 1.78E−02 
               
               
                 CE 15:0/supersensitive C-reactive protein(mg/L) 
                 −38.142 
                 4.22E−02 
               
               
                 CE 20:0/PC O-18:0/20:4-alkenyl (PC O-18:1/ 
                 −37.546 
                 2.35E−02 
               
               
                 20:4-alkyl) 
                   
                   
               
               
                 CE 19:2/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −36.841 
                 1.40E−02 
               
               
                 LacCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/ 
                 −36.267 
                 1.27E−06 
               
               
                 22:2-OH) 
                   
                   
               
               
                 GlcCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/ 
                 −36.247 
                 4.76E−06 
               
               
                 22:2-OH) 
                   
                   
               
               
                 CE 20:3/supersensitive C-reactive protein(mg/L) 
                 −35.784 
                 3.48E−02 
               
               
                 CE 18:0/supersensitive C-reactive protein(mg/L) 
                 −35.292 
                 3.46E−02 
               
               
                 GlcCer(d18:1/16:0)/SM (d18:1/23:1) (d18:1/ 
                 −35.278 
                 1.97E−05 
               
               
                 22:2-OH) 
                   
                   
               
               
                 Cer(d18:1/18:0)/PC 18:0/22:6 
                 −34.301 
                 3.77E−05 
               
               
                 Cer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/ 
                 −34.006 
                 2.53E−04 
               
               
                 22:2-OH) 
                   
                   
               
               
                 LacCer(d18:1/24:0)/SM (d18:1/23:1) (d18:1/ 
                 −33.961 
                 1.38E−05 
               
               
                 22:2-OH) 
                   
                   
               
               
                 CE 22:2/PC O-18:0/20:4-alkenyl (PC O-18:1/ 
                 −33.340 
                 2.11E−02 
               
               
                 20:4-alkyl) 
                   
                   
               
               
                 CE 16:1/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −32.963 
                 4.56E−04 
               
               
                 LacCer(d18:1/16:0)/SM (d18:1/23:1) (d18:1/ 
                 −31.627 
                 1.65E−05 
               
               
                 22:2-OH) 
                   
                   
               
               
                 CE 22:2/DAG 16:0/18:1 
                 −31.231 
                 1.31E−02 
               
               
                 GlcCer(d18:1/24:0)/SM (d18:1/23:1) (d18:1/ 
                 −31.159 
                 7.13E−05 
               
               
                 22:2-OH) 
                   
                   
               
               
                 Cer(d18:1/18:0)/PC O-18:0/20:4-alkenyl (PC  
                 −30.696 
                 8.50E−05 
               
               
                 O-18:1/20:4-alkyl) 
                   
                   
               
               
                 LacCer(d18:1/22:0)/SM (d18:1/23:1) (d18:1/ 
                 −30.368 
                 6.54E−04 
               
               
                 22:2-OH) 
                   
                   
               
               
                 CE 14:0/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −30.267 
                 1.74E−03 
               
               
                 CE 18:3/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −30.194 
                 7.31E−05 
               
               
                 Cer(d18:1/16:0)/triglycerides (EDTA) (mg/dL) 
                 −30.006 
                 9.36E−07 
               
               
                 Cer(d18:1/16:0)/triglycerides (EDTA) (mg/dL) 
                 −30.006 
                 9.36E−07 
               
               
                 Cer(d18:1/18:0)/triglycerides (EDTA) (mg/dL) 
                 −27.662 
                 1.79E−07 
               
               
                 Cer(d18:1/18:0)/DAG 16:0/18:1 
                 −26.271 
                   2E−05 
               
               
                 Cer(d18:1/18:0)/apolipoprotein C-III (mg/dL) 
                 −24.685 
                 2.36E−05 
               
               
                 CE 20:3/HDL cholesterol (EDTA) (mg/dL) 
                 −24.559 
                 3.19E−05 
               
               
                 CE 20:3/apolipoprotein A-1 (mg/dl) 
                 −24.189 
                  3.2E−05 
               
               
                 Increased 
                   
                   
               
               
                 PC 18:0/22:6/SM (d18:1/24:0) (d18:1/23:1-OH) 
                 33.954 
                 4.87E−02 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4a 
               
               
                   
               
               
                 Preferred embodiments of biomarkers of PCSK9 −/−  mice on regular chow. 
               
               
                 PCSK9 −/−  vs. wildtype mice on regular chow diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 TAG 60:12 
                 −64.990 
                 2.508E−04 
                 TAG 54:3 
                 106.898 
                 3.787E−02 
               
               
                 Glc/GalCer(d18:1/20:0) 
                 −61.304 
                 5.021E−07 
                 TAG 54:4 
                 83.059 
                 3.721E−02 
               
               
                 Glc/GalCer(d18:1/18:0) 
                 −60.966 
                 7.032E−14 
                 TAG 52:4 
                 73.643 
                 2.485E−02 
               
               
                 Glc/GalCer(d18:1/22:0) 
                 −58.750 
                 2.897E−06 
                 TAG 56:5 
                 71.820 
                 4.739E−02 
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −56.593 
                 4.968E−12 
                 TAG 54:5 
                 70.166 
                 1.239E−02 
               
               
                 Total Glc/GalCer 
                 −55.866 
                 6.261E−07 
                 TAG 52:5 
                 69.317 
                 4.417E−02 
               
               
                 SM (d18:1/16:0) (d18:1/15:1-OH) 
                 −54.947 
                 2.058E−16 
                 TAG 54:6 
                 52.606 
                 1.530E−02 
               
               
                 LacCer(d18:1/16:0) 
                 −54.523 
                 8.517E−10 
                 TAG 54:7 
                 40.748 
                 4.048E−02 
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −53.028 
                 6.967E−05 
                   
                   
                   
               
               
                 Total LacCer 
                 −51.984 
                 4.137E−08 
                   
                   
                   
               
               
                 CE 16:1 
                 −51.509 
                 3.221E−05 
               
               
                   
               
               
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
                 Lipid-lipid concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Glc/GalCer(d18:1/18:0)/TAG 52:4 
                 −82.312 
                 4.937E−05 
                 TAG 58:10/TAG 60:12 
                 139.279 
                 6.521E−07 
               
               
                   
                   
                   
                 LPC 18:2/SM (d18:1/16:0) 
                   
                   
               
               
                   
                   
                   
                 (d18:1/15:1-OH) 
                 98.384 
                 7.626E−12 
               
               
                   
                   
                   
                 LPC 18:2/LacCer(d18:1/16:0) 
                 94.112 
                 3.915E−10 
               
               
                   
                   
                   
                 Cer(d18:0/24:0)/FC 
                 89.233 
                 0.000E+00 
               
               
                   
                   
                   
                 Cer(d18:0/22:0)/Cer(d18:1/22:0) 
                 82.249 
                 1.926E−07 
               
               
                   
                   
                   
                 Cer(d18:1/16:0)/Glc/ 
                 63.539 
                 2.374E−09 
               
               
                   
                   
                   
                 GalCer(d18:1/16:0) 
                   
                   
               
               
                   
                   
                   
                 Cer(d18:0/24:0)/PC 16:0/18:2 
                 59.693 
                 0.000E+00 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4b 
               
               
                   
               
               
                 Preferred embodiments of biomarkers of PCSK9 +/−  on regular chow. 
               
               
                 PCSK9 +/−  vs. wildtype mice on regular chow diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Glc/GalCer(d18:1/22:0) 
                 −38.116 
                 1.072E−03 
                 TAG 53:3 
                 98.444 
                   
               
               
                 Glc/GalCer(d18:1/20:0) 
                 −36.166 
                 1.126E−03 
                 TAG 49:2 
                 81.946 
                   
               
               
                 Total Glc/GalCer 
                 −34.200 
                 8.552E−04 
                 TAG 54:3 
                 57.889 
                   
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −34.067 
                 4.625E−03 
                 TAG 54:5 
                 48.831 
                   
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −30.734 
                 1.378E−02 
                 TAG 50:4 
                 46.374 
                   
               
               
                 SM (d18:1/16:0) 
                 −30.205 
                 1.297E−08 
                 TAG 54:8 
                 45.668 
                   
               
               
                 (d18:1/15:1-OH) 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0) 
                 −29.219 
                 7.332E−06 
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −27.063 
                 6.051E−05 
                   
                   
                   
               
               
                 LacCer(d18:1/24:0) 
                 −26.786 
                 5.566E−02 
                   
                   
                   
               
               
                 Total LacCer 
                 −24.058 
                 3.203E−03 
               
               
                   
               
               
                 Lipid-lipid 
                   
                   
                 Lipid-lipid 
                   
                   
               
               
                 concentration ratio 
                 Percentage 
                   
                 concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/22:0)/PC 
                 −12.964 
                 0.000E+00 
                 CE 20:5/Glc/GalCer(d18:1/24:0) 
                 82.189 
                 7.377E−03 
               
               
                 16:0/18:2 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/18:0)/ 
                 −33.636 
                 1.669E−05 
                 PC 18:2/18:2/SM (d18:1/16:0) 
                 63.587 
                 9.679E−06 
               
               
                 PC 16:0/20:4 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                 Glc/GalCer(d18:1/22:0)/ 
                 −51.506 
                 8.433E−04 
                 CE 20:4/SM (d18:1/16:0) 
                 42.452 
                 1.598E−05 
               
               
                 LPC 20:4 
                   
                   
                 (d18:1/15:1-OH) 
                   
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4c 
               
               
                   
               
               
                 Preferred embodiments of biomarkers of PCSK9 −/−  on Western diet. 
               
               
                 PCSK9 −/−  vs. wildtype mice on Western diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/24:0) 
                 −36.791 
                 2.958E−02 
                 Sphingosine d16:1 
                 64.323 
                 1.533E−03 
               
               
                 Cer(d18:1/26:1) 
                 −32.004 
                 2.558E−04 
                 Total SPH 
                 50.856 
                 5.789E−03 
               
               
                 Cer(d18:0/22:0) 
                 −28.800 
                 3.316E−02 
                 Sphingosine d18:1 
                 49.840 
                 7.175E−03 
               
               
                 Glc/GalCer(d18:1/26:1) 
                 −27.701 
                 4.231E−03 
                 Sphinganine d18:0 
                 41.602 
                 8.815E−03 
               
               
                 Glc/GalCer(d18:1/16:0) 
                 −26.867 
                 2.746E−02 
                 Total SPA 
                 41.602 
                 8.815E−03 
               
               
                 Cer(d18:1/26:0) 
                 −26.304 
                 7.863E−03 
                 LPC 16:1 
                 27.750 
                 1.120E−01 
               
               
                 Cer(d18:1/24:0) 
                 −25.281 
                 1.231E−03 
                 Sphingosine-1-phosphate 
                 21.375 
                 5.453E−02 
               
               
                   
                   
                   
                 d18:1 
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −25.124 
                 1.452E−02 
                 Total S1P 
                 21.375 
                 5.453E−02 
               
               
                 CE 16:0 
                 −22.506 
                 1.299E−04 
                 Sphinganine-1-phosphate 
                 16.834 
                 2.617E−01 
               
               
                   
                   
                   
                 d18:0 
                   
                   
               
               
                 Total Glc/GalCer 
                 −22.165 
                 1.058E−01 
                   
                   
                   
               
               
                 CE 18:1 
                 −19.571 
                 3.429E−02 
                   
                   
                   
               
               
                 LacCer(d18:1/22:0) 
                 −19.021 
                 7.362E−02 
                   
                   
                   
               
               
                 Total Cer 
                 −18.476 
                 5.787E−02 
               
               
                   
               
               
                 Lipid-lipid 
                   
                   
                 Lipid-lipid 
                   
                   
               
               
                 concentration ratio 
                 Percentage 
                   
                 concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/24:0)/Cer(d18:1/ 
                 −49.008 
                  8.69E−04 
                 LPC 16:1/TAG 56:5 
                 117.120 
                  4.74E−03 
               
               
                 18:0) 
                   
                   
                   
                   
                   
               
               
                 CE 18:1/Sphingosine d16:1 
                 −49.803 
                  1.16E−05 
                 CE 20:3/PC 16:0/16:0 
                 70.816 
                  7.47E−03 
               
               
                 PC 16:0/16:0/Sphingosine 
                 −50.221 
                  9.85E−06 
                 Cer(d18:1/18:0)/Glc/ 
                 64.951 
                  7.32E−03 
               
               
                 d16:1 
                   
                   
                 GalCer(d18:1/24:0) 
                   
                   
               
               
                 Cer(d18:1/24:0)/Sphingosine 
                 −51.685 
                  4.14E−06 
                   
                   
                   
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                 Cer(d18:0/24:0)/Sphingosine 
                 −58.674 
                  4.36E−04 
                   
                   
                   
               
               
                 d16:1 
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4d 
               
               
                   
               
               
                 Preferred embodiments of PCSK9 +/−  on Western diet biomarkers. 
               
               
                 PCSK9 +/−  vs. wildtype mice on Western diet 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Lipid 
                 Percentage 
                   
                 Lipid 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/22:0) 
                 −58.820 
                 3.240E−05 
                 Sphingosine d16:1 
                 107.934 
                 4.509E−07 
               
               
                 Cer(d18:0/24:0) 
                 −57.003 
                 5.688E−04 
                 Total SPH 
                 75.244 
                 4.162E−05 
               
               
                 Glc/GalCer(d18:1/24:0) 
                 −54.076 
                 9.882E−07 
                 Sphingosine d18:1 
                 72.779 
                 7.344E−05 
               
               
                 Glc/GalCer(d18:1/26:1) 
                 −51.762 
                 5.315E−07 
                 Sphinganine-1-phosphate d18:0 
                 70.319 
                 1.192E−05 
               
               
                 Cer(d18:0/24:1) 
                 −51.729 
                 4.012E−03 
                 Total SA1P 
                 70.319 
                 1.192E−05 
               
               
                 Glc/GalCer(d18:1/24:1) 
                 −48.741 
                 6.131E−04 
                 Sphinganine d18:0 
                 62.423 
                 7.539E−05 
               
               
                 Cer(d18:1/24:0) 
                 −47.795 
                 2.927E−08 
                 Total SPA 
                 62.423 
                 7.539E−05 
               
               
                 Cer(d18:1/26:1) 
                 −47.142 
                 2.122E−07 
                 Sphingosine-1-phosphate d18:1 
                 53.308 
                 6.250E−06 
               
               
                 LacCer(d18:1/24:0) 
                 −45.674 
                 2.048E−04 
                 Total S1P 
                 53.308 
                 6.250E−06 
               
               
                 Glc/GalCer(d18:1/26:0) 
                 −43.777 
                 3.426E−04 
               
               
                   
               
               
                 Lipid-lipid 
                   
                   
                 Lipid-lipid 
                   
                   
               
               
                 concentration ratio 
                 Percentage 
                   
                 concentration ratio 
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
                 Increased 
                 Change 
                 p-value 
               
               
                   
               
               
                 Cer(d18:0/24:1)/FC 
                 −56.408 
                 0.000E+00 
                 CE 18:3/Glc/GalCer(d18:1/24:0) 
                 157.031 
                 4.734E−09 
               
               
                 Glc/GalCer(d18:1/24:0)/ 
                 −78.076 
                 1.085E−10 
                 CE 18:2/Glc/GalCer(d18:1/24:0) 
                 171.724 
                 5.882E−09 
               
               
                 Sphingosine d16:1 
                   
                   
                   
                   
                   
               
               
                 Glc/GalCer(d18:1/24:0)/ 
                 −61.038 
                 2.913E−10 
                 Cer(d18:1/18:0)/Glc/ 
                 155.600 
                 1.811E−08 
               
               
                 PC 16:0/20:4 
                   
                   
                 GalCer(d18:1/24:0) 
                   
                   
               
               
                   
                   
                   
                 CE 20:4/Glc/GalCer(d18:1/24:0) 
                 219.850 
                 4.296E−08 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Preferred embodiments of human biomarkers. 
               
               
                 Human R46L vs. control 
               
             
          
           
               
                   
                 Percentage 
                   
               
               
                 Decreased 
                 Change 
                 p-value 
               
               
                   
               
             
          
           
               
                 Lipid 
                   
                   
               
               
                 CE 16:1 
                 −23.1181918 
                 1.91E−04 
               
               
                 CE 20:3 
                 −22.8724155 
                 3.59E−05 
               
               
                 Cer(d18:1/16:0) 
                 −22.1234175 
                 5.98E−04 
               
               
                 Cer(d18:1/18:0) 
                 −21.9814365 
                 5.13E−04 
               
               
                 LacCer(d18:1/18:0) 
                 −21.8121857 
                 3.98E−04 
               
               
                 GlcCer(d18:1/18:0) 
                 −18.6701034 
                 2.55E−03 
               
               
                 LacCer(d18:1/16:0) 
                 −17.8457737 
                 1.20E−04 
               
               
                 GlcCer(d18:1/16:0) 
                 −17.6341642 
                 2.83E−03 
               
               
                 CE 18:1 
                 −17.5455244 
                 2.72E−04 
               
               
                 CE 16:0 
                 −14.8193897 
                 5.06E−04 
               
               
                 Lipid-lipid concentration ratio 
                   
                   
               
               
                 CE 22:2/SM (d18:1/23:1) (d18:1/22:2-OH) 
                 −50.105 
                 5.44E−04 
               
               
                 Cer(d18:1/16:0)/SM (d18:1/23:1) (d18:1/ 
                 −39.696 
                 1.28E−04 
               
               
                 22:2-OH) 
                   
                   
               
               
                 LacCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/ 
                 −36.267 
                 1.27E−06 
               
               
                 22:2-OH) 
                   
                   
               
               
                 GlcCer(d18:1/18:0)/SM (d18:1/23:1) (d18:1/ 
                 −36.247 
                 4.76E−06 
               
               
                 22:2-OH) 
                   
                   
               
               
                 GlcCer(d18:1/16:0)/SM (d18:1/23:1) (d18:1/ 
                 −35.278 
                 1.97E−05 
               
               
                 22:2-OH) 
                   
                   
               
               
                 Cer(d18:1/18:0)/PC 18:0/22:6 
                 −34.301 
                 3.77E−05 
               
               
                 Cer(d18:1/16:0)/triglycerides (EDTA) (mg/dL) 
                 −30.006 
                 9.36E−07 
               
               
                 Cer(d18:1/18:0)/triglycerides (EDTA) (mg/dL) 
                 −27.662 
                 1.79E−07 
               
               
                 Cer(d18:1/18:0)/DAG 16:0/18:1 
                 −26.271 
                 2.00E−05 
               
               
                 Cer(d18:1/18:0)/apolipoprotein C-III (mg/dL) 
                 −24.685 
                 2.36E−05 
               
               
                 CE 20:3/HDL cholesterol (EDTA) (mg/dl) 
                 −24.559 
                 3.19E−05 
               
               
                 CE 20:3/apolipoprotein A-1 (mg/dL) 
                 −24.189 
                 3.20E−05 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Examples of possible fatty acid combinations per TAG brutto species. 
               
             
          
           
               
                 TAG brutto  
                   
               
               
                 species 
                 Possible FA combinations 
               
               
                   
               
               
                 TAG 49:2  
                 TAG 49:2 total (12:0/18:2/19:0)(15:0/16:0/18:2)(15:0/16:1/ 
               
               
                 total 
                 18:1) 
               
               
                 TAG 50:2  
                 TAG 50:2 total (12:0/18:0/20:2)(12:0/18:1/20:1)(12:0/18:2/ 
               
               
                 total 
                 20:0)(14:0/16:0/20:2)(14:0/16:1/20:1)(14:0/18:0/18:2)(14:0/ 
               
               
                   
                 18:1/18:1)(15:0/15:0/20:2)(15:0/17:0/18:2)(16:0/16:0/ 
               
               
                   
                 18:2)(16:0/16:1/18:1)(16:1/16:1/18:0) 
               
               
                 TAG 50:3  
                 TAG 50:3 total (12:0/18:0/20:3)(12:0/18:1/20:2)(12:0/18:2/ 
               
               
                 total 
                 20:1)(12:0/18:3/20:0)(14:0/16:0/20:3)(14:0/16:1/20:2)(14:0/ 
               
               
                   
                 18:0/18:3)(14:0/18:1/18:2)(16:0/16:0/18:3)(16:0/16:1/ 
               
               
                   
                 18:2)(16:1/16:1/18:1) 
               
               
                 TAG 50:4  
                 TAG 50:4 total (12:0/18:0/20:4)(12:0/18:1/20:3)(12:0/18:2/ 
               
               
                 total 
                 20:2)(12:0/18:3/20:1)(14:0/16:0/20:4)(14:0/16:1/20:3)(14:0/ 
               
               
                   
                 16:2/20:2)(14:0/18:1/18:3)(14:0/18:2/18:2)(16:0/16:1/ 
               
               
                   
                 18:3)(16:0/16:2/18:2)(16:1/16:1/18:2)(16:1/16:2/18:1)(16:2/ 
               
               
                   
                 16:2/18:0) 
               
               
                 TAG 50:5  
                 TAG 50:5 total (14:0/16:1/20:4)(14:0/18:2/18:3)(16:0/16:2/ 
               
               
                 total 
                 18:3)(16:1/16:1/18:3)(16:1/16:2/18:2)(16:2/16:2/18:1 
               
               
                 TAG 51:4  
                 TAG 51:4 total (15:0/18:1/18:3)(15:0/18:2/18:2)(16:1/17:0/ 
               
               
                 total 
                 18:3) 
               
               
                 TAG 52:2  
                 TAG 52:2 total (14:0/18:0/20:2)(14:0/18:1/20:1)(15:0/17:0/ 
               
               
                 total 
                 20:2)(16:0/16:0/20:2)(16:0/16:1/20:1)(16:0/18:0/18:2)(16:0/ 
               
               
                   
                 18:1/18:1)(16:1/18:0/18:1)(17:0/17:0/18:2) 
               
               
                 TAG 52:3  
                 TAG 52:3 total (14:0/16:1/22:2)(14:0/16:2/22:1)(14:0/18:1/ 
               
               
                 total 
                 20:2)(14:0/18:2/20:1)(14:0/18:3/20:0)(16:0/16:1/20:2)(16:0/ 
               
               
                   
                 16:2/20:1)(16:0/18:0/18:3)(16:0/18:1/18:2)(16:1/16:1/ 
               
               
                   
                 20:1)(16:1/16:2/20:0)(16:1/18:0/18:2)(16:1/18:1/18:1)(16:2/ 
               
               
                   
                 18:0/18:1)(17:0/17:0/18:3) 
               
               
                 TAG 52:4  
                 TAG 52:4 total (14:0/16:0/22:4)(14:0/16:1/22:3)(14:0/16:2/ 
               
               
                 total 
                 22:2)(14:0/18:0/20:4)(14:0/18:1/20:3)(14:0/18:3/20:1)(16:0/ 
               
               
                   
                 16:0/20:4)(16:0/16:1/20:3)(16:0/16:2/20:2)(16:0/18:1/ 
               
               
                   
                 18:3)(16:0/18:2/18:2)(16:1/16:1/20:2)(16:1/16:2/20:1)(16:1/ 
               
               
                   
                 18:0/18:3)(16:1/18:1/18:2)(16:2/16:2/20:0)(16:2/18:0/18:2) 
               
               
                 TAG 52:5  
                 TAG 52:5 total (14:0/16:0/22:5)(14:0/16:2/22:3)(14:0/18:1/ 
               
               
                 total 
                 20:4)(14:0/18:2/20:3)(14:0/18:3/20:2)(16:0/16:0/20:5)(16:0/ 
               
               
                   
                 16:1/20:4)(16:0/16:2/20:3)(16:0/18:2/18:3)(16:1/16:1/ 
               
               
                   
                 20:3)(16:1/16:2/20:2)(16:1/18:1/18:3)(16:1/18:2/18:2)(16:2/ 
               
               
                   
                 16:2/20:1)(16:2/18:1/18:2) 
               
               
                 TAG 52:6  
                 TAG 52:6 total (14:0/16:0/22:6)(14:0/18:1/20:5)(14:0/18:2/ 
               
               
                 total 
                 20:4)(16:0/16:1/20:5)(16:0/16:2/20:4)(16:0/18:3/18:3)(16:1/ 
               
               
                   
                 16:1/20:4)(16:1/18:2/18:3)(16:2/18:1/18:3)(16:2/18:2/18:2) 
               
               
                 TAG 53:3  
                 TAG 53:3 total (15:0/18:2/20:1)(16:1/18:2/19:0)(17:0/18:1/ 
               
               
                 total 
                 18:2) 
               
               
                 TAG 53:4  
                 TAG 53:4 total (16:1/17:0/20:3)(17:0/18:2/18:2) 
               
               
                 total 
                   
               
               
                 TAG 54:3  
                 TAG 54:3 total (16:0/16:0/22:3)(16:0/18:1/20:2)(16:0/18:2/ 
               
               
                 total 
                 20:1)(16:1/18:0/20:2)(16:1/18:1/20:1)(16:1/18:2/20:0)(18:0/ 
               
               
                   
                 18:1/18:2)(18:1/18:1/18:1) 
               
               
                 TAG 54:4  
                 TAG 54:4 total (16:0/16:0/22:4)(16:0/16:1/22:3)(16:0/16:2/ 
               
               
                 total 
                 22:2)(16:0/18:0/20:4)(16:0/18:1/20:3)(16:0/18:2/20:2)(16:0/ 
               
               
                   
                 18:3/20:1)(16:1/16:1/22:2)(16:1/16:2/22:1)(16:1/18:0/ 
               
               
                   
                 20:3)(16:1/18:1/20:2)(16:1/18:2/20:1)(16:1/18:3/20:0)(16:2/ 
               
               
                   
                 16:2/22:0)(16:2/18:0/20:2)(16:2/18:1/20:1)(18:0/18:1/ 
               
               
                   
                 18:3)(18:0/18:2/18:2) 
               
               
                 TAG 54:5  
                 TAG 54:5 total (16:0/16:1/22:4)(16:0/16:2/22:3)(16:0/18:1/ 
               
               
                 total 
                 20:4)(16:0/18:2/20:3)(16:0/18:3/20:2)(16:1/16:1/22:3)(16:1/ 
               
               
                   
                 16:2/22:2)(16:1/18:0/20:4)(16:1/18:1/20:3)(16:1/18:2/ 
               
               
                   
                 20:2)(16:1/18:3/20:1)(16:2/16:2/22:1)(16:2/18:0/20:3)(16:2/ 
               
               
                   
                 18:1/20:2)(16:2/18:2/20:1)(18:0/18:2/18:3) 
               
               
                 TAG 54:6  
                 TAG 54:6 total (16:0/16:0/22:6)(16:0/16:1/22:5)(16:0/16:2/ 
               
               
                 total 
                 22:4)(16:0/18:1/20:5)(16:0/18:2/20:4)(16:0/18:3/20:3)(16:1/ 
               
               
                   
                 16:1/22:4)(16:1/16:2/22:3)(16:1/18:0/20:5)(16:1/18:1/ 
               
               
                   
                 20:4)(16:1/18:2/20:3)(16:1/18:3/20:2)(16:2/16:2/22:2)(16:2/ 
               
               
                   
                 18:0/20:4)(16:2/18:1/20:3) 
               
               
                 TAG 54:7  
                 TAG 54:7 total (16:0/16:1/22:6)(16:0/18:2/20:5)(16:0/18:3/ 
               
               
                 total 
                 20:4)(16:1/16:1/22:5)(16:1/18:1/20:5)(16:1/18:2/20:4)(16:1/ 
               
               
                   
                 18:3/20:3)(18:1/18:3/18:3)(18:2/18:2/18:3) 
               
               
                 TAG 54:8  
                 TAG 54:8 total (16:0/18:3/20:5)(16:1/18:2/20:5)(16:1/18:3/ 
               
               
                 total 
                 20:4)(18:2/18:3/18:3) 
               
               
                 TAG 55:3  
                 TAG 55:3 total (18:1/18:2/19:0) 
               
               
                 total 
                   
               
               
                 TAG 56:3  
                 TAG 56:3 total (16:0/18:1/22:2)(16:0/18:2/22:1)(16:0/20:1/ 
               
               
                 total 
                 20:2)(16:1/18:0/22:2)(16:1/18:1/22:1)(16:1/20:0/20:2)(16:1/ 
               
               
                   
                 20:1/20:1)(18:0/18:1/20:2)(18:0/18:2/20:1)(18:1/18:1/ 
               
               
                   
                 20:1)(18:1/18:2/20:0) 
               
               
                 TAG 56:4  
                 TAG 56:4 total (16:0/18:1/22:3)(16:0/18:2/22:2)(16:0/18:3/ 
               
               
                 total 
                 22:1)(16:0/20:1/20:3)(16:0/20:2/20:2)(16:1/18:1/22:2)(16:1/ 
               
               
                   
                 18:2/22:1)(16:1/20:0/20:3)(16:1/20:1/20:2)(18:0/18:1/ 
               
               
                   
                 20:3)(18:0/18:2/20:2)(18:0/18:3/20:1)(18:1/18:1/20:2)(18:1/ 
               
               
                   
                 18:2/20:1)(18:1/18:3/20:0)(18:2/18:2/20:0) 
               
               
                 TAG 56:5  
                 TAG 56:5 total (16:0/18:0/22:5)(16:0/18:1/22:4)(16:0/18:2/ 
               
               
                 total  
                 22:3)(16:0/18:3/22:2)(16:0/20:1/20:4)(16:0/20:2/20:3)(18:0/ 
               
               
                   
                 18:1/20:4)(18:0/18:2/20:3)(18:0/18:3/20:2)(18:1/18:1/ 
               
               
                   
                 20:3)(18:1/18:2/20:2)(18:1/18:3/20:1)(18:2/18:2/20:1)(18:2/ 
               
               
                   
                 18:3/20:0) 
               
               
                 TAG 56:6  
                 TAG 56:6 total (16:0/18:0/22:6)(16:0/18:1/22:5)(16:0/18:2/ 
               
               
                 total 
                 22:4)(16:0/18:3/22:3)(16:0/20:1/20:5)(16:0/20:2/20:4)(16:0/ 
               
               
                   
                 20:3/20:3)(18:0/18:2/20:4)(18:0/18:3/20:3)(18:1/18:1/ 
               
               
                   
                 20:4)(18:1/18:2/20:3)(18:1/18:3/20:2)(18:2/18:2/20:2)(18:2/ 
               
               
                   
                 18:3/20:1) 
               
               
                 TAG 56:7  
                 TAG 56:7 total (16:0/18:1/22:6)(16:0/18:2/22:5)(16:0/18:3/ 
               
               
                 total 
                 22:4)(16:0/20:2/20:5)(16:0/20:3/20:4)(16:1/18:1/22:5)(16:1/ 
               
               
                   
                 18:2/22:4)(16:1/20:2/20:4)(16:1/20:3/20:3)(18:1/18:1/ 
               
               
                   
                 20:5)(18:1/18:2/20:4)(18:1/18:3/20:3)(18:2/18:2/20:3)(18:2/ 
               
               
                   
                 18:3/20:2) 
               
               
                 TAG 56:8  
                 TAG 56:8 total (16:0/18:2/22:6)(16:0/18:3/22:5)(16:0/20:3/ 
               
               
                 total 
                 20:5)(16:0/20:4/20:4)(16:1/18:2/22:5)(16:1/20:3/20:4) (18:0/ 
               
               
                   
                 18:3/20:5)(18:2/18:2/20:4)(18:2/18:3/20:3) 
               
               
                 TAG 56:9  
                 TAG 56:9 total (16:0/18:3/22:6)(16:0/20:4/20:5)(16:1/18:2/ 
               
               
                 total 
                 22:6)(16:1/20:4/20:4)(16:2/18:1/22:6)(18:1/18:3/20:5)(18:2/ 
               
               
                   
                 18:2/20:5)(18:2/18:3/20:4) 
               
               
                 TAG 58:7  
                 TAG 58:7 total (18:0/18:2/22:5)(18:1/18:1/22:5)(18:1/18:2/ 
               
               
                 total 
                 22:4) (18:2/18:2/22:3) 
               
               
                 TAG 58:8  
                 TAG 58:8 total (16:0/20:4/22:4)(18:0/18:2/22:6) (18:0/20:4/ 
               
               
                 total 
                 20:4)(18:1/18:1/22:6)(18:1/18:2/22:5)(18:2/18:2/22:4) 
               
               
                 TAG 58:9  
                 TAG 58:9 total (18:1/18:2/22:6) (18:1/20:3/20:5) (18:1/20:4/ 
               
               
                 total  
                 20:4)(18:2/18:2/22:5)(18:2/20:3/20:4) 
               
               
                 TAG 58:10  
                 TAG 58:10 total (16:0/20:4/22:6)(16:0/20:5/22:5)(18:0/20:5/ 
               
               
                 total 
                 20:5)(18:1/18:3/22:6)(18:1/20:4/20:5)(18:2/18:2/22:6) (18:2/ 
               
               
                   
                 18:3/22:5) (18:2/20:4/20:4) 
               
               
                 TAG 60:12  
                 TAG 60:12 total (20:4/20:4/20:4) (18:2/20:4/22:6) 
               
               
                 total 
               
               
                   
               
             
          
         
       
     
         [0192]    In summary, this study provides novel lipid markers for determining the efficacy and specificity of PCSK9 inhibitors and silencers. Since measurement of LDL cholesterol only is not sufficient for providing information about possible adverse drug reactions, the lipidomic biomarkers are more specific and sensitive markers for efficacy of PCSK9 inhibitors and silencers. 
         [0193]    In view of the above, it will be appreciated that the present invention also encompasses the following items: