USE OF CERAMIDES AND LPLs IN DIAGNOSING CVD

Abstract

The present invention inter alia provides a method, and use thereof, of predicting CV events such as AMI, ACS, stroke, and CV death by determining the concentration of at least one ceramide of Formula I or one lysophospholipid of Formula II and/or III and at least one lysophospholipid of Formula IV, V, VI, VII and/or VIII in a biological sample and comparing those concentrations to a control. Finding an increased concentration of the at least one Formula I ceramide or Formula II and/or III lysophospholipid and a decreased concentration of the at least one Formula IV, V, VI, VII and/or VIII lysophospholipid indicates that the subject has an increased risk of developing one or more CV events. The present disclosure also provides a method, and use thereof, of diagnosing subjects suffering acute ischemia. Also provided are kits and compositions comprising the same for use in predicting and/or diagnosing CV events.

1 .- 29 . (canceled) 
     
     
         30 . A method for determining whether a human subject is at risk to develop one or more cardiovascular (CV) events, wherein the method comprises
 (a) determining in a biological sample obtained from the human subject a concentration of
 (i) at least one ceramide of Formula I: 
   
       
         
           
           
               
               
           
         
         wherein R 1  is a mono-unsaturated alkyl chain having 13 carbon atoms or a saturated, mono-unsaturated or di-unsaturated alkyl chain having 15 carbon atoms, and wherein R 2  is a saturated alkyl chain having 15-21 carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 or 25 carbon atoms, and wherein the biological sample is an artery tissue, a blood sample, a blood serum sample, a blood plasma sample or a lipoprotein fraction obtained therefrom, or a urine sample, or
 (ii) at least one LPI of Formula II and/or III: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a fatty acyl group; 
         b) determining in the biological sample obtained from the human subject a concentration of
 (i) at least one LPC of Formula IV and/or V: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a fatty acyl group,
 (ii) at least one LPA of Formula VI and/or VII: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a fatty acyl group, or
 (iii) at least one LSM of Formula VIII: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a mono-unsaturated alkyl chain having 13 carbon atoms or a saturated, mono-unsaturated or di-unsaturated alkyl chain having 15 carbon atoms; 
         wherein each of the LPI, the LPC, the LPA and the LSM is a lysophospholipid (LPL), 
         (c) comparing the concentration of the at least one ceramide of Formula I or the at least one LPL of Formula II and/or III and the concentration of the at least one LPL of Formula IV, V, VI, VII and/or VIII to a control sample; and 
         (d) determining that the human subject has an increased risk of developing one or more CV events, if the biological sample contains an increased concentration of the at least one ceramide of Formula I or at least one LPL of Formula II and/or III and a decreased concentration of the at least one LPL of Formula IV, V, VI, VII and/or VIII, as compared to the control sample. 
       
     
     
         31 . A method of detecting in a biological sample obtained from a subject the concentration of at least one ceramide and at least one LPL, wherein the method comprises the steps of:
 (a) determining in a biological sample obtained from the subject the concentration of
 (i) at least one ceramide of Formula I: 
   
       
         
           
           
               
               
           
         
         wherein R 1  is a mono-unsaturated alkyl chain having 13 carbon atoms or a saturated, mono-unsaturated or di-unsaturated alkyl chain having 15 carbon atoms, and wherein R 2  is a saturated alkyl chain having 15-21 carbon atoms, a mono-unsaturated alkyl chain having 21-23 carbon atoms, or a di-unsaturated alkyl chain having 23 or 25 carbon atoms; or
 (ii) at least one LPI of Formula II and/or III: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a fatty acyl group; 
         (b) determining in the biological sample from the subject the concentration of
 (i) at least one LPC of Formula IV and/or V: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a fatty acyl group,
 (ii) at least one LPA of Formula VI and/or VII: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a fatty acyl group, or
 (iii) at least one LSM of Formula VIII: 
 
       
       
         
           
           
               
               
           
         
         wherein R is a mono-unsaturated alkyl chain having 13 carbon atoms or a saturated, mono-unsaturated or di-unsaturated alkyl chain having 15 carbon atoms; 
         wherein each of the LPI, the LPC, the LPA and the LSM is a lysophospholipid (LPL), and 
         (c) optionally comparing the concentration of the at least one ceramide of Formula I or the at least one LPL of Formula II and/or III and the concentration of the at least one LPL of Formula IV, V, VI, VII and/or VIII to a control sample. 
       
     
     
         32 . The method of  claim 30 , wherein the concentrations of the at least one ceramide of Formula I and the at least one LPL of Formula II, III, IV, V, VI, VII and/or VIII are determined using a mass spectrometry instrument, and optionally wherein the mass spectrometry instrument is coupled to a direct sample infusion method or to a high performance separation method. 
     
     
         33 . The method of  claim 30 , wherein concentrations of at least 2, at least 3, at least 4, at least 5, or at least 6 ceramides of Formula I and/or at least 2, at least 3, at least 4, at least 5, or at least 6 LPLs of Formula II, III, IV, V, VI, VII and/or VIII are determined. 
     
     
         34 . The method of  claim 30 , wherein the subject has an increased risk of developing one or more CV events, if the biological sample contains an increased Z value as compared to a control sample, according to the following equation: Z=a log (ceramide or LPL (II/III))+b log (LPL (IV-VIII)), wherein a, b E R. 
     
     
         35 . The method of  claim 30 , wherein the concentration of at least one of the following lipids of Formula I, II and/or III are determined: Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:1) and LPI 20:4. 
     
     
         36 . The method of  claim 30 , wherein the concentration of at least one of the following lipids of Formula IV, V and/or VIII are determined: LPC 14:0, LPC 18:0, LPC 16:0 and LysoSM(d18:1). 
     
     
         37 . The method of  claim 30 , wherein the one or more CV events are one or more of acute myocardial infarction (AMI), acute coronary syndrome (ACS), stroke, or CV death. 
     
     
         38 . The method of  claim 30 , further comprising a step of extracting lipids from the biological sample prior to determining the concentration of the at least one ceramide of Formula I and the at least one LPL of Formula II, III, IV, V, VI, VII and/or VIII. 
     
     
         39 . The method of  claim 30 , further comprising a step of spiking the biological sample with at least one isotope-labelled ceramide of Formula I and at least one isotope-labelled LPL of Formula II, III, IV, V, VI, VII and/or VIII prior to determining the concentration of the at least one ceramide of Formula I and the at least one LPL of Formula II, III, IV, V, VI, VII and/or VIII, wherein the isotope is optionally deuterium. 
     
     
         40 . The method of  claim 30 , wherein the biological sample and the control sample comprise at least one isotope-labelled ceramide of Formula I and at least one isotope-labelled LPL of Formula II, III, IV, V, VI, VII and/or VIII. 
     
     
         41 . The method of  claim 30 , wherein the biological sample is an artery tissue. 
     
     
         42 . The method of  claim 30 , wherein the method further comprises:
 (e) diagnosing the human subject as having an increased risk of developing one or more CV events from the results in step (d);   (f) administering a treatment to the human subject diagnosed in step (e).   
     
     
         43 . The method of  claim 42 , wherein the treatment is administering a drug and/or providing therapeutic and/or behavioral modification and/or a surgical operation to the human subject. 
     
     
         44 . The method of  claim 42 , wherein the treatment is a lipid lowering drug treatment. 
     
     
         45 . A method of treating or preventing one or more CV events in a human subject identified as being at risk to develop one or more CV events, the method comprising:
 administering to the human subject a treatment, wherein prior to administering the treatment, the human subject has been identified as being at risk to develop the one or more CV events by the method according to  claim 30 .   
     
     
         46 . The method of  claim 31 , wherein the concentrations of at least one of the following lipids of Formula I, II and/or III is detected: Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:1) and LPI 20:4 and wherein the concentrations of at least one of the following lipids of Formula IV, V and/or VIII is detected: LPC 14:0, LPC 18:0, LPC 16:0 and LysoSM(d18:1); and wherein the concentrations of the detected lipids are compared to a control sample. 
     
     
         47 . The method of  claim 31 , wherein the determining in step (a) comprises assaying the concentration of the at least one ceramide or the at least one LPI using mass spectrometry and wherein said determining in step (b) comprises assaying the concentration of the at least one LPC and/or the at least one LPA and/or the at least one LSM using mass spectrometry. 
     
     
         48 . The method of  claim 45 , wherein the treatment is a lipid lowering drug treatment. 
     
     
         49 . The method of  claim 31 , further comprising a step of spiking the biological sample with at least one isotope-labelled ceramide of Formula I and at least one isotope-labelled LPL of Formula II, III, IV, V, VI, VII and/or VIII prior to determining the concentration of the at least one ceramide of Formula I and the at least one LPL of Formula II, III, IV, V, VI, VII and/or VIII, wherein the isotope is optionally deuterium. 
     
     
         50 . The method of  claim 31 , wherein the biological sample and optionally the control sample comprise at least one isotope-labelled ceramide of Formula I and at least one isotope-labelled LPL of Formula II, III, IV, V, VI, VII and/or VIII. 
     
     
         51 . The method of  claim 30 , wherein the R of at least one of Formulas II-VII has 13-21 carbon atoms and 0-6 double bonds. 
     
     
         52 . The method of  claim 31 , wherein the R of at least one of Formulas II-VII has 13-21 carbon atoms and 0-6 double bonds. 
     
     
         53 . The method of  claim 31 , wherein the biological sample is an artery tissue, a blood sample, a blood serum sample, a blood plasma sample or a lipoprotein fraction obtained therefrom, or a urine sample.