	US 12,270,816 B2
	Lipidomic biomarkers for identification of high-risk coronary artery disease patients
Reijo Laaksonen, Lampaeaelae (FI); Kim Ekroos, Helsinki (FI); Reini Hurme, Espoo (FI); and Riikka Katainen, Helsinki (FI)
Assigned to ZORA BIOSCIENCES OY, Espoo (FI)
Filed by ZORA BIOSCIENCES OY, Espoo (FI)
Filed on Feb. 17, 2021, as Appl. No. 17/177,503.
Application 17/177,503 is a continuation of application No. 16/291,845, filed on Mar. 4, 2019, granted, now 10,955,427.
Application 16/291,845 is a continuation of application No. 15/803,252, filed on Nov. 3, 2017, granted, now 10,261,101, issued on Apr. 16, 2019.
Application 15/803,252 is a continuation of application No. 14/875,087, filed on Oct. 5, 2015, granted, now 9,841,431, issued on Dec. 12, 2017.
Application 14/875,087 is a continuation of application No. 13/805,319, granted, now 9,201,080, issued on Dec. 1, 2015, previously published as PCT/EP2011/060253, filed on Jun. 20, 2011.
Claims priority of provisional application 61/356,675, filed on Jun. 21, 2010.
Claims priority of application No. 10006399 (EP), filed on Jun. 20, 2010.
Prior Publication US 2021/0239719 A1, Aug. 5, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/92 (2006.01)

CPC G01N 33/92 (2013.01) [G01N 2405/04 (2013.01); G01N 2405/06 (2013.01); G01N 2570/00 (2013.01); G01N 2800/324 (2013.01); G01N 2800/50 (2013.01); G01N 2800/52 (2013.01)]

14 Claims

1. A method for determining one or more lipid concentration(s), lipid-lipid concentration ratio(s) and/or lipid-clinical concentration ratio(s) in a sample by mass spectrometry (MS), comprising:

(a) extracting lipids from the sample; and

(b) separating the lipids by liquid chromatography (LC) prior to the MS determination; and

(c) determining the one or more lipid concentration(s), lipid-lipid concentration ratio(s) and/or lipid-clinical concentration ratio(s) in the sample by MS,

wherein the one or more lipid(s) is (are) selected from:

Ceramide (Cer)(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:1), Cer(d18:1/26:1), Lactosylceramide (LacCer)(d18:1/18:0), LacCer(d18:1/20:0), LacCer(d18:1/22:0), LacCer(d18:1/24:0), LacCer(d18:1/24:1), Galactosyl- and glucosylceramide (GlcCer) (d18:1/18:0), GlcCer(d18:1/20:0), GlcCer(d18:1/24:1), GlcCer(d18:1/26:1), ether-linked Phosphatidylserine (PS O)-18:2/16:0-alkenyl, PS O-16:0/18:2-alkenyl, PS O-16:1/18:2-alkyl, PS O-18:0/18:2-alkenyl (PS O-18:1/18:2-alkyl), ether-linked phosphatidylcholine (PC-O)-32:0 (KDdiA-PC), phosphatidylcholine (PC) 18:0/20:4, PC 16:0/20:4, PC 18:0/20:3, PC 16:0/20:3, PC 16:0/22:6, PC 16:0/18:2, PC 18:0/18:1, PC 18:1/18:2, LPC 18:1, PC O-40:3, Sphingomyelin (SM) (d18:1/14:0) (d18:1/13:1-OH), SM (d18:1/23:0) (d18:1/22:1-OH), SM (d18:1/24:0) (d18:1/23:1-OH), Cer(d18:0/22:0), Cer(d18:0/24:0) and PC 16:0/16:0;

wherein the one or more lipid-lipid concentration ratio(s) is (are) selected from:

Cer(d18:1/18:0)/PC 16:0/18:1, Cer(d18:1/20:0)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/18:1, Cer(d18:1/24:1)/PC 16:0/18:2, Cer(d18:1/18:0)/PC 18:0/18:1, Cer(d18:1/20:0)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/18:1, Cer(d18:1/24:1)/PC 18:0/18:2, Cer(d18:1/18:0)/PC 18:1/18:1, Cer(d18:1/20:0)/PC 18:1/18:1, Cer(d18:1/24:1)/PC 18:1/18:1, Cer(d18:1/24:1)/PC 18:1/18:2, Cer(d18:1/18:0)/PC 16:0/20:3, Cer(d18:1/20:0)/PC 16:0/20:3, Cer(d18:1/22:0)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 16:0/20:3, Cer(d18:1/24:1)/PC 18:0/20:3, Cer(d18:1/18:0)/PC 16:0/20:4, Cer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/22:0)/PC 16:0/20:4, Cer(d18:1/24:1)/PC 16:0/20:4, Cer(d18:1/18:0)/PC 18:0/20:4, Cer(d18:1/20:0)/PC 18:0/20:4, Cer(d18:1/22:0)/PC 18:0/20:4, Cer(d18:1/24:1)/PC 18:0/20:4, Cer(d18:1/24:1)/PC O-40:3, Cer(d18:1/26:0)/PC O-40:0, PC 16:0/16:0/PC 16:0/20:4, PC O-18:0/18:2-alkyl/PC O-36:5, PC O-32:0 (KDdiA-PC)/PC O-38:5, Cer(d18:1/18:0)/Lysophosphatidylcholine (LPC) 16:0, Cer(d18:1/16:0)/LPC 18:1, Cer(d18:1/18:0)/LPC 18:1, Cer(d18:1/24:1)/LPC 18:1, Cer(d18:1/22:0)/LPC 18:2, Cer(d18:1/24:1)/LPC 18:2, LacCer(d18:1/18:0)/PC 16:0/18:1, LacCer(d18:1/20:0)/PC 16:0/18:1, LacCer(d18:1/18:0)/PC 18:0/18:1, LacCer(d18:1/20:0)/PC 18:0/18:1, LacCer(d18:1/18:0)/PC 18:1/18:1, LacCer(d18:1/20:0)/PC 18:1/18:1, LacCer(d18:1/18:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/PC 18:1/18:2, LacCer(d18:1/20:0)/PC 18:2/18:2, LacCer(d18:1/18:0)/PC 16:0/20:3, LacCer(d18:1/20:0)/PC 16:0/20:3, LacCer(d18:1/22:0)/PC 16:0/20:3, LacCer(d18:1/24:0)/PC 16:0/20:3, LacCer(d18:1/18:0)/PC 18:0/20:3, LacCer(d18:1/20:0)/PC 18:0/20:3, LacCer(d18:1/22:0)/PC 18:0/20:3, LacCer(d18:1/20:0)/PC 16:0/20:4, LacCer(d18:1/22:0)/PC 16:0/20:4, LacCer(d18:1/20:0)/PC 18:0/20:4, GlcCer(d18:1/20:0)/PC 16:0/20:4, Cer(d18:1/18:0)/SM (d18:1/17:2-OH), Cer(d18:1/18:0)/SM (d18:1/18:1), Cer(d18:1/18:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/20:0)/SM (d18:1/14:0) (d18:1/13:1-OH), Cer(d18:1/24:1)/SM (d18:1/17:1-OH), Cer(d18:1/24:1)/SM (d18:1/18:0), Cer(d18:1/24:1)/SM (d18:1/23:0) (d18:1/22:1-OH), Cer(d18:1/24:1)/SM (d18:1/24:0) (d18:1/23:1-OH), LacCer(d18:1/20:0)/SM (d18:1/17:1-OH), LacCer(d18:1/20:0)/SM (d18:1/18:0) and LacCer(d18:1/22:0)/SM (d18:1/14:0) (d18:1/13:1-OH) Cer(d18:1/24:1)/Total PC, Cer(d18:1/18:0)/Total LPC, Cer(d18:1/20:0)/Total LPC, Cer(d18:1/24:1)/Total LPC, Cer(d18:1/18:0)/Total Cholesterol Ester (CE), Cer(d18:1/24:1)/Total CE, Total Cer/Total PC, Total LacCer/Total PC, LacCer(d18:1/22:0)/Total PC, LacCer(d18:1/22:0)/Total LPC, LacCer(d18:1/24:1)/Total PC, LacCer(d18:1/24:1)/Total LPC, LacCer(d18:1/24:1)/Total PC O, SM (d18:1/17:0) (d18:1/16:1-OH)/Total PC O, Cer(d18:0/22:0)/Cer(d18:1/18:0), Cer(d18:0/22:0)/Cer(d18:1/20:0), Cer(d18:0/22:0)/Cer(d18:1/22:0), Cer(d18:0/22:0)/Cer(d18:1/24:1), Cer(d18:0/24:0)/Cer(d18:1/16:0), Cer(d18:0/24:0)/Cer(d18:1/18:0), Cer(d18:0/24:0)/Cer(d18:1/20:0), Cer(d18:0/24:0)/Cer(d18:1/22:0), Cer(d18:0/24:0)/Cer(d18:1/24:1), PC 18:1/18:2/PS O-16:0/18:2-alkenyl, PC 18:1/18:2/PS O-16:1/18:2-alkyl, PC 16:0/20:3/PS O-16:0/18:2-alkenyl, PC 16:0/20:3/PS O-16:1/18:2-alkyl, PC 18:0/20:3/PS O-16:0/18:2-alkenyl, PC 18:0/20:3/PS O-16:1/18:2-alkyl, PC 18:0/20:3/PS O-18:2/16:0-alkenyl, PC 16:0/20:4/PS O-16:0/18:2-alkenyl, PC 16:0/20:4/PS O-16:1/18:2-alkyl, PC 18:0/20:4/PS O-16:0/18:2-alkenyl, PC 18:0/20:4/PS O-16:1/18:2-alkyl, PC O-40:3/PS O-18:2/16:0-alkenyl, LPC 18:2/PS O-16:0/18:2-alkenyl, LPC 18:2/PS O-16:1/18:2-alkyl, LPC 16:0/LacCer(d18:1/20:0), LPC 16:0/LacCer(d18:1/22:0), LPC 16:0/LacCer(d18:1/24:0), LPC 16:0/LacCer(d18:1/24:1), LPC 18:1/LacCer(d18:1/20:0), LPC 18:2/LacCer(d18:1/20:0), (d18:1/24:0), Cer(d18:0/24:0)/LacCer(d18:1/24:0), Cer(d18:0/24:0)/SM (d18:1/17:0) (d18:1/16:1-OH), Cer(d18:0/22:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/22:0)/PS O-16:1/18:2-alkyl, Cer(d18:0/24:0)/PS O-16:0/18:2-alkenyl, Cer(d18:0/24:0)/Total Cer, Cer(d18:0/22:0)/Total CE, Cer(d18:0/24:0)/Total CE, Cer(d18:0/24:1)/Total CE, PC 16:0/20:4/Total diacylglycerol (DAG) and LPC 16:0/Total LacCer; and/or

wherein the one or more lipid-clinical concentration ratio(s) is (are) selected from:

Cer(d18:1/16:0)/apolipoprotein A-I, Cer(d18:1/16:0)/Low Density Lipoprotein (LDL) cholesterol, Cer(d18:1/16:0)/triglycerides, Cer(d18:1/16:0)/total cholesterol, Cer(d18:1/18:0)/apolipoprotein A-I, Cer(d18:1/18:0)/apolipoprotein B, Cer(d18:1/18:0)/HDL cholesterol, Cer(d18:1/18:0)/total-c/HDL-c, Cer(d18:1/18:0)/total cholesterol, Cer(d18:1/18:0)/triglycerides, Cer(d18:1/18:0)/LDL cholesterol, Cer(d18:1/20:0)/apolipoprotein A-I, Cer(d18:1/20:0)/apolipoprotein B, Cer(d18:1/20:0)/High Density Lipoprotein (HDL) cholesterol, Cer(d18:1/20:0)/total cholesterol, Cer(d18:1/20:0)/triglycerides, Cer(d18:1/20:0)/total-cholesterol (total-c)/High Density Lipoprotein-c (HDL-c), Cer(d18:1/20:0)/LDL cholesterol, Cer(d18:1/22:0)/apolipoprotein A-I, Cer(d18:1/22:0)/apolipoprotein B, Cer(d18:1/22:0)/LDL cholesterol, Cer(d18:1/22:0)/triglycerides, Cer(d18:1/22:0)/total cholesterol, Cer(d18:1/24:0)/apolipoprotein A-I, Cer(d18:1/24:0)/apolipoprotein B, Cer(d18:1/24:0)/LDL cholesterol, Cer(d18:1/24:0)/total cholesterol, Cer(d18:1/24:1)/apolipoprotein A-I, Cer(d18:1/24:1)/apolipoprotein B, Cer(d18:1/24:1)/HDL cholesterol, Cer(d18:1/24:1)/Low Density Lipoprotein-c (LDL-c)/HDL-c, Cer(d18:1/24:1)/total-c/HDL-c, Cer(d18:1/24:1)/LDL cholesterol, Cer(d18:1/24:1)/total cholesterol, Cer(d18:1/24:1)/triglycerides, Total Cer/apolipoprotein A-I, Total Cer/apolipoprotein B, Total Cer/LDL cholesterol, Total Cer/total cholesterol, Total Cer/triglycerides, PC O-32:0 (KDdiA-PC)/apolipoprotein A-I, PC O-32:0 (KDdiA-PC)/triglycerides, PC O-34:1/apolipoprotein B, PC O-34:1/triglycerides, LacCer(d18:1/18:0)/apolipoprotein A-I, LacCer(d18:1/18:0)/apolipoprotein B, LacCer(d18:1/18:0)/total cholesterol, LacCer(d18:1/18:0)/HDL cholesterol, LacCer(d18:1/18:0)/LDL cholesterol, LacCer(d18:1/18:0)/total-c/HDL-c, LacCer(d18:1/18:0)/triglycerides, LacCer(d18:1/20:0)/apolipoprotein A-I, LacCer(d18:1/20:0)/apolipoprotein B, LacCer(d18:1/20:0)/total cholesterol, LacCer(d18:1/20:0)/HDL cholesterol, LacCer(d18:1/20:0)/LDL cholesterol, LacCer(d18:1/20:0)/total-c/HDL-c, LacCer(d18:1/20:0)/LDL-c/HDL-c, LacCer(d18:1/20:0)/apoA1/apoB, LacCer(d18:1/20:0)/triglycerides, LacCer(d18:1/22:0)/apolipoprotein A-I, LacCer(d18:1/22:0)/apolipoprotein B, LacCer(d18:1/22:0)/total cholesterol, LacCer(d18:1/22:0)/HDL cholesterol, LacCer(d18:1/22:0)/LDL cholesterol, LacCer(d18:1/22:0)/total-c/HDL-c, LacCer(d18:1/22:0)/apoA1/apoB, LacCer(d18:1/22:0)/triglycerides, LacCer(d18:1/24:0)/apoA1/apoB, LacCer(d18:1/24:1)/apolipoprotein A-I, LacCer(d18:1/24:1)/apolipoprotein B, LacCer(d18:1/24:1)/total cholesterol, LacCer(d18:1/24:1)/triglycerides, Cer(d18:0/22:0)/apolipoprotein B, Cer(d18:0/22:0)/LDL-c/HDL-c, Cer(d18:0/22:0)/total-c/HDL-c, Cer(d18:0/24:0)/apolipoprotein A-I, Cer(d18:0/24:0)/apolipoprotein B, Cer(d18:0/24:0)/total cholesterol, Cer(d18:0/24:0)/HDL cholesterol, Cer(d18:0/24:0)/LDL-c/HDL-c, Cer(d18:0/24:0)/total-c/HDL-c, Cer(d18:0/24:0)/LDL cholesterol, PC 18:0/18:1/LDL-c/HDL-c, PC 16:0/20:3/apolipoprotein B, PC 16:0/20:3/HDL cholesterol, PC 16:0/20:3/total-c/HDL-c, PC 16:0/20:3/LDL-c/HDL-c, PC 18:0/20:3/total-c/HDL-c, PC 18:0/20:3/LDL-c/HDL-c, PC 16:0/20:4/apolipoprotein A-I, PC 16:0/20:4/apolipoprotein B, PC 16:0/20:4/total cholesterol, PC 16:0/20:4/LDL cholesterol, PC 16:0/20:4/total-c/HDL-c, PC 16:0/20:4/LDL-c/HDL-c, PC 18:0/20:4/apoA1/apoB, PC O-38:6/apolipoprotein A-I, LPC 18:2/apolipoprotein B, LPC 18:2/total cholesterol, LPC 18:2/HDL cholesterol, LPC 18:2/LDL cholesterol, LPC 18:2/LDL-c/HDL-c and LPC 18:2/apoA1/apoB, wherein the sample is from a subject suspected to be at risk for one or more CVD complications, wherein the one or more CVD complications comprise myocardial infarction (MI) and/or death.