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WO2015085225A1 - Lipid biomarker signatures for lung injury diagnosis - Google Patents

Lipid biomarker signatures for lung injury diagnosis Download PDF

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Publication number
WO2015085225A1
WO2015085225A1 PCT/US2014/068882 US2014068882W WO2015085225A1 WO 2015085225 A1 WO2015085225 A1 WO 2015085225A1 US 2014068882 W US2014068882 W US 2014068882W WO 2015085225 A1 WO2015085225 A1 WO 2015085225A1
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galcer
glc
cer
lung
control sample
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French (fr)
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Hector De Leon
Stephanie Boue
Julia HOENG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD

Definitions

  • the present invention relates to lipid biomarkers that are useful for the diagnosis of lung injury, such as emphysema.
  • the present invention also relates to methods of diagnosing or prognosing lung injury, such as emphysema.
  • the present invention further relates to methods of treating or preventing lung injury by inhibiting the expression or activity of genes related to lipid metabolism or transport.
  • the present invention also relates to methods of identifying compounds useful for treating or preventing lung injury.
  • Emphysema is a lung condition that occurs when the alveoli are gradually destroyed, rendering an emphysema patient progressively short of breath.
  • Emphysema is part of a group of diseases collectively called Chronic Obstructive Pulmonary Disease (COPD) and is characterized by parenchymal destruction, loss of alveolar attachments and decrease in elastic recoil.
  • COPD chronic Obstructive Pulmonary Disease
  • COPD is the third leading cause of death in the U.S. and is projected to be the fourth leading cause of death worldwide by 2030.
  • over €20 billion is spent treating COPD, while approximately $18 billion is spent in the U.S.
  • Emphysema can be caused by long-term exposure to lung irritants in the environment, such as air pollution, chemical fumes, dust and tobacco smoke.
  • emphysema Because long-term exposure to lung irritants is typically required, most individuals who suffer from emphysema are more than 40 years old when symptoms first present. Such symptoms include an ongoing cough or a cough that produces an excess of mucus; shortness of breath, especially with physical activity; wheezing and chest tightness. Early detection of emphysema can be difficult because symptoms slowly worsen over time, and most affected individuals do not notice early symptoms because they are mild or easy to correct by lifestyle adjustment.
  • Emphysema is typically diagnosed by signs and symptoms, including medical history, family history and test results. Diagnostic tests for emphysema include lung function tests, such as spirometry, including lung volume
  • the diagnostic tests for emphysema require the disease to have progressed to the point that lung function is moderately affected.
  • the present invention is directed to lipid biomarkers for classifying, diagnosing or grading emphysema.
  • a first aspect of the invention provides a method of diagnosing an individual as being at risk for or having lung injury comprising detecting the level of two or more lipid biomarkers in a test sample obtained from the individual; and comparing the level of the two or more lipid biomarkers in the test sample to the level of the two or more lipid biomarkers in a control sample, wherein, if the level of the two or more lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk of having lung injury.
  • the two or more lipid biomarkers may independently be selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a glycerophosphoethanolamine, a glycerophosphoglycerol, a glycerophosphoinositol, a glycerophosphoserine, an acidic glycosphingolipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
  • the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0); CE(18: 1); CE(18:2); CE(18:3); CE(20:3) CE(20:4); CE(20:5); CE(22:0);
  • CE(22:5); CE(22:6); and CE(24:2) preferably CE(20:4) or CE(22:5).
  • the eicosanoid may be selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-
  • DHET 6-keto-PGFl alpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12- DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET;
  • 15-HEPE 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD 2 ); Prostaglandin E2 (PGE 2 ); PGF2alpha; TXB 2 ; and
  • TXB 3 preferably, 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD 2 ; PGE 2 ; or TXB 3 .
  • the glycerophosphocholine may be selected from phosphatidylcholine
  • PC PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18:1); PC(16:0/16:0);
  • PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4) preferably,
  • the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2);
  • the glycerophosphoethanolamine is selected from
  • PE phosphatidylethanolamine
  • the ceramide (Cer) may be selected from Cer(dl8:0/16:0);
  • the neutral glycosphingolipid is selected from glucosyl/galactosyl Cer (Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
  • Gb3 globotriaosylceramide
  • the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl8:l/16:0);
  • the acidic glycosphingolipid may be selected from GMl(dl8:l/16:0); GMl(dl8:l/24:0); GMl(dl8:l/24:l); GM3(dl8:l/16:0); GM3(dl8:l/18:0);
  • the sphingoid base may be selected from sphingosine- 1 -phosphate (SlP)(dl8:l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine
  • SPA sphingosine
  • the lipid biomarker is selected from 5-HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16:1);
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PGD2; PGE2; PC(14:0/14:0);
  • LacCer(dl 8: 1/24: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1);
  • Glc/GalCer(dl8:0/24: l); and Cer(dl 8:0/18:0) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of arachidonic acid; PC(16:0/18:0);
  • PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16:0/18:0); PC(16:0/20:4);
  • Cer(dl8: l/16:0); Cer(dl8:l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
  • the individual suffers from or is at risk of having lung injury if the level of three or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of four or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of five or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of ten or more lipid biomarkers is different in the test sample than in the control sample.
  • the method may further comprise detecting the level of the two or more lipid biomarkers in the control sample.
  • the test sample may be selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • the test sample is obtained from a large airway of the individual, such as a bronchial biopsy, or the lung of the individual, such as a lung biopsy, including a biopsy of the parenchyma.
  • control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • the control sample may be obtained from a large airway or lung of an individual not affected with lung injury, such as from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury.
  • the control sample is obtained, prior to the onset of lung injury, from the individual at risk for or having the emphysema.
  • the control sample is obtained from an individual that does not suffer from lung injury.
  • the level of the two or more lipid biomarkers in the test sample and the level of the two or more lipid biomarkers in the control sample may be detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography.
  • the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry.
  • the chromatography is thin layer chromatography, solid-phase extraction
  • HPLC high performance liquid chromatography
  • hydrophilic interaction liquid chromatography hydrophilic interaction liquid chromatography
  • ultra-performance liquid chromatography ultra-performance liquid chromatography
  • the lung injury is emphysema or COPD.
  • a method of diagnosing an individual as being at risk for or having lung injury comprising
  • the two or more lipid biomarkers are independently selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
  • glycerophosphoinositol a glycerophosphoserine
  • an acidic glycosphingolipid a ceramide
  • a neutral glycosphingolipid a phosphosphingolipid
  • a phosphosphingolipid a sphingoid base
  • sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1);
  • the sterol is CE(20:4) or CE(22:5).
  • the eicosanoid is selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-DHET; 6-keto- PGFlalpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12-DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET; 15-HEPE; 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD 2 ); Prostaglandin E2 (PGE 2 ); PGF2alpha; TXB 2 ; and TXB 3 .
  • eicosanoid is selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD 2 ; PGE 2 ; and TXB 3 .
  • glycerophosphocholine is selected from phosphatidylcholine (PC)(14:0/14:0);
  • glycerophosphocholine is selected from PC(16:0/16:0); and PC(16: 1/16: 1).
  • the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2); PG(18:1/18: 1); PG(18: 1/18:2); and PG(18:2/18:2).
  • glycerophosphoglycerol is selected from PG(18: 1/18: 1), PG(18: 1/18:2) and PG(18:2/18:2).
  • PE PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); PE(16:0/20:4); PE(16:0/22:4); PE(18:0/18:0); PE(18:0/20:4); PE(18:0/22:4); PE(18:1/18: 1); PE(18: l/20:4); and PE(22:6/22:6).
  • glycerophosphoethanolamine is selected from PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); and PE(16:0/20:4).
  • ceramide is selected from Cer(dl8:0/16:0); Cer(dl 8:0/18:0); Cer(dl8:0/18: l);
  • Glc/GalCer (dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl 8:0/24: 1);
  • lactosylCer (LacCer)(dl 8:0/16:0); LacCer(dl8: l/16:0); LacCer(dl8: l/18:0); LacCer(dl8:l/20:0); LacCer(dl 8: 1/22:0); LacCer(dl 8: 1/23:0);
  • glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0);
  • glycosphingolipid is selected from GMl(dl8:l/16:0); GM3(dl8:l/16:0); and GM3(dl8:l/24:0).
  • sphingoid base is selected from sphingosine-1 -phosphate (SlP)(dl8:l);
  • lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4);
  • SPH(d20: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway or a lung.
  • Cer(dl8: l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
  • test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • test sample is obtained from a large airway or a lung of the individual.
  • test sample is obtained from a bronchial biopsy or a lung biopsy.
  • control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • control sample is obtained from a large airway or a lung of an individual not affected with lung injury.
  • control sample is obtained from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury.
  • control sample is obtained from the individual at risk for or having the emphysema prior to onset of the lung injury.
  • control sample is obtained from an individual that does not suffer from lung injury.
  • MALDI desorption/ionization
  • chromatography is thin layer chromatography, solid-phase extraction chromatography, high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography, or ultra-performance liquid chromatography.
  • Figure 1A provides representative images of lung tissue from cigarette smoke (CS)-exposed, Sham, and Cessation animals at the 2-, 3-, and 7-month time points. Tissues were stained with haematoxylin and eosin (H&E).
  • Figure IB provides a representative image of multinucleated giant cells found in lung tissue from CS-exposed animals. Tissues were stained with alcian blue periodic acid Schiff reagent (AB-PAS).
  • Figure 2 provides histopathological findings and histomorphometry in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months.
  • B The number of multinucleated giant cells are shown as the mean numbers ⁇ SEM.
  • Figure 3 provides pulmonary lipid profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung lipid species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 4 provides relative percentage differences in lung molecular PCs and PGs concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05,
  • Figure 5 A provides relative percentage differences in lung molecular PCs and PGs concentrations between mice exposed to smoke (CS) and fresh air
  • FIG. 5B provides relative percentage differences in lung molecular concentrations of PEs between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months.
  • Figure 5C provides relative percentage differences in plasma molecular concentrations of PCs between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 6 provides pulmonary ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 7 provides pulmonary glucosyl/galactosyl ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular glucosyl/galactosyl ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 8 provides pulmonary lactosyl ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular lactosyl ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 9 provides pulmonary GM1 ganglioside species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular GM1 ganglioside species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 10 provides pulmonary GM3 ganglioside species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular GM3 ganglioside species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 11 provides pulmonary sphingoid base species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular sphingoid base species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05,
  • Figure 12 provides pulmonary globotriaosylceramide species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular globotriaosylceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 13 provides pulmonary cholesterol ester species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular cholesterol ester species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, **p ⁇ 0.01, and ***p ⁇ 0.001.
  • Figure 14 provides plasma cholesterol ester species profiles in mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in plasma molecular cholesterol ester species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05,
  • Figure 15 provides a CONSORT diagram for the clinical study.
  • the flow chart of the study shows the number of subjects enrolled in the study and the number of completers indicating reasons for subject recruitment, as recommended by the Consolidated Standards of Reporting Trials guidelines.
  • Figure 16 shows median relative differences in lipid concentrations categorized by lipid class in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 17 provides median relative differences in diradylglycerol (DAG) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 18 provides box-whisker plots and scatter grams of individual diradylglycerol concentrations in the serum of current smokers and never-smokers, respectively.
  • Panel A represents the log-transformed results for DAG(16:0/18: 1), and panel B those for DAG(18: 1/18: 1) serum levels in nmol/mL.
  • Box-whisker plots reflect the first and third quartile (lower and upper boundary of box, respectively), median (green line), and minimum and maximum values (lower and upper whisker, respectively) for the corresponding study group.
  • Outliers are represented by open circles.
  • Serum lipid concentrations are further represented by dots, where red dots indicate study subjects taking lipid-modifying drugs and blue dots those who do not.
  • Figure 19 provides median relative differences in
  • PC glycerophosphatidylcholine
  • Figure 20 provides median relative differences in triacylglycerol (TAG) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • TAG triacylglycerol
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 21 provides median relative differences in lactosylceramide concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 22 provides median relative differences in glucosyl/galactosyl ceramide concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05.
  • Figure 23 provides median relative differences in
  • PE glycerophosphoethanolamine
  • Figure 24 provides median relative differences in eicosanoid (EICO) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • EICO eicosanoid
  • Figure 25 provides median relative differences in ceramide (Cer) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 26 shows median relative differences in glycophosphosphingolipid (SM) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05.
  • Figure 27 shows median relative differences in sterol (CE) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively.
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 28 shows median relative differences in lipid concentrations categorized by lipid class in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 29 shows median relative differences in sterol (CE) concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom.
  • Figure 30 provides box-whisker plots and scattergrams of individual sterol concentrations in the serum of current smokers and never-smokers, respectively.
  • Panel A represents the results for CE(16:0), panel B those for CE(18:2), panel C those for CE(19:0), and panel D represents the log-transformed results for CE(20:5) serum levels in nmol/mL.
  • Box-whisker plots reflect the first and third quartile (lower and upper boundary of box, respectively), median (green line), and minimum and maximum values (lower and upper whisker, respectively) for the corresponding study group.
  • Outliers are represented by open circles.
  • Serum lipid concentrations are further represented by dots, where red dots indicate study subjects taking lipid-modifying drugs and blue dots those who do not.
  • Figure 31 shows median relative differences in phosphosphingolipid concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively.
  • COPD COPD
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • Figure 32 provides median relative differences in
  • glycerophosphatidylcholine concentrations in the serum of smokers with mild COPD COPD
  • COPD COPD
  • NS never-smokers
  • FS former smokers
  • CS current smokers
  • Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p ⁇ 0.05, and **p ⁇ 0.01.
  • biomarker refers to a characteristic whose presence, absence or level indicates a biological state. Typically, the properties of biomarkers indicate a normal process, a pathogenic process or a response to a pharmaceutical or therapeutic intervention.
  • a biomarker can be a cell, a gene, a gene product, an enzyme, a hormone, a protein, a peptide, an antibody, a nucleic acid molecule, a metabolite, a lipid, a free fatty acid, cholesterol or some other chemical compound.
  • a biomarker can be a morphologic biomarker (for example, a histological change, DNA ploidy, malignancy-associated changes in the cell nucleus and premalignant lesions) or a genetic biomarker (for example, DNA mutations, DNA adducts and apoptotic index).
  • a morphologic biomarker for example, a histological change, DNA ploidy, malignancy-associated changes in the cell nucleus and premalignant lesions
  • a genetic biomarker for example, DNA mutations, DNA adducts and apoptotic index
  • COPD Chironic Obstructive Pulmonary Disease
  • COPD refers to a complex disease that results in progressive loss of lung function.
  • COPD is typically characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways.
  • COPD can include the occurrence of chronic bronchitis or emphysema, both of which result in airway narrowing.
  • Clinically, COPD is typically detected by limited airflow in lung function tests.
  • COPD is typically irreversible and gets progressively worse over time. Symptoms of COPD include chronic cough, chronic sputum production, dyspnea, rhonchi, wheezing, chest tightness, tiredness and decreased airflow in lung function tests.
  • COPD ulcerative colitis
  • BODE index is a scoring system that measures FEV1, body-mass index, 6-minute walk distance, and a modified MRC (Medical Research Council) dyspnea scale to estimate outcomes in COPD.
  • control sample refers to a sample against which a test sample is compared in order to diagnose, prognose, classify or grade the test sample.
  • a control sample may be healthy tissue or may be a well-characterized sample, such as from an individual suffering from COPD, including but not limited to, GOLD stage 1, GOLD stage 2, GOLD stage 3, or GOLD stage 4 COPD.
  • a control sample can be analyzed concurrently with or separately from the test sample, including before or after analyzing the test sample.
  • the data from the analysis of a control sample may be stored, e.g., in a computer readable medium or in a manual, for comparison against test samples analyzed in the future, or as data for training network-based or machine-learning methods.
  • a control sample may be developed as a medical standard for comparison. For example, analysis of control samples has developed medical standards for normal fed and fasted blood glucose levels; normal, at risk, and hypertensive blood pressures, and normal resting heart rates.
  • the term "control sample” includes samples that provided a medical standard. Accordingly, a test sample may be compared against a medical standard generated from control samples. For example, production of a variant of a lipid may be indicative of a change medical condition. Alternatively, a change in production level of a lipid may be indicative of a change in medical condition.
  • a control sample may be lung tissue, such as tissue obtained by biopsy from a healthy individual, or some other sample.
  • a control sample may be sputum, saliva, bronchial washing, bronchial aspirates, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • Tissue specimens such as those obtained by biopsy, may be fixed (e.g., formaldehyde-fixed paraffin-embedded (FFPE)).
  • FFPE formaldehyde-fixed paraffin-embedded
  • the control sample may be obtained from a tissue bank.
  • the control sample may also be obtained from a cadaver or an organ donor.
  • fatty acid refers to a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides or phospholipids. When they are not attached to other molecules, they are known as "free" fatty acids.
  • FEV1 force expiratory volume in one second
  • FEV1 refers to the volume of air that can forcibly be blown out in one second, after full inspiration.
  • Average values for FEV1 in healthy individuals depend on sex and age and have been well-characterized in the art.
  • FEV1 and the FEV1 to FVC ration (FEV1/FVC) are used clinically to grade COPD. In healthy adults
  • FEV1/FVC should be approximately 75-80%.
  • obstructive diseases such as myeloma
  • FEV1 is diminished because of increased airway resistance to expiratory flow. While the FVC may be decreased as well, due to the premature closure of airway in expiration, FEV1 is typically more affected because of the increased airway resistance, so the FEV1/FVC ratio reflects the degree of airway closure compared to lung volume.
  • FVC force vital capacity
  • the term "individual” refers to a vertebrate, preferably a mammal.
  • the mammal can be, without limitation, a mouse, a rat, a cat, a dog, a horse, a pig, a cow, a non-human primate or a human.
  • the individual is a human.
  • the term "individual at risk for lung injury” refers to an individual who is predisposed to lung injury, such as COPD, including emphysema. Predisposition to lung injury may be due to one or more genetic or environmental factors. For example, an individual related to a COPD patient is more likely to get COPD than an individual who is not related to a COPD patient. Further, exposure to environmental factors such as radon gas, asbestos, tobacco smoke, and air pollution can increase the risk for lung injury and predispose an individual to lung injury.
  • the term "individual having a lung injury” or “individual suffering from injury” refers to an individual experiencing progressive loss of lung function, typically characterized by alveoli destruction.
  • Lung injury can be bronchial or emphysematous and may be detected by analyzing clinical, functional, and radiological findings or detecting relevant biomarkers.
  • lipid refers to a class of organic compounds that are fatty acids or their derivatives and are, typically, insoluble in water but soluble in organic solvents. Lipids may be divided into eight categories: fatty acids, glycero lipids, glycerophospho lipids, sphingo lipids, saccharo lipids, polyketides sterol lipids and prenol lipids. Fatty acids and fatty acid derivatives may be identified using a notation giving the number of carbon atoms and of double bonds (separated by a colon).
  • palmitic acid which has sixteen carbon atoms and no double bonds
  • oleic acid which has eighteen carbons and one double bond
  • Some lipids comprise a head group with one or more fatty acid tails.
  • phosphatidylcholines comprise a choline head groups and two fatty acid tails, one saturated and one unsaturated. Such lipids may be identified by their fatty acid tails.
  • PC(16:0/18: 1) refers to a phosphtidylcholine lipid with a palmitic acid tail and an oleic acid tail.
  • lipid signature refers to a group of lipids produced by a cell or a tissue, whose combined production pattern may be indicative of, e.g., a normal state, an at-risk state, a diseased state, a treated state or a recovery state.
  • a lipid signature may be characterized by which lipids are produced or at what level each lipid is produced.
  • Lipid signatures are particularly useful in diagnosing, prognosing, classifying or grading complex diseases states, which result from the combination of several genetic and environmental factors.
  • the lipid signatures disclosed herein may be used, e.g., for the diagnosis, prognosis, classification and/or grading of lung injury, such as emphysema, in an individual.
  • MALDI-TOF matrix-assisted laser
  • Time-of-flight (TOF) mass spectrometry refers to a method in which an ion's mass-to-charge ratio is determined via the time that it takes an ionized particle to reach a detector at a known distance.
  • saturated refers to a compound, such as a fatty acid, that has no double or triple bonds or ring. In saturated hydrocarbons, every carbon atom is attached to two hydrogen atoms, except those at the ends of the chain, which bear three hydrogen atoms.
  • :0 refers to a saturated fatty acid. For example, 16:0 (palmitic acid) refers to a saturated fatty acid comprising sixteen carbon atoms.
  • test sample refers to a sample obtained from an individual at risk for, having or suffering from lung injury.
  • a test sample may be any sample suspected of containing or exhibiting a biomarker.
  • test sample is analyzed and compared to a control sample, including medical standards developed from control samples, to diagnose, prognose, classify or grade lung injury in the individual.
  • a test sample may be obtained from lung tissue, such as tissue obtained by biopsy from a tumor, or other biological tissue.
  • a test sample may be sputum, saliva, bronchial washing, bronchial aspirates, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • Tissue specimens, such as those obtained by biopsy may be fixed (e.g., formaldehyde-fixed paraffin-embedded (FFPE)).
  • FFPE formaldehyde-fixed paraffin-embedded
  • the term "unsaturated” refers to a compound, such as a fatty acid, that contains carbon-carbon double bonds or triple bonds.
  • a chain of carbons such as a fatty acid
  • a double or triple bond will cause a kink in the chain.
  • Unsaturated fats tend to be liquid at room temperature, rather than solid, due to the kinks in the chain, which prevent the molecules from packing closely together to form a solid. These fats are typically called oils and are present in fish and plants.
  • the degree of unsaturation refers to the number of double and triple bonds in the fatty acid. In certain fatty acid nomenclature, the number following the colon refers to a saturated fatty acid.
  • 18: 1 (oleic acid) refers to a fatty acid comprising eighteen carbon atoms and one double bond (i.e., one degree of unsaturation).
  • 18:2 (linoleic acid) refers to a fatty acid comprising eighteen carbon atoms and two double bonds (i.e., two degrees of unsaturation).
  • lipid biomarkers useful for diagnosing, prognosing, classifying or grading lung injury, such as COPD, including emphysema.
  • the lipid biomarkers may independently be selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
  • glycerophospho inositol a glycerophosphoserine, an acidic glycosphingo lipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
  • the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0);
  • CE(22:5); CE(22:6); and CE(24:2) preferably CE(20:4) or CE(22:5).
  • CE(20:4) or CE(22:5) are downregulated by CS.
  • the eicosanoid may be selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-
  • DHET 6-keto-PGFl alpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12- DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET;
  • 15-HEPE 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD 2 ); Prostaglandin E2 (PGE 2 ); PGF2alpha; TXB 2 ; and
  • TXB 3 preferably, 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD 2 ; PGE 2 ; or TXB 3 .
  • the eicosanoid is be selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; or TXB 3 .
  • the eicosanoid may be TXB 3 .
  • the eicosanoid is not PGD 2 or PGE 2 .
  • the glycerophosphocholine may be selected from phosphatidylcholine
  • PC PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18:1); PC(16:0/16:0);
  • PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4) preferably, PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16: 1); or
  • the glycerophosphocholine is PC(16:0/16:0) or
  • PC(16: 1/16: 1) which are both upregulated in lung tissue following CS exposure.
  • PC 18:0/20:4; 18: 1/20:4; and 18:2/20:4 are downregulated in plasma following CS exposure.
  • the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2);
  • PG(18:2/18:2) are upregulated in lung tissue following CS exposure.
  • the glycerophosphoethanolamine is selected from phosphatidylethanolamine (PE)( 16:0/16:0); PE(16:0/18:1); PE(16:0/18:2);
  • PE(16:0/18:1); PE(16:0/18:2); and PE(16:0/20:4) are upregulated in lung tissue following CS exposure.
  • the ceramide (Cer) may be selected from Cer(dl8:0/16:0);
  • the ceramide may be selected from
  • the neutral glycosphingolipid is selected from glucosyl/galactosyl Cer (Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0);
  • Glc/GalCer (dl8:l/26:l); lactosylCer (LacCer)(dl8:0/16:0); LacCer(dl8:l/16:0);
  • Gb3 globotriaosylceramide
  • the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
  • the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0);
  • the acidic glycosphingolipid may be selected from GMl(dl8: l/16:0);
  • the acidic glycosphingolipid is selected from GMl(dl8: l/16:0); GM3(dl8: l/16:0); and GM3(dl8:l/24:0), which are each upregulated in lung tissue following CS exposure.
  • the GM1 class of lipids i.e., "Sum(Gl)”
  • the GM3 class of lipids i.e., "Sum(G3)
  • a change in Sum(Gl) or Sum(G3) may indicate severe lung injury.
  • the sphingoid base may be selected from sphingosine-1 -phosphate (SlP)(dl8: l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: 1).
  • the sphingoid base may be selected from sphingosine-1 -phosphate (SlP)(dl8: l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6: l); SPH(dl8
  • the SIP class of lipids i.e., "Sum(SlP)" are upregulated in lung tissue following CS exposure.
  • the SA1P class of lipids i.e., "Sum(SAlP)" are upregulated in lung tissue following CS exposure.
  • the SPA class of lipids are upregulated in lung tissue following CS exposure.
  • the SPH class of lipids i.e., "Sum(SPH)" are upregulated in lung tissue following CS exposure.
  • the sphingoid base may be selected from Sum(SlP); Sum(SAlP); Sum(SPA); and Sum(SPH).
  • the sphingoid base is not a sphingosine-1 -phosphate.
  • the lipid biomarker is selected from CE(20:4);
  • the lipid biomarker is selected from CE(20:4); CE(22:5); 5-HETE; 8,9-DHET;
  • the lipid biomarker is not PGD 2 or PGE 2 . In some embodiments, the lipid biomarker is not a sphingosine-1 -phosphate.
  • the lipid biomarker is selected from 5-HETE; 5,6-
  • DHET 8-HETE; 11,12-DHET; 12-HEPE; 12-oxoETE; 14,15-DHET; arachidonic acid; eicosapentaenoic acid; PGD2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/18: 1);
  • the lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4); Cer(dl8: l/26: 1); Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
  • lipid biomarkers have increased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • lipid biomarkers 20 or more, 25 or more, 30 or more, or 35 or more of the lipid biomarkers have increased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • the lipid biomarkers that are up- regulated in an individual suffering from or at risk for lung injury may be selected from PGD2; PGE2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1);
  • Lipid biomarkers such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0), may be up-regulated in blood samples from an individual suffering from or at risk for lung injury.
  • lipid biomarkers such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0
  • lipid biomarkers such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0
  • lipid biomarkers such as
  • LacCer(dl 8: 1/23:0); LacCer(dl8:l/24:0); or LacCer(dl8:l/24:l), may be up- regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • SAlP(dl8:0); SPA(dl8:0); SPH(dl6:l); SPH(dl8:l); SPH(dl8:2); or SPH(d20:l) is up-regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • SPA(dl8:0); SPH(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: l) is up- regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • At least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 of lipid biomarkers have decreased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 35 or more of the lipid biomarkers have decreased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • the lipid biomarkers that are down- regulated in an individual suffering form or at risk for lung injury may be selected from PC(16:0/18:0); PC(16:0/20:4); PC(16:0/22:6); PC(18:0/18.1); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl8:l/16:0);
  • Lipid biomarkers such as PC(16:0/18:0); PC(16:0/20:4);
  • Cer(dl8: l/16:0); Cer(dl8: l/18:0); or Cer(dl8: l/18: l), may be down-regulated in blood samples from an individual suffering from or at risk for lung injury.
  • lipid biomarkers such as arachidonic acid; PC( 16:0/18:0);
  • PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) or LacCer(dl 8.1/20.0), may be down-regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
  • the lipid biomarker are up-regulated to a certain degree in a sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • each up-regulated lipid biomarker may, independently, be up-regulated at least 1.5-fold, at least 2-fold, at least 2.5- fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1,000-fold or more compared to the control sample.
  • the lipid biomarkers are down-regulated to a certain degree in a sample from an individual suffering from or at risk for lung injury compared to a control sample.
  • each down-regulated lipid biomarker may, independently, be down-regulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1,000-fold or more compared to the control sample.
  • the lipid biomarkers of the invention may be used in methods of diagnosing, prognosing, classifying or grading lung injury in biological sample or an individual.
  • the lung injury may be COPD, including emphysema.
  • One aspect of the invention provides a method of diagnosing, classifying or grading lung injury in an individual at risk for or suffering from a lung injury.
  • the method comprises classifying a test sample as injured or non- injured, such as emphysematous or non-emphysematous or COPD or non-COPD.
  • the method comprises measuring the levels of at least 2 lipid biomarkers described above in a test sample; and comparing those measurements to the level of the at least two lipid biomarker in a control sample to obtain a classification of the test sample as injured or non-injured, such as emphysematous or non-emphysematous or COPD or non-COPD.
  • the levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 lipid biomarkers described above are measured.
  • the methods of the invention comprise obtaining a test sample from an individual, determining the absence, presence or level of one or more of the lipid biomarkers described above in the test sample, comparing said absence, presence or level to the absence, presence or level of the same lipid biomarker(s) in a control sample, and diagnosing the individual as having or being at risk for lung injury based on the comparison.
  • the invention provides a method for monitoring the progress or recovery of a lung injury in an individual, said method comprising determining at suitable time intervals in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the invention provides a method for monitoring the progress or recovery of a lung injury treatment in an individual, said method comprising determining at suitable time intervals before, during, or after therapy (for example, at different time points during the treatment) in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the invention provides a method for monitoring lung injury in an individual resulting from exposure to air-borne pollutants, said method comprising determining at suitable time intervals in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the invention provides a method for monitoring changes in the severity of a lung injury in an individual, said method comprising determining at suitable time intervals before, during, or after changing the method or pattern of nicotine consumption (for example, at different time points during smoking cessation or switching from a combusted tobacco product, e.g., cigarette, to a heated tobacco product or an electronic cigarette) in one or more samples taken from said individual differential production levels of the lipid biomarkers described above.
  • the individual is a cigarette smoker; the individual is a former cigarette smoker; the individual was a cigarette smoker who has stopped smoking cigarette for at least 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36 month(s) prior to the measurements; the individual is or the individual was a cigarette smoker who has switched to using a heated tobacco product or a nicotine- containing product which can include an electronic cigarette or a nicotine patch, for at least 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36 month(s) prior to the measurements instead of smoking cigarette.
  • the method comprises detecting the level of at least 2 lipid biomarkers described above in a test sample obtained from the individual; and comparing the level of the at least 2 lipid biomarkers in the test sample to the level of the at least 2 lipid biomarkers in a control sample. In some embodiments, if the level of the at least 2 lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk for lung injury. In some embodiments, the level of the at least 2 lipid biomarkers is higher in the test sample than in the control sample. Optionally, the level of the lipid biomarkers is lower in the test sample than in the control sample.
  • the method further comprises detecting the level of the lipid biomarkers in the control sample.
  • the levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 lipid biomarkers are detected.
  • the test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • the test sample is obtained from a large airway of the individual, such as a bronchial biopsy, or the lung of the individual, such as a lung biopsy, including a biopsy of the parenchyma of the individual.
  • control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
  • control sample is obtained from a large airway or a lung of an individual not affected with a lung injury, such as from a bronchial biopsy or a lung biopsy of the individual not affected with the lung injury.
  • control sample is obtained from the individual at risk for or having lung injury prior to onset of the lung injury.
  • the control sample is obtained from an individual that does not suffer from a lung injury.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PGD2; PGE2; PC(14:0/14:0);
  • LacCer(dl 8: 1/24: 1) is higher in the test sample than in the control sample, wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1);
  • Glc/GalCer(dl8:0/24: l); and Cer(dl 8:0/18:0) is higher in the test sample than in the control sample, wherein the test sample and the control sample are blood samples.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of arachidonic acid; PC(16:0/18:0);
  • PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
  • the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16:0/18:0); PC(16:0/20:4);
  • Cer(dl8: l/16:0); Cer(dl8:l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, wherein the test sample and the control sample are blood samples.
  • Detection of the lipid biomarkers described herein in a test sample or a control sample may be performed by any method known in the art.
  • the methods of the invention rely on the detection of the presence or absence of lipid biomarker, or the qualitative or quantitative assessment of either over- or under-production of a lipid biomarker in a population of cells or a tissue in a test sample relative to a standard (for example, a control sample).
  • the level of one or more lipid biomarkers in the test sample and the level of one or more lipid biomarkers in the control sample may be detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography.
  • the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry.
  • the chromatography is thin layer chromatography, solid-phase extraction
  • HPLC high performance liquid chromatography
  • hydrophilic interaction liquid chromatography hydrophilic interaction liquid chromatography
  • ultra-performance liquid chromatography ultra-performance liquid chromatography
  • the lung injury is emphysema or COPD.
  • Example 1 Exposure of mice to cigarette smoke [0110] The study design included 3 groups of C57BL/6 mice: Sham (fresh air- exposed), CS (exposed to mainstream smoke from 3R4F, a reference cigarette from the University of Kentucky and smoking cessation. Animals from the Sham and CS groups were exposed to fresh air and cigarette smoke, respectively, for up to seven months. To model the effects of smoking cessation, animals from the cessation group (CESS) were first exposed to CS for 2 months and then switched to filtered air for 5 additional months.
  • CESS cessation group
  • mice were bred under specific pathogen-free conditions were obtained from Charles River, USA and were 8-10 weeks old at exposure initiation. Mice were individually identified by the subcutaneous implantation of transponders and were housed and whole- body exposed in the animal laboratory under specific pathogen-free conditions. Random allocation of mice to experimental groups was conducted prior to exposure. Animals were fed a standard chow diet (T2914C irradiated rodent diet, Harlan). Filtered tap water was supplied ad libitum and changed daily.
  • NACLAR National Advisory Committee For Laboratory Animal Research
  • mice were observed daily for mortality, morbidity, and signs of injury. Body weight was measured twice per week during the exposure period. The number of animals analyzed for each endpoint is given in Table 1.
  • carboxyhemoglobin (COHb) levels were determined as a marker of smoke uptake at 3, 4 and 6 months.
  • mice were exposed in whole-body exposure chambers to mainstream cigarette smoke (CS) or fresh air (sham) for a period of two, three, or seven months.
  • CS mainstream cigarette smoke
  • sham fresh air
  • CS-uptake was confirmed by monitoring blood carboxyhemoglobin (COHb) at 3 time points and nicotine and cotinine
  • the left lung was instilled with 4% paraformaldehyde in PBS (pH 7.4) and fixed for 24 hours before standard paraffin-embedding, sectioning and staining. Briefly, a small cannula (1.6 mm diameter) was gently introduced into the trachea and the proximal left bronchus and fixed to the latter with a ligature.
  • Fixative was delivered to the left lung by gravity at 15 cm water pressure. After the instillation procedure was completed and the lungs were filled, the cannula was carefully removed and the ligature tightened to prevent fixative flowing out of the lung. The lung was then fixed by immersion in the same fixation solution for 24 hours. Histopathological evaluation was performed at 5 different levels of the lung, displaying the central and peripheral aspects of the parenchyma. For each lung section, a separate evaluation was performed. Mean scores of the 5 data sets were calculated for each animal and for each endpoint. The mean values per animal were used for the exposure group- based analysis.
  • H&E haematoxylin and eosin
  • ABS alcian blue periodic acid schiff s
  • BA resorcin fuchsin
  • Unpigmented and pigmented macrophages are free cells in the alveolar lumen not showing any cytoplasmatic pigmentation or containing fine-granular, brownish to yellow cytoplasmic pigmentation, which may be due to CS particles inhaled, respectively.
  • Pigmented macrophage nests consist of multiple macrophages in the alveolar lumen clustered in small groups within adjacent alveoli.
  • Multinucleated giant cells are very large macrophages (size ranging from 18 to 28 ⁇ ) that are positively stained after AB-PAS incubation (Figure IB), likely due to uptake of excessive amounts of surfactant produced at the alveolar surface in response to CS inhalation.
  • CS-exposure was associated with increased mean chord length (Figure 2C), increased destructive index (defined as the percentage of emphysematous tissue over normal tissue, Figure 2D), and fewer bronchiolar attachments (Figure 2E) relative to sham-exposed animals from month two onwards.
  • Some destruction of the lung tissue could be observed with increasing severity in the sham group, likely due to aging.
  • the damage caused by the initial two month of CS-exposure is not reversible, as the post-cessation mice show disease progression. Progression rate is however slower post-cessation than in continuously exposed mice.
  • Microlab Star robot Gangliosides were extracted according to the method described by Fong et al (Fong et al, 2009, Lipids 44, pp. 867-874) with minor modifications, and eicosanoids were extracted as described by Deems et al (Deems et al, 2007, Methods in Enzymol, 432, pp.59-82).
  • lipid extracts were analyzed using a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500) equipped with a robotic nanoflow ion source (NanoMate HD).
  • QTRAP 5500 a hybrid triple quadrupole/linear ion trap mass spectrometer equipped with an ultra-high pressure liquid chromatography (UHPLC) system (CTC HTC PAL autosampler and Rheos Allegro pump) using a multiple reaction monitoring (MRM) -based method in negative ion mode.
  • UHPLC ultra-high pressure liquid chromatography
  • MRM multiple reaction monitoring
  • the mass spectrometry data files were processed using LipidViewTM VI .1 and MultiQuantTM 2.0 Software (Ab Sciex, Massachusetts, USA) to generate a list of lipid names and peak areas. Lipids were normalized to their respective internal standard and the tissue weight. The concentrations of molecular lipids are presented as nmol/mg wet tissue for lung samples. For statistical analysis, a Wilcoxon rank-sum test was conducted for each lipid for comparing the study groups. Monte-Carlo estimation of exact p-values was performed. Multiple testing was controlled with FDR q-values.
  • PC phosphatidylcholine
  • PC-0 and PC-P PC plasmalogen
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • PC( 16:0/16: 1) Figure 5 A
  • the median concentration difference for these species was up to 240% (p ⁇ 0.01), while in the majority of PC molecules little or no change in concentration in response to CS exposure was observed throughout the experiment.
  • the upregulation of PGs was driven mainly by minor species PG(18:1/18: 1), PG(18:1/18:2) and PG(18:2/18:2), upregulated up to 400%
  • LacCer demonstrated a similar pattern as other ceramides, namely that long-chain Gb3 species are most significantly upregulated (Figure 12).
  • Smoke exposure elevated the levels of saturated/mono-unsaturated cholesterol esters in the lung, while levels of poly-unsaturated species were downregulated (Figure 13).
  • Polyunsaturated cholesterol ester species, especially CE20:4 and CE22:5, in the plasma were also down-regulated by cigarette smoke ( Figure 14).
  • the study used a parallel-group, case-controlled study design in order to determine the differential expression of molecular and physiological biomarkers in subjects with COPD (COPD) when compared to healthy (no COPD) current smokers (CS), healthy (no COPD) former smokers (FS) and healthy never-smokers (NS).
  • COPD COPD
  • CS healthy current smokers
  • FS healthy (no COPD) former smokers
  • NS healthy never-smokers
  • AEs Adverse Events
  • concomitant medication details were also recorded on an ongoing basis in the subjects' eCRF.
  • the inclusion and exclusion criteria for this study can be found in Table 4 at www.clinicaltrials.gov using identifier NCT01780298.
  • Subjects attended the center for visit 3 within 3 to 14 days after visit 2. Eligibility was reassessed against the inclusion/exclusion criteria prior to any other procedures and subjects underwent the procedures as indicated in Table 4. Subjects attended the study center for visit 4 within 3 to 14 days after visit 3 and underwent the procedures as described in Figure 15. A follow-up telephone call was made to subjects within 3 to 10 days after visit 4 to record adverse events (AEs) and concomitant medication details and give smoking cessation advice to current smokers. Table 4. Demographics, smoking history, spirometric parameters and cardiorespiratory vital signs across the study's evaluable population. Data are presented as median ran e .
  • BMI Body mass index
  • FEV1 forced expiratory volume in 1 second
  • Lipids were extracted using a modified Folch lipid extraction procedure (Ekroos, K. (2008). Unraveling Glycerophospholipidomes by Lipidomics. (F. Wang, Ed.) (pp. 369-384). Totowa, NJ: Humana Press) performed on a Hamilton Microlab Star robot. Extract samples were spiked with known amounts of non- endogenous synthetic internal standards.
  • lipid extracts were analyzed using a QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) equipped with a robotic nanoflow ion source (NanoMate HD, Advion Biosciences) according to Stahlman and colleagues (Stahlman M, Ejsing CS, Tarasov K, Perman J, Boren J, Ekroos K (2009) High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry.
  • QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer Applied Biosystems
  • SM sphingomyelins
  • DAG diradylglycerols
  • TAG triacylglycerols
  • Targeted eicosanoid and sphingolipid lipidomics were performed using UHPLC (CTC Analytics AG)-coupled QTRAP 5500 mass spectrometry using multiple reaction monitoring (MRM) in positive ion mode.
  • the MS data files were processed using LipidViewTM 1.1 (AB Sciex) or MultiQuantTM 2.0 (AB Sciex) for generating a list of lipid names and peak areas. Lipids were normalized to their respective internal standard (the peak area of the endogenous lipid was divided by the peak area of the corresponding internal standard) and sample volume, yielding concentrations of molecular lipids in ⁇ .
  • the differences and relative differences between the groups were estimated using Hodges-Lehmann estimator (the median value of the cross-pairwise differences between individuals of the two groups). A rank- sum Wilcoxon test was performed to calculate p values. Differences with a p value below 0.05 were considered statistically significant.
  • lipidomics results at the lipid class level are summarized in Figure 16 and indicate small but significant increases in serum diradylglycerols (DAG), neutral glycosphingolipids (LacCer), glycerophospholipids including glycerophosphocholines (PC) and
  • PE glycerophosphoethanolamines
  • TAG triglycerides
  • glycerophosphatidylcholine molecules were generally higher in the serum of current smokers compared to that of never-smokers resulting in an overall up- regulation of this lipid class in disease-free smokers. While not significantly different, serum levels of PC(18:0/20:5) increased noticeably in former smokers relative to current smokers, while the levels of all other members of this lipid class affected by smoking decreased following smoking cessation and mostly returned to levels observed in never-smokers ( Figure 16 and Figure 19).
  • Serum TAG levels were also significantly higher in current compared to never-smokers (Figure 16). This was mainly due to a concerted increase in palmitoleic acid- and oleic acid-containing glycerol triesters in the serum of current smokers ( Figure 20). However, with the exception of the stearic acid ester
  • TAG(52: 1) serum TAG levels exhibited no significant differences when comparing current with former and former with never-smokers, respectively, indicating that potential smoking-related changes in TAG profiles may be reversible upon smoking cessation.
  • Glc/GalCer(dl8:l/18:0) and Glc/GalCer(dl 8: 1/24: 1) were also significantly elevated in the serum of current relative to never-smokers. With the exception of Glc/GalCer(dl8: l/16:0) and Glc/GalCer(dl8: l/18:0), most of Glc/GalCer(dl8: l/16:0) and Glc/GalCer(dl8: l/18:0), most
  • PE glycerophosphoethanolamines
  • two - PE(16:0/20:4) and PE(18: 1/18: 1) - were significantly elevated in the serum of current smokers relative to never-smokers ( Figure 16 and Figure 23). While levels of both molecules were decreased in former smokers compared to current smokers, only the difference in serum PE(16:0/20:4) levels was found to be significant.
  • serum levels of all affected PE species in former smokers were very similar to those seen in never-smokers, suggesting that the effect of cigarette smoke exposure may be reversible upon cessation.
  • SM(dl 8: 1/24: 1) did not, potentially reflecting irreversible smoking-related effects on the serum lipidome.
  • Lipidomics analysis in serum was also performed to examine the effects of the development of mild COPD (GOLD stages I and II; COPD vs. CS, COPD vs. NS). Lipidomics results at the lipid class level are summarized in Figure 28 and indicate small but significant decreases in serum sterols (CE) and
  • SM glycosphingophospho lipids

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Abstract

The present invention relates to lipid biomarkers that are useful for diagnosing, classifying and prognosing lung injury, such as emphysema. The invention also relates to diagnostic methods using these lipid biomarkers. The invention further relates to lipid-processing genes that are useful for treating, diagnosing, classifying and prognosing lung injury, such as emphysema. The invention also relates to therapeutic methods by inhibiting these genes or the proteins they encode.

Description

Lipid Biomarker Signatures for Lung Injury Diagnosis
Field of the invention
[0001] The present invention relates to lipid biomarkers that are useful for the diagnosis of lung injury, such as emphysema. The present invention also relates to methods of diagnosing or prognosing lung injury, such as emphysema. The present invention further relates to methods of treating or preventing lung injury by inhibiting the expression or activity of genes related to lipid metabolism or transport. The present invention also relates to methods of identifying compounds useful for treating or preventing lung injury. Background of the Invention
[0002] Emphysema is a lung condition that occurs when the alveoli are gradually destroyed, rendering an emphysema patient progressively short of breath.
Emphysema is part of a group of diseases collectively called Chronic Obstructive Pulmonary Disease (COPD) and is characterized by parenchymal destruction, loss of alveolar attachments and decrease in elastic recoil. COPD is the third leading cause of death in the U.S. and is projected to be the fourth leading cause of death worldwide by 2030. In Europe, over€20 billion is spent treating COPD, while approximately $18 billion is spent in the U.S.
[0003] Emphysema can be caused by long-term exposure to lung irritants in the environment, such as air pollution, chemical fumes, dust and tobacco smoke.
Because long-term exposure to lung irritants is typically required, most individuals who suffer from emphysema are more than 40 years old when symptoms first present. Such symptoms include an ongoing cough or a cough that produces an excess of mucus; shortness of breath, especially with physical activity; wheezing and chest tightness. Early detection of emphysema can be difficult because symptoms slowly worsen over time, and most affected individuals do not notice early symptoms because they are mild or easy to correct by lifestyle adjustment.
[0004] Emphysema is typically diagnosed by signs and symptoms, including medical history, family history and test results. Diagnostic tests for emphysema include lung function tests, such as spirometry, including lung volume
measurement, and lung diffusion capacity; chest X-rays, chest CT scans and arteriole blood gas tests. Accordingly, the diagnostic tests for emphysema require the disease to have progressed to the point that lung function is moderately affected. Thus, there is a need for a diagnostic test that can identify emphysema in patients at early stages. A need also persists to understand the molecular mechanisms of emphysema, which may allow for the design or optimization of therapies to treat the disease, instead of just the symptoms.
Summary of the invention
[0005] The present invention is directed to lipid biomarkers for classifying, diagnosing or grading emphysema. A first aspect of the invention provides a method of diagnosing an individual as being at risk for or having lung injury comprising detecting the level of two or more lipid biomarkers in a test sample obtained from the individual; and comparing the level of the two or more lipid biomarkers in the test sample to the level of the two or more lipid biomarkers in a control sample, wherein, if the level of the two or more lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk of having lung injury. The two or more lipid biomarkers may independently be selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a glycerophosphoethanolamine, a glycerophosphoglycerol, a glycerophosphoinositol, a glycerophosphoserine, an acidic glycosphingolipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
[0006] In some embodiments, the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0); CE(18: 1); CE(18:2); CE(18:3); CE(20:3) CE(20:4); CE(20:5); CE(22:0);
CE(22:5); CE(22:6); and CE(24:2), preferably CE(20:4) or CE(22:5).
[0007] The eicosanoid may be selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-
DHET; 6-keto-PGFl alpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12- DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET;
15-HEPE; 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD2); Prostaglandin E2 (PGE2); PGF2alpha; TXB2; and
TXB3, preferably, 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PGE2; or TXB3.
[0008] The glycerophosphocholine may be selected from phosphatidylcholine
(PC)(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18:1); PC(16:0/16:0);
PC(16:0/16: 1); PC(16:0/17: 1); PC(16:0/18:0); PC(16:0/18: 1); PC(16:0/18:2);
PC(16:0/20:0); PC(16:0/20: 1); PC(16:0/20:2); PC(16:0/20:4); PC(16:0/22:4);
PC(16:0/22:5); PC(16:0/22:6); PC(16: 1/16: 1); PC(16: 1/18:0); PC(16: 1/18: 1); PC(16: 1/18:2); PC(16: 1/20:4); PC(17:0/18:2); PC(18:0/18: 1); PC(18:0/18:2);
PC(18:0/20:4); PC(18:0/22:4); PC( 18:0/22:6); PC(18: 1/18: 1); PC(18: 1/18:2);
PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4), preferably,
PC(16:0/16:0) or PC(16: 1/16: 1).
[0009] In some embodiments, the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2);
PG(18: 1/18:1); PG(18: 1/18:2); and PG(18:2/18:2), such as PG(18: 1/18: 1), PG(18:1/18:2) or PG(18:2/18:2).
[0010] Optionally, the glycerophosphoethanolamine is selected from
phosphatidylethanolamine (PE)( 16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2);
PE(16:0/20:4); PE(16:0/22:4); PE(18:0/18:0); PE(18:0/20:4); PE(18:0/22:4);
PE(18: 1/18: 1); PE(18: l/20:4); and PE(22:6/22:6), for example PE(16:0/16:0);
PE(16:0/18: 1); PE(16:0/18:2); and PE(16:0/20:4).
[0011] The ceramide (Cer) may be selected from Cer(dl8:0/16:0);
Cer(dl 8:0/18:0); Cer(dl8:0/18: l); Cer(dl 8:0/20:0); Cer(dl8:0/22:0);
Cer(dl8:0/24:0); Cer(dl8:0/24: 1); Cer(dl 8:0/26: 1); Cer(dl8: l/16:0);
Cer(dl8: l/18:0); Cer(dl8: l/18: l); Cer(dl 8: 1/20:0); Cer(dl8: l/22:0);
Cer(dl8: l/22: 1); Cer(dl 8: 1/23:0); Cer(dl 8: 1/24:0); Cer(dl8: l/24: 1); Cer(dl8:l/26:0); and Cer(dl8:l/26:1), preferably Cer(dl8:0/16:0);
Cer(dl8:0/24:0); Cer(dl8:0/24:1); and Cer(dl8:l/26:1).
[0012] In some embodiments, the neutral glycosphingolipid is selected from glucosyl/galactosyl Cer (Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
Glc/GalCer(dl8:0/24:l); Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl8:l/18:0);
Glc/GalCer(dl8:l/20:0); Glc/GalCer(dl 8: 1/22:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl8:l/24:0); Glc/GalCer(dl 8: 1/24:1); Glc/GalCer(dl 8: 1/26:0);
Glc/GalCer(dl8:l/26:l); lactosylCer (LacCer)(dl8:0/16:0); LacCer(dl8:l/16:0); LacCer(dl8:l/18:0); LacCer(dl8:l/20:0); LacCer(dl 8: 1/22:0);
LacCer(dl 8: 1/23:0); LacCer(dl 8: 1/24:0); LacCer(dl 8: 1/24:1);
globotriaosylceramide (Gb3)(dl8:l/16:0); Gb3(dl8:l/18:0); Gb3(dl8:l/20:0);
Gb3(dl8:l/22:0); Gb3(dl8:l/22:1); Gb3(dl 8: 1/23:0); Gb3(dl8:l/24:0); and
Gb3(dl8:l/24:1). Optionally, the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl8:l/16:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24: 1); Glc/GalCer(dl 8 : 1/26:0);
Glc/GalCer(dl8:l/26:l); LacCer(dl8:l/16:0); LacCer(dl8:l/24:0);
Gb3(dl8:l/16:0); and Gb3(dl8:l/24:0).
[0013] The acidic glycosphingolipid may be selected from GMl(dl8:l/16:0); GMl(dl8:l/24:0); GMl(dl8:l/24:l); GM3(dl8:l/16:0); GM3(dl8:l/18:0);
GM3(dl8:l/20:0); GM3(dl8:l/21:0); GM3(dl8:l/22:0); GM3(dl8:l/22:l);
GM3(dl8:l/23:0); GM3(dl8:l/24:0); GM3(dl8:l/24:l) and GM3(dl8:l/24:2), such as GMl(dl8:l/16:0); GM3(dl8:l/16:0); and GM3(dl8:l/24:0).
[0014] The sphingoid base may be selected from sphingosine- 1 -phosphate (SlP)(dl8:l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine
(SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6:l); SPH(dl8:l); SPH(dl8:2); and SPH(d20:l).
[0015] In some embodiments, the lipid biomarker is selected from 5-HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16:1);
PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4); Cer(dl8:l/26:1);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
GMl(dl8: l/16:0); and GM3(dl8: l/24:0).
[0016] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of PGD2; PGE2; PC(14:0/14:0);
PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16:0); PC(16:0/16: 1);
PC(16:0/17: 1); PC(16:0/18: 1); PC(16:0/18:2); PC(16:0/20:4); PC(16:0/22:4);
PC(16:0/22:5); PC(16:0/22:6); PC(16: 1/16: 1); PC(16: 1/18:0); PC(16: 1/18: 1);
PC(16: 1/18:2); PC(16: 1/20:4); PC(18: 1/18: 1); PC(18: 1/18:2); PC(18: l/20:4);
PC(18:2/18:2); PG(16:0/16:0); PG(16:0/18:1); PG(16:0/18:2); PG(18: 1/18: 1); PG(18:1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0); Cer(dl8:0/22:0); Cer(dl 8:0/24:0);
Cer(dl8:0/24: 1); Cer(dl8:0/26: 1); Cer(dl8: l/16:0); Cer(dl8: l/22:0);
Cer(dl8: l/24:0); Cer(dl8: l/24: 1); Cer(dl 8: 1/26:0); Cer(dl8: l/26: 1);
Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl 8:0/20:0);
Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl 8:0/24: 1);
Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8: l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl 8: 1/23:0); LacCer(dl 8: 1/24:0); and
LacCer(dl 8: 1/24: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
[0017] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1);
Glc/GalCer(dl8:0/24: l); and Cer(dl 8:0/18:0) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
[0018] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of arachidonic acid; PC(16:0/18:0);
PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
[0019] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16:0/18:0); PC(16:0/20:4);
PC(16:0/22:6); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl8:0/22:0);
Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8: l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Cer(dl8: l/16:0); Cer(dl8:l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
[0020] Optionally, the individual suffers from or is at risk of having lung injury if the level of three or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of four or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of five or more lipid biomarkers is different in the test sample than in the control sample. In some embodiments, the individual suffers from or is at risk of having lung injury if the level of ten or more lipid biomarkers is different in the test sample than in the control sample.
[0021] The method may further comprise detecting the level of the two or more lipid biomarkers in the control sample.
[0022] The test sample may be selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. Optionally, the test sample is obtained from a large airway of the individual, such as a bronchial biopsy, or the lung of the individual, such as a lung biopsy, including a biopsy of the parenchyma.
[0023] In some embodiments, the control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. The control sample may be obtained from a large airway or lung of an individual not affected with lung injury, such as from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury. Optionally, the control sample is obtained, prior to the onset of lung injury, from the individual at risk for or having the emphysema. In some embodiments, the control sample is obtained from an individual that does not suffer from lung injury.
[0024] The level of the two or more lipid biomarkers in the test sample and the level of the two or more lipid biomarkers in the control sample may be detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography. In some embodiments, the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry. Optionally the chromatography is thin layer chromatography, solid-phase extraction
chromatography, high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography, or ultra-performance liquid chromatography.
[0025] In some embodiments, the lung injury is emphysema or COPD.
[0026] Particular embodiments of the invention are set forth in the following numbered paragraphs:
1. A method of diagnosing an individual as being at risk for or having lung injury comprising
(1) detecting the level of two or more lipid biomarkers in a test sample obtained from the individual; and
(2) comparing the level of the two or more lipid biomarkers in the test sample to the level of the two or more lipid biomarkers in a control sample,
wherein, if the level of the two or more lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk of having lung injury;
wherein the two or more lipid biomarkers are independently selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
glycerophosphoethanolamine, a glycerophosphoglycerol, a
glycerophosphoinositol, a glycerophosphoserine, an acidic glycosphingolipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
2. The method according to paragraph 1, wherein the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1);
CE(18:0); CE(18: 1); CE(18:2); CE(18:3); CE(20:3) CE(20:4); CE(20:5);
CE(22:0); CE(22:5); CE(22:6); and CE(24:2).
3. The method according to paragraph 2, wherein the sterol is CE(20:4) or CE(22:5). 4. The method according to any one of paragraphs 1-3, wherein the eicosanoid is selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-DHET; 6-keto- PGFlalpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12-DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET; 15-HEPE; 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD2); Prostaglandin E2 (PGE2); PGF2alpha; TXB2; and TXB3.
5. The method according to paragraph 4, wherein the eicosanoid is selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PGE2; and TXB3.
6. The method according to any one of paragraphs 1-5, wherein the glycerophosphocholine is selected from phosphatidylcholine (PC)(14:0/14:0);
PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16:0); PC(16:0/16: 1);
PC(16:0/17: 1); PC(16:0/18:0); PC(16:0/18: 1); PC(16:0/18:2); PC(16:0/20:0);
PC(16:0/20: 1); PC(16:0/20:2); PC(16:0/20:4); PC(16:0/22:4); PC(16:0/22:5);
PC(16:0/22:6); PC(16: 1/16: 1); PC(16: 1/18:0); PC(16: 1/18: 1); PC(16: 1/18:2); PC(16: 1/20:4); PC(17:0/18:2); PC(18:0/18: 1); PC(18:0/18:2); PC(18:0/20:4);
PC(18:0/22:4); PC(18:0/22:6); PC(18: 1/18: 1); PC(18: 1/18:2); PC(18: l/20:4);
PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4).
7. The method according to paragraph 6, wherein the glycerophosphocholine is selected from PC(16:0/16:0); and PC(16: 1/16: 1). 8. The method according to any one of paragraphs 1-7, wherein the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2); PG(18:1/18: 1); PG(18: 1/18:2); and PG(18:2/18:2).
9. The method according to paragraph 8, wherein the glycerophosphoglycerol is selected from PG(18: 1/18: 1), PG(18: 1/18:2) and PG(18:2/18:2).
10. The method according to any one of paragraphs 1-9, wherein the glycerophosphoethanolamine is selected from phosphatidylethanolamine
(PE)(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); PE(16:0/20:4); PE(16:0/22:4); PE(18:0/18:0); PE(18:0/20:4); PE(18:0/22:4); PE(18:1/18: 1); PE(18: l/20:4); and PE(22:6/22:6).
11. The method according to paragraph 10, wherein the
glycerophosphoethanolamine is selected from PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); and PE(16:0/20:4).
12. The method according to any one of paragraphs 1-11, wherein the ceramide (Cer) is selected from Cer(dl8:0/16:0); Cer(dl 8:0/18:0); Cer(dl8:0/18: l);
Cer(dl8:0/20:0); Cer(dl8:0/22:0); Cer(dl 8:0/24:0); Cer(dl8:0/24: 1);
Cer(dl8:0/26: 1); Cer(dl8: l/16:0); Cer(dl8: l/18:0); Cer(dl8: l/18: l);
Cer(dl8: l/20:0); Cer(dl8: l/22:0); Cer(dl8: l/22: 1); Cer(dl 8: 1/23:0);
Cer(dl8: l/24:0); Cer(dl8: l/24: 1); Cer(dl 8: 1/26:0); and Cer(dl8: l/26: 1). 13. The method according to paragraph 12, wherein the ceramide is selected from Cer(dl8:0/16:0); Cer(dl8:0/24:0); Cer(dl 8:0/24: 1); and Cer(dl8: l/26: 1).
14. The method according to any one of paragraphs 1-13, wherein the neutral glycosphingo lipid is selected from glucosyl/galactosyl Cer
(Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl 8:0/24: 1);
Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8: l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
lactosylCer (LacCer)(dl 8:0/16:0); LacCer(dl8: l/16:0); LacCer(dl8: l/18:0); LacCer(dl8:l/20:0); LacCer(dl 8: 1/22:0); LacCer(dl 8: 1/23:0);
LacCer(dl8:l/24:0); LacCer(dl8:l/24:l); globotriaosylceramide
(Gb3)(dl8:l/16:0); Gb3(dl8:l/18:0); Gb3(dl8:l/20:0); Gb3(dl8:l/22:0);
Gb3(dl8:l/22:1); Gb3(dl8:l/23:0); Gb3(dl8:l/24:0); and Gb3(dl8:l/24:1). 15. The method according to paragraph 14, wherein the neutral
glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8:l/16:0); LacCer(dl8:l/24:0); Gb3(dl8:l/16:0); and Gb3(dl8:l/24:0). 16. The method according to any one of paragraphs 1-15, wherein the acidic glycosphingolipid is selected from GMl(dl8:l/16:0); GMl(dl8:l/24:0);
GMl(dl8:l/24:l); GM3(dl8:l/16:0); GM3(dl8:l/18:0); GM3(dl8:l/20:0);
GM3(dl8:l/21 :0); GM3(dl8:l/22:0); GM3(dl8:l/22:l); GM3(dl8:l/23:0);
GM3(dl8:l/24:0); GM3(dl8:l/24:l) and GM3(dl8:l/24:2). 17. The method according to paragraph 16, wherein the acidic
glycosphingolipid is selected from GMl(dl8:l/16:0); GM3(dl8:l/16:0); and GM3(dl8:l/24:0).
18. The method according to any one of paragraphs 1-17, wherein the sphingoid base is selected from sphingosine-1 -phosphate (SlP)(dl8:l);
SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6:l); SPH(dl8:l); SPH(dl8:2); and
SPH(d20:l).
19. The method according to any one of paragraphs 1-18, wherein the lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4);
Cer(dl8:l/26:1); Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8:l/16:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24: 1); Glc/GalCer(dl 8 : 1/26:0);
Glc/GalCer(dl8:l/26:l); GMl(dl8:l/16:0); and GM3(dl8:l/24:0). 20. The method according to any one of paragraphs 1-19, wherein the level of one or more of PGD2; PGE2; PC(16:0/16:0); PC(16: 1/16: 1); PG(16:0/16:0);
PG(18:1/18: 1); PG(18: 1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0); Cer(dl 8:0/24:0); Cer(dl8:0/24: 1); Cer(dl8:l/26: 1); Glc/GalCer(dl 8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl8:l/24:0); Gb3(dl8: l/16:0); Gb3(dl8: l/24:0); GMl(dl8: l/16:0); GM3(dl8: l/16:0); GM3(dl8: l/24:0); SlP(dl8: l); SlP(dl8:2); SAlP(dl8:0); SPA(dl8:0); SPH(dl6: l); SPH(dl8: l); SPH(dl8:2); and
SPH(d20: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway or a lung.
21. The method according to any one of paragraphs 1-20, wherein the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1); Glc/GalCer(dl 8:0/24: 1); and Cer(dl8:0/18:0) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
22. The method according to any one of paragraphs 1-21, wherein the level of one or more of arachidonic acid; PC(16:0/18:0); PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway or a lung.
23. The method according to any one of paragraphs 1-22, wherein the level of one or more of PC(16:0/18:0); PC(16:0/20:4); PC(16:0/22:6); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl8:l/16:0);
Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0); Glc/GalCer(dl 8: 1/22:0);
Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0); Cer(dl8: l/16:0);
Cer(dl8: l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
24. The method according to any one of paragraphs 1-23, wherein the individual suffers from or is at risk of having lung injury if the level of three or more lipid biomarkers is different in the test sample than in the control sample. 25. The method according to paragraph 24, wherein the individual suffers from or is at risk of having lung injury if the level of four or more lipid biomarkers is different in the test sample than in the control sample.
26. The method according to paragraph 25, wherein the individual suffers from or is at risk of having lung injury if the level of five or more lipid biomarkers is different in the test sample than in the control sample.
27. The method according to paragraph 26, wherein the individual suffers from or is at risk of having lung injury if the level of ten or more lipid biomarkers is different in the test sample than in the control sample. 28. The method according to any one of paragraphs 1-27, wherein the method further comprises detecting the level of the two or more lipid biomarkers in the control sample.
29. The method according to any one of paragraphs 1-28, wherein the test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
30. The method according to paragraph 29, wherein the test sample is obtained from a large airway or a lung of the individual.
31. The method according to paragraph 30, wherein the test sample is obtained from a bronchial biopsy or a lung biopsy.
32. The method according to any one of paragraphs 1-28, wherein the control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. 33. The method according to paragraph 32, wherein the control sample is obtained from a large airway or a lung of an individual not affected with lung injury. 34. The method according to paragraph 33, wherein the control sample is obtained from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury.
35. The method according to paragraph 32, wherein the control sample is obtained from the individual at risk for or having the emphysema prior to onset of the lung injury.
36. The method according to paragraph 32, wherein the control sample is obtained from an individual that does not suffer from lung injury.
37. The method according to any one of paragraphs 1-36, wherein the level of the two or more lipid biomarkers in the test sample and the level of the two or more lipid biomarkers in the control sample are detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography.
38. The method according to paragraph 37, wherein the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry.
39. The method according to paragraph 38, wherein the chromatography is thin layer chromatography, solid-phase extraction chromatography, high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography, or ultra-performance liquid chromatography.
40. The method according to any one of paragraphs 1-39, wherein the lung injury is emphysema or COPD.
Brief Description of the Drawings
[0027] Figure 1A provides representative images of lung tissue from cigarette smoke (CS)-exposed, Sham, and Cessation animals at the 2-, 3-, and 7-month time points. Tissues were stained with haematoxylin and eosin (H&E). Figure IB provides a representative image of multinucleated giant cells found in lung tissue from CS-exposed animals. Tissues were stained with alcian blue periodic acid Schiff reagent (AB-PAS).
[0028] Figure 2 provides histopathological findings and histomorphometry in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. (A) Histopathological findings in the lungs of mice exposed to filtered air (sham), CS or fresh air after an initial exposure to CS for 2 months. Shown are the mean severity scores ± SEM. Observations were scored according to a defined grading system: 0 = no finding; 1 = slight; 2 = slight/moderate; 3 = moderate; 4 = moderate/marked; 5 = marked. Data were analyzed using Cochran- Mantel-Haenszel statistics. (B) The number of multinucleated giant cells are shown as the mean numbers ± SEM. Data were analyzed using AN OVA followed by Tukey post hoc test. (C) Mean chord length measurements are shown as the mean ± SEM. (D) Destructive indices are shown as the mean ± SEM. (E) The number of bronchiolar attachments is shown as the mean ± SEM. *p < 0.05 as compared to SHAM, # p < 0.05 as compared to CS.
[0029] Figure 3 provides pulmonary lipid profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung lipid species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0030] Figure 4 provides relative percentage differences in lung molecular PCs and PGs concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05,
**p<0.01, and ***p<0.001.
[0031] Figure 5 A provides relative percentage differences in lung molecular PCs and PGs concentrations between mice exposed to smoke (CS) and fresh air
(SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months. Figure 5B provides relative percentage differences in lung molecular concentrations of PEs between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months. Figure 5C provides relative percentage differences in plasma molecular concentrations of PCs between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0032] Figure 6 provides pulmonary ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0033] Figure 7 provides pulmonary glucosyl/galactosyl ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular glucosyl/galactosyl ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0034] Figure 8 provides pulmonary lactosyl ceramide lipid species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular lactosyl ceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001. [0035] Figure 9 provides pulmonary GM1 ganglioside species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular GM1 ganglioside species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0036] Figure 10 provides pulmonary GM3 ganglioside species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular GM3 ganglioside species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0037] Figure 11 provides pulmonary sphingoid base species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular sphingoid base species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05,
**p<0.01, and ***p<0.001.
[0038] Figure 12 provides pulmonary globotriaosylceramide species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular globotriaosylceramide species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001. [0039] Figure 13 provides pulmonary cholesterol ester species profiles in the lungs of mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in lung molecular cholesterol ester species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0040] Figure 14 provides plasma cholesterol ester species profiles in mice exposed to CS, or to a cessation protocol and to control fresh air for 2, 3, or 7 months. Relative percentage differences in plasma molecular cholesterol ester species concentrations between mice exposed to smoke (CS) and fresh air (SHAM) at 2, 3, and 7 months and between mice from the cessation and sham groups at 3 and 7 months are shown. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05,
**p<0.01, and ***p<0.001.
[0041] Figure 15 provides a CONSORT diagram for the clinical study. The flow chart of the study shows the number of subjects enrolled in the study and the number of completers indicating reasons for subject recruitment, as recommended by the Consolidated Standards of Reporting Trials guidelines.
[0042] Figure 16 shows median relative differences in lipid concentrations categorized by lipid class in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
[0043] Figure 17 provides median relative differences in diradylglycerol (DAG) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01. [0044] Figure 18 provides box-whisker plots and scatter grams of individual diradylglycerol concentrations in the serum of current smokers and never-smokers, respectively. Panel A represents the log-transformed results for DAG(16:0/18: 1), and panel B those for DAG(18: 1/18: 1) serum levels in nmol/mL. Box-whisker plots reflect the first and third quartile (lower and upper boundary of box, respectively), median (green line), and minimum and maximum values (lower and upper whisker, respectively) for the corresponding study group. Outliers are represented by open circles. Serum lipid concentrations are further represented by dots, where red dots indicate study subjects taking lipid-modifying drugs and blue dots those who do not.
[0045] Figure 19 provides median relative differences in
glycerophosphatidylcholine (PC) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05.
[0046] Figure 20 provides median relative differences in triacylglycerol (TAG) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
[0047] Figure 21 provides median relative differences in lactosylceramide concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
[0048] Figure 22 provides median relative differences in glucosyl/galactosyl ceramide concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05. [0049] Figure 23 provides median relative differences in
glycerophosphoethanolamine (PE) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05.
[0050] Figure 24 provides median relative differences in eicosanoid (EICO) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, **p<0.01, and ***p<0.001.
[0051] Figure 25 provides median relative differences in ceramide (Cer) concentrations in the serum of healthy, current smokers (CS) compared to never- smokers (NS), former smokers (FS) compared to current smokers and to never- smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
[0052] Figure 26 shows median relative differences in glycophosphosphingolipid (SM) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05.
[0053] Figure 27 shows median relative differences in sterol (CE) concentrations in the serum of healthy, current smokers (CS) compared to never-smokers (NS), former smokers (FS) compared to current smokers and to never-smokers, respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
[0054] Figure 28 shows median relative differences in lipid concentrations categorized by lipid class in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01. [0055] Figure 29 shows median relative differences in sterol (CE) concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom.
Significance of values as *p<0.05, and **p<0.01.
[0056] Figure 30 provides box-whisker plots and scattergrams of individual sterol concentrations in the serum of current smokers and never-smokers, respectively. Panel A represents the results for CE(16:0), panel B those for CE(18:2), panel C those for CE(19:0), and panel D represents the log-transformed results for CE(20:5) serum levels in nmol/mL. Box-whisker plots reflect the first and third quartile (lower and upper boundary of box, respectively), median (green line), and minimum and maximum values (lower and upper whisker, respectively) for the corresponding study group. Outliers are represented by open circles. Serum lipid concentrations are further represented by dots, where red dots indicate study subjects taking lipid-modifying drugs and blue dots those who do not.
[0057] Figure 31 shows median relative differences in phosphosphingolipid concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
[0058] Figure 32 provides median relative differences in
glycerophosphatidylcholine concentrations in the serum of smokers with mild COPD (COPD) compared to never-smokers (NS), former smokers (FS) and current smokers (CS), respectively. Color intensity refers to the magnitude of change, intensity scale depicted at the bottom. Significance of values as *p<0.05, and **p<0.01.
Detailed Description of the invention
[0059] In order that the invention described herein may be fully understood, the following detailed description is set forth.
[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples are illustrative only, and are not intended to be limiting. All publications, patents and other documents mentioned herein are incorporated by reference in their entirety.
[0061] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.
[0062] As used herein, "about" means within a statistically meaningful range of a value such as a stated concentration range, time frame, molecular weight, temperature or pH. Such a range can be within an order of magnitude, typically within 10% and more typically within 5% of a given value or range. The allowable variation encompassed by the term "about" will depend upon the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Whenever a range is recited within this application, every whole number integer within the range is also contemplated as an embodiment of the invention.
[0063] The term "biomarker" refers to a characteristic whose presence, absence or level indicates a biological state. Typically, the properties of biomarkers indicate a normal process, a pathogenic process or a response to a pharmaceutical or therapeutic intervention. A biomarker can be a cell, a gene, a gene product, an enzyme, a hormone, a protein, a peptide, an antibody, a nucleic acid molecule, a metabolite, a lipid, a free fatty acid, cholesterol or some other chemical compound. A biomarker can be a morphologic biomarker (for example, a histological change, DNA ploidy, malignancy-associated changes in the cell nucleus and premalignant lesions) or a genetic biomarker (for example, DNA mutations, DNA adducts and apoptotic index).
[0064] The term "Chronic Obstructive Pulmonary Disease" or "COPD" refers to a complex disease that results in progressive loss of lung function. COPD is typically characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways. COPD can include the occurrence of chronic bronchitis or emphysema, both of which result in airway narrowing. Clinically, COPD is typically detected by limited airflow in lung function tests. COPD is typically irreversible and gets progressively worse over time. Symptoms of COPD include chronic cough, chronic sputum production, dyspnea, rhonchi, wheezing, chest tightness, tiredness and decreased airflow in lung function tests. Individuals suffering from very severe COPD can develop respiratory failure and present with cyanosis, headaches, drowsiness, and/or asterixis. COPD is a progressive disease and prognosis of the disease can be predicted by severe airflow obstruction, poor exercise capacity, shortness of breath, being significantly underweight or overweight, respiratory failure, cor pulmonale, and frequent acute exacerbations. COPD prognosis can be evaluated using the BODE index, which is a scoring system that measures FEV1, body-mass index, 6-minute walk distance, and a modified MRC (Medical Research Council) dyspnea scale to estimate outcomes in COPD.
[0065] The term "control sample" refers to a sample against which a test sample is compared in order to diagnose, prognose, classify or grade the test sample. A control sample may be healthy tissue or may be a well-characterized sample, such as from an individual suffering from COPD, including but not limited to, GOLD stage 1, GOLD stage 2, GOLD stage 3, or GOLD stage 4 COPD. A control sample can be analyzed concurrently with or separately from the test sample, including before or after analyzing the test sample. The data from the analysis of a control sample may be stored, e.g., in a computer readable medium or in a manual, for comparison against test samples analyzed in the future, or as data for training network-based or machine-learning methods. A control sample may be developed as a medical standard for comparison. For example, analysis of control samples has developed medical standards for normal fed and fasted blood glucose levels; normal, at risk, and hypertensive blood pressures, and normal resting heart rates. As used herein, the term "control sample" includes samples that provided a medical standard. Accordingly, a test sample may be compared against a medical standard generated from control samples. For example, production of a variant of a lipid may be indicative of a change medical condition. Alternatively, a change in production level of a lipid may be indicative of a change in medical condition. A control sample may be lung tissue, such as tissue obtained by biopsy from a healthy individual, or some other sample. For example, a control sample may be sputum, saliva, bronchial washing, bronchial aspirates, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. Tissue specimens, such as those obtained by biopsy, may be fixed (e.g., formaldehyde-fixed paraffin-embedded (FFPE)). The control sample may be obtained from a tissue bank. The control sample may also be obtained from a cadaver or an organ donor.
[0066] The term "fatty acid" refers to a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides or phospholipids. When they are not attached to other molecules, they are known as "free" fatty acids.
[0067] The term "forced expiratory volume in one second" or "FEV1" refers to the volume of air that can forcibly be blown out in one second, after full inspiration. Average values for FEV1 in healthy individuals depend on sex and age and have been well-characterized in the art. FEV1 and the FEV1 to FVC ration (FEV1/FVC) are used clinically to grade COPD. In healthy adults
FEV1/FVC should be approximately 75-80%. In obstructive diseases, such as
COPD, FEV1 is diminished because of increased airway resistance to expiratory flow. While the FVC may be decreased as well, due to the premature closure of airway in expiration, FEV1 is typically more affected because of the increased airway resistance, so the FEV1/FVC ratio reflects the degree of airway closure compared to lung volume.
[0068] The term "forced vital capacity" or "FVC" refers to the volume of air that can forcibly be blown out after full inspiration, measured in liters.
[0069] The term "individual" refers to a vertebrate, preferably a mammal. The mammal can be, without limitation, a mouse, a rat, a cat, a dog, a horse, a pig, a cow, a non-human primate or a human. Preferably, the individual is a human.
[0070] The term "individual at risk for lung injury" refers to an individual who is predisposed to lung injury, such as COPD, including emphysema. Predisposition to lung injury may be due to one or more genetic or environmental factors. For example, an individual related to a COPD patient is more likely to get COPD than an individual who is not related to a COPD patient. Further, exposure to environmental factors such as radon gas, asbestos, tobacco smoke, and air pollution can increase the risk for lung injury and predispose an individual to lung injury.
[0071] The term "individual having a lung injury" or "individual suffering from injury" refers to an individual experiencing progressive loss of lung function, typically characterized by alveoli destruction. Lung injury can be bronchial or emphysematous and may be detected by analyzing clinical, functional, and radiological findings or detecting relevant biomarkers.
[0072] The term "lipid" refers to a class of organic compounds that are fatty acids or their derivatives and are, typically, insoluble in water but soluble in organic solvents. Lipids may be divided into eight categories: fatty acids, glycero lipids, glycerophospho lipids, sphingo lipids, saccharo lipids, polyketides sterol lipids and prenol lipids. Fatty acids and fatty acid derivatives may be identified using a notation giving the number of carbon atoms and of double bonds (separated by a colon). For example, palmitic acid, which has sixteen carbon atoms and no double bonds, may be referred to as 16:0, and oleic acid, which has eighteen carbons and one double bond, may be referred to as 18:1. Some lipids comprise a head group with one or more fatty acid tails. For example,
phosphatidylcholines comprise a choline head groups and two fatty acid tails, one saturated and one unsaturated. Such lipids may be identified by their fatty acid tails. For example, "PC(16:0/18: 1)" refers to a phosphtidylcholine lipid with a palmitic acid tail and an oleic acid tail.
[0073] The term "lipid signature" refers to a group of lipids produced by a cell or a tissue, whose combined production pattern may be indicative of, e.g., a normal state, an at-risk state, a diseased state, a treated state or a recovery state. A lipid signature may be characterized by which lipids are produced or at what level each lipid is produced. Lipid signatures are particularly useful in diagnosing, prognosing, classifying or grading complex diseases states, which result from the combination of several genetic and environmental factors. The lipid signatures disclosed herein may be used, e.g., for the diagnosis, prognosis, classification and/or grading of lung injury, such as emphysema, in an individual.
[0074] The term "MALDI-TOF" refers to matrix-assisted laser
desorption/ionization time of flight mass spectroscopy. Matrix-assisted laser desorption/ionization (MALDI) is a two step process that uses laser-triggered desorption of protonated and deprotonated matrix materials to protonate or deprotonate analyte molecules (e.g., DNA, RNA, and proteins). Time-of-flight (TOF) mass spectrometry refers to a method in which an ion's mass-to-charge ratio is determined via the time that it takes an ionized particle to reach a detector at a known distance.
[0075] The terms "protein," "polypeptide" and "peptide" are used
interchangeably and indicate at least one molecular chain of amino acids linked through covalent or non-covalent bonds. The terms do not refer to a specific length of the molecular chain. Peptides, oligopeptides and proteins are included within the definition of "polypeptide". The terms include post-translational modifications of the molecule, e.g., phosphorylation, glycosylation and acetylation. The terms also include protein fragments, fusion proteins, mutant proteins and variant proteins.
[0076] The term "saturated" refers to a compound, such as a fatty acid, that has no double or triple bonds or ring. In saturated hydrocarbons, every carbon atom is attached to two hydrogen atoms, except those at the ends of the chain, which bear three hydrogen atoms. In certain fatty acid nomenclature, ":0" refers to a saturated fatty acid. For example, 16:0 (palmitic acid) refers to a saturated fatty acid comprising sixteen carbon atoms.
[0077] The term "SELDI-TOF" refers surface-enhanced laser
desorption/ionization time of flight mass spectroscopy. Surface-enhanced laser desorption/ionization (SELDI) is a variant of MALDI that uses a target with a biochemical affinity for the analyte. Time-of-flight (TOF) mass spectrometry refers to a method in which an ion's mass-to-charge ratio is determined by measuring the time that it takes an ionized particle to reach a detector at a known distance. [0078] The term "test sample" refers to a sample obtained from an individual at risk for, having or suffering from lung injury. A test sample may be any sample suspected of containing or exhibiting a biomarker. The test sample is analyzed and compared to a control sample, including medical standards developed from control samples, to diagnose, prognose, classify or grade lung injury in the individual. A test sample may be obtained from lung tissue, such as tissue obtained by biopsy from a tumor, or other biological tissue. For example, a test sample may be sputum, saliva, bronchial washing, bronchial aspirates, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. Tissue specimens, such as those obtained by biopsy, may be fixed (e.g., formaldehyde-fixed paraffin-embedded (FFPE)).
[0079] The term "unsaturated" refers to a compound, such as a fatty acid, that contains carbon-carbon double bonds or triple bonds. In a chain of carbons, such as a fatty acid, a double or triple bond will cause a kink in the chain. Unsaturated fats tend to be liquid at room temperature, rather than solid, due to the kinks in the chain, which prevent the molecules from packing closely together to form a solid. These fats are typically called oils and are present in fish and plants. The degree of unsaturation refers to the number of double and triple bonds in the fatty acid. In certain fatty acid nomenclature, the number following the colon refers to a saturated fatty acid. For example, 18: 1 (oleic acid) refers to a fatty acid comprising eighteen carbon atoms and one double bond (i.e., one degree of unsaturation). Similarly, 18:2 (linoleic acid) refers to a fatty acid comprising eighteen carbon atoms and two double bonds (i.e., two degrees of unsaturation).
Lipid Biomarkers [0080] One aspect of the invention provides lipid biomarkers useful for diagnosing, prognosing, classifying or grading lung injury, such as COPD, including emphysema. The lipid biomarkers may independently be selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
glycerophosphoethanolamine, a glycerophosphoglycerol, a
glycerophospho inositol, a glycerophosphoserine, an acidic glycosphingo lipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
[0081] In some embodiments, the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0);
CE(18: 1); CE(18:2); CE(18:3); CE(20:3) CE(20:4); CE(20:5); CE(22:0);
CE(22:5); CE(22:6); and CE(24:2), preferably CE(20:4) or CE(22:5). Optionally, CE(20:4) or CE(22:5) are downregulated by CS.
[0082] The eicosanoid may be selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-
DHET; 6-keto-PGFl alpha; 8-HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12- DHET; 12-HEPE; 12-HETE; 12-oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET;
15-HEPE; 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD2); Prostaglandin E2 (PGE2); PGF2alpha; TXB2; and
TXB3, preferably, 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PGE2; or TXB3. In some embodiments, the eicosanoid is be selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; or TXB3.
The eicosanoid may be TXB3. Optionally, the eicosanoid is not PGD2 or PGE2.
[0083] The glycerophosphocholine may be selected from phosphatidylcholine
(PC)(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18:1); PC(16:0/16:0);
PC(16:0/16: 1); PC(16:0/17: 1); PC(16:0/18:0); PC(16:0/18: 1); PC(16:0/18:2); PC(16:0/20:0); PC(16:0/20: 1); PC(16:0/20:2); PC(16:0/20:4); PC(16:0/22:4);
PC(16:0/22:5); PC(16:0/22:6); PC(16: 1/16: 1); PC(16: 1/18:0); PC(16: 1/18: 1);
PC(16: 1/18:2); PC(16: 1/20:4); PC(17:0/18:2); PC(18:0/18: 1); PC(18:0/18:2);
PC(18:0/20:4); PC(18:0/22:4); PC( 18:0/22:6); PC(18: 1/18: 1); PC(18: 1/18:2);
PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4), preferably, PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16: 1); or
PC(16: 1/16: 1). Most preferably, the glycerophosphocholine is PC(16:0/16:0) or
PC(16: 1/16: 1), which are both upregulated in lung tissue following CS exposure.
Optionally, PC 18:0/20:4; 18: 1/20:4; and 18:2/20:4 are downregulated in plasma following CS exposure.
[0084] In some embodiments, the glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2);
PG(18: 1/18:1); PG(18: 1/18:2); and PG(18:2/18:2), such as PG(18: 1/18: 1), PG(18:1/18:2) or PG(18:2/18:2). Optionally, PG(18:1/18:1), PG(18:1/18:2) and
PG(18:2/18:2) are upregulated in lung tissue following CS exposure.
[0085] Optionally, the glycerophosphoethanolamine is selected from phosphatidylethanolamine (PE)( 16:0/16:0); PE(16:0/18:1); PE(16:0/18:2);
PE(16:0/20:4); PE(16:0/22:4); PE(18:0/18:0); PE(18:0/20:4); PE(18:0/22:4);
PE(18:1/18:1); PE(18:l/20:4); and PE(22:6/22:6), for example PE(16:0/16:0);
PE(16:0/18:1); PE(16:0/18:2); and PE(16:0/20:4). Optionally, PE(16:0/16:0);
PE(16:0/18:1); PE(16:0/18:2); and PE(16:0/20:4) are upregulated in lung tissue following CS exposure.
[0086] The ceramide (Cer) may be selected from Cer(dl8:0/16:0);
Cer(dl 8:0/18:0); Cer(dl8:0/18:l); Cer(dl 8:0/20:0); Cer(dl8:0/22:0);
Cer(dl8:0/24:0); Cer(dl8:0/24:1); Cer(dl 8:0/26:1); Cer(dl8:l/16:0);
Cer(dl8:l/18:0); Cer(dl8:l/18:l); Cer(dl 8: 1/20:0); Cer(dl8:l/22:0);
Cer(dl8:l/22:1); Cer(dl 8: 1/23:0); Cer(dl 8: 1/24:0); Cer(dl8:l/24:1);
Cer(dl8:l/26:0); and Cer(dl8:l/26:1), preferably Cer(dl8:0/16:0);
Cer(dl8:0/22:0); Cer(dl8:0/24:0); Cer(dl 8:0/24:1); Cer(dl8:0/26:1);
Cer(dl8:l/16:0); Cer(dl8:l/24:0); Cer(dl8:l/24:1); Cer(dl8:l/26:0); or
Cer(dl8:l/26:1). Most preferably, the ceramide may be selected from
Cer(dl8:0/16:0); Cer(dl8:0/24:0); Cer(dl 8:0/24:1); and Cer(dl8:l/26:1), which are each upregulated in lung tissue following CS exposure.
[0087] In some embodiments, the neutral glycosphingolipid is selected from glucosyl/galactosyl Cer (Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0);
Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
Glc/GalCer(dl8:0/24:l); Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl8:l/18:0); Glc/GalCer(dl8:l/20:0); Glc/GalCer(dl 8: 1/22:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl8:l/24:0); Glc/GalCer(dl 8: 1/24:1); Glc/GalCer(dl 8: 1/26:0);
Glc/GalCer(dl8:l/26:l); lactosylCer (LacCer)(dl8:0/16:0); LacCer(dl8:l/16:0);
LacCer(dl8:l/18:0); LacCer(dl8:l/20:0); LacCer(dl 8: 1/22:0);
LacCer(dl 8: 1/23:0); LacCer(dl 8: 1/24:0); LacCer(dl 8: 1/24:1);
globotriaosylceramide (Gb3)(dl8:l/16:0); Gb3(dl8:l/18:0); Gb3(dl8:l/20:0);
Gb3(dl8:l/22:0); Gb3(dl8:l/22:1); Gb3(dl 8: 1/23:0); Gb3(dl8:l/24:0); and
Gb3(dl8:l/24:1). Optionally, the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl 8:0/24:0);
Glc/GalCer(dl8:0/24: l); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/22:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24:0); Glc/GalCer(dl 8 : 1/24: 1);
Glc/GalCer(dl8: l/26:0); Glc/GalCer(dl 8: 1/26: 1); LacCer(dl8: l/16:0);
LacCer(dl 8: 1/23:0); LacCer(dl8:l/24:0); LacCer(dl8: l/24: l); Gb3(dl8:l/16:0);
Gb3(dl8: l/22:1); Gb3(dl 8: 1/23:0); Gb3(dl8: l/24:0); and Gb3(dl8: l/24: 1). Most preferably, the neutral glycosphingolipid is selected from Glc/GalCer(dl8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl8:l/24:0); Gb3(dl8: l/16:0); and Gb3(dl8: l/24:0), which are each upregulated in lung tissue following CS exposure.
[0088] The acidic glycosphingolipid may be selected from GMl(dl8: l/16:0);
GMl(dl8: l/24:0); GMl(dl8: l/24: l); GM3(dl8: l/16:0); GM3(dl8: l/18:0);
GM3(dl8: l/20:0); GM3(dl8: l/21 :0); GM3(dl8: l/22:0); GM3(dl8: l/22: l);
GM3(dl8: l/23:0); GM3(dl8: l/24:0); GM3(dl8: l/24: l) and GM3(dl8: l/24:2), such as GMl(dl8: l/16:0); GM3(dl8: l/16:0); GM3(dl8: l/24:0); GM3(dl8: l/24: l) and GM3(dl8: l/24:2). Most preferably, the acidic glycosphingolipid is selected from GMl(dl8: l/16:0); GM3(dl8: l/16:0); and GM3(dl8:l/24:0), which are each upregulated in lung tissue following CS exposure. The GM1 class of lipids (i.e., "Sum(Gl)") and the GM3 class of lipids (i.e., "Sum(G3)") remain statistically different following cessation. Accordingly, a change in Sum(Gl) or Sum(G3) may indicate severe lung injury.
[0089] The sphingoid base may be selected from sphingosine-1 -phosphate (SlP)(dl8: l); SlP(dl8:2); sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: 1). Optionally, the sphingoid base may be selected from
SAlP(dl8:0); SPA(dl8:0); SPA(d20:0); SPH(dl6:l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: l), preferably, SAlP(dl8:0). In some embodiments, the SIP class of lipids (i.e., "Sum(SlP)") are upregulated in lung tissue following CS exposure. In some embodiments, the SA1P class of lipids (i.e., "Sum(SAlP)") are upregulated in lung tissue following CS exposure. In some embodiments, the SPA class of lipids (i.e., "Sum(SPA)") are upregulated in lung tissue following CS exposure. In some embodiments, the SPH class of lipids (i.e., "Sum(SPH)") are upregulated in lung tissue following CS exposure. Accordingly, the sphingoid base may be selected from Sum(SlP); Sum(SAlP); Sum(SPA); and Sum(SPH). Optionally, the sphingoid base is not a sphingosine-1 -phosphate.
[0090] In some embodiments, the lipid biomarker is selected from CE(20:4);
CE(22:5); 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid;
PGD2; PGE2; TXB3; PC(16:0/16:0); PC(16: 1/16: 1); PG(18:1/18: 1), PG(18:1/18:2);
PG(18:2/18:2); PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); PE(16:0/20:4);
Cer(dl8:0/16:0); Cer(dl8:0/24:0); Cer(dl 8:0/24: 1); Cer(dl8: l/26: 1);
Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl8: l/16:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24: 1); Glc/GalCer(dl 8 : 1/26:0);
Glc/GalCer(dl8: l/26: l); LacCer(dl8: l/16:0); LacCer(dl8: l/24:0);
Gb3(dl8: l/16:0); Gb3(dl8: l/24:0); GMl(dl8: l/16:0); GM3(dl8: l/16:0);
GM3(dl8: l/24:0); SlP(dl8: l); SlP(dl8:2); SAlP(dl8:0); SPA(dl8:0);
SPA(d20:0); SPH(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20:l). Optionally, the lipid biomarker is selected from CE(20:4); CE(22:5); 5-HETE; 8,9-DHET;
11,12-DHET; 14,15-DHET; arachidonic acid; TXB3; PC(16:0/16:0);
PC(16: 1/16: 1); PG(18: 1/18: 1), PG(18: 1/18:2); PG(18:2/18:2); PE(16:0/16:0);
PE(16:0/18: 1); PE(16:0/18:2); PE(16:0/20:4); Cer(dl8:0/16:0); Cer(dl 8:0/24:0); Cer(dl8:0/24: 1); Cer(dl8:l/26: 1); Glc/GalCer(dl 8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl8:l/24:0); Gb3(dl8: l/16:0); Gb3(dl8: l/24:0);
GMl(dl8: l/16:0); GM3(dl8: l/16:0); GM3(dl8: l/24:0); SAlP(dl8:0);
SPA(dl8:0); SPA(d20:0); SPH(dl6: l); SPH(dl8:l); SPH(dl8:2); and SPH(d20: l).
In some embodiments, the lipid biomarker is not PGD2 or PGE2. In some embodiments, the lipid biomarker is not a sphingosine-1 -phosphate.
[0091] In some embodiments, the lipid biomarker is selected from 5-HETE; 5,6-
DHET; 8-HETE; 11,12-DHET; 12-HEPE; 12-oxoETE; 14,15-DHET; arachidonic acid; eicosapentaenoic acid; PGD2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/18: 1);
PC(16: 1/16: 1); PC(16: 1/20:4); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4);
PE(18: 1/18: 1); PG(16:0/16:0); PG(16:0/18: 1); Cer(dl8:l/26: 1); Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/22:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24:0); Glc/GalCer(dl 8 : 1/24: 1);
Glc/GalCer(dl8: l/26:0); Glc/GalCer(dl 8: 1/26: 1); LacCer(dl8: l/18:0);
LacCer(dl8: l/20:0); GMl(dl8: l/16:0); GMl(dl8: l/24:0); GM3(dl8: l/24:0); GM3(dl8: l/24: l); and GM3(dl8: l/24:2). These lipids remain significantly different following cessation of exposure to CS. Optionally, the lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16: 1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4); Cer(dl8: l/26: 1); Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl8: l/24: l); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
GMl(dl8: l/16:0); and GM3(dl8: l/24:0).
[0092] In some embodiments, at least 2, at least 3, at least 4, at least 5, at least
10, at least 15, at least 20, at least 25, at least 30 or at least 35 of the lipid biomarkers have increased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample. In some
embodiments, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more,
20 or more, 25 or more, 30 or more, or 35 or more of the lipid biomarkers have increased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample. The lipid biomarkers that are up- regulated in an individual suffering from or at risk for lung injury may be selected from PGD2; PGE2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1);
PC(16:0/16:0); PC(16:0/16: 1); PC(16:0/17: 1); PC(16:0/18: 1); PC(16:0/18:2);
PC(16:0/20:4); PC(16:0/22:4); PC(16:0/22:5); PC(16:0/22:6); PC(16: 1/16: 1);
PC(16: 1/18:0); PC(16: 1/18: 1); PC(16: 1/18:2); PC(16: 1/20:4); PC(18: 1/18: 1); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PG(16:0/16:0); PG(16:0/18:1);
PG(16:0/18:2); PG(18: 1/18: 1); PG(18: 1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0);
Cer(dl 8:0/18:0); Cer(dl8:0/22:0); Cer(dl 8:0/24:0); Cer(dl8:0/24: 1);
Cer(dl8:0/26: 1); Cer(dl8: l/16:0); Cer(dl 8: 1/22:0); Cer(dl8: l/24:0);
Cer(dl8: l/24: 1); Cer(dl8: l/26:0); Cer(dl8: l/26: 1); Glc/GalCer(dl 8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl 8:0/20:0); Glc/GalCer(dl 8:0/22:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl 8:0/24: 1); Glc/GalCer(dl8: l/16:0);
Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0); Glc/GalCer(dl 8: 1/22:0); Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24:0); Glc/GalCer(dl 8 : 1/24: 1);
Glc/GalCer(dl8:l/26:0); Glc/GalCer(dl 8: 1/26:1); LacCer(dl8:l/16:0);
LacCer(dl 8: 1/23:0); LacCer(dl8:l/24:0); and LacCer(dl8:l/24:l). Lipid biomarkers, such as PC(16: 1/18: 1); PC(18:1/18:1); Glc/GalCer(dl8:0/24:l); or Cer(dl 8:0/18:0), may be up-regulated in blood samples from an individual suffering from or at risk for lung injury. Optionally, lipid biomarkers, such as
PGD2; PGE2; PC(14:0/14:0); PC(14:0/16:0); PC(14:0/16:1); PC(14:0/18:1);
PC(16:0/16:0); PC(16:0/16:1); PC(16:0/17:1); PC(16:0/18:1); PC(16:0/18:2);
PC(16:0/20:4); PC(16:0/22:4); PC(16:0/22:5); PC(16:0/22:6); PC(16:1/16:1); PC(16:1/18:0); PC(16:1/18:1); PC(16:1/18:2); PC(16: 1/20:4); PC(18: 1/18: 1);
PC(18:1/18:2); PC(18:l/20:4); PC(18:2/18:2); PG(16:0/16:0); PG(16:0/18:1);
PG(16:0/18:2); PG(18:1/18:1); PG(18:1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0);
Cer(dl8:0/22:0); Cer(dl8:0/24:0); Cer(dl 8:0/24:1); Cer(dl8:0/26:1);
Cer(dl8:l/16:0); Cer(dl8:l/22:0); Cer(dl 8: 1/24:0); Cer(dl8:l/24:1);
Cer(dl8:l/26:0); Cer(dl8:l/26:1); Glc/GalCer(dl 8:0/16:0);
Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl 8:0/20:0); Glc/GalCer(dl 8:0/22:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl 8:0/24:1); Glc/GalCer(dl8:l/16:0);
Glc/GalCer(dl8:l/18:0); Glc/GalCer(dl 8: 1/20:0); Glc/GalCer(dl 8: 1/22:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24:0); Glc/GalCer(dl 8 : 1/24: 1);
Glc/GalCer(dl8:l/26:0); Glc/GalCer(dl 8: 1/26:1); LacCer(dl8:l/16:0);
LacCer(dl 8: 1/23:0); LacCer(dl8:l/24:0); or LacCer(dl8:l/24:l), may be up- regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury. In some embodiments, the level of one or more of PGD2; PGE2; PC(16:0/16:0); PC(16:1/16:1);
PG(16:0/16:0); PG(18:1/18:1); PG(18:1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0);
Cer(dl8:0/24:0); Cer(dl 8:0/24:1); Cer(dl8:l/26:1); Glc/GalCer(dl 8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8:l/16:0); LacCer(dl8:l/24:0); Gb3(dl8:l/16:0); Gb3(dl8:l/24:0);
GMl(dl8:l/16:0); GM3(dl8:l/16:0); GM3(dl8:l/24:0); SlP(dl8:l); SlP(dl8:2);
SAlP(dl8:0); SPA(dl8:0); SPH(dl6:l); SPH(dl8:l); SPH(dl8:2); or SPH(d20:l) is up-regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury. Optionally, the level of one or more of PC(16:0/16:0); PC(16: 1/16: 1); PG(16:0/16:0);
PG(18:1/18: 1); PG(18: 1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0); Cer(dl 8:0/24:0); Cer(dl8:0/24: 1); Cer(dl8:l/26: 1); Glc/GalCer(dl 8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl8:l/24:0); Gb3(dl8: l/16:0); Gb3(dl8: l/24:0); GMl(dl8: l/16:0); GM3(dl8: l/16:0); GM3(dl8: l/24:0); SAlP(dl8:0);
SPA(dl8:0); SPH(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20: l) is up- regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
[0093] In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 of lipid biomarkers have decreased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample. In some embodiments, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 35 or more of the lipid biomarkers have decreased production in an sample from an individual suffering from or at risk for lung injury compared to a control sample. The lipid biomarkers that are down- regulated in an individual suffering form or at risk for lung injury may be selected from PC(16:0/18:0); PC(16:0/20:4); PC(16:0/22:6); PC(18:0/18.1); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl8:l/16:0);
Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0); Glc/GalCer(dl 8: 1/22:0);
Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0); Cer(dl8: l/16:0);
Cer(dl8: l/18:0); Cer(dl8: l/18: l); LacCer(dl8.1/18:0); LacCer(dl 8.1/20.0); and arachidonic acid. Lipid biomarkers, such as PC(16:0/18:0); PC(16:0/20:4);
PC(16:0/22:6); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl 8:0/22:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8: l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Cer(dl8: l/16:0); Cer(dl8: l/18:0); or Cer(dl8: l/18: l), may be down-regulated in blood samples from an individual suffering from or at risk for lung injury.
Optionally, lipid biomarkers, such as arachidonic acid; PC( 16:0/18:0);
PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) or LacCer(dl 8.1/20.0), may be down-regulated in a large airway, such as in a sample from a bronchial biopsy, or a lung, for example in a sample from a lung biopsy, including a biopsy of the parenchyma, of an individual suffering from or at risk for lung injury.
[0094] In some embodiments, the lipid biomarker are up-regulated to a certain degree in a sample from an individual suffering from or at risk for lung injury compared to a control sample. For example, each up-regulated lipid biomarker may, independently, be up-regulated at least 1.5-fold, at least 2-fold, at least 2.5- fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1,000-fold or more compared to the control sample. Similarly, in some embodiments, the lipid biomarkers are down-regulated to a certain degree in a sample from an individual suffering from or at risk for lung injury compared to a control sample. For example, each down-regulated lipid biomarker may, independently, be down-regulated at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least 1,000-fold or more compared to the control sample.
Methods of Using Lipid Biomarkers [0095] The lipid biomarkers of the invention may be used in methods of diagnosing, prognosing, classifying or grading lung injury in biological sample or an individual. The lung injury may be COPD, including emphysema. One aspect of the invention provides a method of diagnosing, classifying or grading lung injury in an individual at risk for or suffering from a lung injury. In some embodiments the method comprises classifying a test sample as injured or non- injured, such as emphysematous or non-emphysematous or COPD or non-COPD. In some embodiments, the method comprises measuring the levels of at least 2 lipid biomarkers described above in a test sample; and comparing those measurements to the level of the at least two lipid biomarker in a control sample to obtain a classification of the test sample as injured or non-injured, such as emphysematous or non-emphysematous or COPD or non-COPD. In some embodiments, the levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 lipid biomarkers described above are measured. In some embodiments, the levels of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 125, or 150 or more lipid biomarkers described above are measured.
[0096] In some embodiments, the methods of the invention comprise obtaining a test sample from an individual, determining the absence, presence or level of one or more of the lipid biomarkers described above in the test sample, comparing said absence, presence or level to the absence, presence or level of the same lipid biomarker(s) in a control sample, and diagnosing the individual as having or being at risk for lung injury based on the comparison. In a further embodiment, the invention provides a method for monitoring the progress or recovery of a lung injury in an individual, said method comprising determining at suitable time intervals in one or more samples taken from said individual differential production levels of the lipid biomarkers described above. In a further embodiment, the invention provides a method for monitoring the progress or recovery of a lung injury treatment in an individual, said method comprising determining at suitable time intervals before, during, or after therapy (for example, at different time points during the treatment) in one or more samples taken from said individual differential production levels of the lipid biomarkers described above. In a further embodiment, the invention provides a method for monitoring lung injury in an individual resulting from exposure to air-borne pollutants, said method comprising determining at suitable time intervals in one or more samples taken from said individual differential production levels of the lipid biomarkers described above. In a further embodiment, the invention provides a method for monitoring changes in the severity of a lung injury in an individual, said method comprising determining at suitable time intervals before, during, or after changing the method or pattern of nicotine consumption (for example, at different time points during smoking cessation or switching from a combusted tobacco product, e.g., cigarette, to a heated tobacco product or an electronic cigarette) in one or more samples taken from said individual differential production levels of the lipid biomarkers described above. In some embodiments, the individual is a cigarette smoker; the individual is a former cigarette smoker; the individual was a cigarette smoker who has stopped smoking cigarette for at least 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36 month(s) prior to the measurements; the individual is or the individual was a cigarette smoker who has switched to using a heated tobacco product or a nicotine- containing product which can include an electronic cigarette or a nicotine patch, for at least 0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36 month(s) prior to the measurements instead of smoking cigarette.
[0097] In some embodiments, the method comprises detecting the level of at least 2 lipid biomarkers described above in a test sample obtained from the individual; and comparing the level of the at least 2 lipid biomarkers in the test sample to the level of the at least 2 lipid biomarkers in a control sample. In some embodiments, if the level of the at least 2 lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk for lung injury. In some embodiments, the level of the at least 2 lipid biomarkers is higher in the test sample than in the control sample. Optionally, the level of the lipid biomarkers is lower in the test sample than in the control sample. In some embodiments, the method further comprises detecting the level of the lipid biomarkers in the control sample. In some embodiments, the levels of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 lipid biomarkers are detected. In some embodiments, the levels of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 125, or 150 or more lipid biomarkers are detected.
[0098] In some embodiments, the test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. Optionally, the test sample is obtained from a large airway of the individual, such as a bronchial biopsy, or the lung of the individual, such as a lung biopsy, including a biopsy of the parenchyma of the individual.
[0099] In some embodiments, the control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy. Optionally, the control sample is obtained from a large airway or a lung of an individual not affected with a lung injury, such as from a bronchial biopsy or a lung biopsy of the individual not affected with the lung injury. In some embodiments, the control sample is obtained from the individual at risk for or having lung injury prior to onset of the lung injury. In other embodiments, the control sample is obtained from an individual that does not suffer from a lung injury.
[0100] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of PGD2; PGE2; PC(14:0/14:0);
PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16:0); PC(16:0/16: 1);
PC(16:0/17: 1); PC(16:0/18: 1); PC(16:0/18:2); PC(16:0/20:4); PC(16:0/22:4);
PC(16:0/22:5); PC(16:0/22:6); PC(16: 1/16: 1); PC(16: 1/18:0); PC(16: 1/18: 1);
PC(16: 1/18:2); PC(16: 1/20:4); PC(18: 1/18: 1); PC(18: 1/18:2); PC(18: l/20:4);
PC(18:2/18:2); PG(16:0/16:0); PG(16:0/18:1); PG(16:0/18:2); PG(18: 1/18: 1); PG(18:1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0); Cer(dl8:0/22:0); Cer(dl 8:0/24:0);
Cer(dl8:0/24: 1); Cer(dl8:0/26: 1); Cer(dl8: l/16:0); Cer(dl8: l/22:0);
Cer(dl8: l/24:0); Cer(dl8: l/24: 1); Cer(dl 8: 1/26:0); Cer(dl8: l/26: 1); Glc/GalCer(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl 8:0/20:0);
Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl 8:0/24: 1);
Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8: l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Glc/GalCer(dl8: l/24: l); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl 8: 1/23:0); LacCer(dl 8: 1/24:0); and
LacCer(dl 8: 1/24: 1) is higher in the test sample than in the control sample, wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
[0101] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16: 1/18: 1); PC(18:1/18: 1);
Glc/GalCer(dl8:0/24: l); and Cer(dl 8:0/18:0) is higher in the test sample than in the control sample, wherein the test sample and the control sample are blood samples.
[0102] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of arachidonic acid; PC(16:0/18:0);
PC(18:0/18.1); Cer(dl8: l/18:0); LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, wherein the test sample and the control sample are obtained from a large airway, such as a bronchial biopsy, or the lung, such as a lung biopsy, including a biopsy of the parenchyma.
[0103] In some embodiments, the individual suffers from or is at risk of having lung injury if the level of one or more of PC(16:0/18:0); PC(16:0/20:4);
PC(16:0/22:6); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl 8:0/22:0);
Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8: l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Cer(dl8: l/16:0); Cer(dl8:l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, wherein the test sample and the control sample are blood samples.
[0104] Detection of the lipid biomarkers described herein in a test sample or a control sample may be performed by any method known in the art. [0105] In one aspect, the methods of the invention rely on the detection of the presence or absence of lipid biomarker, or the qualitative or quantitative assessment of either over- or under-production of a lipid biomarker in a population of cells or a tissue in a test sample relative to a standard (for example, a control sample).
[0106] The level of one or more lipid biomarkers in the test sample and the level of one or more lipid biomarkers in the control sample may be detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography. In some embodiments, the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry. Optionally the chromatography is thin layer chromatography, solid-phase extraction
chromatography, high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography, or ultra-performance liquid chromatography.
[0107] In some embodiments, the lung injury is emphysema or COPD.
Examples
[0108] In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
[0109] Unless otherwise indicated, data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the Student t-test when only 2 groups were compared, or an ANOVA followed by Fisher's Least
Significance Difference (LSD) for pairwise comparisons when more than 2 groups were compared. In this later case, results are reported as differences between groups with associated 95%-confidence interval (CI) as estimated by the LSD method. All computations were performed with SAS version 9.2 (SAS Institute Inc., Cary, NC, USA).
Example 1 Exposure of mice to cigarette smoke [0110] The study design included 3 groups of C57BL/6 mice: Sham (fresh air- exposed), CS (exposed to mainstream smoke from 3R4F, a reference cigarette from the University of Kentucky and smoking cessation. Animals from the Sham and CS groups were exposed to fresh air and cigarette smoke, respectively, for up to seven months. To model the effects of smoking cessation, animals from the cessation group (CESS) were first exposed to CS for 2 months and then switched to filtered air for 5 additional months.
[0111] All procedures involving animals were performed with approval of an Institutional Animal Care and Use Committee, in compliance with the National Advisory Committee For Laboratory Animal Research (NACLAR) Guidelines on the Care and Use of Animals for Scientific Purposes. Female C57BL/6 mice bred under specific pathogen-free conditions were obtained from Charles River, USA and were 8-10 weeks old at exposure initiation. Mice were individually identified by the subcutaneous implantation of transponders and were housed and whole- body exposed in the animal laboratory under specific pathogen-free conditions. Random allocation of mice to experimental groups was conducted prior to exposure. Animals were fed a standard chow diet (T2914C irradiated rodent diet, Harlan). Filtered tap water was supplied ad libitum and changed daily. The animal holding and procedure rooms were maintained at 21.8 +/- 0.4°C and 21.6 °C and at a relative humidity of 55.5 +/- 0.7% and 51.0 +/- 1.2% respectively. Mice were observed daily for mortality, morbidity, and signs of injury. Body weight was measured twice per week during the exposure period. The number of animals analyzed for each endpoint is given in Table 1.
Table 1. Number of re licates er rou for each anal sis
Figure imgf000041_0001
[0112] The reference cigarettes (3R4F) were obtained from the Tobacco
Research and Development Center (University of Kentucky, KY, USA). Smoking parameters including puff duration and volume were conducted in conformity with the Health Canada Intense (HCI) smoking regimen. Total particulate matter (TPM) levels for CS-exposed groups were targeted at 750 μg/l. Animals were
acclimatized for 16 days prior to full exposure after which they were exposed to fresh, conditioned air or to CS at concentrations of 750 μg/l TPM for 4 hours per day, 5 days per week. During this administration animals were exposed to fresh filtered air for 30 minutes after completing the first hour of smoke exposure and for 60 minutes after the second and third exposure hours. Plasma
carboxyhemoglobin (COHb) levels were determined as a marker of smoke uptake at 3, 4 and 6 months.
[0113] Reproducibility of the smoke generation was insured by regular analyses of the test atmosphere during the study. The test atmosphere was tested for components including TPM, carbon monoxide (CO), nicotine, aldehydes and particle size distribution.
[0114] To investigate the effects of cigarette smoke and smoking cessation in the lung, mice were exposed in whole-body exposure chambers to mainstream cigarette smoke (CS) or fresh air (sham) for a period of two, three, or seven months. Moreover, a smoking cessation arm consisted of a group of mice which were withdrawn from CS exposure after two months and further exposed to fresh air for one or five months. CS-uptake was confirmed by monitoring blood carboxyhemoglobin (COHb) at 3 time points and nicotine and cotinine
concentration in plasma at the latest time point. COHb returns very rapidly in the cessation to sham levels (Table 2). Decreased body weights were recorded for smoke-exposed animals (CS group and Cessation group up to 2 months). After smoke withdrawal, body weights in the cessation group rapidly normalized to sham-levels (Table 3).
Table 2. Biomarkers of exposure - Carboxyhemoglobin (COHb) concentration (%), nicotine and cotinine concentrations in plasma
(mean ± sem).
Figure imgf000042_0001
Values expressed as mean ± sem. <LOD: all values in a group are below lower limit of detection (LOD). If not all values in a group were below LOD (0.7ng/ml for nicotine and cotinine), values below LOD were replaced by LOD/2. N=8 to 12 Table 3. Body Weight Progression (average weight per group in g). 95% confidence interval is indicated. *: different to sham, #:
different to CS at the same time oint <0.05 .
Figure imgf000043_0001
Example 2 Histological Changes in the Lungs of Mice Exposed to CS
[0115] The left lung was instilled with 4% paraformaldehyde in PBS (pH 7.4) and fixed for 24 hours before standard paraffin-embedding, sectioning and staining. Briefly, a small cannula (1.6 mm diameter) was gently introduced into the trachea and the proximal left bronchus and fixed to the latter with a ligature.
Fixative was delivered to the left lung by gravity at 15 cm water pressure. After the instillation procedure was completed and the lungs were filled, the cannula was carefully removed and the ligature tightened to prevent fixative flowing out of the lung. The lung was then fixed by immersion in the same fixation solution for 24 hours. Histopathological evaluation was performed at 5 different levels of the lung, displaying the central and peripheral aspects of the parenchyma. For each lung section, a separate evaluation was performed. Mean scores of the 5 data sets were calculated for each animal and for each endpoint. The mean values per animal were used for the exposure group- based analysis. Each lung section was stained with haematoxylin and eosin (H&E), alcian blue periodic acid schiff s (AB-PAS) and a resorcin fuchsin. The morphometric endpoints, mean chord length (Lm), destructive index (DI) and bronchiolar attachment (BA) were determined using the Visiopharm software (Visiopharm, Horlshom, Denmark), version 4.9.2.0
(Visiopharm Integrator System) on a section of the left lung running parallel to the main bronchus. For the semi-quantitative evaluation of all histopathological endpoints, a severity score was applied ranging from 0 (finding not present) to 5 (severe alteration).
[0116] To characterize and grade smoke-induced damage in the lung tissue, lung sections (see, e.g., Figure 1) were histologically evaluated for the two, three, and seven-month time points. The overall histopathological assessment revealed typical cigarette smoke-related effects in the lungs of exposed animals, which were evidenced by the infiltration of inflammatory cells (neutrophils and macrophages, Figure 2A-B), along with changes to the lung morphology (Figure 2C-E).
Unpigmented and pigmented macrophages are free cells in the alveolar lumen not showing any cytoplasmatic pigmentation or containing fine-granular, brownish to yellow cytoplasmic pigmentation, which may be due to CS particles inhaled, respectively. Pigmented macrophage nests consist of multiple macrophages in the alveolar lumen clustered in small groups within adjacent alveoli. Multinucleated giant cells are very large macrophages (size ranging from 18 to 28 μιη) that are positively stained after AB-PAS incubation (Figure IB), likely due to uptake of excessive amounts of surfactant produced at the alveolar surface in response to CS inhalation.
[0117] All macrophages in the alveolar lumen were elevated in CS-exposed groups and showed an increase between two and seven months. Following cessation, an immediate decrease at the three-month time point (i.e., one month after cessation) was observed for unpigmented macrophages, which returned to sham levels by seven months. Decreases in pigmented macrophages and pigmented macrophage nests were slower and did not reach sham levels at seven months
(Figure 2A). Whereas no multinucleated giant cell was observed in sham-exposed mice, their count increased rapidly in CS-exposed mice, and disappeared remarkably fast from mice in the cessation group (where only few were still observed just one month after cessation). At the seven month time point, no multinucleated giant cell was observed in the cessation group, mirroring exactly the situation in the sham group (Figure 2B).
[0118] Further demonstration of progression of the disease state was
quantitatively assessed by morphometric analysis of the lung tissue: CS-exposure was associated with increased mean chord length (Figure 2C), increased destructive index (defined as the percentage of emphysematous tissue over normal tissue, Figure 2D), and fewer bronchiolar attachments (Figure 2E) relative to sham-exposed animals from month two onwards. Some destruction of the lung tissue could be observed with increasing severity in the sham group, likely due to aging. The evaluation at different time points, i.e. right before cessation, after one and five months of cessation, shows that CS-induced histopathological changes were significantly alleviated after smoking cessation. The damage caused by the initial two month of CS-exposure is not reversible, as the post-cessation mice show disease progression. Progression rate is however slower post-cessation than in continuously exposed mice.
Example 3 Lipoidomic Profiles Change in the Lungs of Mice Exposed to CS
[0119] Lung tissue samples were pulverized with a CP02 CryoPrep Dry
Pulverization System (Covaris), and resuspended in ice-cold methanol containing 0.1% butyl-hydroxy-toluene (BHT) at a concentration of 100 mg/ml. Cer, cerebrosides, glycerolipids, glycerophospholipids and cholesteryl esters were extracted using a modified Folch lipid extraction performed on a Hamilton
Microlab Star robot. Gangliosides were extracted according to the method described by Fong et al (Fong et al, 2009, Lipids 44, pp. 867-874) with minor modifications, and eicosanoids were extracted as described by Deems et al (Deems et al, 2007, Methods in Enzymol, 432, pp.59-82).
[0120] In Shotgun Lipidomics, lipid extracts were analyzed using a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500) equipped with a robotic nanoflow ion source (NanoMate HD). Targeted molecular lipids were analyzed on a hybrid triple quadrupole/linear ion trap mass spectrometer (5500 QTRAP) equipped with an ultra-high pressure liquid chromatography (UHPLC) system (CTC HTC PAL autosampler and Rheos Allegro pump) using a multiple reaction monitoring (MRM) -based method in negative ion mode.
[0121] The mass spectrometry data files were processed using LipidView™ VI .1 and MultiQuant™ 2.0 Software (Ab Sciex, Massachusetts, USA) to generate a list of lipid names and peak areas. Lipids were normalized to their respective internal standard and the tissue weight. The concentrations of molecular lipids are presented as nmol/mg wet tissue for lung samples. For statistical analysis, a Wilcoxon rank-sum test was conducted for each lipid for comparing the study groups. Monte-Carlo estimation of exact p-values was performed. Multiple testing was controlled with FDR q-values.
[0122] To investigate the effects of smoke exposure on lung lipid metabolism, a lipidomics analysis of the lung tissue was performed at the time of smoke exposure cessation, and one and five months later. Lipidomics results at the lipid class level are summarized in Figure 3. CS induced a small, but significant increase in sterols at the 2 month exposure time point, however this increase was no longer observed with prolonged CS exposure.
[0123] Unexpectedly, smoke exposure resulted in lower concentrations of the sum of eicosanoid species in the lung (Figures 3 and 4). This tendency at the lipid class level was largely driven by the reduction of arachidonic acid (AA) at all 3 time points in CS-exposed mice. Several arachidonic acid metabolites of lipoxygenase 5 (LOX5) and cytochrome P450 oxidase (CYP) pathways (e.g. 5- HETE, and a few DHET species) were also significantly decreased by smoke exposure (Figure 4). In contrast, prostaglandin D2 (PGD2) levels peaked dramatically (+395%, p=0.0001) as early as after 2 months smoke exposure.
Moreover, concentrations of two other cyclooxygenase (COX) metabolites, PGE2 and thromboxane B3 (TXB3) increased significantly, albeit to a lesser extent than PGD2. Smoke-induced changes in eicosanoid levels were progressively relieved, and the levels of all measured eicosanoids were back to sham levels after 5 months of smoking cessation (Figures 3 and 4).
[0124] Smoke exposure elevated the levels of phosphatidylcholine (PC), PC plasmalogen (PC-0 and PC-P), phosphatidylglycerol (PG), and
phosphatidylethanolamine (PE) lipid classes at all time points (Figure 3). These increases are likely linked to surfactant production by aveolar type II cells in response to lung injury. Among PCs, most significant changes were among the myristic acid (14:0)-containing molecules, as well as PC(16: 1/16: 1), and
PC( 16:0/16: 1) (Figure 5 A), some of which were not detected until the latest time point due to low abundance. The median concentration difference for these species was up to 240% (p<0.01), while in the majority of PC molecules little or no change in concentration in response to CS exposure was observed throughout the experiment. The upregulation of PGs was driven mainly by minor species PG(18:1/18: 1), PG(18:1/18:2) and PG(18:2/18:2), upregulated up to 400%
(p<0.001) at the late time point (Figure 5A). The levels of the levels of 16:0 fatty acid-containing PE species were also significantly upregulated (Figure 5B). PC species with poly-unsaturated fatty acyls, however, were downregulated in the plasma following exposure to CS (Figure 5C). The changes observed in these lipid classes generally returned to sham levels after smoking cessation (Figures 3 and 5).
[0125] The data indicated most significant and constant changes in several sphingolipid classes, including Cer, glucosyl/galactosyl Cer (Glc/GalCer), lactosylCer (LacCer), and GM1 gangliosides. Different sphingoid bases including SIP, SA1P, SPA and SPH were also significantly and progressively elevated in mice exposed to cigarette smoke (Figure 3). Interestingly, a length chain-specific sphingolipid pattern of Cer (Figure 6), glucosyl Cer (Figure 7), and lactosylCer (Figure 8) in mice exposed to cigarette smoke could be observed: very long chain and 16:0 fatty acid- containing species were significantly upregulated, while little (Cer and Glc/GalCer) or opposite (LacCer) changes were observed in species containing medium chain length (CI 8-20) fatty acids. A similar profile was also observed in the precursor (i.e. dihydro) forms of the ceramide and
glucosylceramide molecules. The increases in these lipid levels was strongly attenuated or returned to sham levels after only one month of smoking cessation (Figures 3, 6, 7, and 8).
[0126] Similarly, the levels of 16:0 fatty acid-containing GM1 gangliosides were significantly upregulated (Figure 9). Following cessation, the levels of 16:0 fatty acid-containing GM1 gangliosides remained elevated, potentially signifying lasting injury. The levels of the levels of 16:0 fatty acid-containing and very long chain (particularly 24-carbon chains) GM3 gangliosides were also significantly upregulated (Figure 10). Additionally, sphingoid bases were significantly upregulated (Figure 11). Globotriaosylceramide (Gb3), which is derived from
LacCer, demonstrated a similar pattern as other ceramides, namely that long-chain Gb3 species are most significantly upregulated (Figure 12). [0127] Smoke exposure elevated the levels of saturated/mono-unsaturated cholesterol esters in the lung, while levels of poly-unsaturated species were downregulated (Figure 13). Polyunsaturated cholesterol ester species, especially CE20:4 and CE22:5, in the plasma were also down-regulated by cigarette smoke (Figure 14).
Example 4 Lipidomic Profiles in Blood Samples of Human Individuals
[0128] The study used a parallel-group, case-controlled study design in order to determine the differential expression of molecular and physiological biomarkers in subjects with COPD (COPD) when compared to healthy (no COPD) current smokers (CS), healthy (no COPD) former smokers (FS) and healthy never-smokers (NS). This approach was taken in order to determine if biomarkers can be used to determine if negative impacts of smoking on lung health can be detected before the onset of clinical symptoms (irreversible airflow obstruction) that diagnose COPD.
[0129] Recruitment was ongoing throughout the 18-month duration of the study to ensure the pre-determined sample size of 60 subjects per group. Each subject visited the clinic on four occasions throughout the course of the study. After the four clinical visits, all subjects were contacted by the Principal Investigator (PI) or a designated representative by telephone to ensure their well-being. Physiological and clinical data were collected throughout the course of the study and recorded in an electronic case report form (eCRF) linked to a SAS database using the standardized Active Directory Application Mode (ADAM) solution. All subjects enrolled in the study had the study procedures explained to them before they agreed to sign an informed consent form (ICF). All subjects were free to withdraw their consent at any time without any reason and for the smokers, smoking cessation advice was given at each visit. The occurrence of Adverse Events (AEs) and concomitant medication details were also recorded on an ongoing basis in the subjects' eCRF. The inclusion and exclusion criteria for this study can be found in Table 4 at www.clinicaltrials.gov using identifier NCT01780298.
Population and Outline Study Plan
[0130] Male and female subjects aged between 41 and 70 years were enrolled with a completed total subject number of 60 per group. If a subject was withdrawn (for medical or personal reasons) the subject was replaced. At the end of the study (18 months after the start, Nov 2012) sputum, blood, nasal samples were collected and a number of physiological and clinical measurements taken from 240 subjects. Each subject in each of the 3 control groups, namely the healthy smokers, never- smokers and former smokers, were matched to subjects in the COPD group by age (±5 years), ethnicity, gender, and all smoking subjects had a smoking history of at least 10 pack-years. The summary of the study enrolment and recruitment success as well as a summary of the samples collected from each of the 240 subjects and the physiological and clinical measurements that were taken are summarized in Figure 15 as recommended by the guidelines set out by CONSORT (Consolidated Standards of Reporting Trials) .
[0131] After completion of visit 1, each subject attended the study center within 4 to 28 days for visit 2. Since subjects underwent TLCO measurements
(measurement of the transfer factor for carbon monoxide) at visit 2, and subjects with normal lung function (post-bronchodilator FEV1 >80% of the predicted normal, with no evidence of airway obstruction, and an FEV1/FVC ratio of >70% at visit 1) but an abnormal TLCO (<60% at visit 2) were withdrawn at the end of visit 2.
[0132] Subjects attended the center for visit 3 within 3 to 14 days after visit 2. Eligibility was reassessed against the inclusion/exclusion criteria prior to any other procedures and subjects underwent the procedures as indicated in Table 4. Subjects attended the study center for visit 4 within 3 to 14 days after visit 3 and underwent the procedures as described in Figure 15. A follow-up telephone call was made to subjects within 3 to 10 days after visit 4 to record adverse events (AEs) and concomitant medication details and give smoking cessation advice to current smokers. Table 4. Demographics, smoking history, spirometric parameters and cardiorespiratory vital signs across the study's evaluable population. Data are presented as median ran e .
Figure imgf000050_0001
BMI: Body mass index; FEV1: forced expiratory volume in 1 second
Lipidomics
[0133] Blood for lipidomics analysis was collected during visit 3 in an 8.5 mL serum-separating tube (SST), allowed to clot at room temperature for 30 min and centrifuged. Serum samples were aliquotted at stored at -80°C until further use. Analyses of lipid species were performed in serum samples from a subset of study subjects including 40 never-smokers, 40 former smokers, 40 smokers, and 40 COPD patients (Table 5) by Zora Biosciences Oy (Espoo, Finland). able 5. Demographics of study sample subset for lipidomics ana ysis
Figure imgf000051_0001
[0134] Lipids were extracted using a modified Folch lipid extraction procedure (Ekroos, K. (2008). Unraveling Glycerophospholipidomes by Lipidomics. (F. Wang, Ed.) (pp. 369-384). Totowa, NJ: Humana Press) performed on a Hamilton Microlab Star robot. Extract samples were spiked with known amounts of non- endogenous synthetic internal standards. For the identification and quantitation of cholesteryl esters (CE), phosphatidyl lipids (PL), lysophospholipids (LPL), sphingomyelins (SM), diradylglycerols (DAG) and triacylglycerols (TAG) by shotgun lipidomics, lipid extracts were analyzed using a QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems) equipped with a robotic nanoflow ion source (NanoMate HD, Advion Biosciences) according to Stahlman and colleagues (Stahlman M, Ejsing CS, Tarasov K, Perman J, Boren J, Ekroos K (2009) High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry. Journal of Chromatography B 877: 2664-2672). Molecular lipids were analyzed in both positive and negative modes using Multiple Precursor Ion Scanning (MPIS)-based methods (Ejsing CS, Duchoslav E, Sampaio J, Simons K, Bonner R, Thiele C, Ekroos K, Shevchenko A (2006) Automated Identification and Quantification of Glycerophospholipid Molecular Species by Multiple Precursor Ion Scanning. Analytical Chemistry 78: 6202-6214; Ekroos K, Chernushevich IV, Simons K, Shevchenko A (2002) Quantitative Profiling of Phospholipids by Multiple Precursor Ion Scanning on a Hybrid Quadrupole Time-of-Flight Mass Spectrometer. Analytical Chemistry 74: 941-949. Triacylglycerols (TAG) were analyzed using Precursor Ion Scanning (PIS)- and Neutral Loss scanning (NL)-based methods. The molecular lipid species were identified and quantified in semi-absolute or absolute amounts. Targeted eicosanoid and sphingolipid lipidomics were performed using UHPLC (CTC Analytics AG)-coupled QTRAP 5500 mass spectrometry using multiple reaction monitoring (MRM) in positive ion mode. The MS data files were processed using LipidView™ 1.1 (AB Sciex) or MultiQuant™ 2.0 (AB Sciex) for generating a list of lipid names and peak areas. Lipids were normalized to their respective internal standard (the peak area of the endogenous lipid was divided by the peak area of the corresponding internal standard) and sample volume, yielding concentrations of molecular lipids in μΜ. The differences and relative differences between the groups were estimated using Hodges-Lehmann estimator (the median value of the cross-pairwise differences between individuals of the two groups). A rank- sum Wilcoxon test was performed to calculate p values. Differences with a p value below 0.05 were considered statistically significant.
[0135] To investigate the effects of cigarette smoke exposure (CS vs. NS, FS vs. CS), lipidomics analysis in serum was performed. Lipidomics results at the lipid class level are summarized in Figure 16 and indicate small but significant increases in serum diradylglycerols (DAG), neutral glycosphingolipids (LacCer), glycerophospholipids including glycerophosphocholines (PC) and
glycerophosphoethanolamines (PE), and triglycerides (TAG) in current smokers compared to never-smokers. Further, individual members of other lipid classes including eicosanoids, sphingolipids and sterols among others also exhibited significant increases and decreases in the serum of current smokers when compared to serum levels of never-smokers (Figure 16).
[0136] Increases in DAG levels were driven by smoking-related elevation of the 18: 1 fatty acid-containing diradylglycerols DAG(16:0/18:1) and DAG(18: 1/18: 1) serum levels in current smokers, and these levels remained high, although not statistically significantly so, in former smokers compared to never-smokers
(Figure 17). Moreover, the observed differences between current and never- smokers were still significant following adjustment for the use of lipid-modifying drugs (Figures 18A and B). Interestingly, serum DAG levels remained elevated in former smokers in comparison to never-smokers, albeit not significantly, indicating a prolonged and potentially irreversible effect of smoke exposure on the circulatory lipidome (Figure 16). [0137] With the exception of PC(18:0/20:5), levels of multiple
glycerophosphatidylcholine molecules were generally higher in the serum of current smokers compared to that of never-smokers resulting in an overall up- regulation of this lipid class in disease-free smokers. While not significantly different, serum levels of PC(18:0/20:5) increased noticeably in former smokers relative to current smokers, while the levels of all other members of this lipid class affected by smoking decreased following smoking cessation and mostly returned to levels observed in never-smokers (Figure 16 and Figure 19).
[0138] Serum TAG levels were also significantly higher in current compared to never-smokers (Figure 16). This was mainly due to a concerted increase in palmitoleic acid- and oleic acid-containing glycerol triesters in the serum of current smokers (Figure 20). However, with the exception of the stearic acid ester
TAG(52: 1), serum TAG levels exhibited no significant differences when comparing current with former and former with never-smokers, respectively, indicating that potential smoking-related changes in TAG profiles may be reversible upon smoking cessation.
[0139] In general, serum lactosylceramide (LacCer) levels were higher in current compared to never-smokers, albeit only statistically significantly different in the case of LacCer(18: l/16:0), resulting in a net increase in the serum levels of this lipid class (Figure 16 and Figure 21). Interestingly, LacCer levels were significantly decreased in former smokers who had quit smoking at least 5 years prior to enrolment compared to current smokers, with the biggest changes observed for LacCer(18: l/16:0) and LacCer(18: l/18:0) indicating potential reversibility of smoking-associated changes in at least these 2 members of this lipid class.
[0140] In addition, several other species of the neutral glycosphingolipid class including Glc/GalCer(dl8:l/18:0) and Glc/GalCer(dl 8: 1/24: 1) were also significantly elevated in the serum of current relative to never-smokers. With the exception of Glc/GalCer(dl8: l/16:0) and Glc/GalCer(dl8: l/18:0), most
Glc/GalCer serum levels remained elevated in former smokers compared to never- smokers, although the differences were not statistically significant (Figure 22).
[0141] Of the three glycerophosphoethanolamines (PE) species detected, two - PE(16:0/20:4) and PE(18: 1/18: 1) - were significantly elevated in the serum of current smokers relative to never-smokers (Figure 16 and Figure 23). While levels of both molecules were decreased in former smokers compared to current smokers, only the difference in serum PE(16:0/20:4) levels was found to be significant. Of note, serum levels of all affected PE species in former smokers were very similar to those seen in never-smokers, suggesting that the effect of cigarette smoke exposure may be reversible upon cessation.
[0142] Other lipid molecules exhibiting significant differences in serum levels of current relative to never-smokers included members of the eicosanoid class such as 9-HODE and 13-HODE which were decreased, and arachidonic acid (AA), 11,12- DHET and 14,15-DHET which were increased (Figure 16 and Figure 24). Of interest, in former smokers, the levels of the affected eicosanoid appeared to be "restored" to serum levels observed in never-smokers.
[0143] Significant differences were further found in serum levels of various ceramide (Cer) molecules including Cer(dl 8:0/18:0), Cer(dl8:0/20:0),
Cer(dl8: l/18:0), and Cer(dl8: l/22:1). Minor differences not reaching statistical significance were also observed for other ceramides. Although the serum levels of all affected Cer decreased upon smoking cessation, these changes were only significant for Cer(dl8: l/16:0), Cer(dl8: l/18:0) and Cer(dl8: l/20:0), and serum levels remained elevated, albeit not significantly, in former smokers compared to never-smokers (Figure 25).
[0144] Moreover, serum levels of two glycophosphosphingolipids (SM) exhibited significant differences when comparing current to never-smokers
(Figure 16 and Figure 26). While the SM(dl 8:0/18:0) serum levels decreased in former smokers nearing levels seen in never-smokers, serum levels of
SM(dl 8: 1/24: 1) did not, potentially reflecting irreversible smoking-related effects on the serum lipidome.
[0145] Several sterols (CE) were also affected by smoking, exhibiting
significantly higher levels in the serum of current relative to never-smokers. While serum cholesteryl oleate (CE(18:1)) and meadoate (CE(20:3)) levels decreased significantly upon smoking cessation, serum levels of cholesteryl eicsoate
(CE(20:4)) remained significantly elevated in former relative to never-smokers, potentially reflecting irreversible effects of cigarette smoke exposure on the circulatory lipidome (Figure 27).
[0146] Lipidomics analysis in serum was also performed to examine the effects of the development of mild COPD (GOLD stages I and II; COPD vs. CS, COPD vs. NS). Lipidomics results at the lipid class level are summarized in Figure 28 and indicate small but significant decreases in serum sterols (CE) and
glycosphingophospho lipids (SM).
[0147] Specifically, there were significantly lower cholesteryl palmitoate (CE(16:0)), linoleoate (CE(18:2)), nonadecanoate (CE(19:0)), eicosapentaenoate (CE(20:5)), and docosahexaenoate (CE(22:6)) levels in the serum of COPD patients than in that of smokers without COPD (Figure 29). None of these sterols were found to be affected by smoking (Figure 27); however, the observed differences were significantly influenced by BMI, the use of lipid-modifying drugs and/or alcohol intake, respectively (Figure 30 A-D).
[0148] In addition, serum levels of a number of glycosphingophospholipids including the sphingosine (SPH)-containing SPH(dl8:0) and SPH(dl8: l) sphingomyelins (SM) SM(dl 8:0/16:0), SM(dl8: l/16:0), SM(dl8: 1/18:0), SM(dl 8: 1/23:0), SM(dl 8: 1/24:0), and SM(dl8: l/23: l), were also significantly decreased in COPD patients relative to disease-free smokers (Figure 28 and
Figure 31). This was also true following adjustment for intake of lipid-modifying drugs and other potential confounders.
[0149] Further decomposition of the lipidomics data also highlighted
significantly decreased levels of individual glycerophosphatidylcholme molecules PC(16:0/18:2), PC(16:0/22:6), and PC(18:0/22:6) in the serum of patients with mild COPD compared to serum levels in healthy smokers (Figure 32). Again, these differences remained significant even after adjusting for potential confounders including the use of lipid-modifying drugs.

Claims

We claim:
1. A method of diagnosing an individual as being at risk for or having lung injury comprising
(1) detecting the level of two or more lipid biomarkers in a test sample obtained from the individual; and
(2) comparing the level of the two or more lipid biomarkers in the test sample to the level of the two or more lipid biomarkers in a control sample,
wherein, if the level of the two or more lipid biomarkers is different in the test sample than in the control sample, then the individual suffers from or is at risk of having lung injury;
wherein the two or more lipid biomarkers are independently selected from a sterol, a diradylglycerol, an eicosanoid, a glycerophosphocholine, a
glycerophosphoethanolamine, a glycerophosphoglycerol, a
glycerophosphoinositol, a glycerophosphoserine, an acidic glycosphingolipid, a ceramide, a neutral glycosphingolipid, a phosphosphingolipid, and a sphingoid base.
2. The method according to claim 1, wherein the sterol is selected from cholesterol ester (CE)(14:0); CE(15:0); CE(16:0); CE(16: 1); CE(17:0); CE(17: 1); CE(18:0); CE(18: 1); CE(18:2); CE(18:3); CE(20:3) CE(20:4); CE(20:5);
CE(22:0); CE(22:5); CE(22:6); and CE(24:2).
3. The method according to claim 2, wherein the sterol is CE(20:4) or CE(22:5).
4. The method according to any one of claims 1-3, wherein the eicosanoid is selected from 5-HEPE; 5-HETE; 5-oxoETE; 5,6-DHET; 6-keto-PGFl alpha; 8-
HETE; 8,9-DHET; 9-HODE; 11-HETE; 11,12-DHET; 12-HEPE; 12-HETE; 12- oxoETE; 13-HODE; 13-HOTrE; 14,15-DHET; 15-HEPE; 15-HETE; arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; Prostaglandin D2 (PGD2); Prostaglandin E2 (PGE2); PGF2alpha; TXB2; and TXB3.
5. The method according to claim 4, wherein the eicosanoid is selected from 5-HETE; 8,9-DHET; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PGE2; and TXB3.
6. The method according to any one of claims 1-5, wherein the
glycerophosphocholine is selected from phosphatidylcholine (PC)(14:0/14:0); PC(14:0/16:0); PC(14:0/16: 1); PC(14:0/18: 1); PC(16:0/16:0); PC(16:0/16: 1); PC(16:0/17: 1); PC(16:0/18:0); PC(16:0/18: 1); PC(16:0/18:2); PC(16:0/20:0); PC(16:0/20: 1); PC(16:0/20:2); PC(16:0/20:4); PC(16:0/22:4); PC(16:0/22:5); PC(16:0/22:6); PC(16: 1/16: 1); PC(16: 1/18:0); PC(16: 1/18: 1); PC(16: 1/18:2); PC(16: 1/20:4); PC(17:0/18:2); PC(18:0/18: 1); PC(18:0/18:2); PC(18:0/20:4); PC(18:0/22:4); PC(18:0/22:6); PC(18: 1/18: 1); PC(18: 1/18:2); PC(18: l/20:4); PC(18: l/22:6); PC(18:2/18:2); and PC(18:2/20:4).
7. The method according to claim 6, wherein the glycerophosphocholine is selected from PC(16:0/16:0); and PC(16: 1/16: 1).
8. The method according to any one of claims 1-7, wherein the
glycerophosphoglycerol is selected from phosphatidylglycerol (PG)(16:0/16:0); PG(16:0/18: 1); PG(16:0/18:2); PG(18:1/18: 1); PG(18: 1/18:2); and PG(18:2/18:2).
9. The method according to claim 8, wherein the glycerophosphoglycerol is selected from PG(18: 1/18:1), PG(18: 1/18:2) and PG(18:2/18:2).
10. The method according to any one of claims 1-9, wherein the
glycerophosphoethanolamine is selected from phosphatidylethanolamine
(PE)(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); PE(16:0/20:4); PE(16:0/22:4); PE(18:0/18:0); PE(18:0/20:4); PE(18:0/22:4); PE(18:1/18: 1); PE(18: l/20:4); and PE(22:6/22:6).
11. The method according to claim 10, wherein the
glycerophosphoethanolamine is selected from PE(16:0/16:0); PE(16:0/18: 1); PE(16:0/18:2); and PE(16:0/20:4).
12. The method according to any one of claims 1-11, wherein the ceramide (Cer) is selected from Cer(dl8:0/16:0); Cer(dl 8:0/18:0); Cer(dl8:0/18:l);
Cer(dl8:0/20:0); Cer(dl8:0/22:0); Cer(dl 8:0/24:0); Cer(dl8:0/24:1);
Cer(dl8:0/26:1); Cer(dl8:l/16:0); Cer(dl8:l/18:0); Cer(dl8:l/18:l);
Cer(dl8:l/20:0); Cer(dl8:l/22:0); Cer(dl8:l/22:1); Cer(dl 8: 1/23:0);
Cer(dl8:l/24:0); Cer(dl8:l/24:1); Cer(dl 8: 1/26:0); and Cer(dl8:l/26:1).
13. The method according to claim 12, wherein the ceramide is selected from Cer(dl8:0/16:0); Cer(dl8:0/24:0); Cer(dl 8:0/24:1); and Cer(dl8:l/26:1).
14. The method according to any one of claims 1-13, wherein the neutral glycosphingo lipid is selected from glucosyl/galactosyl Cer
(Glc/GalCer)(dl8:0/16:0); Glc/GalCer(dl 8:0/18:0); Glc/GalCer(dl8:0/20:0); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl 8:0/24:0); Glc/GalCer(dl 8:0/24:1);
Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl8:l/18:0); Glc/GalCer(dl 8: 1/20:0);
Glc/GalCer(dl8:l/22:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
lactosylCer (LacCer)(dl 8:0/16:0); LacCer(dl8:l/16:0); LacCer(dl8:l/18:0);
LacCer(dl8:l/20:0); LacCer(dl 8: 1/22:0); LacCer(dl 8: 1/23:0);
LacCer(dl8:l/24:0); LacCer(dl8:l/24:l); globotriaosylceramide
(Gb3)(dl8:l/16:0); Gb3(dl8:l/18:0); Gb3(dl8:l/20:0); Gb3(dl8:l/22:0);
Gb3(dl8:l/22:1); Gb3(dl8:l/23:0); Gb3(dl8:l/24:0); and Gb3(dl8:l/24:1).
15. The method according to claim 14, wherein the neutral glycosphingo lipid is selected from Glc/GalCer(dl 8:0/16:0); Glc/GalCer(dl8:0/24:0);
Glc/GalCer(dl8:l/16:0); Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:1);
Glc/GalCer(dl8:l/26:0); Glc/GalCer(dl 8: 1/26:1); LacCer(dl8:l/16:0);
LacCer(dl8:l/24:0); Gb3(dl8:l/16:0); and Gb3(dl8: 1/24:0).
16. The method according to any one of claims 1-15, wherein the acidic glycosphingolipid is selected from GMl(dl8:l/16:0); GMl(dl8:l/24:0);
GMl(dl8:l/24:l); GM3(dl8:l/16:0); GM3(dl8:l/18:0); GM3(dl8:l/20:0);
GM3(dl8:l/21 :0); GM3(dl8:l/22:0); GM3(dl8:l/22:l); GM3(dl8:l/23:0);
GM3(dl8:l/24:0); GM3(dl8:l/24:l) and GM3(dl8:l/24:2).
17. The method according to claim 16, wherein the acidic glycosphingolipid is selected from GMl(dl8:l/16:0); GM3(dl8: l/16:0); and GM3(dl8: l/24:0).
18. The method according to any one of claims 1-17, wherein the sphingoid base is selected from sphingosine-1 -phosphate (SlP)(dl8:l); SlP(dl8:2);
sphinganine-1 -phosphate (SAlP)(dl8:0); sphinganine (SPA)(dl8:0); SPA(d20:0); sphingosine (SPH)(dl6: l); SPH(dl8: l); SPH(dl8:2); and SPH(d20:l).
19. The method according to any one of claims 1-18, wherein the lipid biomarker is selected from 5 -HETE; 11,12-DHET; 14,15-DHET; arachidonic acid; PGD2; PC(16:1/16: 1); PE(16:0/16:0); PE(16:0/18:2); PE(16:0/20:4);
Cer(dl8: l/26: 1); Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0);
Glc/GalCer(dl 8 : 1/23:0); Glc/GalCer(dl 8 : 1/24: 1); Glc/GalCer(dl 8 : 1/26:0);
Glc/GalCer(dl8: l/26: l); GMl(dl8: l/16:0); and GM3(dl8: l/24:0).
20. The method according to any one of claims 1-19, wherein the level of one or more of PGD2; PGE2; PC(16:0/16:0); PC(16: 1/16: 1); PG(16:0/16:0);
PG(18:1/18: 1); PG(18: 1/18:2); PG(18:2/18:2); Cer(dl8:0/16:0); Cer(dl 8:0/24:0); Cer(dl8:0/24: 1); Cer(dl8:l/26: 1); Glc/GalCer(dl 8:0/16:0);
Glc/GalCer(dl8:0/24:0); Glc/GalCer(dl8: l/16:0); Glc/GalCer(dl 8: 1/23:0);
Glc/GalCer(dl 8: 1/24: 1); Glc/GalCer(dl 8: 1/26:0); Glc/GalCer(dl 8: 1/26: 1);
LacCer(dl8: l/16:0); LacCer(dl8:l/24:0); Gb3(dl8: l/16:0); Gb3(dl8: l/24:0);
GMl(dl8: l/16:0); GM3(dl8: l/16:0); GM3(dl8: l/24:0); SlP(dl8: l); SlP(dl8:2); SAlP(dl8:0); SPA(dl8:0); SPH(dl6: l); SPH(dl8: l); SPH(dl8:2); and
SPH(d20: 1) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway or a lung.
21. The method according to any one of claims 1-20, wherein the level of one or more of PC(16: l/18: l); PC(18: 1/18: 1); Glc/GalCer(dl8:0/24:l); and
Cer(dl8:0/18:0) is higher in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
22. The method according to any one of claims 1-21, wherein the level of one or more of arachidonic acid; PC(16:0/18:0); PC(18:0/18.1); Cer(dl8: l/18:0);
LacCer(dl8.1/18:0) and LacCer(dl 8.1/20.0) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are obtained from a large airway or a lung.
23. The method according to any one of claims 1-22, wherein the level of one or more of PC(16:0/18:0); PC(16:0/20:4); PC(16:0/22:6); PC(18:0/18:2);
PC(18:0/20:4); PC(18:0/22:6); PC(18: 1/18:2); PC(18: l/20:4); PC(18:2/18:2); PC(18:2/20:4); Glc/GalCer(dl8:0/22:0); Glc/GalCer(dl8:l/16:0);
Glc/GalCer(dl8: l/18:0); Glc/GalCer(dl 8: 1/20:0); Glc/GalCer(dl 8: 1/22:0);
Glc/GalCer(dl 8: 1/23:0); Glc/GalCer(dl 8: 1/24:0); Cer(dl8: l/16:0);
Cer(dl8: l/18:0); and Cer(dl8: l/18: l) is lower in the test sample than in the control sample, and wherein the test sample and the control sample are blood samples.
24. The method according to any one of claims 1-23, wherein the individual suffers from or is at risk of having lung injury if the level of three or more lipid biomarkers is different in the test sample than in the control sample.
25. The method according to claim 24, wherein the individual suffers from or is at risk of having lung injury if the level of four or more lipid biomarkers is different in the test sample than in the control sample.
26. The method according to claim 25, wherein the individual suffers from or is at risk of having lung injury if the level of five or more lipid biomarkers is different in the test sample than in the control sample.
27. The method according to claim 26, wherein the individual suffers from or is at risk of having lung injury if the level of ten or more lipid biomarkers is different in the test sample than in the control sample.
28. The method according to any one of claims 1-27, wherein the method further comprises detecting the level of the two or more lipid biomarkers in the control sample.
29. The method according to any one of claims 1-28, wherein the test sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
30. The method according to claim 29, wherein the test sample is obtained from a large airway or a lung of the individual.
31. The method according to claim 30, wherein the test sample is obtained from a bronchial biopsy or a lung biopsy.
32. The method according to any one of claims 1-28, wherein the control sample is selected from sputum, saliva, bronchial brushings, exhaled breath, bronchial biopsy, lung biopsy, nasal scrapings and lung tissue obtained during bronchoscopy.
33. The method according to claim 32, wherein the control sample is obtained from a large airway or a lung of an individual not affected with lung injury.
34. The method according to claim 33, wherein the control sample is obtained from a bronchial biopsy or a lung biopsy of the individual not affected with lung injury.
35. The method according to claim 32, wherein the control sample is obtained from the individual at risk for or having the emphysema prior to onset of the lung injury.
36. The method according to claim 32, wherein the control sample is obtained from an individual that does not suffer from lung injury.
37. The method according to any one of claims 1-36, wherein the level of the two or more lipid biomarkers in the test sample and the level of the two or more lipid biomarkers in the control sample are detected by mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, dual polarization interferometry or chromatography.
38. The method according to claim 37, wherein the mass spectrometry is electrospray ionization mass spectrometry, matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry, or atmospheric pressure chemical ionization mass spectrometry.
39. The method according to claim 38, wherein the chromatography is thin layer chromatography, solid-phase extraction chromatography, high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography, or ultra-performance liquid chromatography.
40. The method according to any one of claims 1-39, wherein the lung injury is emphysema or COPD.
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