WO2022101442A1 - Biomarkers for severe pulmonary condition - Google Patents

Biomarkers for severe pulmonary condition Download PDF

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Publication number
WO2022101442A1
WO2022101442A1 PCT/EP2021/081580 EP2021081580W WO2022101442A1 WO 2022101442 A1 WO2022101442 A1 WO 2022101442A1 EP 2021081580 W EP2021081580 W EP 2021081580W WO 2022101442 A1 WO2022101442 A1 WO 2022101442A1
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cer
subject
pulmonary
biomarker
combination
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PCT/EP2021/081580
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French (fr)
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Reijo Laaksonen
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Zora Biosciences Oy
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Publication of WO2022101442A1 publication Critical patent/WO2022101442A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present disclosure is related to biomarkers for predicting severe pulmonary conditions.
  • it provides a novel in vitro method for assessing the severity of a pulmonary condition of a subject using lipid biomarkers.
  • the present biomarkers can be used in methods for predicting the progress of a pulmonary condition, selecting and administering an appropriate treatment and preventing the occurrence of serious complications of a pulmonary condition.
  • Pulmonary conditions are some of the most common medical conditions in the world. Lungs are a complex system bringing in oxygen, delivering oxygen into circulation and releasing carbon dioxide. A lung disease can occur when there is a problem in any part of this system. Pulmonary conditions cause a wide range of different complications and severe forms are life-threatening. There are three main types of pulmonary conditions: conditions affecting airways, conditions affecting the structure of the lung tissue, and conditions affecting circulation in the lungs.
  • Pulmonary conditions affecting airways such as asthma, chronic obstructive pulmonary disease (COPD) and bronchitis, usually cause a narrowing or blockage of the airways.
  • Pulmonary conditions affecting lung tissue may be caused due to scarring or inflammation of the pulmonary tissue.
  • This type of lung diseases include for example pulmonary fibrosis, lung cancer and acute respiratory distress syndrome (ARDS).
  • Pulmonary conditions affecting circulation may be caused by clotting, scarring or inflammation of the blood vessels.
  • Pulmonary conditions affecting circulation include for example thrombosis, pulmonary embolism and pulmonary hypertension. Pulmonary conditions that affect the efficiency of respiration such as respiratory failure or respiratory distress syndrome may affect the circulation as well, thus causing circulatory problems such as thrombosis.
  • the severity of pulmonary conditions vary considerably between the patients. Some individuals suffering from a pulmonary condition may be symptomless, whereas some develop a severe, even life-threatening, form of the condition.
  • the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 has shown the importance of early detection of patients who are suffering from a severe form of the disease.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the early identification and effective treatment of the patients suffering from a severe condition is crucial for both the survival of the patients and sustainability of healthcare systems. In order to effectively target healthcare resources, it is essential to be able to identify the patiens who will be needing immediate enhanced care. Predicting the progression of the disease supports selecting an appropriate treatment and early initiation of the treatment which benefits the patient's recovery and survival.
  • Patients suffering from a severe form of a pulmonary condition are currently diagnosed based on mainly respiratory function tests and medical imaging. However, at the time of diagnosis the patients are already at the severe phase of the condition and need urgent enhanced care. Some patients develop symptoms only when significant lung damage has already occurred, and the symptoms usually become worse over time. There are currently no methods for predicting the progress of a pulmonary condition and identifying the patients who will develop a severe condition at an early stage. Therefore, in order to enhance the treatment and recovery of the patients, new methods are needed for early identification of the high-risk patients before the manifestation of severe symptoms.
  • the novel blood-based methods of the present disclosure enable early identification and treatment of the patients who are at high risk of developing a severe pulmonary condition and/or its complications.
  • the methods disclosed herein are more convenient and accessible than the currently used imaging and respiratory function methods which require specific equipment.
  • the biomarkers of the present disclosure offer superior performance for risk stratification of pulmonary patients. They also allow predicting and preventing the occurrence of serious complications of a pulmonary condition, including thrombosis.
  • novel pulmonary biomarkers determined from a biological sample have been identified.
  • the present disclosure is based on the finding that certain lipid molecules are expressed at higher levels and certain molecules are expressed at lower levels, when compared to a control, in biological samples, e.g., blood of subjects suffering from or having a risk to develop a severe pulmonary condition. Concentrations of these molecules and combinations derived thereof were found to be surprisingly superior predictive biomarkers of severe pulmonary conditions, as described herein.
  • the present disclosure provides novel biomarkers, biomarker combinations and associated diagnostic methods and uses for the identification and treatment of subjects suffering from or having a risk of developing a severe pulmonary condition. Such methods and uses comprise monitoring specific lipid concentrations and combinations of specific lipid concentrations in a sample from a subject and comparing such concentrations to those in a control.
  • the novel pulmonary biomarkers of the present disclosure are presented in Table 1. Table 1. Novel pulmonary biomarkers. Increased and decreased directions of change refer to a change in a quantity, amount, abundance, level or concentration of the biomarker in a sample from a subject when the subject is suffering from, or has a risk of developing, a severe pulmonary condition, when compared to a control. Abbreviations are described in the detailed description of the present disclosure. PE 35:2_sn2 PC 36:0 LPE 16:0_snl SM 44:1
  • a method for assessing the severity of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • the present disclosure is directed to a method for selecting an appropriate treatment for a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
  • the present disclosure is directed to a method for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for determining the risk of a subject to develop a severe pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for determining the risk of a subject to develop thrombosis, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • the present disclosure is directed to a method of evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
  • the method of evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in the subject may further comprise after the determining step, changing, supplementing, or keeping the same an already administered treatment in the subject based on the concentrations of the at least one biomarker obtained in the determining step.
  • the present disclosure is directed to a method of treating a pulmonary condition or preventing the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising administering to the subject a treatment, if the subject has been identified as having a risk of developing a severe pulmonary condition.
  • the method may further comprise determining the risk of the subject to develop a severe pulmonary condition according to the methods described herein prior to administering the treatment.
  • the method of treating a pulmonary condition or preventing the progress of a pulmonary condition in a subject to more severe form of the condition comprises administering to the subject a treatment, wherein the subject has been identified as having a risk of developing a severe pulmonary condition according to the methods described herein.
  • the present disclosure is directed to a method of treating a serious complication of a pulmonary condition or preventing the occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising administering to the subject a treatment, if the subject has been identified as needing a treatment.
  • the method may further comprise determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition according to the methods described herein prior to administering the treatment.
  • the method of treating a serious complication of a pulmonary condition or preventing the occurrence of a serious complication, or its progression, of a pulmonary condition in a subject comprises administering to the subject a treatment, wherein the subject has been identified as needing a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition according to the methods described herein.
  • the present disclosure is directed to a method of treating thrombosis or preventing thrombosis, the method comprising administering to the subject a treatment, if the subject has been identified as having a risk of developing thrombosis.
  • the method may further comprise determining the risk of the subject to develop thrombosis according to the methods described herein prior to administering the treatment.
  • the method of treating thrombosis or preventing thrombosis in a subject comprises administering to the subject a treatment, wherein the subject has been identified as having a risk of developing thrombosis according to the methods described herein.
  • a method for detecting at least one biomarker selected from Table 1 in a sample from a subject suffering from or suspected of suffering from a pulmonary condition, the method comprising assaying the sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • the method of detecting comprises detecting in a sample from a subject a changed concentration of the at least one biomarker selected from Table 1, in comparison to a control.
  • the method of detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1 comprises using a standard combination comprising at least one lipid selected from Table 1.
  • the standard combination comprises at least one isotope-labelled lipid selected from Table 1.
  • the isotope of the at least one isotope-labelled lipid selected from Table 1 is deuterium, 13 C or 15 N.
  • a method for obtaining or collecting data for assessing the severity of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • the present disclosure is directed to a method for obtaining or collecting data for selecting an appropriate treatment for a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
  • the present disclosure is directed to a method for obtaining or collecting data for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for obtaining or collecting data for determining the risk of a subject to develop a severe pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for obtaining or collecting data for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for obtaining or collecting data for determining the risk of a subject to develop thrombosis, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for obtaining or collecting data for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a method for obtaining or collecting data for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
  • a method for generating quantitative data for assessing the severity of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • the present disclosure is directed to a method for generating quantitative data for selecting an appropriate treatment for a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • the present disclosure is directed to a method for generating quantitative data for preventing the occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • a method for generating quantitative data for determining the risk of a subject to develop a severe pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • a method for generating quantitative data for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • a method for generating quantitative data for determining the risk of a subject to develop thrombosis, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • a method for generating quantitative data for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • a method for generating quantitative data for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
  • a pulmonary marker combination is provided for the methods of all aspects of the disclosure, the pulmonary marker combination comprising at least two biomarkers selected from Table 1.
  • the pulmonary marker combination comprises at least one biomarker ratio selected from Table 3. In some embodiments, the pulmonary marker combination comprises at least two biomarkers selected from Table 1 and at least one biomarker ratio selected from Table 3. In some embodiments, the pulmonary marker combination comprises any of the scores selected from Tables 4-10. In certain embodiments, the pulmonary marker combination may be e.g. a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1. In some embodiments, the combination may be calculated from the concentrations of the at least two biomarkers selected from Table 1.
  • the pulmonary marker combination may be a concentration ratio of the at least two biomarkers selected from Table 1.
  • the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
  • the pulmonary marker combination further comprises other biomarkers, including, but not limited to, CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof.
  • the pulmonary marker combination further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
  • the marker combination may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
  • the marker combination is a continuous or discrete scoring systems based on the biomarkers selected from Table 1.
  • the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
  • a use of a pulmonary marker combination for assessing the severity of a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a pulmonary marker combination for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
  • a use of a pulmonary marker combination for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a pulmonary marker combination for determining the risk of a subject to develop a severe pulmonary condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a pulmonary marker combination for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a pulmonary marker combination for determining the risk of a subject to develop thrombosis, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a pulmonary marker combination for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a pulmonary marker combination for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
  • a standard combination for the methods of all aspects of the disclosure, wherein the standard combination comprises at least two lipids selected from Table 1.
  • the standard combination comprises 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 11, or at least 12, or more lipids selected from Table 1.
  • the standard combination comprises at least one lipid selected from the lipid ratios of Table 3.
  • the standard combination comprises at least two lipids selected from Table 1 and at least one lipid selected from the lipid ratios of Table 3.
  • the standard combination comprises at least one component of the scores selected from Tables 4-10.
  • the standard combination comprises at least one isotope-labelled lipid selected from Table 1.
  • the isotope of the at least one isotope-labelled lipid selected from Table 1 is deuterium, 13 C or 15 N.
  • a standard combination for assaying a concentration of at least one biomarker selected from Table 1 in a sample from a subject in a preparation of a reagent, kit or composition for the methods of all aspects of the disclosure, wherein the standard combination comprises at least two lipids selected from Table 1.
  • a use of a standard combination for detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1, wherein the standard combination comprises at least two lipids selected from Table 1.
  • a use of a standard combination for assessing the severity of a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a standard combination for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
  • a use of a standard combination for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a standard combination for determining the risk of a subject to develop a severe pulmonary condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a standard combination for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a standard combination for determining the risk of a subject to develop thrombosis, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a standard combination for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
  • a use of a standard combination for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for assessing the severity of a pulmonary condition of a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for selecting an appropriate treatment for a pulmonary condition of a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for determining the risk of a subject to develop a severe pulmonary condition, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for determining the risk of a subject to develop thrombosis, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
  • a use of one or more reagent in a preparation of a reagent, kit or composition for detecting at least one biomarker selected from Table 1 in a sample from a subject, wherein the use comprises assaying the sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
  • the use of one or more reagent in a preparation of a reagent, kit or composition for detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1 comprises using a standard combination comprising at least two lipids selected from Table 1.
  • the standard combination comprises at least two isotope-labelled lipids selected from Table 1.
  • the isotopes of the at least two isotope-labelled lipids selected from Table 1 are deuteriums, 13 C or 15 N.
  • the reagent is used for assaying a sample to determine a concentration of at least one biomarker selected from Table 1.
  • the reagent comprises any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s), preparation(s), or any combination thereof used for the methods and uses of the present disclosure.
  • the reagent is a combination or mixture of any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s) and preparation(s) used in the methods and uses of the present disclosure.
  • one or more of any components of a reagent is isotope-labelled.
  • the isotope of the isotope-labelled component is deuterium, 13 C or 15 N.
  • the reagent comprises at least one, 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 11, at least 12, or more lipids selected from Table 1. In certain embodiments of all aspects of the disclosure, the reagent comprises at least one, 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 11, at least 12, or more isotope-labelled lipids selected from Table 1.
  • the isotope-labelled lipids of the reagent are selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(d 16: 1/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dll8:0/16:0), Cer(d
  • PC 39:5 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
  • the isotope-labelled lipid(s) of the reagent is (are) d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7-Cer(dl8:l/24:l), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
  • the reagent is used in a preparation of a reagent, kit or composition which are used for performing the methods and uses of the present disclosure.
  • a composition or kit is provided for performing any of the methods or uses according to the present disclosure, wherein the composition or kit comprises at least one biomarker selected from Table 1.
  • the at least one biomarker is 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 11, at least 12, or more biomarkers selected from Table 1.
  • the composition or kit comprises at least one biomarker ratio selected from Table 3.
  • the composition or kit comprises at least one biomarker selected from Table 1 and at least one biomarker ratio selected from Table 3.
  • the composition or kit comprises at least one component of the scores selected from Tables 4-10.
  • the composition or kit comprises at least one isotope-labelled biomarker selected from Table 1.
  • the isotope of the at least one isotope-labelled biomarker is deuterium, 13 C or 15 N.
  • the at least one isotope-labelled biomarker is 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 11, at least 12, or more isotope-labelled biomarkers selected from Table 1.
  • the isotope of the at least one, 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 11, at least 12, or more isotope-labelled biomarkers is/are deuterium, 13 C or 15 N.
  • the composition or kit comprises at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 isotopelabelled lipids selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(d 16: 1/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0
  • the isotope-labelled lipid(s) of the composition or kit is (are) d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7- Cer(dl8:l/24:1), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
  • composition or kit may further include standard(s), control(s), reagent(s), solution(s), solvent(s), container(s), use instruction(s) for the methods or uses disclosed herein, and/or other element(s) for performing the methods or uses disclosed herein.
  • the composition or kit includes element(s) for collecting a blood sample, for example, a dried blood spot on a filter.
  • the composition or kit includes element(s) for collecting plasma or serum samples.
  • plasma or serum samples are collected on a filter card that separates the plasma or serum from a blood sample.
  • plasma or serum sample is dried on a card or filter.
  • the composition or kit may be a test kit for use in a laboratory or a home use test kit (over-the-counter test).
  • the composition or kit may be a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), either purchased from a commercial manufacturer or prepared in-house in a laboratory, used for performing any of the methods or uses according to the present disclosure.
  • the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), either purchased from a commercial manufacturer or prepared in-house in a laboratory, used for assaying a concentration of at least one biomarker selected from Table 1.
  • composition or kit may be used in assays performed with various chemical and high-resolution analytical techniques, as appropriate.
  • Suitable analytical techniques include, but are not limited to, mass spectrometry (MS) and nuclear magnetic resonance (NMR). Any high-resolution technique capable of resolving individual biomarkers can be used to collect the information on the biomarker in question, such as the concentration of a biomarker profile from the biological sample. Typically, the information is collected using mass spectrometry.
  • the MS analysis can be coupled to another high performance separation method, such as gas chromatography (GC), two- dimensional gas chromatography (GCxGC), liquid chromatography (LC), two-dimensional liquid chromatography (LCxLC), high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
  • GC gas chromatography
  • GCxGC two- dimensional gas chromatography
  • LC liquid chromatography
  • LCxLC two-dimensional liquid chromatography
  • HPLC high performance liquid chromatography
  • UHPLC ultra-high performance liquid chromatography
  • the at least one biomarker selected from Table 1 is 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 11, at least 12, or more biomarkers selected from Table 1.
  • the at least one biomarker is at least one biomarker ratio selected from Table 3. In certain embodiments of all aspects of the present disclosure, the at least one biomarker ratio is 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 11, at least 12, or more biomarker ratios selected from Table 3.
  • the at least one biomarker is any combination of at least one biomarker selected from Table 1 and/or at least one biomarker ratio selected from Table 3. In certain embodiments, the at least one biomarker is any of the scores selected from Tables 4-10 and/or any component of the scores selected from Tables 4-10. In certain embodiments, the at least one biomarker is any combination of the biomarkers selected from Tables 1-10.
  • the at least one biomarker may be e.g. a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1.
  • the biomarker may be calculated from the concentrations of the at least two biomarkers selected from Table
  • the biomarker may be a concentration ratio of the at least two biomarkers selected from Table 1.
  • the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
  • the at least one biomarker further comprises other biomarkers, including, but not limited to, CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof.
  • the at least one biomarker further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
  • the at least one biomarker may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
  • the at least one biomarker is a continuous or discrete scoring systems based on the biomarkers selected from Table 1.
  • the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
  • the present disclosure provides methods of treatment, the methods comprising administering a treatment to a subject, wherein the subject has been identified as being in need of the treatment according to the methods described herein.
  • the methods and uses further comprise administering a treatment to the subject.
  • a treatment may comprise any therapeutic treatment typically given to a subject suffering from a pulmonary condition and/or needing immediate or intensive care.
  • a treatment may be, e.g., hospitalization, intensive care, life support care, noninvasive or invasive medical care, monitoring and/or supporting bodily functions, administering a drug, administering supplemental oxygen (e.g.
  • a drug may comprise any pharmaceutical typically given to a subject suffering from a pulmonary condition and/or its symptoms.
  • a drug may be, e.g., an anti-inflammatory agent, antibiotic, antiviral drug, corticosteroid, analgesic, sedative, antithrombotic, anticoagulant or antiplatelet drug.
  • a drug may be, e.g., a functional inhibitor of acid sphingomyelinase (FIASMA), molnupiravir, dexamethasone, a ceramide synthase or an inhibitor of a ceramide synthase.
  • FIASMA acid sphingomyelinase
  • FIASMA acid sphingomyelinase
  • molnupiravir molnupiravir
  • dexamethasone a ceramide synthase or an inhibitor of a ceramide synthase.
  • the methods of treating or preventing a pulmonary condition, its serious complications or thrombosis, of a subject further comprise identifying the subject as in need of the treatment or prevention, for example, by requesting a test or receiving the test results, for example, from a commercial laboratory, which provides the results of an assay useful for determining the concentration of the at least one biomarker selected from Table 1, and administering to the subject a treatment, for example, a therapeutically effective dose of a drug, if the subject has an increased or decresed concentration of the at least one biomarker selected from Table 1 in the sample, as compared to the control.
  • the methods and uses further comprise a step of adding at least one isotope-labelled biomarker selected from Table 1 to the sample prior to assaying the concentration of the at least one biomarker selected from Table 1.
  • the at least one isotopelabelled biomarker may be, but is not limited to, deuterium-labelled biomarker selected from Table 1.
  • the at least one isotope-labelled biomarker may also be 13 C-labelled or 15 N-labelled.
  • the methods and uses further comprise a step of adding at least one, 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 11, at least 12, or more isotope-labelled biomarkers selected from Table 1 to the sample.
  • the at least one, 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 11, at least 12, or more isotope-labelled biomarkers may be, but are not limited to, deuterium-labelled, Relabelled or 15 N-labelled biomarkers selected from Table 1.
  • the isotope-labelled biomarker is selected from the following isotope-labelled lipids: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(d 18: 1/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16::0), Cer(dl8:l/
  • PC 39:0 PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
  • the isotope-labelled biomarker(s) is (are) d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7-Cer(dl8:l/24:l), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
  • the determination of a concentration of at least one biomarker selected from Table 1 is typically performed using an assay.
  • such assay is, or involves, mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy or dual polarisation interferometry, a high performance separation method such as liquid chromatography (LC), gas chromatography (GC), two-dimensional liquid chromatography (LCxLC), two-dimensional gas chromatography (GCxGC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC) or ultra performance liquid chromatography (UPLC), an immunoassay such as an ELISA and/or an assay with a binding moiety capable of specifically binding the analyte.
  • LC liquid chromatography
  • GC gas chromatography
  • LCxLC two-dimensional liquid chromatography
  • GCxGC two-dimensional gas chromatography
  • HPLC high performance liquid chromatography
  • UHPLC ultra high performance liquid chromatography
  • UPLC ultra performance liquid chromatography
  • the assay is mass spectrometry (MS).
  • MS instrument can be coupled to a direct sample infusion method, such as a robotic nanoflow ion source device, or to a high performance separation method such as HPLC, UHPLC, or UPLC.
  • the concentrations of the 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 11, at least 12, or more biomarkers selected from Table 1 are determined by one assay.
  • the one assay is performed with mass spectrometry (MS).
  • the method or use further comprises determining other biomarkers, including, but not limited to, C-reactive protein (CRP), high sensitivity CRP (hs-CRP), a cytokine, troponin T (TNT or TnT), troponin I (TNI or Tnl), B-type natriuretic peptide (BNP), pro B-type natriuretic peptide (proBNP), N-terminal pro B-type natriuretic peptide (NT-proBNP), cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof.
  • CRP C-reactive protein
  • hs-CRP high sensitivity CRP
  • a cytokine a cytokine
  • TNT or TnT troponin T
  • TnI troponin I
  • BNP B-type natriuretic peptide
  • proBNP pro B-type natriuretic peptide
  • the method or use further comprises using other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
  • other personal information or health data such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
  • a concentration ratio is calculated from the concentrations of the 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 11, at least 12, or more biomarkers selected from Table 1.
  • a concentration ratio is calculated from at least 2 of the following lipids: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(d 18: 1/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl6
  • a concentration ratio is selected from any of the lipid ratios referred to in Table 3.
  • the concentration ratio is a Cer/Cer, Cer/PC, PC/Cer and/or PC/PC ratio, e.g., Cer(dl8:l/16:0)/PC 16:0/22:5, Cer(dl8:l/18:0)/PC 14:0/22:6, Cer(dl8:l/24:l)/Cer(dl8:l/24:0), Cer(dl8:l/18:0)/PC 36:6, Cer(dl8:l/16:0)/PC 38:5,
  • the marker combination may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
  • the marker combination is a continuous or discrete scoring systems based on the Cer, PC, Cer/PC, Cer/Cer, PC/Cer, PC/PC or other pulmonary biomarkers.
  • the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
  • the marker combination is a scoring system (for example, but not limited to, points 0-15, 0-12, 0-9 or 0-6), where the points are given based on the quartiles (Q.1-Q.4) of a study population. For example, if a lipid biomarker of a person belongs to the highest quartile, the person will receive 3 points. The same evaluation will be performed for the other components of the score, and the points are then summed up to create the score.
  • a scoring system for example, but not limited to, points 0-15, 0-12, 0-9 or 0-6
  • the points are given based on the quartiles (Q.1-Q.4) of a study population. For example, if a lipid biomarker of a person belongs to the highest quartile, the person will receive 3 points. The same evaluation will be performed for the other components of the score, and the points are then summed up to create the score.
  • the score is based on 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 11, at least 12, or more lipids selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(d 16: 1/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0),
  • PC 39:5 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
  • the score is constructed according to Tables 4-10.
  • the pulmonary condition is caused by severe acute respiratory syndrome coronavirus 2 (SARS-Co-2).
  • SARS-Co-2 severe acute respiratory syndrome coronavirus 2
  • ARDS acute respiratory distress syndrome
  • BMI body mass index
  • BNP B-type natriuretic peptide
  • 13 c carbon-13 isotope
  • CAD coronary artery disease
  • CE cholesterylester
  • Cer ceramide
  • COVID-19 coronavirus disease 2019
  • CRP C-reactive protein
  • DAG diacylglycerol
  • ELISA enzyme-linked immunosorbent assay
  • FA is fatty acid
  • Gb3 is globotriasoylceramide
  • GC is gas chromatography
  • GCxGC two- dimensional gas chromatography
  • 2 H is deuterium
  • HPLC high performance liquid chromatography
  • HR is hazard ratio
  • LacCer lactosylceramide
  • LC liquid chromatography
  • FIASMA is a functional inhibitor of acid sphingomyelinase.
  • a pulmonary condition may be an acute or chrocic condition.
  • a pulmonary condition may be, e.g., a respiratory/pulmonary/lung infection, respiratory/pulmonary/lung inflammation, respiratory/pulmonary/lung damage, injury or distress, pneumonia, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, emphysema, bronchitis, pulmonary edema, asthma, lung cancer, pulmonary embolism or pulmonary hypertension.
  • a pulmonary condition may be caused by, e.g., an infection, inflammation, apoptosis, smoking, secondhand smoking, air pollution, exposure to dust, smoke or fumes, and/or genetic factors.
  • a respiratory infection or inflammation may be caused by, e.g., a pathogen, smoking, secondhand smoking, air pollution, exposure to dust, smoke or fumes and/or genetic factors.
  • a "severe pulmonary condition” and a “serious pulmonary condition” are used interchangeably herein to refer to any clinically severe, critical or life-threatening pulmonary condition which may require immediate enhanced care, hospitalization or care in the intensive care unit.
  • a severe pulmonary condition may be, e.g., pneumonia, pulmonary fibrosis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary embolism or lung cancer.
  • the condition may or may not be acute or the condition may or may not be chronic.
  • a “complication”, “sequela”, “symptom”, “consequence”, “outcome” and “adverse event” are used interchangeably herein to refer to any symptom caused by a pulmonary condition.
  • the severity of the complications vary significantly from mild to severe, life-threatening, symptoms.
  • a complication may be, e.g., cough, sore throat, shortness of breath, difficulty breathing, chest pain, pain when breathing, pain in the airways, chest pressure or tightness, fever, chills, headache, tiredness, nasal congestion, runny nose, muscle and joint pain, rash, nausea, diarrhea, vomiting, loss of taste or smell, eye problems, mucus production, expectoration, wheezing, gastrointestinal symptoms, oxygen deficiency, pneumonia, pulmonary fibrosis, thrombosis, pulmonary embolism, hypertension, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g.
  • ARDS acute respiratory distress syndrome
  • a "serious complication”, “serious sequela”, “serious symptom”, “serious consequence”, “serious outcome”, “serious adverse event”, “severe complication”, “severe sequela”, “severe symptom”, “severe consequence”, “severe outcome” and “severe adverse event” are used interchangeably herein to refer to any clinically severe, critical or life-threatening symptom of a pulmonary condition.
  • a serious complication may require immediate enhanced care or treatment, hospitalization or care in the intensive care unit.
  • a serious complication may be, e.g.
  • pulmonary fibrosis e.g. pulmonary fibrosis, thrombosis, pulmonary embolism, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g. acute kidney failure), sepsis, acute or chronic cardiovascular damage (e.g. myocardial injury, myocarditis, arrhytmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure), seizure, clinical deterioration or death.
  • ARDS acute respiratory distress syndrome
  • cardiovascular damage e.g. myocardial injury, myocarditis, arrhytmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure
  • seizure e.g. myocardial injury, myocarditis, arrhytmias, cardiomyopathy, acute coronary syndrome, myocardi
  • An underlying medical condition may be, e.g., diabetes, a cardiovascular disease (e.g. heart failure, atherosclerosis, coronary artery disease, cardiomyopathy or hypertension), severe obesity, kidney disease, liver disease, sickle cell disease, weakened immune system, lung disease or neurological condition.
  • a cardiovascular disease e.g. heart failure, atherosclerosis, coronary artery disease, cardiomyopathy or hypertension
  • severe obesity kidney disease, liver disease, sickle cell disease, weakened immune system, lung disease or neurological condition.
  • thrombosis refers to formation of a blood clot, a thrombus, within a blood vessel.
  • the blood clot prevents blood from flowing normally through the circulatory system.
  • Thrombosis may occur in veins (venous thrombosis) or in arteries (arterial thrombosis).
  • Venous thrombosis leads to congestion of the affected part of the body, while arterial thrombosis affects the blood supply and leads to damage of the tissue supplied by that artery.
  • a thrombus in the lungs either formed in the lungs, or originating from another part of the body, may cause a severe pulmonary condition when blocking the blood flow in the lungs.
  • Pulmonary embolism refers to a blood clot that has travelled to the lungs. Pulmonary embolism is a severe pulmonary condition that can block the blood flow to the lungs, causing serious damage to the lungs and affecting a subject's ability to breath. "Deep vein thrombosis” occurs when a blood clot forms in a major vein, e.g. in the leg. Pulmonary conditions that affect the efficiency of respiration may also affect the circulation and cause circulatory problems such as thrombosis.
  • a “treatment” and “therapy” are used interchangeably in the present disclosure and may comprise any therapeutic treatment typically given to a subject suffering from a pulmonary condition and/or needing immediate or intensive care.
  • a treatment may be, e.g., hospitalization, intensive care, life support care, noninvasive or invasive medical care, monitoring and/or supporting bodily functions, administering a drug, administering supplemental oxygen (e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation), extracorporeal membrane oxygenation, a surgical procedure or lung transplantation.
  • supplemental oxygen e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation
  • extracorporeal membrane oxygenation e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation
  • a “drug”, “pharmaceutical”, “medicament”, “medicine” and “medication” are used interchangeably in the present disclosure and may comprise any pharmaceutical typically given to a subject suffering from a pulmonary condition and/or its symptoms.
  • a drug may be, e.g., an anti-inflammatory agent, antibiotic, antiviral drug, corticosteroid, analgesic, sedative, antithrombotic, anticoagulant or antiplatelet drug.
  • a drug may be, e.g., a functional inhibitor of acid sphingomyelinase (FIASMA), molnupiravir, dexamethasone, a ceramide synthase or an inhibitor of a ceramide synthase.
  • FIASMA acid sphingomyelinase
  • molnupiravir molnupiravir
  • dexamethasone a ceramide synthase or an inhibitor of a ceramide synthase.
  • pathogen is an infectious microorganism or agent that causes disease or illness to its host.
  • a pathogen may be a virus, bacterium, protozoan, prion, viroid, fungus or any other eukaryotic or prokaryotic invader of host.
  • a “virus” may be, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D, any subtype or variant of type A, B, C or D influenza virus or any other virus capable of infecting mammalian subjects.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV middle east respiratory syndrome coronavirus
  • any other coronavirus or a subtype or variant of a coronavirus any influenza virus A, B, C or D, any subtype or variant of type A, B, C or D influenza virus or any other virus capable of infecting mammalian subjects.
  • the terms "subject”, “patient” and “individual” are used interchangeably herein to refer to any mammalian subject for whom prediction, diagnosis or therapy is desired, particularly humans.
  • the subject may be a healthy individual with no previous signs or symptoms of a pulmonary condition. Or, the subject may have previously suffered or is suffering from a pulmonary condition.
  • the subject may also be suspected of suffering from a pulmonary condition and/or being at high risk of developing a severe pulmonary condition and/or its serious complications.
  • the subject may also be suffering from a pulmonary condition and is at high risk of progressing to a more severe form of the condition.
  • the subject may or may not have previously suffered from a pulmonary condition.
  • the subject may be a healthy individual with no previous signs or symptoms of thrombosis. Or, the subject may have previously suffered or is suffering from thrombosis. The subject may also be suspected of suffering from thrombosis and/or being at high risk of developing thrombosis.
  • the subject may or may not be infected by a virus or the subject may or may not have previously been infected by a virus.
  • the subject may or may not be infected by SARS-CoV-2, SARS-CoV, MERS-CoV, any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D or any subtype or variant of type A, B, C or D influenza virus.
  • the subject may or may not have previously been infected by SARS-CoV-2, SARS-CoV, MERS-CoV, any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D, or any subtype or variant of type A, B, C or D influenza virus.
  • the subject may or may not be suffering from coronavirus disease (COVID-19) or the subject may or may not have previously suffered from COVID-19.
  • COVID-19 coronavirus disease
  • the subject may or may not be hospitalized.
  • the subject may or may not be under a treatment in a hospital.
  • the subject may or may not be hospitalized and/or under a treatment due to a pulmonary condition.
  • the subject may or may not be hospitalized and/or under a treatment due to a viral infection.
  • the subject may or may not be hospitalized and/or under a treatment due to COVID-19.
  • the subject may or may not be apparently healthy.
  • the subject may or may not suffer an acute or chronic medical condition(s).
  • the subject may or may not have a diagnosed or undiagnosed underlying medical condition(s).
  • the subject may or may not be under a treatment for an underlying medical condition(s).
  • the subject may or may not suffer from a cardiovascular or metabolic disease, such as atherosclerosis, CAD, diabetes and fatty liver disease.
  • a cardiovascular or metabolic disease such as atherosclerosis, CAD, diabetes and fatty liver disease.
  • the subject may or may not have normal levels of other pulmonary biomarkers, and/or may or may not have any traditional risk factors of pulmonary conditions.
  • the subject may be or may have been under a treatment or medication, or may not have had any previous treatment or medication.
  • a sample is a biological sample obtained from a subject or a group or population of subjects.
  • the sample may be a blood sample, a serum sample, a plasma sample, a saliva sample, a urine sample, a tissue sample or a fraction thereof, such as a lipoprotein fraction.
  • Blood serum and plasma samples are typical.
  • the sample can be prepared with techniques well known in the art.
  • the blood sample is a dried blood spot.
  • the blood sample is dried on a filter.
  • the blood sample is dried on a filter card which separates plasma or serum from the blood sample.
  • the sample is a dried plasma or serum sample.
  • a "control" may be a control sample.
  • a control may also be a concentration determined from a sample from a single healthy individual.
  • the control may also be a sample that represents a combination of samples from a generalized population of healthy individuals.
  • the control may be a control value, a score, a threshold, a limit, a limit value or a set of data concerning the biomarker in a sample previously determined, calculated or extrapolated, or may have yet to be determined, calculated or extrapolated, or may also be taken from the literature.
  • the control sample may be previously obtained from the same subject or the control value may be obtained from a previously taken sample from the same subject.
  • the control sample or the control value may be obtained from another subject.
  • a “control” as used herein, i.e., a control value or a control sample, is typically representative of a group of subjects or a population of subjects.
  • "representative” means that the lipid concentration(s) reflected by said control value to which a comparison is made in the context of the present disclosure correspond(s) to the average concentration value(s) of said lipid concentration(s) in corresponding individual samples from the subjects of said group or population.
  • a control sample representsative means that the lipid concentration(s) in said control sample to which a comparison is made in the context of the present disclosure correspond(s) to the average concentration(s) of said lipid concentration(s) in corresponding individual samples from the subjects of said group or population.
  • the concentrations of all lipid concentrations in said control sample correspond to the average concentrations of said lipid concentrations in corresponding individual samples from the subjects of said group or population.
  • An individual with such values can be considered a "healthy individual" for the purposes of the present disclosure.
  • a control sample can be particularly suitably compared to the subject's sample if it has been obtained from the same type of biological tissue or source in the same, or essentially the same, manner.
  • the subject's sample is a serum sample or a plasma sample
  • a corresponding control sample will likewise be a serum sample or a plasma sample, respectively.
  • a control sample from a group of subjects or a control sample from a population of subjects in the sense of the present disclosure is obtained by mixing equal amounts of samples directly obtained from the subjects of said group or population, or by mixing equal amounts of fractions, constituents or reaction products thereof.
  • a useful control value for the purposes of the present disclosure is typically one that has been, or is, obtained using any one of the suitable control samples described herein.
  • a control value or a control sample can be from a healthy individual, a generalized population of healthy individuals, a pulmonary patient that has remained free of any severe condition or serious complication, or a group of pulmonary patients that have remained free of any severe condition or serious complication.
  • a control value or a control sample may be obtained from a symptomless pulmonary patient or a pulmonary patient who has mild symptoms, or a group of symptomless pulmonary patients or a group of pulmonary patients who have mild symptoms.
  • a control value or a control sample may also be obtained from a symptomless COVID-19 patient or a COVID-19 patient who has mild symptoms, or a group of symptomless COVID-19 patients or a group of COVID-19 patients who have mild symptoms.
  • a control may be a control value, e.g., a score, a threshold or a combination value of the concentrations of the biomarkers selected from Table 1.
  • a control may be a control value, e.g., a score, a threshold or or a combination value of the biomarker ratios selected from Table 3.
  • a control may be a control value, e.g., a score, a threshold or a combination value of the concentrations of the at least one biomarker selected from Table 1 and the at least one biomarker ratio selected from Table 3.
  • a control may be a control value, e.g., a score, a threshold or a combination value obtained from the scores selected from Tables 4-10.
  • the increased concentration of the at least one biomarker is 0.01 - 10000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the increased concentration of the at least one biomarker is 1 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the increased concentration of the at least one biomarker is 10 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some further embodiments, the increased concentration of the at least one biomarker is 25 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
  • the decreased concentration of the at least one biomarker is 0.01 - 10 000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the decreased concentration of the at least one biomarker is 1 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the decreased concentration of the at least one biomarker is 10 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some further embodiments, the decreased concentration of the at least one biomarker is 25 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
  • lipids are named according to the following nomenclature:
  • ceramides Cer
  • lactocylceramides LacCer
  • globotriasoylceramides Gb3
  • SB sphingoid base
  • FA fatty acid
  • phosphatidylcholines is presented either as brutto species or molecular species.
  • a first number refers to the total number of carbon atoms in the FA chains of the molecule and the second number refers to the total number of the carbon-carbon double bonds of in the FA chains of the molecule.
  • molecular nomenclature a first pair of numbers corresponds to the first FA chain and a second pair of numbers corresponds to the second FA chain of the molecule.
  • the first number of each pair refers to the number of carbon atoms in the FA chain
  • the second number refers to the number of carbon-carbon double bonds of the FA chain.
  • the PC may be a brutto PC or a corresponding molecular PC.
  • lysophosphatidylcholines LPC
  • lysophosphatidylethanolamines LPE
  • lysophosphatidylinositols LPI
  • lysoalkylphosphatidylcholines LPC O-
  • lysoalkenylphosphatidylcholine LPC P-
  • lysoalkenylphosphatidylethanolamines LPE P-
  • acylcarnitines is presented with a number corresponding to the FA chain of the molecule.
  • the first number refers to the number of carbon atoms in the FA chain
  • the second number refers to the number of carbon-carbon double bonds of the FA chain.
  • phosphatidylethanolamines PE
  • phosphatidylglycerols PG
  • phosphatidylinositols PI
  • alkyl-linked phosphatidylcholines PC O-
  • alkenyl-linked phosphatidylcholines PC P-
  • alkyl-linked phosphatidylethanolamines PE O-
  • alkenyl-linked phosphatidylethanolamines PE P-
  • DAG diacylglycerols
  • SM sphingomyelins
  • CE cholesterolesters
  • TAG triacylglycerols
  • snl and sn2 indicate thesnl and sn2 positions of the glycerol backbone, respectively, to which the fatty acid moiety is attached.
  • MHDA methylhexadecanoic acid
  • a "pulmonary marker combination” refers to a specific combination of lipid biomarkers used for the methods and uses of the present disclosure.
  • the pulmonary marker combination comprises at least two biomarkers selected from Table 1.
  • the pulmonary marker combination comprises at least one biomarker ratio.
  • the biomarker ratio is selected from Table 3.
  • the pulmonary marker combination comprises at least two biomarkers selected from Table 1 and at least one biomarker ratio, and optionally the biomarker ratio is selected from Table 3.
  • the pulmonary marker combination comprises a score. In some embodiments, the score is selected from Tables 4-10.
  • the pulmonary marker combination may be e.g.
  • the combination may be calculated from the concentrations of the at least two biomarkers selected from Table 1.
  • the pulmonary marker combination may be a concentration ratio of the at least two biomarkers selected from Table 1.
  • the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
  • the pulmonary marker combination further comprises other biomarkers, including, but not limited to, CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof.
  • the pulmonary marker combination further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
  • the marker combination may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
  • the marker combination is a continuous or discrete scoring systems based on the biomarkers selected from Table 1.
  • the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
  • pulmonary marker combination pulmonary biomarker combination, marker combination, biomarker combination, lipid marker combination, lipid combination and combination may be used interchangeable.
  • cytokine refers to one or more of any interleukin (IL), interferon (INF), tumor negrosis factor (TNF), transforming growth factor (TGF), chemokine, lymphokine and/or monokine.
  • An interleukin may be, but is not limited to, IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 or any IL from IL19 to IL36.
  • an interferon may be, but is not limited to, an INF type I, such as INF-a and INF-P, INF type II, such as INF-y, or INF type III.
  • a tumor negrosis factor may be, but is not limited to, TNF-a or TNF-p.
  • a "standard combination" refers to a specific combination of lipid standards used for the methods and uses of the present disclosure.
  • the standard combination comprises at least two lipids selected from Table 1.
  • the standard combination comprises at least one lipid selected from the lipid ratios of Table 3.
  • the standard combination comprises at least two lipids selected from Table 1 and at least one lipid selected from the lipid ratios of Table 3.
  • the standard combination comprises at least one component of the scores selected from Tables 4-10. In some embodiments, one or more of the components of the standard combination is isotope-labelled. In some embodiments, the isotope of the isotope-labelled component is deuterium ( 2 H). In some embodiments, the isotope of the isotope-labelled component is 13 C or 15 N.
  • compositions or kit may be any combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for the methods and uses of the present disclosure.
  • the composition or kit may comprise at least one at least one biomarker selected from Table 1.
  • the at least one biomarker is 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 11, at least 12, or more biomarkers selected from Table 1.
  • the composition or kit comprises at least one biomarker ratio selected from Table 3.
  • the composition or kit comprises at least one biomarker selected from Table 1 and at least one biomarker ratio selected from Table 3.
  • the composition or kit comprises at least one component of the scores selected from Tables 4-10.
  • one or more of the components of the composition or kit is isotope-labelled.
  • the isotope of the isotope-labelled component is deuterium.
  • the isotope of the isotope-labelled component is 13 C or 15 N.
  • composition or kit may further include standard(s), control(s), reagent(s), solution(s), solvent(s), container(s), use instruction(s) for the methods or uses disclosed herein, and/or other element(s) for performing the methods or uses disclosed herein.
  • the composition or kit includes element(s) for collecting a blood sample, for example, a dried blood spot on a filter.
  • the composition or kit includes element(s) for collecting a plasma or serum sample.
  • the element separates plasma or serum from a blood sample.
  • the plasma or serum sample is dried on a filter card.
  • the composition or kit may be purchased from a commercial manufacturer or prepared in-house in a laboratory.
  • the composition or kit may be a test kit for used in a laboratory or a home use test kit (over- the-counter test).
  • the composition or kit may be a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), either purchased from a commercial manufacturer or prepared in-house in a laboratory, used for performing any of the methods or uses according to the present disclosure.
  • the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for assaying a concentration of at least one biomarker selected from Table 1.
  • the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for assaying at least one biomarker ratio selected from Table 3. In certain embodiments, the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for assaying any of the scores selected from Tables 4-10.
  • composition or kit may be used in assays performed with various chemical and high-resolution analytical techniques, such as, but not limited to, mass spectrometry (MS) and nuclear magnetic resonance (NMR).
  • MS analysis can be coupled to another high performance separation method, such as gas chromatography (GC), two-dimensional gas chromatography (GCxGC), liquid chromatography (LC), two-dimensional liquid chromatography (LCxLC), high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
  • GC gas chromatography
  • GCxGC gas chromatography
  • LC liquid chromatography
  • HPLC high performance liquid chromatography
  • UHPLC ultra-high performance liquid chromatography
  • a "reagent” may be any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s), preparation(s), or any combination thereof used for the methods and uses of the present disclosure.
  • a reagent may also be a combination or mixture of any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s) and preparation(s) used in the methods and uses of the present disclosure.
  • one or more of any components of a reagent is isotope-labelled.
  • the isotope of the isotope-labelled component is deuterium.
  • the isotope of the isotope-labelled component is 13 C or 15 N.
  • a reagent is used for assaying in a sample a concentration of at least one biomarker selected from Table 1. In some embodiments, a reagent is used for assaying in a sample at least one biomarker ratio selected from Table 3. In some embodiments, a reagent is used for assaying in a sample any of the scores selected from Tables 4-10.
  • a reagent is used in a preparation of a reagent, kit or composition for performing the methods and uses of the present disclosure.
  • obtaining data For the purposes of the present disclosure, the terms “obtaining data”, “collecting data”, “obtaining information” and “collecting information” may be used interchangeably.
  • sample refers to a measurement of a quantity, amount, abundance, level or concentration of the biomarkers of the present disclosure in a sample using a laboratory apparatus, equipment or device, such as, but not limited to, mass spectrometry.
  • a measurement may be quantitative or relative determination of an amount of a biomarker in a sample. For quantitative determination, either the absolute or precise amount of the biomarker in a sample is determined. The relative amount or level of a biomarker in a sample, may alternatively be determined, e.g., the biomarker amount in the sample is determined to be increased or decreased with respect to a control as described herein.
  • biomarker As used herein, a “biomarker”, “marker”, “pulmonary marker”, “pulmonary biomarker”, “lipid marker”, “lipid biomarker” and “lipid” may be used interchangeably.
  • an "increased or decreased" direction of change refers to a change in a quantity, amount, abundance, level or concentration of the biomarker in a sample from a subject when the subject is suffering from or is having a risk of developing a pulmonary condition or thrombosis, when compared to a control.
  • a "risk” refers to a low, intermediate or high risk of developing a pulmonary condition or thrombosis.
  • Serum samples were obtained from 39 adult subjects who were suffering from COVID-19, 9 subjects suffered from a severe form of COVID-19 and 30 non-severe form of COVID-19.
  • Ceramide and phospholipid quantification from serum samples was performed using a validated high- throughput mass spectrometry approach. 10 pL of serum or plasma was spiked with deuterated internal standards, D7-Cer(dl8:l/16:0), D7-Cer(dl8:l/18:0), D7-Cer(dl8:l/24:0), D7-Cer(dl8:l/24:1), D9- PC((14:0/22:6), D9-PC(16:0/16:0) and D9-PC(16:0/22:5) and extracted in isopropanokethyl acetate (8:2, v/v) using a Hamilton MICROLAB® STAR robot.
  • LC gradient was used: 0.5 min at 80% B, linear increase of B from 80% to 84% in 0.5 min, 84% to 89% B in 1.4 min, 89% to 100% in O.lmin, 2.0 min at 100% B, 100% to 80% B in 0.1 min and 0.4 min equilibration at 80% prior to the next injection.
  • Flow rate was 500 pl/min and column temperature 60°C.
  • the individual ceramides and phospholipids were quantified in MRM mode. Quantification was assessed through calibration line samples comprising of known amounts of synthetic Cer(dl8:l/16:0), Cer(d 18: 1/18:0), Cer(dl8:l/24:0), Cer(dl8:l/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) and corresponding deuterated standards. The peak area ratios of each ceramide to its corresponding deuterated form were calculated and plotted against the added ceramide concentration and fitted by linear regression analysis. Final ceramide concentrations were presented in pM.
  • Mobile phases consisted of (A) 10 mM ammonium acetate in LC-MS grade water with 0.1% formic acid, and (B) 10 mM ammonium acetate in acetonitrile: 2- propanol (3:4, V/V) with 0.1% formic acid (FA).
  • the following LC gradient was used: 0.3 min at 45% B, linear increase of B from 45% to 95% in 10 min, 95% to 100% B in 0.1 min, 2.5 min at 100% B, 100% to 45% B in 0.1 min and 1.5 min equilibration at 45% prior to the next injection.
  • Flow rate was 600 pl/min and column temperature 60°C.
  • MS analysis a targeted approach in the positive ion mode was used.
  • the data were collected using a scheduled multiple reaction monitoring (sMRMTM) algorithm.
  • the lipidomic data were processed using Analyst and MultiQuant 3.0 software (AB Sciex), and the area or height ratios of the analyte and its corresponding IS peak were normalized with the IS amount and the sample volume.
  • Table 2 presents the predictive value of the pulmonary biomarkers. Median relative change and p-value are presented. Positive median relative change shows an increased concentration of a biomarker and negative a decreased concentration when a subject is suffering from or has a risk to develop a severe pulmonary condition.
  • PE P-36 l -6089% 5.27E-05 PE 38:3 -4837% 7.42E-02
  • Table 3 shows the predictive value of examples of pulmonary biomarker ratios. Median relative change and p-value are presented.
  • PC 40:5/PC 34:4 311 2.83E-07 PC 40:5/PC 38:2 93
  • PC 38:4/PC 38:6 192 6.32E-07 PC 38:4/PC(14:0/22:6) 173
  • PC 40:6/PC 36:6 312 6.32E-07 PC 40:2/PC 40:1 80
  • PC 40:2/PC 40:8 192 3.39E-06 Cer(dl8:l/20:0)/PC 0-32:0 58
  • PC 40:2/PC 38:2 142 5.27E-05 PC 40:6/PC 40:4 48
  • Tables 4-9 present simplifying scoring systems (points 0-12, 0-9 and 0-6), where the points are given based on the quartiles (Q.1-Q.4) of a general population. For example, in Table 4, if Cer(dl8:l/16:0) concentration of a person belongs to the highest quartile, the person will receive 2 points. The same evaluation will be performed for the other components of the score, and the points are then summed up to create the score.
  • Score 1 an example of a scoring system based on lipids.
  • Score 2 Another example of a scoring system based on lipids.
  • Score 3 another example of a scoring system based on lipids.
  • Score 4 another example of a scoring system based on lipids.
  • Table 10 presents an example of the predictive value of the scoring systems.
  • Scores 1-6 refer to the scoring systems presented in Tables 4-9. P-values are presented for the scores.

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Abstract

The present disclosure is related to biomarkers for predicting severe pulmonary conditions. In particular, it provides a novel in vitro method for assessing the severity of a pulmonary condition of a subject using lipid biomarkers. In addition, the present biomarkers can be used in methods for predicting the progress of a pulmonary condition, selecting an appropriate treatment and preventing the occurrence of serious complications of a pulmonary condition.

Description

BIOMARKERS FOR SEVERE PULMONARY CONDITION
FIELD OF THE INVENTION
The present disclosure is related to biomarkers for predicting severe pulmonary conditions. In particular, it provides a novel in vitro method for assessing the severity of a pulmonary condition of a subject using lipid biomarkers. In addition, the present biomarkers can be used in methods for predicting the progress of a pulmonary condition, selecting and administering an appropriate treatment and preventing the occurrence of serious complications of a pulmonary condition.
BACKGROUND
Pulmonary conditions are some of the most common medical conditions in the world. Lungs are a complex system bringing in oxygen, delivering oxygen into circulation and releasing carbon dioxide. A lung disease can occur when there is a problem in any part of this system. Pulmonary conditions cause a wide range of different complications and severe forms are life-threatening. There are three main types of pulmonary conditions: conditions affecting airways, conditions affecting the structure of the lung tissue, and conditions affecting circulation in the lungs.
Pulmonary conditions affecting airways, such as asthma, chronic obstructive pulmonary disease (COPD) and bronchitis, usually cause a narrowing or blockage of the airways. Pulmonary conditions affecting lung tissue may be caused due to scarring or inflammation of the pulmonary tissue. This type of lung diseases include for example pulmonary fibrosis, lung cancer and acute respiratory distress syndrome (ARDS). Pulmonary conditions affecting circulation may be caused by clotting, scarring or inflammation of the blood vessels. Pulmonary conditions affecting circulation include for example thrombosis, pulmonary embolism and pulmonary hypertension. Pulmonary conditions that affect the efficiency of respiration such as respiratory failure or respiratory distress syndrome may affect the circulation as well, thus causing circulatory problems such as thrombosis.
The severity of pulmonary conditions vary considerably between the patients. Some individuals suffering from a pulmonary condition may be symptomless, whereas some develop a severe, even life-threatening, form of the condition. The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 has shown the importance of early detection of patients who are suffering from a severe form of the disease. The early identification and effective treatment of the patients suffering from a severe condition is crucial for both the survival of the patients and sustainability of healthcare systems. In order to effectively target healthcare resources, it is essential to be able to identify the patiens who will be needing immediate enhanced care. Predicting the progression of the disease supports selecting an appropriate treatment and early initiation of the treatment which benefits the patient's recovery and survival.
Patients suffering from a severe form of a pulmonary condition are currently diagnosed based on mainly respiratory function tests and medical imaging. However, at the time of diagnosis the patients are already at the severe phase of the condition and need urgent enhanced care. Some patients develop symptoms only when significant lung damage has already occurred, and the symptoms usually become worse over time. There are currently no methods for predicting the progress of a pulmonary condition and identifying the patients who will develop a severe condition at an early stage. Therefore, in order to enhance the treatment and recovery of the patients, new methods are needed for early identification of the high-risk patients before the manifestation of severe symptoms.
The novel blood-based methods of the present disclosure enable early identification and treatment of the patients who are at high risk of developing a severe pulmonary condition and/or its complications. The methods disclosed herein are more convenient and accessible than the currently used imaging and respiratory function methods which require specific equipment. The biomarkers of the present disclosure offer superior performance for risk stratification of pulmonary patients. They also allow predicting and preventing the occurrence of serious complications of a pulmonary condition, including thrombosis.
SUMMARY
In the present disclosure, novel pulmonary biomarkers determined from a biological sample have been identified. The present disclosure is based on the finding that certain lipid molecules are expressed at higher levels and certain molecules are expressed at lower levels, when compared to a control, in biological samples, e.g., blood of subjects suffering from or having a risk to develop a severe pulmonary condition. Concentrations of these molecules and combinations derived thereof were found to be surprisingly superior predictive biomarkers of severe pulmonary conditions, as described herein.
The present disclosure provides novel biomarkers, biomarker combinations and associated diagnostic methods and uses for the identification and treatment of subjects suffering from or having a risk of developing a severe pulmonary condition. Such methods and uses comprise monitoring specific lipid concentrations and combinations of specific lipid concentrations in a sample from a subject and comparing such concentrations to those in a control. The novel pulmonary biomarkers of the present disclosure are presented in Table 1. Table 1. Novel pulmonary biomarkers. Increased and decreased directions of change refer to a change in a quantity, amount, abundance, level or concentration of the biomarker in a sample from a subject when the subject is suffering from, or has a risk of developing, a severe pulmonary condition, when compared to a control. Abbreviations are described in the detailed description of the present disclosure.
Figure imgf000004_0001
PE 35:2_sn2 PC 36:0 LPE 16:0_snl SM 44:1
PE 40:3 PC 28:1 LPE 18:2_sn2 SM 40:0
PE 40:4 PC 35:0 LPE 18:l_snl SM 33:1
LacCer(dl8:l/16:0) PC 31:1 LPE P-18:l SM 38:3
LacCer(dl8:l/18:0) PC 36:2 LPE P-20:0 SM 42:0
LacCer(dl8:2/16:0) PC 35:5 LPE 18:l_sn2 SM 34:2
LacCer(dl8:2/18:0) PC 37:1 LPE P-16:0 SM 33:2
LacCer(dl8:l/20:0) PC 38:3 LPE P-18:0 SM 38:2
LacCer(dl8:l/24:l) PC 31:0 LPE 18:0_sn2 SM 35:2
LacCer(dl6:l/16:0) PC 36:1 LPE 18:0_snl SM 33:0
AcylCarnitine(14:l) PC 34:0 LPI 18:l_snl SM 44:3
AcylCarnitine(16:l) PC 34:2 LPI 18:l_sn2 SM 34:0
AcylCarnitine(18:l) PC 33:2 LPE 20:4_snl SM 35:1
AcylCarnitine(14:0) PC 32:1 PE P-38:4 DAG 32:1
AcylCarnitine(12:0) PC 39:4 PE 0-36:3 DAG 30:0
AcylCarnitine(16:0) PC 40:5 PE 0-36:4 DAG 32:2
AcylCarnitine(18:2) PC 33:3 PE P-36:3 DAG 32:2
SM 36:2 PC 40:4 PE P-38:3 DAG 32:1
SM 36:0 PC 35:4 PE 0-36:5 DAG 38:4
SM 36:1 PC 38:0 PE 0-38:4 DAG 36:5
SM 42:2 PC 40:1 PE 0-40:6 TAG(14:0/16:l/18:l)
SM 40:3 PC 37:3 PE P-36:2 TAG(14:0/16:0/18:l)
SM 44:2 PC 37:4_snl PE 0-38:3 TAG(14:0/16:l/18:2)
SM 38:0 PC 37:4_sn2 PE P-36:5 TAG(14:l/16:0/18:l)
SM 36:3 PC 36:4 PE P-34:2 TAG(16:l/16:l/18:0)
SM 34:1 PC 33:1 PI 38:3 TAG(14:0/16:0/18:2)
SM 42:3 PC 39:6_sn2 PE P-36:l TAG(14:0/18:0/18:l)
DAG 38:4 PC 37:6 PE 0-40:4 TAG(16:1/16:1/16:1)
DAG 38:5 PC 39:0 PE 0-34:2 TAG(14:l/16:l/18:0)
DAG 36:2 PC 38:1 PE 0-36:2 TAG(14:1/18:1/18:1)
DAG 38:6 PC 34:1 PE 0-34:1 TAG(14:l/18:0/18:2)
DAG 34:1 PC 35:l_sn2 PE 0-38:6 TAG(16:0/16:0/18:0)
DAG 36:3 PC 39:5 PE P-38:5 TAG(14:0/17:0/18:l)
DAG 36:4 PC 40:3 PI 38:1 TAG(16:0/16:l/18:l)
DAG 36:4 LPC 14:0_sn2 PE 0-36:1 TAG( 15:0/16:0/18:1)
DAG 38:6 LPC 22:0_snl PI 36:3 TAG(16:0/16:0/16:0)
DAG 36:2 LPC 17:l_snl PE P-34:l TAG(16:0/16:l/17:0)
DAG 34:2 LPC 19:0_snl PE P-36:4 TAG(14:0/18:2/18:2)
DAG 36:1 LPC 24:0_snl PI 38:5 TAG(18:0/18:0/18:l)
DAG 34:2 LPC 14:0_snl PE P-40:6 TAG(16:0/17:0/18:l)
DAG 36:4 LPC 18:2_snl PI 40:5 TAG(16:1/16:1/18:1)
TAG(18:l/18:l/20:4) LPC 19:0_sn2 PI 36:5 TAG(16:0/17:0/18:2)
TAG(18:l/18:l/22:6) LPC 22:0_sn2 PI 38:2 TAG(16:0/18:0/18:l)
TAG(16:0/18:l/18:2) LPC 24:0_sn2 PE 0-38:5 TAG( 15:0/18: 1/18:1)
TAG(16:0/18:l/18:l) LPC(15-MHDA)_sn2 PI 40:4 TAG(18:1/18:2/18:2)
TAG(18:0/18:l/18:l) LPC 17:l_sn2 PI 36:1 TAG(16:l/17:0/18:l)
TAG(18:1/18:1/18:1) LPC 20:0_snl PE P-38:6 CE 18:3
TAG( 17:0/18: 1/18:1) LPC 20:0_sn2 PE 36:3 CE 24:6
TAG(18:1/18:1/18:2) LPC 18:l_snl PE 36:2 CE 16:2
Figure imgf000006_0001
In a first aspect of the present disclosure, a method is provided for assessing the severity of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In one aspect, the present disclosure is directed to a method for selecting an appropriate treatment for a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
In another aspect, the present disclosure is directed to a method for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a method is provided for determining the risk of a subject to develop a severe pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a method is provided for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a method is provided for determining the risk of a subject to develop thrombosis, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control. In yet another aspect of the present disclosure, a method is provided for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In another aspect, the present disclosure is directed to a method of evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
In certain embodiments, the method of evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in the subject may further comprise after the determining step, changing, supplementing, or keeping the same an already administered treatment in the subject based on the concentrations of the at least one biomarker obtained in the determining step.
In another aspect, the present disclosure is directed to a method of treating a pulmonary condition or preventing the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising administering to the subject a treatment, if the subject has been identified as having a risk of developing a severe pulmonary condition. The method may further comprise determining the risk of the subject to develop a severe pulmonary condition according to the methods described herein prior to administering the treatment. Alteratively, the method of treating a pulmonary condition or preventing the progress of a pulmonary condition in a subject to more severe form of the condition, comprises administering to the subject a treatment, wherein the subject has been identified as having a risk of developing a severe pulmonary condition according to the methods described herein.
In another aspect, the present disclosure is directed to a method of treating a serious complication of a pulmonary condition or preventing the occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising administering to the subject a treatment, if the subject has been identified as needing a treatment. The method may further comprise determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition according to the methods described herein prior to administering the treatment. Alteratively, the method of treating a serious complication of a pulmonary condition or preventing the occurrence of a serious complication, or its progression, of a pulmonary condition in a subject, comprises administering to the subject a treatment, wherein the subject has been identified as needing a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition according to the methods described herein.
In yet another aspect, the present disclosure is directed to a method of treating thrombosis or preventing thrombosis, the method comprising administering to the subject a treatment, if the subject has been identified as having a risk of developing thrombosis. The method may further comprise determining the risk of the subject to develop thrombosis according to the methods described herein prior to administering the treatment. Alteratively, the method of treating thrombosis or preventing thrombosis in a subject, comprises administering to the subject a treatment, wherein the subject has been identified as having a risk of developing thrombosis according to the methods described herein.
In another aspect of the present disclosure, a method is provided for detecting at least one biomarker selected from Table 1 in a sample from a subject suffering from or suspected of suffering from a pulmonary condition, the method comprising assaying the sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In certain embodiments, the method of detecting comprises detecting in a sample from a subject a changed concentration of the at least one biomarker selected from Table 1, in comparison to a control.
In certain embodiments, the method of detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1 comprises using a standard combination comprising at least one lipid selected from Table 1. In certain embodiments, the standard combination comprises at least one isotope-labelled lipid selected from Table 1. In certain embodiments, the isotope of the at least one isotope-labelled lipid selected from Table 1 is deuterium, 13C or 15N.
In one aspect of the present disclosure, a method is provided for obtaining or collecting data for assessing the severity of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In another aspect, the present disclosure is directed to a method for obtaining or collecting data for selecting an appropriate treatment for a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
In another aspect, the present disclosure is directed to a method for obtaining or collecting data for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a method is provided for obtaining or collecting data for determining the risk of a subject to develop a severe pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a method is provided for obtaining or collecting data for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a method is provided for obtaining or collecting data for determining the risk of a subject to develop thrombosis, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a method is provided for obtaining or collecting data for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control. In yet another aspect of the present disclosure, a method is provided for obtaining or collecting data for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
In one aspect of the present disclosure, a method is provided for generating quantitative data for assessing the severity of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In another aspect, the present disclosure is directed to a method for generating quantitative data for selecting an appropriate treatment for a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In another aspect, the present disclosure is directed to a method for generating quantitative data for preventing the occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In another aspect of the present disclosure, a method is provided for generating quantitative data for determining the risk of a subject to develop a severe pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In yet another aspect of the present disclosure, a method is provided for generating quantitative data for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In yet another aspect of the present disclosure, a method is provided for generating quantitative data for determining the risk of a subject to develop thrombosis, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In yet another aspect of the present disclosure, a method is provided for generating quantitative data for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1. In yet another aspect of the present disclosure, a method is provided for generating quantitative data for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the method comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
In one aspect of the disclosure, a pulmonary marker combination is provided for the methods of all aspects of the disclosure, the pulmonary marker combination comprising at least two biomarkers selected from Table 1.
In certain embodiments of all aspects of the disclosure, the pulmonary marker combination comprises at least one biomarker ratio selected from Table 3. In some embodiments, the pulmonary marker combination comprises at least two biomarkers selected from Table 1 and at least one biomarker ratio selected from Table 3. In some embodiments, the pulmonary marker combination comprises any of the scores selected from Tables 4-10. In certain embodiments, the pulmonary marker combination may be e.g. a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1. In some embodiments, the combination may be calculated from the concentrations of the at least two biomarkers selected from Table 1. In some embodiments, the pulmonary marker combination may be a concentration ratio of the at least two biomarkers selected from Table 1. In some embodiments, the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
In some embodiments, the pulmonary marker combination further comprises other biomarkers, including, but not limited to, CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof. In some embodiments, the pulmonary marker combination further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof. In some embodiments, the marker combination may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
In some embodiments, the marker combination is a continuous or discrete scoring systems based on the biomarkers selected from Table 1. For discrete scoring, the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
In one aspect of the present disclosure, a use of a pulmonary marker combination is provided for assessing the severity of a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a use of a pulmonary marker combination is provided for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
In another aspect of the present disclosure, a use of a pulmonary marker combination is provided for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a use of a pulmonary marker combination is provided for determining the risk of a subject to develop a severe pulmonary condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control. In yet another aspect of the present disclosure, a use of a pulmonary marker combination is provided for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a pulmonary marker combination is provided for determining the risk of a subject to develop thrombosis, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a pulmonary marker combination is provided for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a pulmonary marker combination is provided for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
In one aspect of the disclosure, a standard combination is provided for the methods of all aspects of the disclosure, wherein the standard combination comprises at least two lipids selected from Table 1. In some embodiments, the standard combination comprises 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 11, or at least 12, or more lipids selected from Table 1. In some embodiments, the standard combination comprises at least one lipid selected from the lipid ratios of Table 3. In some embodiments, the standard combination comprises at least two lipids selected from Table 1 and at least one lipid selected from the lipid ratios of Table 3. In some embodiments, the standard combination comprises at least one component of the scores selected from Tables 4-10.
In certain embodiments of all aspects of the disclosure, the standard combination comprises at least one isotope-labelled lipid selected from Table 1. In certain embodiments of all aspects of the disclosure, the isotope of the at least one isotope-labelled lipid selected from Table 1 is deuterium, 13C or 15N.
In another aspect of the disclosure, a standard combination is provided for assaying a concentration of at least one biomarker selected from Table 1 in a sample from a subject in a preparation of a reagent, kit or composition for the methods of all aspects of the disclosure, wherein the standard combination comprises at least two lipids selected from Table 1.
In another aspect of the disclosure, a use of a standard combination is provided for detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1, wherein the standard combination comprises at least two lipids selected from Table 1.
In one aspect of the present disclosure, a use of a standard combination is provided for assessing the severity of a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a use of a standard combination is provided for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
In another aspect of the present disclosure, a use of a standard combination is provided for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a use of a standard combination is provided for determining the risk of a subject to develop a severe pulmonary condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a standard combination is provided for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a standard combination is provided for determining the risk of a subject to develop thrombosis, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a standard combination is provided for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of a standard combination is provided for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
In one aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for assessing the severity of a pulmonary condition of a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for selecting an appropriate treatment for a pulmonary condition of a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control, and selecting an appropriate treatment for the pulmonary condition of the subject.
In another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for determining the risk of a subject to develop a severe pulmonary condition, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for determining the risk of a subject to develop thrombosis, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1, and determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, the use comprising assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1 and determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
In yet another aspect of the present disclosure, a use of one or more reagent in a preparation of a reagent, kit or composition is provided for detecting at least one biomarker selected from Table 1 in a sample from a subject, wherein the use comprises assaying the sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
In certain embodiments, the use of one or more reagent in a preparation of a reagent, kit or composition for detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1 comprises using a standard combination comprising at least two lipids selected from Table 1. In certain embodiments, the standard combination comprises at least two isotope-labelled lipids selected from Table 1. In certain embodiments, the isotopes of the at least two isotope-labelled lipids selected from Table 1 are deuteriums, 13C or 15N. In certain embodiments, the reagent is used for assaying a sample to determine a concentration of at least one biomarker selected from Table 1.
In certain embodiments, the reagent comprises any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s), preparation(s), or any combination thereof used for the methods and uses of the present disclosure. In certain embodiments, the reagent is a combination or mixture of any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s) and preparation(s) used in the methods and uses of the present disclosure.
In some embodiments, one or more of any components of a reagent is isotope-labelled. In some embodiments, the isotope of the isotope-labelled component is deuterium, 13C or 15N.
In certain embodiments of all aspects of the disclosure, the reagent comprises at least one, 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 11, at least 12, or more lipids selected from Table 1. In certain embodiments of all aspects of the disclosure, the reagent comprises at least one, 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 11, at least 12, or more isotope-labelled lipids selected from Table 1.
In certain embodiments of all aspects of the present disclosure, the isotope-labelled lipids of the reagent are selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(d 16: 1/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC
36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC
37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC
35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC
35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
In certain embodiments of all aspects of the present disclosure, the isotope-labelled lipid(s) of the reagent is (are) d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7-Cer(dl8:l/24:l), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
In certain embodiments, the reagent is used in a preparation of a reagent, kit or composition which are used for performing the methods and uses of the present disclosure. In one aspect of the present disclosure, a composition or kit is provided for performing any of the methods or uses according to the present disclosure, wherein the composition or kit comprises at least one biomarker selected from Table 1.
In certain embodiments of the composition or kit, the at least one biomarker is 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 11, at least 12, or more biomarkers selected from Table 1. In certain embodiments, the composition or kit comprises at least one biomarker ratio selected from Table 3. In certain embodiments, the composition or kit comprises at least one biomarker selected from Table 1 and at least one biomarker ratio selected from Table 3. In certain embodiments, the composition or kit comprises at least one component of the scores selected from Tables 4-10.
In certain embodiments, the composition or kit comprises at least one isotope-labelled biomarker selected from Table 1. In certain embodiments, the isotope of the at least one isotope-labelled biomarker is deuterium, 13C or 15N. In certain embodiments of the composition or kit, the at least one isotope-labelled biomarker is 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 11, at least 12, or more isotope-labelled biomarkers selected from Table 1. In certain embodiments, the isotope of the at least one, 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 11, at least 12, or more isotope-labelled biomarkers is/are deuterium, 13C or 15N.
In certain embodiments of all aspects of the disclosure, the composition or kit comprises at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 isotopelabelled lipids selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(d 16: 1/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8,
PC 30:0, PC 36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2,
PC 35:5, PC 37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3,
PC 40:4, PC 35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1,
PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2. In certain embodiments of all aspects of the present disclosure, the isotope-labelled lipid(s) of the composition or kit is (are) d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7- Cer(dl8:l/24:1), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
The composition or kit may further include standard(s), control(s), reagent(s), solution(s), solvent(s), container(s), use instruction(s) for the methods or uses disclosed herein, and/or other element(s) for performing the methods or uses disclosed herein. In some embodiments, the composition or kit includes element(s) for collecting a blood sample, for example, a dried blood spot on a filter. In some embodiments, the composition or kit includes element(s) for collecting plasma or serum samples. In some embodiments, plasma or serum samples are collected on a filter card that separates the plasma or serum from a blood sample. In some embodiments, plasma or serum sample is dried on a card or filter.
The composition or kit may be a test kit for use in a laboratory or a home use test kit (over-the-counter test). The composition or kit may be a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), either purchased from a commercial manufacturer or prepared in-house in a laboratory, used for performing any of the methods or uses according to the present disclosure.
In certain embodiments, the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), either purchased from a commercial manufacturer or prepared in-house in a laboratory, used for assaying a concentration of at least one biomarker selected from Table 1.
The composition or kit may be used in assays performed with various chemical and high-resolution analytical techniques, as appropriate. Suitable analytical techniques according to the present methods and uses include, but are not limited to, mass spectrometry (MS) and nuclear magnetic resonance (NMR). Any high-resolution technique capable of resolving individual biomarkers can be used to collect the information on the biomarker in question, such as the concentration of a biomarker profile from the biological sample. Typically, the information is collected using mass spectrometry. The MS analysis can be coupled to another high performance separation method, such as gas chromatography (GC), two- dimensional gas chromatography (GCxGC), liquid chromatography (LC), two-dimensional liquid chromatography (LCxLC), high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
In certain embodiments of all aspects of the present disclosure, the at least one biomarker selected from Table 1 is 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 11, at least 12, or more biomarkers selected from Table 1.
In certain embodiments of all aspects of the present disclosure, the at least one biomarker is at least one biomarker ratio selected from Table 3. In certain embodiments of all aspects of the present disclosure, the at least one biomarker ratio is 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 11, at least 12, or more biomarker ratios selected from Table 3.
In certain embodiments of all aspects of the present disclosure, the at least one biomarker is any combination of at least one biomarker selected from Table 1 and/or at least one biomarker ratio selected from Table 3. In certain embodiments, the at least one biomarker is any of the scores selected from Tables 4-10 and/or any component of the scores selected from Tables 4-10. In certain embodiments, the at least one biomarker is any combination of the biomarkers selected from Tables 1-10.
In certain embodiments of all aspects of the present disclosure, the at least one biomarker may be e.g. a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1. In some embodiments, the biomarker may be calculated from the concentrations of the at least two biomarkers selected from Table
1. In some embodiments, the biomarker may be a concentration ratio of the at least two biomarkers selected from Table 1. In some embodiments, the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
In some embodiments, the at least one biomarker further comprises other biomarkers, including, but not limited to, CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof. In some embodiments, the at least one biomarker further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof. In some embodiments, the at least one biomarker may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
In some embodiments, the at least one biomarker is a continuous or discrete scoring systems based on the biomarkers selected from Table 1. For discrete scoring, the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
In certain embodiments of all aspects of the present disclosure, the concentrations of at least one, 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 11, at least 12, or more biomarkers selected from Table 1 are assayed, including but not limited to: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC 36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2,
PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC 37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0,
PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC 35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4,
PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6,
PC 38:4 and/or PC 39:2.
In certain aspects, the present disclosure provides methods of treatment, the methods comprising administering a treatment to a subject, wherein the subject has been identified as being in need of the treatment according to the methods described herein. In certain embodiments of all aspects of the present disclosure, the methods and uses further comprise administering a treatment to the subject. A treatment may comprise any therapeutic treatment typically given to a subject suffering from a pulmonary condition and/or needing immediate or intensive care. A treatment may be, e.g., hospitalization, intensive care, life support care, noninvasive or invasive medical care, monitoring and/or supporting bodily functions, administering a drug, administering supplemental oxygen (e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation), extracorporeal membrane oxygenation, a surgical procedure or lung transplantation. A drug may comprise any pharmaceutical typically given to a subject suffering from a pulmonary condition and/or its symptoms. A drug may be, e.g., an anti-inflammatory agent, antibiotic, antiviral drug, corticosteroid, analgesic, sedative, antithrombotic, anticoagulant or antiplatelet drug. A drug may be, e.g., a functional inhibitor of acid sphingomyelinase (FIASMA), molnupiravir, dexamethasone, a ceramide synthase or an inhibitor of a ceramide synthase.
In certain embodiments, the methods of treating or preventing a pulmonary condition, its serious complications or thrombosis, of a subject, further comprise identifying the subject as in need of the treatment or prevention, for example, by requesting a test or receiving the test results, for example, from a commercial laboratory, which provides the results of an assay useful for determining the concentration of the at least one biomarker selected from Table 1, and administering to the subject a treatment, for example, a therapeutically effective dose of a drug, if the subject has an increased or decresed concentration of the at least one biomarker selected from Table 1 in the sample, as compared to the control. In certain embodiments of all aspects of the present disclosure, the methods and uses further comprise a step of adding at least one isotope-labelled biomarker selected from Table 1 to the sample prior to assaying the concentration of the at least one biomarker selected from Table 1. The at least one isotopelabelled biomarker may be, but is not limited to, deuterium-labelled biomarker selected from Table 1. The at least one isotope-labelled biomarker may also be 13C-labelled or 15N-labelled. In certain embodiments of all aspects of the present disclosure, the methods and uses further comprise a step of adding at least one, 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 11, at least 12, or more isotope-labelled biomarkers selected from Table 1 to the sample. The at least one, 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 11, at least 12, or more isotope-labelled biomarkers may be, but are not limited to, deuterium-labelled, Relabelled or 15N-labelled biomarkers selected from Table 1.
In certain embodiments of all aspects of the present disclosure, the isotope-labelled biomarker is selected from the following isotope-labelled lipids: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(d 18: 1/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC
36:5, PC 35:3, PC 40:8, PC 30:0, PC 36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC
35:0, PC 31:1, PC 36:2, PC 35:5, PC 37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC
39:4, PC 40:5, PC 33:3, PC 40:4, PC 35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC
37:6, PC 39:0, PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
In certain embodiments of all aspects of the present disclosure, the isotope-labelled biomarker(s) is (are) d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7-Cer(dl8:l/24:l), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
In certain embodiments of all aspects of the present disclosure, the determination of a concentration of at least one biomarker selected from Table 1 is typically performed using an assay. In certain embodiments, such assay is, or involves, mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy or dual polarisation interferometry, a high performance separation method such as liquid chromatography (LC), gas chromatography (GC), two-dimensional liquid chromatography (LCxLC), two-dimensional gas chromatography (GCxGC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC) or ultra performance liquid chromatography (UPLC), an immunoassay such as an ELISA and/or an assay with a binding moiety capable of specifically binding the analyte. In certain embodiments, the assay is mass spectrometry (MS). The MS instrument can be coupled to a direct sample infusion method, such as a robotic nanoflow ion source device, or to a high performance separation method such as HPLC, UHPLC, or UPLC.
In certain embodiments of all aspects of the present disclosure, the concentrations of the 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 11, at least 12, or more biomarkers selected from Table 1 are determined by one assay. In some embodiments, the one assay is performed with mass spectrometry (MS).
In certain embodiments of all aspects of the present disclosure, the method or use further comprises determining other biomarkers, including, but not limited to, C-reactive protein (CRP), high sensitivity CRP (hs-CRP), a cytokine, troponin T (TNT or TnT), troponin I (TNI or Tnl), B-type natriuretic peptide (BNP), pro B-type natriuretic peptide (proBNP), N-terminal pro B-type natriuretic peptide (NT-proBNP), cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof.
In certain embodiments of all aspects of the present disclosure, the method or use further comprises using other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
In certain embodiments of all aspects of the disclosure, a concentration ratio is calculated from the concentrations of the 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 11, at least 12, or more biomarkers selected from Table 1. In certain embodiments, a concentration ratio is calculated from at least 2 of the following lipids: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(d 18: 1/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC 36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC
36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC 37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC
33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC 35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC
33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2. In certain embodiments of all aspects of the disclosure, a concentration ratio is selected from any of the lipid ratios referred to in Table 3. In certain embodiments, the concentration ratio is a Cer/Cer, Cer/PC, PC/Cer and/or PC/PC ratio, e.g., Cer(dl8:l/16:0)/PC 16:0/22:5, Cer(dl8:l/18:0)/PC 14:0/22:6, Cer(dl8:l/24:l)/Cer(dl8:l/24:0), Cer(dl8:l/18:0)/PC 36:6, Cer(dl8:l/16:0)/PC 38:5,
Cer(dl8:l/18:0)/Cer(dl8:l/16:0), Cer(dl8:l/18:0)/PC(16:0/16:0), Cer(dl8:l/18:0)/LPC(15-MHDA),
Cer(dl8:l/18:0)/PC 32:0, Cer(dl8:l/18:0)/PC(16:0/22:5), Cer(dl8:l/18:0)/Cer(dl8:l/24:0),
Cer(dl8:l/18:0)/PC 38:5, Cer(dl8:l/24:l)/PC(16:0/22:5), Cer(dl8:l/24:l)/LPC(15-MHDA),
Cer(dl8:l/16:0)/LPC(15-MHDA), Cer(dl8:l/24:l)/PC 38:5, Cer(dl8:l/16:0)/Cer(dl8:l/24:0),
Cer(dl8:l/24:l)/PC 36:6, Cer(dl8:l/24:l)/PC(14:0/22:6), Cer(dl8:l/16:0)/PC(14:0/22:6),
Cer(dl8:l/16:0)/PC 36:6, Cer(dl8:l/16:0)/PC(16:0/16:0), Cer(dl8:l/24:l)/PC(16:0/16:0),
Cer(dl8:l/16:0)/PC 32:0 and/or Cer(dl8:l/24:l)/PC 32:0.
In certain embodiments of all aspects of the disclosure, the marker combination may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
In certain embodiments of all aspects of the disclosure, the marker combination is a continuous or discrete scoring systems based on the Cer, PC, Cer/PC, Cer/Cer, PC/Cer, PC/PC or other pulmonary biomarkers. For discrete scoring, the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
In certain embodiments of all aspects of the disclosure, the marker combination is a scoring system (for example, but not limited to, points 0-15, 0-12, 0-9 or 0-6), where the points are given based on the quartiles (Q.1-Q.4) of a study population. For example, if a lipid biomarker of a person belongs to the highest quartile, the person will receive 3 points. The same evaluation will be performed for the other components of the score, and the points are then summed up to create the score.
In certain embodiments of all aspects of the disclosure, the score is based on 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 11, at least 12, or more lipids selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(d 16: 1/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC
36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC
37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC
35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC
35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
In certain embodiments of all aspects of the disclosure, the score is constructed according to Tables 4-10.
In certain embodiments of all aspects of the disclosure, the pulmonary condition is caused by severe acute respiratory syndrome coronavirus 2 (SARS-Co-2).
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations
As used herein, "ARDS" is acute respiratory distress syndrome, "BMI" is body mass index, "BNP" is B-type natriuretic peptide, " 13c" is carbon-13 isotope, "CAD" is coronary artery disease, "CE" is cholesterylester, "Cer" is ceramide, "COPD" is chronic obstructive pulmonary disease, "COVID-19" is coronavirus disease 2019, "CRP" is C-reactive protein, "DAG" is diacylglycerol", "ELISA" is enzyme-linked immunosorbent assay, "FA" is fatty acid, "Gb3" is globotriasoylceramide, "GC" is gas chromatography, "GCxGC" is two- dimensional gas chromatography, "2H" is deuterium, "HPLC" is high performance liquid chromatography, "HR" is hazard ratio, “LacCer" is lactosylceramide, "LC is liquid chromatography, "LCxLC" is two- dimensional liquid chromatography, “LPC" is lysophosphatidylcholine, “LPC O-" is lysoalkylphosphatidylcholine, "LPC P-" is lysoalkenylphosphatidylcholine, "LPE" is lysophosphatidylethanolamine, "LPE P-" is lysoalkenylphosphatidylethanolamine, "LPI" is lysophosphatidylinositol, "MERS-CoV" middle east respiratory syndrome coronavirus, "MHDA" is methylhexadecanoic acid, "MRM" is multiple reaction monitoring, "MS" is mass spectrometry, " 15N" is nitrogen-15 isotope, "NMR" is nuclear magnetic resonance, "NT-proBNP" is N-terminal pro B-type natriuretic peptide, "PC" is phosphatidylcholine, “PC O-" is alkyl-linked phosphatidylcholine, "PC P-" is alkenyl-linked phosphatidylcholine, “PE" is phosphatidylethanolamine, "PE O-" is alkyl-linked phosphatidylethanolamine, "PE P-" is alkenyl-linked phosphatidylethanolamine, “PG" is phosphatidylglycerol, "PI" is phosphatidylinositol, "proBNP" is pro B-type natriuretic peptide, "SARS-CoV" is severe acute respiratory syndrome coronavirus, "SARS-CoV-2" is severe acute respiratory syndrome coronavirus 2, "SB" is sphingoid base, "SM" is sphingomyelin, "TAG" is triacylglycerol", "TC" is total cholesterol, "TG" is triglyseride, "TNI" or "Tnl" is troponin I or high sensitivity troponin I, "TNT" or "TnT" is troponin T or high sensitivity troponin T, "UHPLC" is ultra high performance liquid chromatography, "UPLC" is ultra performance liquid chromatography.
As used herein, "FIASMA" is a functional inhibitor of acid sphingomyelinase.
Definitions
In order that the present invention may be more readily understood, certain terms are defined herein. Additional definitions are set forth throughout the detailed description.
As used herein, a "pulmonary condition", "pulmonary disease", "lung condition", "lung disease", "respiratory condition" and "respiratory disease" are used interchangeably herein to refer to several disorders affecting the lungs and other parts of the respiratory system. A pulmonary condition may be an acute or chrocic condition. A pulmonary condition may be, e.g., a respiratory/pulmonary/lung infection, respiratory/pulmonary/lung inflammation, respiratory/pulmonary/lung damage, injury or distress, pneumonia, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, emphysema, bronchitis, pulmonary edema, asthma, lung cancer, pulmonary embolism or pulmonary hypertension.
A pulmonary condition may be caused by, e.g., an infection, inflammation, apoptosis, smoking, secondhand smoking, air pollution, exposure to dust, smoke or fumes, and/or genetic factors.
A respiratory infection or inflammation may be caused by, e.g., a pathogen, smoking, secondhand smoking, air pollution, exposure to dust, smoke or fumes and/or genetic factors.
As used herein, a "severe pulmonary condition" and a "serious pulmonary condition" are used interchangeably herein to refer to any clinically severe, critical or life-threatening pulmonary condition which may require immediate enhanced care, hospitalization or care in the intensive care unit. A severe pulmonary condition may be, e.g., pneumonia, pulmonary fibrosis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary embolism or lung cancer. The condition may or may not be acute or the condition may or may not be chronic.
As used herein, a "complication", "sequela", "symptom", "consequence", "outcome" and "adverse event" are used interchangeably herein to refer to any symptom caused by a pulmonary condition. The severity of the complications vary significantly from mild to severe, life-threatening, symptoms. A complication may be, e.g., cough, sore throat, shortness of breath, difficulty breathing, chest pain, pain when breathing, pain in the airways, chest pressure or tightness, fever, chills, headache, tiredness, nasal congestion, runny nose, muscle and joint pain, rash, nausea, diarrhea, vomiting, loss of taste or smell, eye problems, mucus production, expectoration, wheezing, gastrointestinal symptoms, oxygen deficiency, pneumonia, pulmonary fibrosis, thrombosis, pulmonary embolism, hypertension, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g. acute kidney injury), sepsis, additional viral or bacterial infection, multisystem inflammatory syndrome, chronic fatigue syndrome, acute or chronic cardiovascular damage (e.g. myocardial injury, myocarditis, arrhytmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure), seizure, clinical deterioration or death.
As used herein, a "serious complication", "serious sequela", "serious symptom", "serious consequence", "serious outcome", "serious adverse event", "severe complication", "severe sequela", "severe symptom", "severe consequence", "severe outcome" and "severe adverse event" are used interchangeably herein to refer to any clinically severe, critical or life-threatening symptom of a pulmonary condition. A serious complication may require immediate enhanced care or treatment, hospitalization or care in the intensive care unit. A serious complication may be, e.g. oxygen deficiency, pneumonia, pulmonary fibrosis, thrombosis, pulmonary embolism, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g. acute kidney failure), sepsis, acute or chronic cardiovascular damage (e.g. myocardial injury, myocarditis, arrhytmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure), seizure, clinical deterioration or death.
People who have one or more underlying medical conditions may have a higher risk of a severe pulmonary condition and/or its serious complications. An underlying condition may be, e.g., diabetes, a cardiovascular disease (e.g. heart failure, atherosclerosis, coronary artery disease, cardiomyopathy or hypertension), severe obesity, kidney disease, liver disease, sickle cell disease, weakened immune system, lung disease or neurological condition.
As used herein, "thrombosis" refers to formation of a blood clot, a thrombus, within a blood vessel. The blood clot prevents blood from flowing normally through the circulatory system. Thrombosis may occur in veins (venous thrombosis) or in arteries (arterial thrombosis). Venous thrombosis leads to congestion of the affected part of the body, while arterial thrombosis affects the blood supply and leads to damage of the tissue supplied by that artery. A thrombus in the lungs, either formed in the lungs, or originating from another part of the body, may cause a severe pulmonary condition when blocking the blood flow in the lungs. "Pulmonary embolism" refers to a blood clot that has travelled to the lungs. Pulmonary embolism is a severe pulmonary condition that can block the blood flow to the lungs, causing serious damage to the lungs and affecting a subject's ability to breath. "Deep vein thrombosis" occurs when a blood clot forms in a major vein, e.g. in the leg. Pulmonary conditions that affect the efficiency of respiration may also affect the circulation and cause circulatory problems such as thrombosis. A "treatment" and "therapy" are used interchangeably in the present disclosure and may comprise any therapeutic treatment typically given to a subject suffering from a pulmonary condition and/or needing immediate or intensive care. A treatment may be, e.g., hospitalization, intensive care, life support care, noninvasive or invasive medical care, monitoring and/or supporting bodily functions, administering a drug, administering supplemental oxygen (e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation), extracorporeal membrane oxygenation, a surgical procedure or lung transplantation.
A "drug", "pharmaceutical", "medicament", "medicine" and "medication" are used interchangeably in the present disclosure and may comprise any pharmaceutical typically given to a subject suffering from a pulmonary condition and/or its symptoms. A drug may be, e.g., an anti-inflammatory agent, antibiotic, antiviral drug, corticosteroid, analgesic, sedative, antithrombotic, anticoagulant or antiplatelet drug. A drug may be, e.g., a functional inhibitor of acid sphingomyelinase (FIASMA), molnupiravir, dexamethasone, a ceramide synthase or an inhibitor of a ceramide synthase.
As used herein, a "pathogen" is an infectious microorganism or agent that causes disease or illness to its host. A pathogen may be a virus, bacterium, protozoan, prion, viroid, fungus or any other eukaryotic or prokaryotic invader of host.
As used herein, A "virus" may be, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D, any subtype or variant of type A, B, C or D influenza virus or any other virus capable of infecting mammalian subjects.
As used herein, the terms "subject", "patient" and "individual" are used interchangeably herein to refer to any mammalian subject for whom prediction, diagnosis or therapy is desired, particularly humans. The subject may be a healthy individual with no previous signs or symptoms of a pulmonary condition. Or, the subject may have previously suffered or is suffering from a pulmonary condition. The subject may also be suspected of suffering from a pulmonary condition and/or being at high risk of developing a severe pulmonary condition and/or its serious complications. The subject may also be suffering from a pulmonary condition and is at high risk of progressing to a more severe form of the condition. The subject may or may not have previously suffered from a pulmonary condition.
The subject may be a healthy individual with no previous signs or symptoms of thrombosis. Or, the subject may have previously suffered or is suffering from thrombosis. The subject may also be suspected of suffering from thrombosis and/or being at high risk of developing thrombosis. The subject may or may not be infected by a virus or the subject may or may not have previously been infected by a virus. The subject may or may not be infected by SARS-CoV-2, SARS-CoV, MERS-CoV, any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D or any subtype or variant of type A, B, C or D influenza virus. The subject may or may not have previously been infected by SARS-CoV-2, SARS-CoV, MERS-CoV, any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D, or any subtype or variant of type A, B, C or D influenza virus.
The subject may or may not be suffering from coronavirus disease (COVID-19) or the subject may or may not have previously suffered from COVID-19.
The subject may or may not be hospitalized. The subject may or may not be under a treatment in a hospital. The subject may or may not be hospitalized and/or under a treatment due to a pulmonary condition. The subject may or may not be hospitalized and/or under a treatment due to a viral infection. The subject may or may not be hospitalized and/or under a treatment due to COVID-19. The subject may or may not be apparently healthy. The subject may or may not suffer an acute or chronic medical condition(s). The subject may or may not have a diagnosed or undiagnosed underlying medical condition(s). The subject may or may not be under a treatment for an underlying medical condition(s).
The subject may or may not suffer from a cardiovascular or metabolic disease, such as atherosclerosis, CAD, diabetes and fatty liver disease. In addition, the subject may or may not have normal levels of other pulmonary biomarkers, and/or may or may not have any traditional risk factors of pulmonary conditions. The subject may be or may have been under a treatment or medication, or may not have had any previous treatment or medication.
As used herein, a "sample" is a biological sample obtained from a subject or a group or population of subjects. The sample may be a blood sample, a serum sample, a plasma sample, a saliva sample, a urine sample, a tissue sample or a fraction thereof, such as a lipoprotein fraction. Blood serum and plasma samples are typical. The sample can be prepared with techniques well known in the art. In certain embodiments, the blood sample is a dried blood spot. In certain embodiments, the blood sample is dried on a filter. In certain embodiments, the blood sample is dried on a filter card which separates plasma or serum from the blood sample. In certain embodiments, the sample is a dried plasma or serum sample.
As used herein, a "control" may be a control sample. A control may also be a concentration determined from a sample from a single healthy individual. The control may also be a sample that represents a combination of samples from a generalized population of healthy individuals. Alternatively, the control may be a control value, a score, a threshold, a limit, a limit value or a set of data concerning the biomarker in a sample previously determined, calculated or extrapolated, or may have yet to be determined, calculated or extrapolated, or may also be taken from the literature. In some embodiments, the control sample may be previously obtained from the same subject or the control value may be obtained from a previously taken sample from the same subject. In other embodiments, the control sample or the control value may be obtained from another subject.
A "control" as used herein, i.e., a control value or a control sample, is typically representative of a group of subjects or a population of subjects. In this context, "representative" means that the lipid concentration(s) reflected by said control value to which a comparison is made in the context of the present disclosure correspond(s) to the average concentration value(s) of said lipid concentration(s) in corresponding individual samples from the subjects of said group or population. Likewise, in the case of a control sample "representative" means that the lipid concentration(s) in said control sample to which a comparison is made in the context of the present disclosure correspond(s) to the average concentration(s) of said lipid concentration(s) in corresponding individual samples from the subjects of said group or population. Typically, the concentrations of all lipid concentrations in said control sample correspond to the average concentrations of said lipid concentrations in corresponding individual samples from the subjects of said group or population. An individual with such values can be considered a "healthy individual" for the purposes of the present disclosure.
A control sample can be particularly suitably compared to the subject's sample if it has been obtained from the same type of biological tissue or source in the same, or essentially the same, manner. For example, if the subject's sample is a serum sample or a plasma sample, a corresponding control sample will likewise be a serum sample or a plasma sample, respectively.
In some embodiments, a control sample from a group of subjects or a control sample from a population of subjects in the sense of the present disclosure is obtained by mixing equal amounts of samples directly obtained from the subjects of said group or population, or by mixing equal amounts of fractions, constituents or reaction products thereof.
It will be appreciated that a useful control value for the purposes of the present disclosure is typically one that has been, or is, obtained using any one of the suitable control samples described herein.
In the context of the present disclosure, a control value or a control sample can be from a healthy individual, a generalized population of healthy individuals, a pulmonary patient that has remained free of any severe condition or serious complication, or a group of pulmonary patients that have remained free of any severe condition or serious complication.
In some embodiments, a control value or a control sample may be obtained from a symptomless pulmonary patient or a pulmonary patient who has mild symptoms, or a group of symptomless pulmonary patients or a group of pulmonary patients who have mild symptoms. A control value or a control sample may also be obtained from a symptomless COVID-19 patient or a COVID-19 patient who has mild symptoms, or a group of symptomless COVID-19 patients or a group of COVID-19 patients who have mild symptoms.
In some embodiments of the methods described herein, a control may be a control value, e.g., a score, a threshold or a combination value of the concentrations of the biomarkers selected from Table 1. In some embodiments, a control may be a control value, e.g., a score, a threshold or or a combination value of the biomarker ratios selected from Table 3. In some embodiments, a control may be a control value, e.g., a score, a threshold or a combination value of the concentrations of the at least one biomarker selected from Table 1 and the at least one biomarker ratio selected from Table 3. In some embodiments, a control may be a control value, e.g., a score, a threshold or a combination value obtained from the scores selected from Tables 4-10.
In some embodiments of the methods described herein, the increased concentration of the at least one biomarker is 0.01 - 10000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the increased concentration of the at least one biomarker is 1 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the increased concentration of the at least one biomarker is 10 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some further embodiments, the increased concentration of the at least one biomarker is 25 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
In some embodiments of the methods described herein, the decreased concentration of the at least one biomarker is 0.01 - 10 000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the decreased concentration of the at least one biomarker is 1 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some embodiments, the decreased concentration of the at least one biomarker is 10 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers. In some further embodiments, the decreased concentration of the at least one biomarker is 25 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
For the purposes of the present disclosure, lipids are named according to the following nomenclature:
As used herein, the nomenclature of ceramides (Cer), lactocylceramides (LacCer) and globotriasoylceramides (Gb3) is presented as a first pair of numbers corresponding to the sphingoid base (SB) and a second pair of numbers corresponding to the fatty acid (FA) chain of the molecule. In SB and FA nomenclature, the first number of each pair refers to the number of carbon atoms in the SB or FA chain, and the second number refers to the number of carbon-carbon double bonds of the SB or FA chain.
As used herein, the nomenclature of phosphatidylcholines (PC) is presented either as brutto species or molecular species. In brutto nomenclature, a first number refers to the total number of carbon atoms in the FA chains of the molecule and the second number refers to the total number of the carbon-carbon double bonds of in the FA chains of the molecule. In molecular nomenclature, a first pair of numbers corresponds to the first FA chain and a second pair of numbers corresponds to the second FA chain of the molecule. In FA nomenclature, the first number of each pair refers to the number of carbon atoms in the FA chain, and the second number refers to the number of carbon-carbon double bonds of the FA chain. In all aspects of the present disclosure, the PC may be a brutto PC or a corresponding molecular PC.
The nomenclature of lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), lysophosphatidylinositols (LPI), lysoalkylphosphatidylcholines (LPC O-), lysoalkenylphosphatidylcholine (LPC P-), lysoalkenylphosphatidylethanolamines (LPE P-) and acylcarnitines is presented with a number corresponding to the FA chain of the molecule. In FA nomenclature, the first number refers to the number of carbon atoms in the FA chain, and the second number refers to the number of carbon-carbon double bonds of the FA chain.
The nomenclature of phosphatidylethanolamines (PE), phosphatidylglycerols (PG), phosphatidylinositols (PI), alkyl-linked phosphatidylcholines (PC O-), alkenyl-linked phosphatidylcholines (PC P-), alkyl-linked phosphatidylethanolamines (PE O-), alkenyl-linked phosphatidylethanolamines (PE P-), diacylglycerols (DAG), sphingomyelins (SM) and cholesterolesters (CE) is presented with a number corresponding to the total number of carbon atoms and total number of carbon-carbon double bonds in the FA chains of the molecule.
The nomenclature of triacylglycerols (TAG) is presented with numbers corresponding to the FA chains of the molecule. In FA nomenclature, the first number refers to the number of carbon atoms in the FA chain, and the second number refers to the number of carbon-carbon double bonds of the FA chain.
The nomenclature of snl and sn2 indicate thesnl and sn2 positions of the glycerol backbone, respectively, to which the fatty acid moiety is attached.
In nomenclature of LPCs, MHDA refers to methylhexadecanoic acid.
As used herein, a "pulmonary marker combination" refers to a specific combination of lipid biomarkers used for the methods and uses of the present disclosure. The pulmonary marker combination comprises at least two biomarkers selected from Table 1. In some embodiments, the pulmonary marker combination comprises at least one biomarker ratio. In some embodiments, the biomarker ratio is selected from Table 3. In some embodiments, the pulmonary marker combination comprises at least two biomarkers selected from Table 1 and at least one biomarker ratio, and optionally the biomarker ratio is selected from Table 3. In some embodiments, the pulmonary marker combination comprises a score. In some embodiments, the score is selected from Tables 4-10. As used herein, the pulmonary marker combination may be e.g. a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1. In some embodiments, the combination may be calculated from the concentrations of the at least two biomarkers selected from Table 1. In some embodiments, the pulmonary marker combination may be a concentration ratio of the at least two biomarkers selected from Table 1. In some embodiments, the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
In some embodiments, the pulmonary marker combination further comprises other biomarkers, including, but not limited to, CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof. In some embodiments, the pulmonary marker combination further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof. In some embodiments, the marker combination may be constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
In some embodiments, the marker combination is a continuous or discrete scoring systems based on the biomarkers selected from Table 1. For discrete scoring, the points may be given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
In the present disclosure, the terms pulmonary marker combination, pulmonary biomarker combination, marker combination, biomarker combination, lipid marker combination, lipid combination and combination may be used interchangeable.
As used herein, a "cytokine" refer to one or more of any interleukin (IL), interferon (INF), tumor negrosis factor (TNF), transforming growth factor (TGF), chemokine, lymphokine and/or monokine. An interleukin may be, but is not limited to, IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 or any IL from IL19 to IL36. An interferon may be, but is not limited to, an INF type I, such as INF-a and INF-P, INF type II, such as INF-y, or INF type III. A tumor negrosis factor may be, but is not limited to, TNF-a or TNF-p. As used herein, a "standard combination" refers to a specific combination of lipid standards used for the methods and uses of the present disclosure. The standard combination comprises at least two lipids selected from Table 1. In some embodiments, the standard combination comprises at least one lipid selected from the lipid ratios of Table 3. In some embodiments, the standard combination comprises at least two lipids selected from Table 1 and at least one lipid selected from the lipid ratios of Table 3. In some embodiments, the standard combination comprises at least one component of the scores selected from Tables 4-10. In some embodiments, one or more of the components of the standard combination is isotope-labelled. In some embodiments, the isotope of the isotope-labelled component is deuterium (2H). In some embodiments, the isotope of the isotope-labelled component is 13C or 15N.
As used herein, a "composition" or "kit" may be any combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for the methods and uses of the present disclosure. The composition or kit may comprise at least one at least one biomarker selected from Table 1. In certain embodiments of the composition or kit, the at least one biomarker is 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 11, at least 12, or more biomarkers selected from Table 1. In certain embodiments, the composition or kit comprises at least one biomarker ratio selected from Table 3. In certain embodiments, the composition or kit comprises at least one biomarker selected from Table 1 and at least one biomarker ratio selected from Table 3. In certain embodiments, the composition or kit comprises at least one component of the scores selected from Tables 4-10.
In some embodiments, one or more of the components of the composition or kit is isotope-labelled. In some embodiments, the isotope of the isotope-labelled component is deuterium. In some embodiments, the isotope of the isotope-labelled component is 13C or 15N.
The composition or kit may further include standard(s), control(s), reagent(s), solution(s), solvent(s), container(s), use instruction(s) for the methods or uses disclosed herein, and/or other element(s) for performing the methods or uses disclosed herein. In some embodiments, the composition or kit includes element(s) for collecting a blood sample, for example, a dried blood spot on a filter. In some embodiments, the composition or kit includes element(s) for collecting a plasma or serum sample. In some embodiments, the element separates plasma or serum from a blood sample. In some embodiments the plasma or serum sample is dried on a filter card.
The composition or kit may be purchased from a commercial manufacturer or prepared in-house in a laboratory. The composition or kit may be a test kit for used in a laboratory or a home use test kit (over- the-counter test). The composition or kit may be a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), either purchased from a commercial manufacturer or prepared in-house in a laboratory, used for performing any of the methods or uses according to the present disclosure. In certain embodiments, the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for assaying a concentration of at least one biomarker selected from Table 1. In certain embodiments, the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for assaying at least one biomarker ratio selected from Table 3. In certain embodiments, the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s) used for assaying any of the scores selected from Tables 4-10.
The composition or kit may be used in assays performed with various chemical and high-resolution analytical techniques, such as, but not limited to, mass spectrometry (MS) and nuclear magnetic resonance (NMR). The MS analysis can be coupled to another high performance separation method, such as gas chromatography (GC), two-dimensional gas chromatography (GCxGC), liquid chromatography (LC), two-dimensional liquid chromatography (LCxLC), high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
As used herein, a "reagent" may be any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s), preparation(s), or any combination thereof used for the methods and uses of the present disclosure. A reagent may also be a combination or mixture of any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s) and preparation(s) used in the methods and uses of the present disclosure.
In some embodiments, one or more of any components of a reagent is isotope-labelled. In some embodiments, the isotope of the isotope-labelled component is deuterium. In some embodiments, the isotope of the isotope-labelled component is 13C or 15N.
In some embodiments, a reagent is used for assaying in a sample a concentration of at least one biomarker selected from Table 1. In some embodiments, a reagent is used for assaying in a sample at least one biomarker ratio selected from Table 3. In some embodiments, a reagent is used for assaying in a sample any of the scores selected from Tables 4-10.
In some embodiments, a reagent is used in a preparation of a reagent, kit or composition for performing the methods and uses of the present disclosure.
For the purposes of the present disclosure, the terms "obtaining data", "collecting data", "obtaining information" and "collecting information" may be used interchangeably.
As used herein, "assaying", "detecting", "analyzing" or "generating quantitative data" refers to a measurement of a quantity, amount, abundance, level or concentration of the biomarkers of the present disclosure in a sample using a laboratory apparatus, equipment or device, such as, but not limited to, mass spectrometry. A measurement may be quantitative or relative determination of an amount of a biomarker in a sample. For quantitative determination, either the absolute or precise amount of the biomarker in a sample is determined. The relative amount or level of a biomarker in a sample, may alternatively be determined, e.g., the biomarker amount in the sample is determined to be increased or decreased with respect to a control as described herein.
As used herein, a "biomarker", "marker", "pulmonary marker", "pulmonary biomarker", "lipid marker", "lipid biomarker" and "lipid" may be used interchangeably.
As used herein, a "combination", "group", "set", "composition" and "mixture" may be used interchangeably.
As used herein, an "increased or decreased" direction of change refers to a change in a quantity, amount, abundance, level or concentration of the biomarker in a sample from a subject when the subject is suffering from or is having a risk of developing a pulmonary condition or thrombosis, when compared to a control.
As used herein, a "risk" refers to a low, intermediate or high risk of developing a pulmonary condition or thrombosis.
The terms "the disclosure, description or invention", "in accordance with the disclosure, description or invention", "according to the disclosure, description or invention", "the present disclosure, description or invention", as used herein, are intended to refer to all aspects and embodiments of the disclosure described and/or claimed herein.
As used herein, the term "comprising" is to be construed as encompassing both "including" and "consisting of".
EXAMPLES
Example 1. Materials and Methods
Description of study cohort and samples
Serum samples were obtained from 39 adult subjects who were suffering from COVID-19, 9 subjects suffered from a severe form of COVID-19 and 30 non-severe form of COVID-19.
Analytical Methods
Liquid chromatography mass spectrometry for ceramides and phospholipids
Ceramide and phospholipid quantification from serum samples was performed using a validated high- throughput mass spectrometry approach. 10 pL of serum or plasma was spiked with deuterated internal standards, D7-Cer(dl8:l/16:0), D7-Cer(dl8:l/18:0), D7-Cer(dl8:l/24:0), D7-Cer(dl8:l/24:1), D9- PC((14:0/22:6), D9-PC(16:0/16:0) and D9-PC(16:0/22:5) and extracted in isopropanokethyl acetate (8:2, v/v) using a Hamilton MICROLAB® STAR robot. The levels of Cer(dl8:l/16:0), Cer(dl8:l/18:0), Cer(dl8:l/24:0), Cer(dl8:l/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) were quantified on a TripleMS 6500 (SCIEX, Concord, Canada) mass spectrometer equipped with an Sciex MPX UHPLC system.
Chromatographic separation was performed on Acquity BEH C18, 2.1 x 75 mm id. 1.7 pm column (Waters, Massachusetts, USA). Mobile phases consisted of (A) 10 mM ammonium acetate in LC-MS grade water with 0.1% formic acid, and (B) 10 mM ammonium acetate in acetonitrile: 2-propanol (3:4, V/V) with 0.1% formic acid (FA). The following LC gradient was used: 0.5 min at 80% B, linear increase of B from 80% to 84% in 0.5 min, 84% to 89% B in 1.4 min, 89% to 100% in O.lmin, 2.0 min at 100% B, 100% to 80% B in 0.1 min and 0.4 min equilibration at 80% prior to the next injection. Flow rate was 500 pl/min and column temperature 60°C.
The individual ceramides and phospholipids were quantified in MRM mode. Quantification was assessed through calibration line samples comprising of known amounts of synthetic Cer(dl8:l/16:0), Cer(d 18: 1/18:0), Cer(dl8:l/24:0), Cer(dl8:l/24:1), PC(14:0/22:6), PC(16:0/16:0) and PC(16:0/22:5) and corresponding deuterated standards. The peak area ratios of each ceramide to its corresponding deuterated form were calculated and plotted against the added ceramide concentration and fitted by linear regression analysis. Final ceramide concentrations were presented in pM.
Liquid chromatography mass spectrometry for other lipids
10 pl of sample was used for the extraction of the lipids using a modified Folch extraction. The analysis was performed on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB Sciex, Concords, Canada) equipped with ultra-high-performance liquid chromatography (UHPLC) (Nexera-X2, Shimadzu, Kyoto, Japan). Chromatographic separation was performed on Acquity BEH C18, 2.1 x 50 mm id. 1.7 pm column (Waters, Massachusetts, USA). Mobile phases consisted of (A) 10 mM ammonium acetate in LC-MS grade water with 0.1% formic acid, and (B) 10 mM ammonium acetate in acetonitrile: 2- propanol (3:4, V/V) with 0.1% formic acid (FA). The following LC gradient was used: 0.3 min at 45% B, linear increase of B from 45% to 95% in 10 min, 95% to 100% B in 0.1 min, 2.5 min at 100% B, 100% to 45% B in 0.1 min and 1.5 min equilibration at 45% prior to the next injection. Flow rate was 600 pl/min and column temperature 60°C. For the MS analysis, a targeted approach in the positive ion mode was used. The data were collected using a scheduled multiple reaction monitoring (sMRM™) algorithm. The lipidomic data were processed using Analyst and MultiQuant 3.0 software (AB Sciex), and the area or height ratios of the analyte and its corresponding IS peak were normalized with the IS amount and the sample volume.
Statistical analyses
All statistical analyses were performed using R software. The groups were compared with median differences and tested using Mann-Whitney U test.
Example 2. Results
Table 2 presents the predictive value of the pulmonary biomarkers. Median relative change and p-value are presented. Positive median relative change shows an increased concentration of a biomarker and negative a decreased concentration when a subject is suffering from or has a risk to develop a severe pulmonary condition.
Table 2. Pulmonary biomarkers, median relative change and p-value. Abbreviations are described in the detailed description of the present disclosure.
Figure imgf000041_0001
PC 34:5 -8702% 1.33E-05 PE 40:6 6007% 4.60E-02
PC P-36:l -4683 % 1.33E-05 PI 36:0 -2972 % 4.60E-02
SM 30:2 -6151% 1.33E-05 LacCer(dl8:2/16:0) 3065% 5.00E-02
LPC 22:0_sn2 -6457% 1.70E-05 Cer(dl6:l/26:0) -3088% 5.42E-02
LPC 24:0_sn2 -4552 % 1.70E-05 DAG 30:0 -4552 % 5.42E-02
PC 32:3 -8638% 1.70E-05 SM 44:0 -2535% 5.42E-02
PC P-38:4 -6624% 1.70E-05 CE 22:3 -4970% 5.88E-02
LPC(15-MHDA)_sn2 -6652% 2.16E-05 LacCer(dl8:2/18:0) 1909% 5.88E-02
PE P-34:2 -7163 % 2.16E-05 LPI 18:l_sn2 -3505 % 5.88E-02
PI 38:3 -6765% 2.16E-05 PC P-40:4 -3257% 5.88E-02
LPC 17:l_sn2 -5371% 2.73E-05 PE 34:2 -3197% 5.88E-02
LPC20:0_snl -6124% 2.73E-05 SM 40:1 -2076% 5.88E-02
LPC P-18:l -5614% 2.73E-05 TAG(14:0/17:0/18:l) -4160% 5.88E-02
PC 36:5 -6426% 2.73E-05 TAG(16:0/16:l/18:l) -1400% 5.88E-02
PC P-36:5 -7553% 2.73E-05 Cer(dl8:0/23:0) -3249% 6.36E-02
CE 16:2 -5910% 3.42E-05 LPC 0-24:1 -2354% 6.36E-02
LPC 20:0_sn2 -5773% 3.42E-05 SM 44:1 -2357% 6.36E-02
PC 35:3 -3655% 3.42E-05 Cer(dl8:l/24:1) 6710% 6.87E-02
PC 40:8 -5800% 3.42E-05 CE 24:0 3957% 6.87E-02
SM 31:1 -6559% 3.42E-05 PC 37:6 -4028% 6.87E-02
PC P-38:2 -6101% 4.25E-05 PI 34:2 -1830% 6.87E-02
LPC 18:l_snl -4447% 5.27E-05 SM 40:0 -1266% 6.87E-02
PC 0-36:1 -4676% 5.27E-05 Cer(dl8:l/16:0) 5257% 7.42E-02
PC 0-38:4 -3849 % 5.27E-05 DAG 32:2 -6239 % 7.42E-02
PE P-36:l -6089% 5.27E-05 PE 38:3 -4837% 7.42E-02
TAG(14:0/16:0/18:l) -5213 % 5.27E-05 PE P-38:2 -3062 % 7.42E-02
CE 14:0 -5509 % 7.95E-05 DAG 36:2 7927 % 8.00E-02
CE 20:2 -4783% 7.95E-05 PC 39:0 -1822% 8.00E-02
PC 0-38:3 -6031% 7.95E-05 PI 32:1 -2767% 8.00E-02
LPC20:3_snl -4380% 9.69E-05 CE 22:4 -1109% 8.62E-02
SM 31:2 -6896 % 9.69E-05 DAG 38:6 8328 % 8.62E-02
Cer(dl6:l/24:0) -5741% 1.18E-04 PE 36:1 -4834% 8.62E-02
LPC 18:2_sn2 -6033% 1.18E-04 PE 38:5 -1818% 8.62E-02
LPC P-16:0 -5876% 1.18E-04 CE 24:2 3641% 9.27E-02
PC 30:0 -4909% 1.18E-04 LacCer(dl8:l/24:0) -2225% 9.27E-02
SM 39:1 -4057% 1.18E-04 SM 33:1 -2440% 9.27E-02
LPC 18:l_sn2 -3902% 1.42E-04 TAG( 15:0/16:0/18:1) -3704% 9.27E-02
PC 0-36:4 -3822% 1.42E-04 TAG(16:0/16:0/16:0) -1593% 9.27E-02
PC 0-38:0 -2572% 1.42E-04 TAG(16:0/16:l/17:0) -3628% 9.27E-02
PE 0-40:4 -5697% 1.42E-04 TAG(18:0/18:l/18:l) 1660% 9.27E-02
SM 32:0 -4973% 1.42E-04 TAG(18:1/18:1/18:1) 2040% 9.27E-02
Cer(dl8:l/18:0) 17623% 1.71E-04 PC 0-40:4 -1649% 9.96E-02
PC 36:7 -6426% 1.71E-04 SM 38:3 -1146% 9.96E-02
PC 0-36:3 -5952% 1.71E-04 Cer(d20:l/22:0) 6204% 1.07E-01
PC P-36:3 -2661 % 1.71E-04 DAG 34:1 5041 % 1.07E-01
PE 0-34:2 -8823% 1.71E-04 LacCer(dl8:2/24:0) -2346% 1.07E-01
PE 0-36:2 -7510% 1.71E-04 LPI 20:4_snl 1780% 1.07E-01
SM 41:2 -3724% 1.71E-04 SM 40:3 1375% 1.07E-01
Cer(dl6:l/23:0) -5724% 2.05E-04 SM 42:0 -3794% 1.07E-01 PE 0-34:1 -6912 % 2.05E-04 Cer(dl8:l/14:0) -2494 % 1.14E-01
PE 0-38:6 -6542 % 2.05E-04 Cer(dl8:2/16:0) 578 % 1.14E-01
PE P-38:5 -6857 % 2.05E-04 PG 36:1 -2481 % 1.14E-01
PI 38:1 -6443 % 2.05E-04 TAG( 17:0/18: 1/18:1) 1646 % 1.14E-01
TAG(14:0/16:l/18:2) -6508 % 2.05E-04 PE 34:3 -3905 % 1.23E-01
LacCer(dl6:l/22:0) -6165 % 2.45E-04 TAG(14:0/18:2/18:2) -3729 % 1.23E-01
PC 38:7 -5842 % 2.45E-04 TAG(18:1/18:1/18:2) 2451 % 1.23E-01
PC P-38:3 -2978 % 2.45E-04 LacCer(dl8:2/24:l) -3102 % 1.31E-01
PE 0-36:1 -6819 % 2.45E-04 LPE 20:4_snl -1766 % 1.40E-01
Gb3(dl8:l/24:0) -4765 % 2.91E-04 PG 34:1 -2449 % 1.49E-01
PC 36:3 -6417 % 2.91E-04 SM 44:2 3650 % 1.49E-01
PC 37:2 -6188 % 2.91E-04 Cer(dl8:0/24:l) 1262 % 1.59E-01
SM 32:1 -3484 % 2.91E-04 DAG 36:3 5025 % 1.59E-01
Gb3(dl8:l/22:0) -3968 % 3.45E-04 LacCer(dl6:l/20:0) -2197 % 1.59E-01
PC 0-36:2 -6312 % 3.45E-04 PC 38:1 -1555 % 1.59E-01
LPC 20:2_snl -5403 % 4.07E-04 PC 40:2 2086 % 1.59E-01
LPC P-18:0 -5756 % 4.07E-04 DAG 36:4 3692 % 1.69E-01
PC 36:6 -6733 % 4.07E-04 LPC 0-22:1 -2900 % 1.69E-01
PC P-38:l -5002 % 4.07E-04 AcylCarnitine(14:0) 815 % 1.80E-01
PC(14:0/22:6) -5956 % 4.79E-04 DAG 36:4 7753 % 1.80E-01
CE 17:1 -5851 % 4.79E-04 LacCer(dl8:l/23:0) -2037 % 1.80E-01
LPC 18:0_snl -2738 % 4.79E-04 PE 39:6_snl -44 % 1.80E-01
PC 35:2 -4997 % 4.79E-04 PI 32:0 -2276 % 1.80E-01
PC 38:2 -4708 % 4.79E-04 PI 36:4 -2084 % 1.80E-01
PC 0-38:1 -3760 % 4.79E-04 TAG(16:1/18:1/18:1) 2212 % 2.03E-01
TAG(18:l/18:l/20:4) 5034 % 4.79E-04 TAG(18:2/18:2/20:4) 5461 % 2.03E-01
TAG(14:l/16:0/18:l) -6762 % 5.62E-04 AcylCarnitine(12:0) 2087 % 2.15E-01
TAG(16:l/16:l/18:0) -6908 % 5.62E-04 Cer(dl8:2/20:0) 2448 % 2.15E-01
LacCer(dl6:l/24:l) -3066 % 6.57E-04 PE 40:7 3115 % 2.28E-01
LPC 18:0_sn2 -3792 % 6.57E-04 CE 20:5 -3954 % 2.41E-01
LPC 20:3_sn2 -4715 % 6.57E-04 PE 35:l_snl -2659 % 2.41E-01
PC 36:0 -3329 % 6.57E-04 CE 24:1 3790 % 2.55E-01
PC 0-40:5 -4968 % 6.57E-04 DAG 38:6 4798 % 2.55E-01
PI 36:3 -6770 % 6.57E-04 LPI 20:4_sn2 785 % 2.55E-01
TAG(14:0/16:0/18:2) -6328 % 6.57E-04 TAG(16:0/18:2/18:2) 2290 % 2.55E-01
LPC(15-MHDA)_snl -4266 % 7.67E-04 PC 0-32:0 -1264 % 2.69E-01
PC 28:1 -5178 % 7.67E-04 PE 36:6 -3036 % 2.69E-01
PC 35:0 -6013 % 7.67E-04 AcylCarnitine(18:0) -3229 % 2.84E-01
PC 38:5 -4305 % 7.67E-04 Cer(dl8:0/24:0) -1322 % 2.84E-01
PE P-34:l -5103 % 7.67E-04 PE 37:4_sn2 4710 % 2.84E-01
PE P-36:4 -6205 % 7.67E-04 PE 42:6 -2341 % 2.84E-01
PI 38:5 -5317 % 7.67E-04 CE 12:0 -2003 % 2.99E-01
SM 41:1 -2748 % 7.67E-04 AcylCarnitine(18:3) -2024 % 3.15E-01
CE 18:0 -4841 % 8.92E-04 CE 22:1 -2465 % 3.15E-01
PC 31:1 -5648 % 8.92E-04 PE 38:4 2530 % 3.15E-01
PC(16:0/22:5) -3245 % 1.03E-03 SM 34:2 -899 % 3.15E-01
Gb3(dl8:l/23:0) -4359 % 1.03E-03 DAG 36:2 3860 % 3.32E-01
LPC 16:0_snl -3372 % 1.03E-03 PE 38:1 -1406 % 3.32E-01
LPC 20:2_sn2 -4734 % 1.03E-03 DAG 34:2 6393 % 3.48E-01 PC 36:2 -4127 % 1.O3E-O3 SM 33:2 -2320 % 3.48E-01
SM 31:0 -5499 % 1.03E-03 CE 24:3 4084 % 3.66E-01
TAG(14:0/18:0/18:l) -6867 % 1.03E-03 Cer(dl8:l/22:0) 624 % 3.66E-01
TAG(18:l/18:l/22:6) 5069 % 1.03E-03 PC P-38:6 -2181 % 3.66E-01
CE 14:1 -7922 % 1.20E-03 TAG(18:0/18:0/18:l) -1259 % 3.66E-01
LacCer(dl8:l/16:0) 2377 % 1.20E-03 PI 38:4 -1478 % 3.84E-01
LPC 22:l_snl -4037 % 1.20E-03 CE 16:0 -2046 % 4.02E-01
LPC 0-18:0 -3227 % 1.20E-03 CE 20:4 2961 % 4.02E-01
PC 35:5 -6498 % 1.20E-03 Cer(dl6:l/24:1) -2227 % 4.02E-01
PC 37:1 -5848 % 1.20E-03 PE 32:0 -941 % 4.02E-01
PE P-40:6 -4365 % 1.20E-03 PE 32:1 -986 % 4.02E-01
PI 40:5 -4590 % 1.20E-03 SM 38:2 -1109 % 4.02E-01
Gb3(dl8:l/18:0) -4647 % 1.38E-03 TAG(16:0/16:0/18:l) 2286 % 4.02E-01
PI 36:5 -5620 % 1.38E-03 CE 20:1 -1269 % 4.21E-01
PI 38:2 -5339 % 1.38E-03 Cer(d20:l/24:0) 3112 % 4.21E-01
LPE 16:0_sn2 -4262 % 1.59E-03 PE 40:8 -991 % 4.21E-01
PC 0-38:5 -2993 % 1.59E-03 CE 22:5 -379 % 4.40E-01
PE 0-38:5 -7150 % 1.59E-03 Cer(dl8:2/26:1) 272 % 4.40E-01
PI 40:4 -6535 % 1.59E-03 PC 34:1 -281 % 4.40E-01
LPE 18:2_snl -7479 % 1.82E-03 PE 42:10 -2049 % 4.60E-01
PC 38:3 -5520 % 1.82E-03 SM 35:2 -1438 % 4.60E-01
Cer(dl8:2/23:0) -3351 % 2.09E-03 DAG 36:1 4085 % 4.80E-01
Gb3(dl8:l/16:0) -3262 % 2.09E-03 LacCer(dl8:2/22:0) -530 % 4.80E-01
LPC 15:0_sn2 -5844 % 2.09E-03 LPC 0-20:1 -1257 % 4.80E-01
LPE 16:0_snl -3091 % 2.09E-03 PE 34:1 -216 % 4.80E-01
LPE 18:2_sn2 -7326 % 2.09E-03 PC(16:0/16:0) -965 % 5.01E-01
PI 36:1 -4973 % 2.09E-03 CE 19:1 -3990 % 5.01E-01
TAG(16:1/16:1/16:1) -6177 % 2.09E-03 AcylCarnitine(13:0) -3762 % 5.22E-01
Gb3(d 18: 1/24:1) -3723 % 2.38E-03 AcylCarnitine(16:0) 389 % 5.22E-01
PC 31:0 -7794 % 2.38E-03 PC 32:0 -998 % 5.22E-01
PC 0-38:6 -4407 % 2.38E-03 CE 22:2 -3352 % 5.44E-01
LPC 17:0_sn2 -5493 % 2.72E-03 DAG 32:2 -2365 % 5.44E-01
PC 36:1 -4695 % 2.72E-03 LacCer(dl6:l/18:0) -3724 % 5.44E-01
PC P-38:5 -3726 % 2.72E-03 SM 38:0 338 % 5.44E-01
Cer(d20:l/24:l) 14616 % 3.09E-03 AcylCarnitine(18:2) 2058 % 5.66E-01
PC 34:0 -3522 % 3.09E-03 PC 40:6 3214 % 5.66E-01
PC 34:2 -2522 % 3.09E-03 SM 33:0 -407 % 5.66E-01
PC 33:2 -5229 % 3.51E-03 TAG(16:0/16:0/18:2) 1188 % 5.66E-01
TAG(14:l/16:l/18:0) -4943 % 3.51E-03 TAG(16:0/17:0/18:l) -1887 % 5.66E-01
Cer(dl8:0/16:0) 6810 % 3.97E-03 CE 24:4 -2020 % 5.88E-01
PC 32:1 -3780 % 3.97E-03 Cer(dl6:l/16:0) 320 % 6.11E-01
PC 39:4 -3116 % 3.97E-03 LacCer(dl8:l/22:0) -1613 % 6.11E-01
PC 40:5 -1662 % 3.97E-03 LPI 18:0_sn2 524 % 6.11E-01
CE 18:2 -4028 % 4.49E-03 LPI 18:2_sn2 1603 % 6.11E-01
LacCer(dl6:l/24:0) -4831 % 4.49E-03 TAG(16:1/16:1/18:1) -1912 % 6.11E-01
LPC 15:0_snl -5663 % 4.49E-03 LacCer(dl8:l/20:0) 3146 % 6.35E-01
PC 33:3 -7943 % 4.49E-03 PE 39:6_sn2 -4499 % 6.35E-01
PE P-38:6 -4438 % 4.49E-03 PI 38:6 -936 % 6.35E-01
SM 40:2 -1375 % 4.49E-03 CE 24:5 1662 % 6.58E-01 TAG(14:1/18:1/18:1) -2450 % 4.49E-03 LacCer(dl8:l/24:l) 1524 % 6.58E-01
AcylCarnitine(14:l) 9988 % 5.06E-03 PC 35:l_sn2 -420 % 6.58E-01
CE 15:0 -4812 % 5.06E-03 PE 0-40:1 960 % 6.58E-01
Cer(dl8:2/26:0) -4705 % 5.06E-03 PE 30:2 -113 % 6.82E-01
LPC 16:0_sn2 -3394 % 5.06E-03 Cer(dl8:2/22:0) -2035 % 7.06E-01
PE 36:3 -6574 % 5.06E-03 LPE 22:6_sn2 130 % 7.06E-01
Gb3(dl8:l/20:0) -4350 % 5.70E-03 DAG 32:1 -1452 % 7.31E-01
LPE 18:l_snl -6386 % 5.70E-03 Cer(dl6:l/20:0) 474 % 7.56E-01
PC 40:4 -2682 % 5.70E-03 Cer(dl8:2/24:1) -559 % 7.56E-01
PE 36:2 -4884 % 5.70E-03 LacCer(dl6:l/16:0) 950 % 7.56E-01
PI 34:1 -4143 % 5.70E-03 LPC 20:4_sn2 -412 % 7.56E-01
CE 16:1 -5081 % 6.40E-03 LPC 22:6_sn2 -52 % 7.56E-01
Cer(dl6:l/22:0) -3787 % 6.40E-03 PI 36:2 -1853 % 7.56E-01
LPE P-18:l -6007 % 6.40E-03 CE 22:0 -148 % 7.81E-01
PE 36:5 -3606 % 6.40E-03 TAG(16:0/17:0/18:2) -1486 % 7.81E-01
SM 36:2 2240 % 6.40E-03 TAG(18:2/18:2/18:2) 2321 % 8.06E-01
LPE P-20:0 -2862 % 7.18E-03 DAG 34:2 2437 % 8.31E-01
DAG 38:4 5738 % 8.03E-03 DAG 38:4 -2236 % 8.31E-01
LPC 20:5_sn2 -4980 % 8.03E-03 LPE 22:6_snl 1252 % 8.31E-01
LPE 18:l_sn2 -5750 % 8.03E-03 PC 38:4 206 % 8.31E-01
PC 0-40:1 -1845 % 8.03E-03 SM 36:3 92 % 8.31E-01
PC P-40:5 -1690 % 8.03E-03 TAG(16:0/18:0/18:l) -11 % 8.31E-01
LPC 20:5_snl -5653 % 8.97E-03 Cer(d20:l/23:0) 1564 % 8.57E-01
LPC P-20:0 -4526 % 8.97E-03 LPC 20:4_snl -1412 % 8.57E-01
LPE P-16:0 -4924 % 8.97E-03 LPI 18:0_snl 274 % 8.57E-01
TAG(16:0/18:l/18:2) 4555 % 8.97E-03 PC 39:6_snl 2687 % 8.57E-01
Cer(dl8:l/26:0) -3680 % 1.00E-02 PC 0-32:1 -1452 % 8.57E-01
LPE P-18:0 -5086 % 1.00E-02 PE 40:5 -268 % 8.57E-01
PI 34:0 -3516 % 1.00E-02 SM 44:3 -114 % 8.57E-01
SM 37:0 -1934 % 1.00E-02 TAG(16:1/18:1/18:2) 990 % 8.57E-01
SM 38:1 -2226 % 1.00E-02 Cer(dl8:0/22:0) -1175 % 8.83E-01
CE 20:3 -3926 % 1.11E-02 PC 39:2 453 % 8.83E-01
LPC 20:l_sn2 -2880 % 1.11E-02 PC 39:5 -1694 % 8.83E-01
PE 38:6 5199 % 1.11E-02 SM 34:0 -983 % 8.83E-01
CE 18:1 -4013 % 1.24E-02 TAG(18:0/18:2/18:2) 47 % 8.83E-01
Cer(dl8:2/24:0) -3281 % 1.24E-02 DAG 36:4 1088 % 9.09E-01
LPC 22:4_sn2 -2548 % 1.24E-02 PC 40:3 -1434 % 9.09E-01
LPE 18:0_sn2 -3050 % 1.24E-02 LPC 22:6_snl -67 % 9.35E-01
SM 36:0 9951 % 1.24E-02 LPI 18:2_snl 455 % 9.35E-01
SM 36:1 3029 % 1.24E-02 PE 30:1 764 % 9.35E-01
Cer(dl8:l/20:0) 5955 % 1.37E-02 PE 35:2_sn2 1672 % 9.35E-01
PC 35:4 -3905 % 1.37E-02 SM 34:1 127 % 9.35E-01
PG 34:2 -3851 % 1.37E-02 TAG( 15:0/18: 1/18:1) -989 % 9.35E-01
Cer(dl8:l/24:0) -2828 % 1.52E-02 TAG(18:1/18:2/18:2) -10 % 9.61E-01
LacCer(dl8:l/18:0) 3462 % 1.52E-02 DAG 36:5 -385 % 9.87E-01
LPC 22:l_sn2 -3673 % 1.52E-02 PC 0-40:2 43 % 9.87E-01
LPC 22:4_snl -3324 % 1.52E-02 SM 42:3 52 % 9.87E-01
LPC 17:0_snl -3398 % 1.68E-02 CE 22:6 616 % l.OOE+OO
LPC 0-20:0 -3907 % 1.68E-02 PE 40:3 3067 % l.OOE+OO
Figure imgf000046_0001
Table 3 shows the predictive value of examples of pulmonary biomarker ratios. Median relative change and p-value are presented.
Table 3. Example of lipid ratios, median relative change and p-value. Abbreviations are described in the detailed description of the present disclosure.
Figure imgf000046_0002
Cer(dl8:l/18:0)/PC 36:7 654 3.78E-08 Cer(dl8:l/20:0)/PC(16:0/16:0) 71
Cer(dl8:l/24:l)/PC 34:3 244 3.78E-08 Cer(dl8:l/20:0)/LPC 18:l_snl 223
Cer(dl8:l/20:0)/PC 34:3 255 3.78E-08 Cer(dl8:l/20:0)/LPC 18:l_sn2 248
Cer(dl8:l/20:0)/PC 36:7 318 3.78E-08 Cer(dl8:l/20:0)/LPC 18:2_sn2 358
PC 39:6_snl/PC(14:0/22:6) 220 3.78E-08 Cer(dl8:l/20:0)/LPC 20:l_snl 118
Cer(dl8:l/18:0)/PC 32:3 909 6.61E-08 Cer(dl8:l/20:0)/LPC 22:4_snl 93
PC 38:4/PC 34:3 109 6.61E-08 Cer(dl8:l/20:0)/PC 35:5 383
PC 39:6_snl/PC 36:6 228 6.61E-08 Cer(dl8:l/20:0)/PC 0-40:5 169
PC 39:6_snl/PC 38:7 106 6.61E-08 Cer(dl8:l/22:0)/LPC 22:0_sn2 238
PC 40:6/PC 38:7 127 6.61E-08 Cer(dl8:l/26:l)/Cer(dl8:l/23:0) 73
Cer(dl8:l/18:0)/PC 32:2 845 1.13E-07 Cer(dl8:l/26:l)/LPC 18:2_sn2 221
Cer(dl8:l/18:0)/PC 35:3 446 1.13E-07 Cer(dl8:l/26:l)/PC 0-38:6 119
Cer(dl8:l/24:l)/PC 35:3 256 1.13E-07 Cer(dl8:l/16:0)/LPC 17:l_snl 311
Cer(dl8:l/24:l)/PC 36:7 302 1.13E-07 Cer(dl8:l/16:0)/PC 31:1 326
Cer(dl8:l/22:0)/PC 34:3 167 1.13E-07 Cer(dl8:l/16:0)/PC 33:1 157
PC 40:5/PC 32:2 209 1.13E-07 Cer(dl8:l/16:0)/PC 0-34:1 84
Cer(dl8:l/16:0)/PC 34:3 220 1.79E-07 Cer(dl8:l/18:0)/PC P-40:6 327
Cer(dl8:l/18:0)/PC 30:2 1696 1.79E-07 Cer(dl8:l/24:l)/PC 0-34:1 77
PC 40:6/PC 32:2 230 1.79E-07 Cer(dl8:l/24:l)/PC 0-40:5 130
Cer(dl8:l/18:0)/PC 33:2 366 2.83E-07 Cer(dl8:l/20:0)/Cer(dl8:l/26:0) 115
Cer(dl8:l/18:0)/PC 35:2 589 2.83E-07 Cer(dl8:l/20:0)/PC 38:0 107
Cer(dl8:l/18:0)/PC 36:1 334 2.83E-07 Cer(dl8:l/20:0)/PC 39:4 168
Cer(dl8:l/18:0)/PC 36:3 582 2.83E-07 Cer(dl8:l/20:0)/PC P-38:4 384
Cer(dl8:l/24:l)/PC 30:2 940 2.83E-07 Cer(dl8:l/22:0)/LPC 17:l_snl 249
Cer(dl8:l/24:l)/PC 32:2 416 2.83E-07 Cer(dl8:l/22:0)/LPC 17:l_sn2 226
Cer(dl8:l/24:l)/PC 37:2 271 2.83E-07 Cer(dl8:l/22:0)/LPC 20:2_sn2 116
Cer(dl8:l/24:l)/PC 38:3 120 2.83E-07 Cer(dl8:l/22:0)/PC 35:2 186
Cer(dl8:l/20:0)/PC 30:2 921 2.83E-07 Cer(dl8:l/26:l)/LPC 20:3_sn2 129
PC 39:6_snl/PC 32:2 205 2.83E-07 Cer(dl8:l/26:l)/PC 38:3 173
PC 40:5/PC 30:2 605 2.83E-07 PC 38:4/PC 38:5 75
PC 40:5/PC 34:4 311 2.83E-07 PC 40:5/PC 38:2 93
PC 40:5/PC 38:7 127 2.83E-07 Cer(dl8:l/16:0)/LPC 20:2_sn2 179
PC 40:6/PC 30:2 621 2.83E-07 Cer(dl8:l/16:0)/PC 36:2 151
Cer(dl8:l/16:0)/PC 32:2 501 4.25E-07 Cer(dl8:l/16:0)/PC 40:8 296
Cer(dl8:l/16:0)/PC 35:3 254 4.25E-07 Cer(dl8:l/16:0)/PC 0-36:4 154
Cer(dl8:l/16:0)/PC 36:7 246 4.25E-07 Cer(dl8:l/16:0)/PC 0-38:1 115
Cer(dl8:l/18:0)/PC 33:1 347 4.25E-07 Cer(dl8:l/16:0)/PC P-36:l 214
Cer(dl8:l/18:0)/PC 33:3 619 4.25E-07 Cer(dl8:l/18:0)/LPC 22:l_snl 413
Cer(dl8:l/18:0)/PC 37:1 274 4.25E-07 Cer(dl8:l/24:l)/PC 38:7 380
Cer(dl8:l/18:0)/PC 37:3 363 4.25E-07 Cer(dl8:l/24:l)/PC 0-40:4 67
Cer(dl8:l/18:0)/PC 38:3 432 4.25E-07 Cer(dl8:l/20:0)/PC(16:0/22:5) 115
Cer(dl8:l/18:0)/PC P-36:4 621 4.25E-07 Cer(dl8:l/20:0)/LPC 18:2_snl 342
Cer(dl8:l/24:l)/PC 36:3 272 4.25E-07 Cer(dl8:l/20:0)/LPC 22:4_sn2 140
Cer(dl8:l/20:0)/PC 32:3 662 4.25E-07 Cer(dl8:l/20:0)/PC 31:0 730
Cer(dl8:l/20:0)/PC 37:2 224 4.25E-07 Cer(dl8:l/20:0)/PC 40:1 130
Cer(dl8:l/22:0)/LPC(15-
Cer(dl8:l/20:0)/PC 38:3 176 4.25E-07 MHDA)_sn2 257
Cer(dl8:l/22:0)/PC 36:7 T1 4.25E-07 Cer(dl8:l/22:0)/PC P-38:4 201
Cer(dl8:l/26:l)/PC P-34:l 198 4.25E-07 Cer(dl8:l/26:l)/LPC 15:0_sn2 198
Figure imgf000047_0001
Cer(dl8:l/26:l)/PC P-36:2 392 4.25E-07 Cer(dl8:l/26:l)/LPC 16:0_snl 115
PC 38:4/PC 30:2 504 4.25E-07 Cer(dl8:l/26:l)/LPC 18:0_snl 80
PC 39:6_snl/PC 34:5 684 4.25E-07 Cer(dl8:l/26:l)/LPC 20:l_sn2 95
PC 40:6/PC 34:4 322 4.25E-07 PC 38:4/PC 36:6 300
Cer(dl8:l/16:0)/PC 37:2 202 6.32E-07 PC 38:4/PC 36:7 271
Cer(dl8:l/18:0)/PC 30:0 402 6.32E-07 PC 40:2/PC 30:0 174
Cer(dl8:l/18:0)/PC 30:1 670 6.32E-07 PC 40:2/PC 38:1 34
Cer(dl8:l/18:0)/PC 32:1 376 6.32E-07 Cer(dl8:l/16:0)/PC P-32:0 223
Cer(dl8:l/18:0)/PC 34:4 1313 6.32E-07 Cer(dl8:l/18:0)/LPC 22:5_sn2 258
Cer(dl8:l/18:0)/PC 37:2 428 6.32E-07 Cer(dl8:l/18:0)/PC 39:6_sn2 465
Cer(dl8:l/18:0)/PC 40:4 H 6.32E-07 Cer(dl8:l/18:0)/PC P-40:3 339
Cer(dl8:l/18:0)/PC 40:5 287 6.32E-07 Cer(dl8:l/24:l)/Cer(dl8:l/23:0) 79
Cer(dl8:l/18:0)/PC 0-40:1 234 6.32E-07 Cer(dl8:l/24:l)/PC 28:1 149
Cer(dl8:l/18:0)/PC P-32:l 584 6.32E-07 Cer(dl8:l/24:l)/PC 35:5 359
Cer(dl8:l/24:l)/PC 38:2 212 6.32E-07 Cer(dl8:l/20:0)/PC 0-38:6 145
Cer(dl8:l/20:0)/PC 36:1 154 6.32E-07 Cer(dl8:l/22:0)/LPC 24:0_sn2 82
Cer(dl8:l/22:0)/PC 30:2 716 6.32E-07 Cer(dl8:l/22:0)/PC 33:3 287
Cer(dl8:l/22:0)/PC 32:2 351 6.32E-07 Cer(dl8:l/22:0)/PC 40:8 234
Cer(dl8:l/26:l)/PC 30:1 266 6.32E-07 Cer(dl8:l/26:l)/Cer(dl8:l/24:0) 60
Cer(dl8:l/26:l)/PC 30:2 717 6.32E-07 Cer(dl8:l/26:l)/PC 40:5 63
Cer(dl8:l/26:l)/PC P-34:2 404 6.32E-07 Cer(dl8:l/26:l)/PC 0-38:4 182
PC 38:4/PC 38:6 192 6.32E-07 PC 38:4/PC(14:0/22:6) 173
PC 39:6_snl/PC 28:0 682 6.32E-07 PC 39:2/PC 31:1 77
PC 39:6_snl/PC 30:2 798 6.32E-07 PC 39:6_snl/PC 35:3 248
PC 39:6_snl/PC 34:4 287 6.32E-07 PC 40:2/PC(16:0/22:5) 116
PC 40:5/PC 28:0 555 6.32E-07 PC 40:2/PC 35:5 324
PC 40:6/PC 36:6 312 6.32E-07 PC 40:2/PC 40:1 80
PC 40:6/PC 40:8 172 6.32E-07 PC 40:6/PC 38:2 131
Cer(dl8:l/16:0)/PC 36:1 144 9.15E-07 Cer(dl8:l/18:0)/PC 0-40:6 586
Cer(dl8:l/16:0)/PC 36:3 206 9.15E-07 Cer(dl8:l/24:l)/LPC 22:4_sn2 102
Cer(dl8:l/16:0)/PC 37:1 149 9.15E-07 Cer(dl8:l/24:l)/PC P-36:3 84
Cer(dl8:l/16:0)/PC 38:3 188 9.15E-07 Cer(dl8:l/20:0)/LPC 18:0_sn2 232
Cer(dl8:l/18:0)/LPC 20:3_snl 461 9.15E-07 Cer(dl8:l/22:0)/LPC 24:0_snl 80
Cer(dl8:l/18:0)/PC 28:0 1506 9.15E-07 Cer(dl8:l/22:0)/PC 30:0 206
Cer(dl8:l/18:0)/PC 31:1 655 9.15E-07 Cer(dl8:l/22:0)/PC 36:0 93
Cer(dl8:l/18:0)/PC 38:2 384 9.15E-07 Cer(dl8:l/26:l)/LPC 20:l_snl 109
Cer(dl8:l/18:0)/PC 0-36:1 339 9.15E-07 Cer(dl8:l/26:l)/LPC 22:4_sn2 103
Cer(dl8:l/26:l)/LPC(15-
Cer(dl8:l/20:0)/PC 32:2 467 9.15E-07 MHDA)_snl 95
Cer(dl8:l/20:0)/PC 35:3 360 9.15E-07 Cer(dl8:l/26:l)/PC 31:1 214
Cer(dl8:l/20:0)/PC 38:2 191 9.15E-07 Cer(dl8:l/26:l)/PC 0-38:5 108
Cer(dl8:l/20:0)/PC 0-38:3 243 9.15E-07 Cer(dl8:l/26:l)/PC P-38:l 167
Cer(dl8:l/20:0)/PC P-34:2 385 9.15E-07 Cer(dl8:l/26:l)/PC P-38:3 93
Cer(dl8:l/20:0)/PC P-38:2 262 9.15E-07 PC 39:2/PC 33:3 191
Cer(dl8:l/26:l)/LPC 22:0_snl 236 9.15E-07 PC 39:6_snl/PC 34:3 200
Cer(dl8:l/26:l)/PC 32:2 535 9.15E-07 PC 39:6_snl/PC 36:5 116
Cer(dl8:l/26:l)/PC 34:3 211 9.15E-07 PC 40:5/PC 33:3 133
Cer(dl8:l/26:l)/PC 34:5 811 9.15E-07 PC 40:6/PC 35:0 115
Cer(dl8:l/26:l)/PC P-32:l 150 9.15E-07 Cer(dl8:l/18:0)/LPC 22:5_snl T1
Figure imgf000048_0001
Cer(dl8:l/26:l)/PC P-36:l 107 9.15E-07 Cer(dl8:l/18:0)/PC P-38:l 357
PC 38:4/PC 32:2 323 9.15E-07 Cer(dl8:l/24:l)/LPC 15:0_sn2 164
PC 40:2/PC 30:2 548 9.15E-07 Cer(dl8:l/20:0)/LPC 18:0_snl 173
PC 40:5/PC 34:5 616 9.15E-07 Cer(dl8:l/20:0)/LPC 20:l_sn2 118
PC 40:5/PC 40:8 147 9.15E-07 Cer(dl8:l/20:0)/PC 32:0 80
PC 40:6/PC(14:0/22:6) 274 9.15E-07 Cer(dl8:l/20:0)/PC P-38:5 119
PC 40:6/PC 34:5 786 9.15E-07 Cer(dl8:l/22:0)/PC 37:3 72
Cer(dl8:l/16:0)/PC 30:2 1152 1.30E-06 Cer(dl8:l/22:0)/PC 40:4 80
Cer(dl8:l/18:0)/LPC 22:4_sn2 213 1.30E-06 Cer(dl8:l/26:l)/LPC 22:l_snl 186
Cer(dl8:l/18:0)/PC 35:0 785 1.30E-06 Cer(dl8:l/26:l)/LPC 22:l_sn2 111
Cer(dl8:l/18:0)/PC 36:0 336 1.30E-06 Cer(dl8:l/26:l)/PC 39:0 48
Cer(dl8:l/18:0)/PC 36:2 319 1.30E-06 Cer(dl8:l/26:l)/PC P-36:3 90
Cer(dl8:l/18:0)/PC 36:5 794 1.30E-06 PC 38:4/PC 38:2 96
Cer(dl8:l/18:0)/PC 38:0 282 1.30E-06 PC 38:4/PC 39:4 58
Cer(dl8:l/18:0)/PC 0-36:3 600 1.30E-06 PC 40:2/PC 39:4 69
Cer(dl8:l/18:0)/PC 0-38:4 380 1.30E-06 Cer(dl8:l/16:0)/LPC 22:0_sn2 446
Cer(dl8:l/18:0)/PC P-36:2 796 1.30E-06 Cer(dl8:l/16:0)/LPC 22:4_sn2 64
Cer(dl8:l/18:0)/PC P-38:2 554 1.30E-06 Cer(dl8:l/16:0)/PC 0-38:5 116
Cer(dl8:l/18:0)/PC P-38:4 725 1.30E-06 Cer(dl8:l/16:0)/PC P-38:4 332
Cer(dl8:l/24:l)/PC 38:6 325 1.30E-06 Cer(dl8:l/20:0)/Cer(dl8:l/14:0) 96
Cer(dl8:l/24:l)/PC P-36:4 204 1.30E-06 Cer(dl8:l/20:0)/LPC 15:0_sn2 307
Cer(dl8:l/20:0)/LPC(15-
Cer(dl8:l/24:l)/PC P-38:2 189 1.30E-06 MHDA)_snl 272
Cer(dl8:l/22:0)/PC 0-38:3 135 1.30E-06 Cer(dl8:l/20:0)/PC 34:2 123
Cer(dl8:l/22:0)/PC P-36:2 216 1.30E-06 Cer(dl8:l/22:0)/LPC 18:l_sn2 106
Cer(dl8:l/22:0)/PC P-38:2 136 1.30E-06 Cer(dl8:l/22:0)/PC 36:6 457
Cer(dl8:l/26:l)/PC 28:0 1019 1.30E-06 Cer(dl8:l/22:0)/PC 0-34:0 58
Cer(dl8:l/26:l)/PC 38:6 453 1.30E-06 Cer(dl8:l/22:0)/PC P-38:3 87
PC 39:6_snl/PC 40:8 120 1.30E-06 Cer(dl8:l/26:l)/PC P-40:6 67
PC 40:2/PC 32:2 473 1.30E-06 PC 40:5/PC 30:0 113
PC 40:6/PC 28:0 610 1.30E-06 PC 40:6/PC 30:0 107
PC 40:6/PC 32:3 454 1.30E-06 Cer(dl8:l/16:0)/LPC 22:4_snl 76
Cer(dl8:l/16:0)/PC 30:1 419 1.81E-06 Cer(dl8:l/16:0)/LPC 24:0_snl 192
Cer(dl8:l/18:0)/LPC 14:0_snl 1221 1.81E-06 Cer(dl8:l/16:0)/PC 31:0 504
Cer(dl8:l/18:0)/LPC 14:0_sn2 1177 1.81E-06 Cer(dl8:l/24:l)/LPC 18:2_snl 216
Cer(dl8:l/18:0)/LPC 20:3_sn2 399 1.81E-06 Cer(dl8:l/20:0)/PC 38:1 74
Cer(dl8:l/18:0)/PC 34:5 2573 1.81E-06 Cer(dl8:l/22:0)/LPC 18:2_sn2 199
Cer(dl8:l/18:0)/PC 38:6 1060 1.81E-06 Cer(dl8:l/22:0)/PC 33:2 84
Cer(dl8:l/18:0)/PC 0-34:1 233 1.81E-06 Cer(dl8:l/26:l)/PC 39:4 106
Cer(dl8:l/18:0)/PC 0-36:2 281 1.81E-06 PC 38:4/PC 36:2 60
Cer(dl8:l/18:0)/PC 0-38:3 493 1.81E-06 PC 39:2/PC 31:0 196
Cer(dl8:l/18:0)/PC P-34:l 543 1.81E-06 PC 39:2/PC 34:3 139
Cer(dl8:l/18:0)/PC P-34:2 694 1.81E-06 PC 39:2/PC 37:2 180
Cer(dl8:l/24:l)/PC 28:0 975 1.81E-06 PC 39:6_snl/PC 39:6_sn2 66
Cer(dl8:l/24:l)/PC 30:1 411 1.81E-06 PC 40:5/PC 35:0 124
Cer(dl8:l/24:l)/PC 36:1 178 1.81E-06 PC 40:5/PC 36:7 257
Cer(dl8:l/20:0)/PC 30:1 412 1.81E-06 PC 40:6/PC 36:7 263
Cer(dl8:l/20:0)/PC 37:1 156 1.81E-06 Cer(dl8:l/16:0)/LPC 18:l_sn2 214
Cer(dl8:l/20:0)/PC 0-36:2 322 1.81E-06 Cer(dl8:l/16:0)/LPC 18:2_sn2 352
Figure imgf000049_0001
Cer(dl8:l/20:0)/PC P-36:2 376 1.81E-06 Cer(dl8:l/16:0)/LPC 24:0_sn2 176
Cer(dl8:l/22:0)/PC 38:3 95 1.81E-06 Cer(dl8:l/16:0)/PC 34:2 115
Cer(dl8:l/22:0)/PC P-34:2 241 1.81E-06 Cer(dl8:l/18:0)/LPC 20:5_sn2 681
Cer(dl8:l/26:l)/PC 40:8 161 1.81E-06 Cer(dl8:l/24:l)/LPC 22:4_snl 117
Cer(dl8:l/26:l)/PC P-36:4 166 1.81E-06 Cer(dl8:l/24:l)/PC 31:0 399
Cer(dl8:l/26:l)/PC P-36:5 257 1.81E-06 Cer(dl8:l/24:l)/PC P-38:l 146
PC 38:4/PC 34:4 339 1.81E-06 Cer(dl8:l/20:0)/PC 0-40:4 127
PC 39:6_snl/PC 32:3 593 1.81E-06 Cer(dl8:l/22:0)/PC(14:0/22:6) 438
PC 40:2/PC 28:0 911 1.81E-06 Cer(dl8:l/22:0)/Cer(dl8:l/23:0) 29
PC 40:2/PC 34:3 152 1.81E-06 Cer(dl8:l/22:0)/PC 31:1 229
PC 40:5/PC(14:0/22:6) 236 1.81E-06 Cer(dl8:l/22:0)/PC P-32:0 87
PC 40:5/PC 30:1 225 1.81E-06 Cer(dl8:l/26:l)/LPC 20:5_sn2 156
PC 40:5/PC 32:3 514 1.81E-06 Cer(dl8:l/26:l)/LPC 22:4_snl 80
PC 40:5/PC 36:6 303 1.81E-06 Cer(dl8:l/26:l)/PC 36:1 151
Cer(dl8:l/16:0)/PC P-36:2 370 2.50E-06 Cer(dl8:l/26:l)/PC 0-34:0 78
Cer(dl8:l/16:0)/PC P-38:2 222 2.50E-06 PC 38:4/PC(16:0/22:5) 51
Cer(dl8:l/18:0)/LPC 17:l_snl 905 2.50E-06 PC 40:2/PC 35:0 190
Cer(dl8:l/18:0)/PC 34:0 303 2.50E-06 Cer(dl8:l/16:0)/LPC 18:2_snl 313
Cer(dl8:l/18:0)/PC 34:2 310 2.50E-06 Cer(dl8:l/16:0)/PC 28:1 258
Cer(dl8:l/18:0)/PC 0-34:0 347 2.50E-06 Cer(dl8:l/16:0)/PC P-38:l 155
Cer(dl8:l/18:0)/PC 0-38:0 367 2.50E-06 Cer(dl8:l/18:0)/LPC 20:5_snl 683
Cer(dl8:l/18:0)/PC 0-38:1 264 2.50E-06 Cer(dl8:l/18:0)/LPC 22:l_sn2 345
Cer(dl8:l/24:l)/PC 37:1 165 2.50E-06 Cer(dl8:l/24:l)/LPC 17:0_sn2 187
Cer(dl8:l/24:l)/PC P-32:l 176 2.50E-06 Cer(dl8:l/22:0)/PC 38:5 138
Cer(dl8:l/24:l)/PC P-36:2 323 2.50E-06 Cer(dl8:l/26:l)/LPC 15:0_snl T1
Cer(dl8:l/20:0)/PC 36:0 140 2.50E-06 Cer(dl8:l/26:l)/LPC 17:0_sn2 174
Cer(dl8:l/20:0)/PC 36:3 322 2.50E-06 Cer(dl8:l/26:l)/PC 31:0 362
Cer(dl8:l/22:0)/PC 35:3 174 2.50E-06 Cer(dl8:l/26:l)/PC 0-38:2 74
Cer(dl8:l/22:0)/PC 36:1 102 2.50E-06 PC 39:2/PC 33:2 37
Cer(dl8:l/22:0)/PC 36:3 157 2.50E-06 PC 39:2/PC 38:5 119
Cer(dl8:l/22:0)/PC 38:2 133 2.50E-06 PC 39:6_snl/PC 33:2 56
Cer(dl8:l/26:l)/LPC 14:0_sn2 351 2.50E-06 PC 39:6_snl/PC 35:2 113
Cer(dl8:l/26:l)/PC 32:3 1064 2.50E-06 PC 40:2/PC 33:3 356
PC 40:2/PC 34:4 448 2.50E-06 PC 40:2/PC 35:2 103
PC 40:2/PC 34:5 784 2.50E-06 PC 40:5/PC 33:2 47
PC 40:2/PC 38:6 299 2.50E-06 Cer(dl8:l/16:0)/LPC 18:l_snl 210
Cer(dl8:l/16:0)/PC 28:0 941 3.39E-06 Cer(dl8:l/16:0)/PC P-36:3 98
Cer(dl8:l/16:0)/PC 32:3 563 3.39E-06 Cer(dl8:l/18:0)/PC 37:6 440
Cer(dl8:l/16:0)/PC 0-38:3 210 3.39E-06 Cer(dl8:l/24:l)/LPC 20:l_snl 119
Cer(dl8:l/18:0)/Cer(dl8:l/26:0) 246 3.39E-06 Cer(dl8:l/22:0)/PC 32:1 129
Cer(dl8:l/18:0)/PC 0-34:2 661 3.39E-06 Cer(dl8:l/22:0)/PC 35:5 237
Cer(dl8:l/18:0)/PC 0-36:4 338 3.39E-06 Cer(dl8:l/22:0)/PC 0-40:5 101
Cer(dl8:l/18:0)/PC 0-38:5 274 3.39E-06 Cer(dl8:l/26:l)/PC 40:4 122
Cer(dl8:l/18:0)/PC P-32:0 444 3.39E-06 PC 38:4/PC 36:0 47
Cer(dl8:l/24:l)/PC 34:4 478 3.39E-06 PC 39:2/PC 36:5 171
Cer(dl8:l/24:l)/PC 36:5 296 3.39E-06 PC 39:6_snl/PC 30:0 108
Cer(dl8:l/24:l)/PC 0-36:3 296 3.39E-06 PC 39:6_snl/PC 31:0 207
Cer(dl8:l/24:l)/PC 0-38:3 194 3.39E-06 PC 39:6_snl/PC 35:0 107
Cer(dl8:l/20:0)/PC 28:0 1015 3.39E-06 PC 40:6/PC 36:3 170
Figure imgf000050_0001
Cer(dl8:l/20:0)/PC 33:3 247 3.39E-06 Cer(dl8:l/16:0)/LPC 15:0_sn2 319
Cer(dl8:l/20:0)/PC 34:5 1360 3.39E-06 Cer(dl8:l/16:0)/LPC 20:l_snl 83
Cer(dl8:l/20:0)/PC 38:6 540 3.39E-06 Cer(dl8:l/16:0)/PC 35:5 514
Cer(dl8:l/20:0)/PC 0-34:2 356 3.39E-06 Cer(dl8:l/16:0)/PC 0-40:5 126
Cer(dl8:l/20:0)/PC 0-38:4 156 3.39E-06 Cer(dl8:l/24:l)/LPC 20:l_sn2 119
Cer(dl8:l/22:0)/PC 37:2 148 3.39E-06 Cer(dl8:l/24:l)/PC 0-38:6 134
Cer(dl8:l/26:l)/LPC 22:0_sn2 266 3.39E-06 Cer(dl8:l/24:l)/PC P-38:5 113
PC 39:2/PC 32:2 206 3.39E-06 Cer(dl8:l/20:0)/LPC 15:0_snl 302
PC 40:2/PC 36:0 78 3.39E-06 Cer(dl8:l/20:0)/PC 34:1 94
PC 40:2/PC 40:8 192 3.39E-06 Cer(dl8:l/20:0)/PC 0-32:0 58
Cer(dl8:l/16:0)/PC P-34:2 361 4.55E-06 Cer(dl8:l/20:0)/PC P-40:4 146
Cer(dl8:l/18:0)/LPC 17:l_sn2 901 4.55E-06 Cer(dl8:l/22:0)/PC 0-40:1 71
Cer(dl8:l/18:0)/LPC 24:0_snl 411 4.55E-06 Cer(dl8:l/26:l)/LPC 17:0_snl 73
Cer(dl8:l/24:l)/LPC 14:0_sn2 406 4.55E-06 PC 38:4/PC 35:5 226
Cer(dl8:l/24:l)/PC 32:3 645 4.55E-06 PC 38:4/PC 40:5 T1
Cer(dl8:l/24:l)/PC 0-36:2 234 4.55E-06 PC 39:2/PC(14:0/22:6) 288
Cer(dl8:l/24:l)/PC P-34:l 175 4.55E-06 PC 39:2/PC 36:6 269
Cer(dl8:l/24:l)/PC P-34:2 273 4.55E-06 PC 40:2/PC 31:0 368
Cer(dl8:l/20:0)/LPC 14:0_sn2 560 4.55E-06 PC 40:5/PC 37:1 82
Cer(dl8:l/20:0)/PC 36:5 420 4.55E-06 PC 40:6/PC 31:0 179
Cer(dl8:l/20:0)/PC 37:3 166 4.55E-06 PC 40:6/PC 31:1 103
Cer(dl8:l/20:0)/PC 0-36:3 348 4.55E-06 Cer(dl8:l/16:0)/Cer(dl8:l/23:0) 113
Cer(dl8:l/20:0)/PC 0-38:0 150 4.55E-06 Cer(dl8:l/24:l)/LPC 15:0_snl 164
Cer(dl8:l/20:0)/PC P-34:l 230 4.55E-06 Cer(dl8:l/24:l)/LPC 17:0_snl 104
Cer(dl8:l/20:0)/PC P-36:4 255 4.55E-06 Cer(dl8:l/24:l)/LPC 18:0_sn2 124
Cer(dl8:l/22:0)/PC 38:6 297 4.55E-06 Cer(dl8:l/20:0)/PC P-40:6 161
Cer(dl8:l/26:l)/LPC 14:0_snl 345 4.55E-06 Cer(dl8:l/22:0)/LPC 18:2_snl 175
Cer(dl8:l/26:l)/LPC 19:0_sn2 196 4.55E-06 Cer(dl8:l/22:0)/PC 38:7 236
Cer(dl8:l/26:l)/LPC 24:0_snl 216 4.55E-06 Cer(dl8:l/22:0)/PC 40:5 77
Cer(dl8:l/26:l)/LPC 24:0_sn2 212 4.55E-06 PC 38:4/PC 35:0 118
Cer(dl8:l/26:l)/PC 34:4 447 4.55E-06 PC 39:2/PC 36:7 239
Cer(dl8:l/26:l)/PC 35:3 113 4.55E-06 PC 39:2/PC 38:7 173
Cer(dl8:l/26:l)/PC 36:5 256 4.55E-06 PC 40:5/PC 36:3 181
Cer(dl8:l/26:l)/PC 40:1 64 4.55E-06 Cer(dl8:l/16:0)/LPC 20:l_sn2 77
PC 39:2/PC 28:0 653 4.55E-06 Cer(dl8:l/16:0)/LPC 22:l_snl 162
PC 40:6/PC 30:1 212 4.55E-06 Cer(dl8:l/16:0)/PC 38:0 71
Cer(dl8:l/16:0)/PC 40:5 114 6.03E-06 Cer(dl8:l/16:0)/PC 38:7 392
Cer(dl8:l/18:0)/Cer(dl8:l/23:0) 235 6.03E-06 Cer(dl8:l/16:0)/PC 0-38:6 160
Cer(dl8:l/18:0)/LPC 19:0_snl 917 6.03E-06 Cer(dl8:l/18:0)/LPC 20:4_sn2 170
Cer(dl8:l/18:0)/LPC 19:0_sn2 776 6.03E-06 Cer(dl8:l/18:0)/PC P-38:6 228
Cer(dl8:l/18:0)/LPC 20:0_snl 881 6.03E-06 Cer(dl8:l/20:0)/PC 39:0 70
Cer(dl8:l/18:0)/LPC 20:2_snl 489 6.03E-06 Cer(dl8:l/20:0)/PC P-40:5 78
Cer(dl8:l/18:0)/LPC 20:2_sn2 394 6.03E-06 Cer(dl8:l/22:0)/PC 34:0 61
Cer(dl8:l/18:0)/LPC 22:4_snl 266 6.03E-06 Cer(dl8:l/22:0)/PC 0-38:5 43
Cer(dl8:l/18:0)/PC 40:1 322 6.03E-06 Cer(dl8:l/26:l)/PC 34:2 75
Cer(dl8:l/18:0)/PC P-36:3 256 6.03E-06 PC 39:2/PC 30:0 100
Cer(dl8:l/24:l)/PC 35:2 74 6.03E-06 PC 39:2/PC 40:8 173
Cer(dl8:l/24:l)/PC 0-34:2 302 6.03E-06 PC 39:6_snl/PC 36:7 239
Cer(dl8:l/24:l)/PC 0-38:4 124 6.03E-06 PC 39:6_snl/PC 37:6 57
Figure imgf000051_0001
Cer(dl8:l/20:0)/PC 32:1 267 6.03E-06 PC 40:2/PC 33:2 80
Cer(dl8:l/20:0)/PC 36:2 155 6.03E-06 PC 40:2/PC 38:3 169
Cer(dl8:l/20:0)/PC 40:4 163 6.03E-06 PC 40:2/PC 40:4 94
Cer(dl8:l/20:0)/PC 0-34:0 120 6.03E-06 PC 40:6/PC 35:2 102
Cer(dl8:l/20:0)/PC 0-36:1 140 6.03E-06 PC 40:6/PC 37:1 114
Cer(dl8:l/20:0)/PC 0-40:1 120 6.03E-06 Cer(dl8:l/24:l)/LPC 22:l_snl 181
Cer(dl8:l/20:0)/PC P-32:l 205 6.03E-06 Cer(dl8:l/24:l)/PC 35:4 128
Cer(dl8:l/22:0)/PC 30:1 275 6.03E-06 Cer(dl8:l/24:l)/PC P-40:6 95
Cer(dl8:l/22:0)/PC 0-34:2 275 6.03E-06 Cer(dl8:l/20:0)/LPC 17:0_snl 224
Cer(dl8:l/26:l)/PC(14:0/22:6) 262 6.03E-06 Cer(dl8:l/20:0)/LPC 17:0_sn2 337
Cer(dl8:l/26:l)/PC 36:6 463 6.03E-06 Cer(dl8:l/20:0)/PC P-38:l 178
Cer(dl8:l/26:l)/PC 36:7 278 6.03E-06 Cer(dl8:l/22:0)/PC 0-38:6 68
Cer(dl8:l/26:l)/PC 0-34:2 283 6.03E-06 Cer(dl8:l/22:0)/PC P-38:l 75
PC 38:4/PC 36:5 148 6.03E-06 Cer(dl8:l/22:0)/PC P-38:5 61
PC 39:2/PC 32:3 361 6.03E-06 Cer(dl8:l/26:l)/LPC 16:0_sn2 100
PC 39:2/PC 34:4 319 6.03E-06 Cer(dl8:l/26:l)/LPC 20:5_snl 197
PC 40:2/PC 37:2 230 6.03E-06 Cer(dl8:l/26:l)/PC 35:4 140
Cer(dl8:l/16:0)/PC 38:6 382 7.93E-06 PC 38:4/PC 30:0 107
Cer(dl8:l/16:0)/PC P-36:4 337 7.93E-06 PC 39:2/PC 38:6 183
Cer(dl8:l/18:0)/LPC 18:l_sn2 517 7.93E-06 Cer(dl8:l/16:0)/LPC 18:0_sn2 182
Cer(dl8:l/18:0)/LPC 22:0_snl 880 7.93E-06 Cer(dl8:l/16:0)/PC 39:4 173
Cer(dl8:l/18:0)/PC 31:0 1289 7.93E-06 Cer(dl8:l/24:l)/PC 34:1 50
Cer(dl8:l/18:0)/PC 35:l_sn2 172 7.93E-06 Cer(dl8:l/22:0)/LPC 18:l_snl 93
Cer(dl8:l/18:0)/PC 0-32:0 198 7.93E-06 Cer(dl8:l/22:0)/PC 28:1 121
Cer(dl8:l/18:0)/PC P-36:l 489 7.93E-06 Cer(dl8:l/26:l)/PC 38:1 41
Cer(dl8:l/18:0)/PC P-38:3 418 7.93E-06 PC 40:2/PC 28:1 178
Cer(dl8:l/24:l)/LPC 20:3_snl 181 7.93E-06 PC 40:2/PC 36:1 94
Cer(dl8:l/24:l)/PC 37:3 117 7.93E-06 PC 40:5/PC 35:2 95
Cer(dl8:l/20:0)/Cer(dl8:l/24:0) 115 7.93E-06 Cer(dl8:l/16:0)/PC 0-40:4 95
Cer(dl8:l/20:0)/LPC 20:0_snl 441 7.93E-06 Cer(dl8:l/16:0)/PC P-38:5 163
Cer(dl8:l/20:0)/LPC 20:0_sn2 446 7.93E-06 Cer(dl8:l/20:0)/LPC 16:0_snl 140
Cer(dl8:l/20:0)/LPC 22:0_snl 431 7.93E-06 Cer(dl8:l/20:0)/PC 37:4_snl 278
Cer(dl8:l/20:0)/PC 34:4 661 7.93E-06 Cer(dl8:l/20:0)/PC 37:4_sn2 278
Cer(dl8:l/22:0)/LPC(15-
Cer(dl8:l/20:0)/PC 35:0 380 7.93E-06 MHDA)_snl 155
Cer(dl8:l/22:0)/PC 28:0 841 7.93E-06 Cer(dl8:l/26:l)/PC P-40:5 57
Cer(dl8:l/22:0)/PC P-34:l 132 7.93E-06 PC 40:2/PC 32:1 114
Cer(dl8:l/26:l)/LPC 17:l_snl 166 7.93E-06 PC 40:5/PC 31:0 173
Cer(dl8:l/26:l)/LPC 17:l_sn2 168 7.93E-06 Cer(dl8:l/18:0)/LPC 20:4_snl 124
Cer(dl8:l/26:l)/PC 37:2 246 7.93E-06 Cer(dl8:l/24:l)/LPC 16:0_snl 60
Cer(dl8:l/26:l)/PC 38:7 195 7.93E-06 Cer(dl8:l/24:l)/LPC 18:0_snl 101
PC 40:2/PC 32:3 985 7.93E-06 Cer(dl8:l/20:0)/PC 35:4 191
PC 40:5/PC 37:2 199 7.93E-06 Cer(dl8:l/22:0)/PC 39:4 67
PC 40:5/PC 38:6 195 7.93E-06 Cer(dl8:l/26:l)/PC 33:1 42
Cer(dl8:l/16:0)/PC 35:0 376 1.03E-05 PC 38:4/PC 28:1 122
Cer(dl8:l/16:0)/PC 0-34:0 89 1.03E-05 PC 38:4/PC 34:0 45
Cer(dl8:l/16:0)/PC 0-36:2 122 1.03E-05 PC 40:6/PC 38:3 116
Cer(dl8:l/16:0)/PC 0-38:4 164 1.03E-05 Cer(dl8:l/16:0)/LPC 15:0_snl 333
Cer(dl8:l/18:0)/LPC 18:l_snl 482 1.03E-05 Cer(dl8:l/16:0)/LPC 18:0_snl 143
Figure imgf000052_0001
Cer(dl8:l/18:0)/LPC 24:0_sn2 399 1.03E-05 Cer(dl8:l/16:0)/PC 40:1 91
Cer(dl8:l/18:0)/PC 34:1 240 1.03E-05 Cer(dl8:l/24:l)/LPC 20:5_sn2 190
Cer(dl8:l/18:0)/PC 38:1 193 1.03E-05 Cer(dl8:l/24:l)/PC 37:4_snl 185
Cer(dl8:l/18:0)/PC 39:4 354 1.03E-05 Cer(dl8:l/24:l)/PC 37:4_sn2 185
Cer(dl8:l/24:l)/PC 33:2 136 1.03E-05 Cer(dl8:l/22:0)/LPC 18:0_sn2 88
Cer(dl8:l/24:l)/PC 0-36:1 94 1.03E-05 Cer(dl8:l/22:0)/PC 33:1 89
Cer(dl8:l/20:0)/LPC 14:0_snl 574 1.03E-05 Cer(dl8:l/26:l)/Cer(dl8:l/14:0) 68
Cer(dl8:l/20:0)/LPC 20:2_snl 218 1.03E-05 Cer(dl8:l/26:l)/PC 37:3 75
Cer(dl8:l/20:0)/LPC 20:3_snl 223 1.03E-05 PC 39:6_snl/PC 36:3 142
Cer(dl8:l/20:0)/LPC 20:3_sn2 183 1.03E-05 PC 40:2/Cer(dl8:l/24:0) 43
Cer(dl8:l/20:0)/PC 34:0 153 1.03E-05 PC 40:2/PC 39:0 52
Cer(dl8:l/20:0)/PC 40:5 153 1.03E-05 PC 40:5/PC 38:3 113
Cer(dl8:l/20:0)/PC P-36:l 236 1.03E-05 Cer(dl8:l/16:0)/Cer(dl8:l/26:0) 77
Cer(dl8:l/20:0)/PC P-36:5 439 1.03E-05 Cer(dl8:l/16:0)/LPC 17:0_sn2 296
Cer(dl8:l/22:0)/Cer(dl8:l/24:0) 53 1.03E-05 Cer(dl8:l/16:0)/PC P-40:6 162
Cer(dl8:l/22:0)/LPC 22:0_snl 216 1.03E-05 Cer(dl8:l/22:0)/PC(16:0/22:5) 42
Cer(dl8:l/22:0)/PC P-36:4 166 1.03E-05 Cer(dl8:l/22:0)/PC 31:0 362
Cer(dl8:l/26:l)/LPC 19:0_snl 223 1.03E-05 Cer(dl8:l/22:0)/PC 34:2 33
Cer(dl8:l/26:l)/LPC 20:0_sn2 285 1.03E-05 PC 38:4/PC 38:3 120
Cer(dl8:l/26:l)/PC 0-36:1 155 1.03E-05 PC 38:4/PC 40:4 51
Cer(dl8:l/26:l)/PC 0-38:0 140 1.03E-05 PC 39:6_snl/PC 38:2 93
PC 39:6_snl/PC 30:1 199 1.03E-05 PC 40:2/PC 31:1 237
PC 40:2/PC 30:1 285 1.03E-05 PC 40:5/PC 31:1 116
PC 40:2/PC 36:7 295 1.03E-05 Cer(dl8:l/16:0)/PC 39:0 64
PC 40:5/PC 34:3 156 1.03E-05 Cer(dl8:l/24:l)/LPC 16:0_sn2 56
PC 40:6/PC 37:2 207 1.03E-05 Cer(dl8:l/20:0)/LPC 20:5_snl 334
Cer(dl8:l/16:0)/LPC 14:0_sn2 722 1.33E-05 Cer(dl8:l/20:0)/LPC 22:l_snl 170
Cer(dl8:l/16:0)/PC 34:4 710 1.33E-05 Cer(dl8:l/22:0)/LPC 15:0_sn2 170
Cer(dl8:l/16:0)/PC 0-36:3 295 1.33E-05 Cer(dl8:l/26:l)/LPC 22:5_snl 72
Cer(dl8:l/18:0)/LPC 15:0_snl 578 1.33E-05 PC 38:4/PC 37:1 93
Cer(dl8:l/18:0)/LPC 15:0_sn2 566 1.33E-05 PC 39:6_snl/PC 37:1 77
Cer(dl8:l/18:0)/LPC 20:0_sn2 920 1.33E-05 PC 40:2/PC 34:2 83
Cer(dl8:l/18:0)/PC 28:1 493 1.33E-05 PC 40:2/PC 40:5 62
Cer(dl8:l/18:0)/PC 40:8 698 1.33E-05 PC 40:6/PC 32:1 79
Cer(dl8:l/24:l)/LPC 14:0_snl 417 1.33E-05 Cer(dl8:l/16:0)/LPC 22:l_sn2 136
Cer(dl8:l/24:l)/LPC 17:l_snl 248 1.33E-05 Cer(dl8:l/16:0)/PC P-40:4 125
Cer(dl8:l/24:l)/LPC 17:l_sn2 250 1.33E-05 Cer(dl8:l/18:0)/PC 39:5 175
Cer(dl8:l/24:l)/LPC 20:3_sn2 138 1.33E-05 Cer(dl8:l/24:l)/LPC 22:l_sn2 155
Cer(dl8:l/24:l)/LPC 22:0_snl 320 1.33E-05 Cer(dl8:l/24:l)/PC 38:1 84
Cer(dl8:l/24:l)/PC 34:5 1262 1.33E-05 Cer(dl8:l/20:0)/LPC 20:5_sn2 291
Cer(dl8:l/24:l)/PC 36:2 136 1.33E-05 Cer(dl8:l/20:0)/PC 35:l_sn2 70
Cer(dl8:l/24:l)/PC P-38:3 122 1.33E-05 Cer(dl8:l/20:0)/PC 0-40:6 213
Cer(dl8:l/20:0)/PC 30:0 312 1.33E-05 Cer(dl8:l/22:0)/LPC 22:4_sn2 61
Cer(dl8:l/22:0)/PC 32:3 766 1.33E-05 Cer(dl8:l/22:0)/PC P-36:3 54
Cer(dl8:l/22:0)/PC 36:2 83 1.33E-05 Cer(dl8:l/26:l)/PC 37:4_snl 91
Cer(dl8:l/22:0)/PC 37:1 108 1.33E-05 Cer(dl8:l/26:l)/PC 37:4_sn2 91
Cer(dl8:l/22:0)/PC 0-36:2 186 1.33E-05 Cer(dl8:l/26:l)/PC P-40:3 76
Cer(dl8:l/26:l)/LPC 20:0_snl 271 1.33E-05 PC 39:2/PC 35:5 248
Figure imgf000053_0001
Cer(dl8:l/26:l)/LPC(15-
MHDA)_sn2 211 1.33E-05 PC 39:6_snl/PC 31:1 136
PC 38:4/PC 28:0 616 1.33E-05 PC 40:2/Cer(dl8:l/26:0) 70
PC 38:4/PC 30:1 197 1.33E-05 Cer(dl8:l/16:0)/PC 37:4_snl 256
PC 38:4/PC 34:5 539 1.33E-05 Cer(dl8:l/16:0)/PC 37:4_sn2 256
PC 39:2/PC 30:2 733 1.33E-05 Cer(dl8:l/24:l)/PC P-40:5 60
PC 39:2/PC 35:3 69 1.33E-05 Cer(dl8:l/20:0)/LPC 16:0_sn2 135
PC 39:6_snl/PC 35:5 192 1.33E-05 Cer(dl8:l/20:0)/PC 39:6_sn2 153
PC 39:6_snl/PC 38:6 249 1.33E-05 Cer(dl8:l/26:l)/LPC 22:5_sn2 72
PC 40:2/PC 36:5 253 1.33E-05 Cer(dl8:l/26:l)/PC 39:6_sn2 126
PC 40:6/PC 34:3 188 1.33E-05 Cer(dl8:l/26:l)/PC 0-40:6 140
PC 40:6/PC 38:6 186 1.33E-05 PC 39:2/PC 37:1 90
Cer(dl8:l/16:0)/LPC 14:0_snl 725 1.70E-05 PC 40:2/Cer(dl8:l/23:0) 66
Cer(dl8:l/16:0)/LPC 20:3_sn2 159 1.70E-05 PC 40:5/PC 28:1 95
Cer(dl8:l/16:0)/PC 40:4 114 1.70E-05 PC 40:6/PC 33:1 43
Cer(dl8:l/18:0)/LPC 17:0_sn2 689 1.70E-05 Cer(dl8:l/24:l)/LPC 20:5_snl 198
Cer(dl8:l/18:0)/LPC 18:2_sn2 718 1.70E-05 Cer(dl8:l/24:l)/PC P-40:4 58
Cer(dl8:l/18:0)/LPC 22:0_sn2 951 1.70E-05 Cer(dl8:l/22:0)/LPC 17:0_sn2 222
Cer(dl8:l/18:0)/PC 35:4 393 1.70E-05 Cer(dl8:l/22:0)/PC P-40:6 52
Cer(dl8:l/18:0)/PC 35:5 959 1.70E-05 PC 40:5/PC 32:1 76
Cer(dl8:l/18:0)/PC 0-32:1 189 1.70E-05 Cer(dl8:l/16:0)/LPC 20:5_sn2 326
Cer(dl8:l/18:0)/PC 0-40:5 372 1.70E-05 Cer(dl8:l/16:0)/PC 34:1 67
Cer(dl8:l/24:l)/PC 32:1 173 1.70E-05 Cer(dl8:l/16:0)/PC 35:4 181
Cer(dl8:l/24:l)/PC 40:4 115 1.70E-05 Cer(dl8:l/18:0)/LPC 22:6_snl 279
Cer(dl8:l/20:0)/LPC 19:0_sn2 436 1.70E-05 Cer(dl8:l/24:l)/PC 0-32:0 38
Cer(dl8:l/20:0)/LPC 20:2_sn2 264 1.70E-05 Cer(dl8:l/20:0)/LPC 22:l_sn2 146
Cer(dl8:l/20:0)/LPC 22:0_sn2 457 1.70E-05 Cer(dl8:l/22:0)/PC 0-38:1 66
Cer(dl8:l/20:0)/PC 33:1 174 1.70E-05 PC 38:4/PC 31:0 286
Cer(dl8:l/20:0)/PC 35:2 245 1.70E-05 PC 40:6/PC 28:1 103
Cer(dl8:l/22:0)/PC 36:5 187 1.70E-05 Cer(dl8:l/16:0)/Cer(dl8:l/14:0) 113
Cer(dl8:l/22:0)/PC 0-38:4 108 1.70E-05 Cer(dl8:l/16:0)/LPC 20:5_snl 317
Cer(dl8:l/26:l)/LPC 20:2_snl 181 1.70E-05 Cer(dl8:l/16:0)/PC 38:1 70
Cer(dl8:l/26:l)/LPC 20:3_snl 97 1.70E-05 Cer(dl8:l/22:0)/LPC 22:4_snl 51
Cer(dl8:l/26:l)/PC 0-36:3 235 1.70E-05 PC 39:2/PC 36:3 156
Cer(dl8:l/26:l)/PC 0-40:5 136 1.70E-05 PC 39:2/PC 38:2 104
PC 39:6_snl/PC 37:2 170 1.70E-05 PC 40:6/PC 36:2 62
PC 40:2/PC 35:3 237 1.70E-05 Cer(dl8:l/16:0)/PC 0-32:0 72
PC 40:2/PC 36:3 172 1.70E-05 Cer(dl8:l/18:0)/LPC 22:6_sn2 250
PC 40:5/PC 38:5 99 1.70E-05 Cer(dl8:l/24:l)/Cer(dl8:l/14:0) 85
PC 40:6/PC 38:5 100 1.70E-05 Cer(dl8:l/24:l)/PC 0-40:6 184
Cer(dl8:l/16:0)/PC 32:1 147 2.16E-05 Cer(dl8:l/22:0)/PC 0-34:1 37
Cer(dl8:l/16:0)/PC 36:5 276 2.16E-05 PC 40:5/PC 36:2 68
Cer(dl8:l/16:0)/PC 0-36:1 151 2.16E-05 Cer(dl8:l/24:l)/PC 40:1 103
Cer(dl8:l/16:0)/PC P-34:l 268 2.16E-05 Cer(dl8:l/24:l)/PC P-40:3 67
Cer(dl8:l/16:0)/PC P-38:3 149 2.16E-05 Cer(dl8:l/20:0)/LPC 22:5_snl 106
Cer(dl8:l/18:0)/Cer(dl8:l/14:0) 252 2.16E-05 Cer(dl8:l/20:0)/PC P-38:6 119
Cer(dl8:l/18:0)/LPC 16:0_snl 297 2.16E-05 Cer(dl8:l/20:0)/PC P-40:3 150
Cer(dl8:l/18:0)/LPC 18:2_snl 744 2.16E-05 Cer(dl8:l/22:0)/LPC 20:5_sn2 160
Cer(dl8:l/18:0)/PC 0-40:4 252 2.16E-05 Cer(dl8:l/26:l)/PC(16:0/16:0) 55
Figure imgf000054_0001
Cer(dl8:l/18:0)/PC P-36:5 935 2.16E-05 PC 38:4/PC 36:1 45
Cer(dl8:l/18:0)/PC P-40:5 205 2.16E-05 PC 40:5/PC 39:4 40
Cer(dl8:l/24:l)/LPC 20:0_snl 386 2.16E-05 Cer(dl8:l/16:0)/PC P-40:5 73
Cer(dl8:l/24:l)/LPC 20:2_snl 193 2.16E-05 Cer(dl8:l/24:l)/Cer(dl8:l/26:0) 83
Cer(dl8:l/20:0)/LPC 24:0_sn2 204 2.16E-05 Cer(dl8:l/24:l)/PC 35:l_sn2 43
Cer(dl8:l/20:0)/PC 31:1 432 2.16E-05 Cer(dl8:l/24:l)/PC 38:0 127
Cer(dl8:l/20:0)/PC 33:2 157 2.16E-05 Cer(dl8:l/20:0)/LPC 22:5_sn2 136
Cer(dl8:l/20:0)/PC 0-36:4 158 2.16E-05 Cer(dl8:l/22:0)/LPC 15:0_snl 164
Cer(dl8:l/22:0)/LPC 14:0_sn2 389 2.16E-05 Cer(dl8:l/22:0)/LPC 22:l_snl 83
Cer(dl8:l/22:0)/LPC 20:3_snl 93 2.16E-05 Cer(dl8:l/26:l)/PC 36:4 183
Cer(dl8:l/22:0)/PC 0-36:3 252 2.16E-05 Cer(dl8:l/26:l)/PC 37:6 110
Cer(dl8:l/22:0)/PC P-36:l 126 2.16E-05 Cer(dl8:l/26:l)/PC 0-34:1 88
Cer(dl8:l/26:l)/PC P-32:0 81 2.16E-05 PC 39:6_snl/PC 28:1 85
Cer(dl8:l/16:0)/LPC 20:3_snl 128 2.73E-05 PC 40:2/PC 35:4 127
Cer(dl8:l/16:0)/PC 30:0 209 2.73E-05 PC 40:2/PC 37:3 95
Cer(dl8:l/16:0)/PC 37:3 109 2.73E-05 PC 40:5/PC 36:0 38
Cer(dl8:l/16:0)/PC 38:2 174 2.73E-05 PC 40:6/PC 37:3 86
Cer(dl8:l/16:0)/PC 0-34:2 380 2.73E-05 PC 40:6/PC 39:6_sn2 105
Cer(dl8:l/16:0)/PC P-32:l 291 2.73E-05 Cer(dl8:l/20:0)/PC 0-38:2 88
Cer(dl8:l/18:0)/LPC 20:l_sn2 314 2.73E-05 Cer(dl8:l/22:0)/LPC 18:0_snl 52
Cer(dl8:l/18:0)/PC 38:7 971 2.73E-05 PC 38:4/PC 31:1 135
Cer(dl8:l/24:l)/PC 33:1 117 2.73E-05 PC 38:4/PC 33:2 61
Cer(dl8:l/24:l)/PC 35:0 340 2.73E-05 PC 38:4/PC 33:3 289
Cer(dl8:l/24:l)/PC 0-34:0 97 2.73E-05 PC 40:2/PC 33:1 58
Cer(dl8:l/24:l)/PC 0-36:4 110 2.73E-05 PC 40:2/PC 37:4_snl 83
Cer(dl8:l/20:0)/LPC(15-
MHDA)_sn2 366 2.73E-05 PC 40:2/PC 37:4_sn2 83
Cer(dl8:l/20:0)/PC 40:8 416 2.73E-05 Cer(dl8:l/16:0)/PC 0-40:6 341
Cer(dl8:l/22:0)/PC 34:4 430 2.73E-05 Cer(dl8:l/24:l)/PC 0-38:2 55
Cer(dl8:l/22:0)/PC 34:5 1077 2.73E-05 Cer(dl8:l/20:0)/PC 36:4 351
Cer(dl8:l/22:0)/PC 0-38:0 98 2.73E-05 Cer(dl8:l/20:0)/PC 0-32:1 88
Cer(dl8:l/26:l)/LPC 18:l_snl 99 2.73E-05 Cer(dl8:l/26:l)/PC 32:0 48
Cer(dl8:l/26:l)/PC 30:0 168 2.73E-05 PC 39:2/PC 38:3 104
Cer(dl8:l/26:l)/PC 36:0 136 2.73E-05 PC 40:5/PC 37:3 67
Cer(dl8:l/26:l)/PC 38:2 146 2.73E-05 Cer(dl8:l/16:0)/PC 36:4 193
Cer(dl8:l/26:l)/PC 38:5 115 2.73E-05 Cer(dl8:l/24:l)/PC 39:6_sn2 132
PC 39:2/PC 35:0 106 2.73E-05 Cer(dl8:l/22:0)/LPC 20:l_sn2 59
PC 39:2/PC 35:2 69 2.73E-05 PC 38:4/PC 32:1 73
Cer(dl8:l/16:0)/PC 34:5 1488 3.42E-05 PC 38:4/PC 35:2 23
Cer(dl8:l/18:0)/PC 36:4 128 3.42E-05 PC 40:2/Cer(dl8:l/14:0) 45
Cer(dl8:l/24:l)/LPC 18:l_sn2 157 3.42E-05 Cer(dl8:l/16:0)/LPC 16:0_snl 118
Cer(dl8:l/24:l)/LPC 19:0_snl 290 3.42E-05 Cer(dl8:l/24:l)/LPC 22:5_snl 53
Cer(dl8:l/24:l)/LPC 19:0_sn2 283 3.42E-05 Cer(dl8:l/24:l)/PC 39:0 62
Cer(dl8:l/24:l)/LPC 20:0_sn2 384 3.42E-05 Cer(dl8:l/20:0)/PC 37:6 153
Cer(dl8:l/24:l)/PC 40:5 112 3.42E-05 Cer(dl8:l/20:0)/PC 40:3 74
Cer(dl8:l/24:l)/PC 0-38:0 164 3.42E-05 Cer(dl8:l/22:0)/PC 0-40:4 47
Cer(dl8:l/24:l)/PC P-32:0 125 3.42E-05 PC 39:2/PC 32:1 75
Cer(dl8:l/24:l)/PC P-38:4 193 3.42E-05 PC 40:5/PC 33:1 28
Cer(dl8:l/20:0)/LPC 19:0_snl 475 3.42E-05 PC 40:5/PC 34:0 26
Figure imgf000055_0001
Cer(dl8:l/20:0)/LPC 24:0_snl 199 3.42E-05 PC 40:6/PC 37:6 80
Cer(dl8:l/20:0)/PC P-32:0 168 3.42E-05 PC 40:6/PC 39:4 38
Cer(dl8:l/26:l)/PC 36:3 261 3.42E-05 Cer(dl8:l/16:0)/LPC 22:5_snl 99
Cer(dl8:l/26:l)/PC 38:0 82 3.42E-05 Cer(dl8:l/22:0)/LPC 17:0_snl 98
Cer(dl8:l/26:l)/PC P-38:4 249 3.42E-05 Cer(dl8:l/22:0)/LPC 20:5_snl 182
PC 38:4/PC 37:2 151 3.42E-05 Cer(dl8:l/22:0)/PC P-40:4 90
PC 40:2/PC(14:0/22:6) 329 3.42E-05 PC 39:2/PC 33:1 26
PC 40:2/PC 36:6 559 3.42E-05 PC 39:6_snl/PC 32:1 108
Cer(dl8:l/16:0)/PC 33:3 335 4.25E-05 PC 39:6_snl/PC 38:3 128
Cer(dl8:l/16:0)/PC 35:2 288 4.25E-05 PC 40:2/PC 36:4 145
Cer(dl8:l/18:0)/PC P-40:4 299 4.25E-05 PC 40:5/PC 36:1 96
Cer(dl8:l/24:l)/PC 30:0 209 4.25E-05 PC 40:5/PC 39:6_sn2 77
Cer(dl8:l/24:l)/PC 33:3 414 4.25E-05 Cer(dl8:l/16:0)/PC 35:l_sn2 69
Cer(dl8:l/24:l)/PC 36:0 132 4.25E-05 Cer(dl8:l/16:0)/PC 39:6_sn2 222
Cer(dl8:l/24:l)/PC P-36:l 174 4.25E-05 Cer(dl8:l/16:0)/PC P-38:6 79
Cer(dl8:l/22:0)/LPC 20:0_sn2 220 4.25E-05 Cer(dl8:l/22:0)/LPC 20:l_snl 60
Cer(dl8:l/22:0)/LPC 20:3_sn2 92 4.25E-05 Cer(dl8:l/22:0)/PC 40:1 55
Cer(dl8:l/22:0)/PC P-32:l 144 4.25E-05 Cer(dl8:l/26:l)/PC 0-40:4 49
Cer(dl8:l/26:l)/PC 35:0 162 4.25E-05 Cer(dl8:l/16:0)/PC P-40:3 141
Cer(dl8:l/26:l)/PC 0-36:2 216 4.25E-05 Cer(dl8:l/18:0)/PC P-40:l 259
Cer(dl8:l/26:l)/PC 0-38:1 103 4.25E-05 Cer(dl8:l/24:l)/LPC 22:5_sn2 86
Cer(dl8:l/26:l)/PC 0-40:1 95 4.25E-05 Cer(dl8:l/26:l)/PC P-40:4 172
Cer(dl8:l/26:l)/PC P-38:2 194 4.25E-05 PC 39:6_snl/PC 39:4 24
PC 38:4/PC 40:8 103 4.25E-05 PC 40:2/PC(16:0/16:0) 30
PC 40:2/PC 38:0 67 4.25E-05 PC 40:2/PC 39:6_sn2 139
PC 40:5/PC 36:5 183 4.25E-05 PC 40:5/PC 37:6 80
Cer(dl8:l/16:0)/LPC 19:0_sn2 438 5.27E-05 PC 40:6/PC 36:0 44
Cer(dl8:l/18:0)/LPC 20:l_snl 310 5.27E-05 Cer(dl8:l/16:0)/LPC 17:0_snl 207
Cer(dl8:l/18:0)/PC 37:4_snl 513 5.27E-05 Cer(dl8:l/24:l)/PC 37:6 97
Cer(dl8:l/18:0)/PC 37:4_sn2 513 5.27E-05 Cer(dl8:l/24:l)/PC 0-32:1 43
Cer(dl8:l/18:0)/PC 39:0 211 5.27E-05 Cer(dl8:l/22:0)/PC 38:0 78
Cer(dl8:l/18:0)/PC 0-38:2 201 5.27E-05 Cer(dl8:l/22:0)/PC 39:6_sn2 93
Cer(dl8:l/20:0)/PC(14:0/22:6) 475 5.27E-05 PC 38:4/Cer(dl8:l/24:0) 26
Cer(dl8:l/20:0)/LPC 17:l_sn2 325 5.27E-05 PC 39:6_snl/PC 37:3 81
Cer(dl8:l/20:0)/PC 36:6 469 5.27E-05 PC 40:2/PC 32:0 25
Cer(dl8:l/22:0)/LPC 14:0_snl 396 5.27E-05 PC 40:6/PC 36:1 93
Cer(dl8:l/22:0)/LPC 20:2_snl 132 5.27E-05 Cer(dl8:l/22:0)/PC 32:0 22
Cer(dl8:l/26:l)/PC(16:0/22:5) 110 5.27E-05 Cer(dl8:l/22:0)/PC 35:4 110
Cer(dl8:l/26:l)/LPC 18:2_snl 172 5.27E-05 Cer(dl8:l/22:0)/PC 0-40:6 165
Cer(dl8:l/26:l)/PC 35:5 324 5.27E-05 PC 38:4/PC 38:0 33
PC 40:2/PC 34:0 72 5.27E-05 PC 40:6/PC 34:0 40
PC 40:2/PC 38:2 142 5.27E-05 PC 40:6/PC 40:4 48
PC 40:2/PC 38:7 236 5.27E-05 Cer(dl8:l/24:l)/PC P-38:6 101
PC 40:6/PC 35:5 239 5.27E-05 Cer(dl8:l/26:l)/LPC 20:4_sn2 61
Cer(dl8:l/16:0)/LPC 20:0_sn2 369 6.49E-05 Cer(dl8:l/26:l)/PC 34:1 40
Cer(dl8:l/16:0)/PC 33:2 207 6.49E-05 PC 39:6_snl/PC 36:2 52
Cer(dl8:l/16:0)/PC 36:0 130 6.49E-05 PC 40:5/PC 40:4 41
Cer(dl8:l/18:0)/LPC 17:0_snl 402 6.49E-05 Cer(dl8:l/16:0)/PC 0-38:2 90
Cer(dl8:l/24:l)/PC 31:1 371 6.49E-05 Cer(dl8:l/24:l)/PC 36:4 288
Figure imgf000056_0001
Figure imgf000057_0001
Tables 4-9 present simplifying scoring systems (points 0-12, 0-9 and 0-6), where the points are given based on the quartiles (Q.1-Q.4) of a general population. For example, in Table 4, if Cer(dl8:l/16:0) concentration of a person belongs to the highest quartile, the person will receive 2 points. The same evaluation will be performed for the other components of the score, and the points are then summed up to create the score.
Table 4. Score 1, an example of a scoring system based on lipids.
Figure imgf000058_0001
Table 5. Score 2, another example of a scoring system based on lipids.
Figure imgf000058_0002
Table 6. Score 3, another example of a scoring system based on lipids.
Figure imgf000058_0003
Table 7. Score 4, another example of a scoring system based on lipids.
Figure imgf000058_0004
Table 8. Score 5, another example of a scoring system based on lipids.
Figure imgf000059_0001
Table 10 presents an example of the predictive value of the scoring systems. In this example, Scores 1-6 refer to the scoring systems presented in Tables 4-9. P-values are presented for the scores.
Table 10. Predictive value of the scoring systems.
Figure imgf000059_0002
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein both in the Examples and in the body of the entire description. Such equivalents are considered to be within the scope of the present disclosure and are intended to be encompassed by the following claims or the items listed above.

Claims

1. A method for assessing the severity of a pulmonary condition of a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
2. A method for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1;
(b) determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control; and
(c) selecting an appropriate treatment for the pulmonary condition of the subject.
3. A method for determining that a subject needs a treatment to preventan occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
4. A method for determining the risk of a subject to develop a severe pulmonary condition, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
5. A method for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
6. A method for determining the risk of a subject to develop thrombosis, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
7. A method for treating a pulmonary condition or preventing the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the method comprises administering to the subject a treatment, if the subject has been identified as having a risk of developing a severe pulmonary condition according to the methods described herein.
8. A method for treating a serious complication of a pulmonary condition or preventing the occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the method comprises administering to the subject a treatment, if the subject has been identified as needing a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition according to the methods described herein.
9. A method for treating thrombosis or preventing thrombosis, wherein the method comprises administering to the subject a treatment, if the subject has been identified as having a risk of developing thrombosis according to the methods described herein.
10. A method for detecting at least one biomarker selected from Table 1 in a sample from a subject suffering from or suspected of suffering from a pulmonary condition, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
11. The method of claim 10, wherein the method comprises detecting in the sample from the subject a changed concentration of the at least one biomarker selected from Table 1, when compared to a control.
12. The method of claims 10 or 11, wherein the method comprises using a standard combination comprising at least one lipid selected from Table 1, and optionally wherein the standard combination comprises at least one isotope-labelled lipid selected from Table 1.
13. A method for obtaining or collecting data for assessing the severity of a pulmonary condition of a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
14. A method for obtaining or collecting data for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1;
(b) determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control; and
(c) selecting an appropriate treatment for the pulmonary condition of the subject.
15. A method for obtaining or collecting data for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
16. A method for obtaining or collecting data for determining the risk of a subject to develop a severe pulmonary condition, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
17. A method for obtaining or collecting data for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
18. A method for obtaining or collecting data for determining the risk of a subject to develop thrombosis, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
19. A method for generating quantitative data for assessing the severity of a pulmonary condition of a subject, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
20. A method for generating quantitative data for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
21. A method for generating quantitative data for preventing the occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
22. A method for generating quantitative data for determining the risk of a subject to develop a severe pulmonary condition, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
23. A method for generating quantitative data for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
24. A method for generating quantitative data for determining the risk of a subject to develop thrombosis, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
25. A pulmonary marker combination, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1.
26. A use of a pulmonary marker combination for assessing the severity of a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject is suffering from a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
27. A use of a pulmonary marker combination for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; (b) determining that the subject needs a treatment for the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control; and
(c) selecting an appropriate treatment for the pulmonary condition of the subject.
28. A use of a pulmonary marker combination for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
29. A use of a pulmonary marker combination for determining the risk of a subject to develop a severe pulmonary condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
30. A use of a pulmonary marker combination for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject will progress to more severe form of the condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
31. A use of a pulmonary marker combination for determining the risk of a subject to develop thrombosis, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject has a risk to develop thrombosis, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
32. A standard combination, wherein the standard combination comprises at least two lipids selected from Table 1.
33. A standard combination for assaying a concentration of at least one biomarker selected from Table 1 in a sample from a subject in a preparation of a reagent, kit or composition, wherein the standard combination comprises at least two lipids selected from Table 1.
34. A use of a standard combination for detecting in a sample from a subject a concentration of at least one biomarker selected from Table 1, wherein the standard combination comprises at least two lipids selected from Table 1.
35. A use of a standard combination for assessing the severity of a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject is suffering from a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
36. A use of a standard combination for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; (b) determining that the subject needs a treatment for the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control; and
(c) selecting an appropriate treatment for the pulmonary condition of the subject.
37. A use of a standard combination for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
38. A use of a standard combination for determining the risk of a subject to develop a severe pulmonary condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
39. A use of a standard combination for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject will progress to more severe form of the condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
40. A use of a standard combination for determining the risk of a subject to develop thrombosis, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject has a risk to develop thrombosis, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
41. A use of one or more reagent in a preparation of a reagent, kit or composition for assessing the severity of a pulmonary condition of a subject, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject is suffering from a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
42. A use of one or more reagent in a preparation of a reagent, kit or composition for selecting an appropriate treatment for a pulmonary condition of a subject, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1;
(b) determining that the subject needs a treatment for the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control; and
(c) selecting an appropriate treatment for the pulmonary condition of the subject.
43. A use of one or more reagent in a preparation of a reagent, kit or composition for determining that a subject needs a treatment to prevent an occurrence of a serious complication, or its progression, of a pulmonary condition of a subject, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject needs the treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
44. A use of one or more reagent in a preparation of a reagent, kit or composition for determining the risk of a subject to develop a severe pulmonary condition, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop a severe pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
45. A use of one or more reagent in a preparation of a reagent, kit or composition for predicting the progress of a pulmonary condition of a subject to more severe form of the condition, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject will progress to more severe form of the condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
46. A use of one or more reagent in a preparation of a reagent, kit or composition for determining the risk of a subject to develop thrombosis, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop thrombosis, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
47. A use of one or more reagent in a preparation of a reagent, kit or composition for detecting at least one biomarker selected from Table 1 in a sample from a subject, wherein the use comprises assaying the sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
48. The use of any one of claims 41-47, wherein the use comprises using a standard combination comprising at least two lipids selected from Table 1, and optionally wherein the standard combination comprises at least two isotope-labelled lipids selected from Table 1.
49. The use of any one of claims 41-48, wherein the reagent is used for assaying a sample to determine a concentration of at least one biomarker selected from Table 1.
50. The use of any one of claims 41-49, wherein the reagent comprises any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s), preparation(s), or any combination thereof.
51. The use of any one of claims 41-50, wherein the reagent comprises a combination of any standard(s), control(s), substance(s), compound(s), solution(s), solvent(s), agent(s), ingredient(s) or preparation(s) used for the methods and uses of any of the preceding claims.
52. The use of any one of claims 41-51, wherein the reagent comprises one or more isotope-labelled component, and optionally wherein the isotope of the isotope-labelled component is deuterium, 13C or 15N.
53. The use of any one of claims 41-52, wherein the reagent comprises at least one, 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 11, at least 12, or more isotope-labelled lipids selected from Table 1.
54. A composition or kit for performing the methods or uses of any one of the preceding claims, wherein the composition or kit comprises at least one biomarker selected from Table 1.
55. The composition or kit of claim 54, wherein the at least one biomarker selected from Table 1 is 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 11, at least 12, or more biomarkers selected from Table 1.
56. The composition or kit of claim 54 or 55, wherein the composition or kit comprises at least one isotope-labelled biomarker selected from Table 1, and optionally wherein the isotope of the at least one isotope-labelled biomarker is deuterium, 13C or 15N .
57. The composition or kit of any one of claims 54-56, wherein the composition or kit further comprises (a) standard(s), control(s), reagent(s), solution(s), solvent(s), container(s), use instruction(s) and/or other element(s) for performing the methods or uses of any one of the preceding claims.
58. The composition or kit of any one of claims 54-57, wherein the composition or kit comprises an element for collecting a blood sample, and optionally wherein the element is for collecting a dried blood spot on a filter.
70
59. The composition or kit of any one of claims 54-58, wherein the composition or kit comprises an element for collecting a plasma or serum sample, and optionally wherein the element separates plasma or serum from a blood sample, and further optionally wherein the plasma or serum sample is dried on a filter card.
60. The composition or kit of any one of claims 54-59, wherein the composition or kit is a test kit for used in a laboratory or a home use test kit.
61. The composition or kit of any one of claims 54-60, wherein the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), used for performing the methods or uses of any one of the preceding claims, and optionally wherein the combination is purchased from a commercial manufacturer or prepared in-house in a laboratory.
62. The composition or kit of any one of claims 54-61, wherein the composition or kit is a combination of any standard(s), control(s), reagent(s), solution(s) or solvent(s), used for assaying a concentration of at least one biomarker selected from Table 1.
63. The composition or kit of any one of claims 54-62, wherein the composition or kit is used in a mass spectrometry (MS) or nuclear magnetic resonance (NMR) assay, and optionally wherein the mass spectrometry (MS) is coupled to gas chromatography (GC), two-dimensional gas chromatography (GCxGC), liquid chromatography (LC), two-dimensional liquid chromatography (LCxLC), high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC).
64. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker selected from Table 1 is 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 11, at least 12, or more biomarkers selected from Table 1.
65. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is any combination of the biomarkers selected from Tables 1-10.
66. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is at least one biomarker ratio, and optionally wherein the biomarker ratio is a biomarker ratio of any biomarkers selected from Tables
1-10.
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67. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is at least one biomarker ratio selected from Table 3, and optionally wherein the at least one biomarker ratio is 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 11, at least 12, or more biomarker ratios selected from Table 3.
68. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1, and optionally wherein the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
69. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is calculated from the concentrations of the at least two biomarkers selected from Table 1, and optionally wherein the biomarker is a concentration ratio of the at least two biomarkers selected from Table 1.
70. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or, neural networks.
71. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is a continuous or discrete scoring system based on the biomarkers selected from Table 1, and optionally wherein for discrete scoring, the points are given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
72. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is a continuous or discrete scoring system based on ceramide (Cer) and/or phosphatidylcholine (PC) biomarkers and/or Cer/Cer, Cer/PC, PC/Cer and/or PC/PC biomarker ratios.
72
73. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is a marker combination and the marker combination is a scoring system where the points are given based on the quartiles of a study population.
74. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is any of the scores selected from Tables 4-10 and/or any component of the scores selected from Tables 4-10.
75. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is a score and the score is based on 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 11, at least 12 or more lipids selected from: Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(d 18: 1/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC
36:5, PC 35:3, PC 40:8, PC 30:0, PC 36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC
35:0, PC 31:1, PC 36:2, PC 35:5, PC 37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC
39:4, PC 40:5, PC 33:3, PC 40:4, PC 35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC
37:6, PC 39:0, PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
76. The method or use of any one of the proceeding claims, wherein the method or use further comprises determining other biomarkers, such as CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof, and optionally wherein the method or use further comprises using other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
77. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is ceramide (Cer), phosphatidylcholine (PC) and/or any combination thereof.
73
78. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker selected from Table 1 comprises Cer(d 18: 1/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC 36:7, PC 38:7, PC
36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC 37:1, PC 38:3, PC
31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC 35:4, PC 38:0, PC
40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC
40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
79. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is at least one biomarker ratio, and optionally wherein the biomarker ratio is a Cer/Cer, Cer/PC, PC/Cer and/or PC/PC ratio.
80. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the at least one biomarker is at least one biomarker ratio and the biomarker ratio comprises Cer(dl8:l/16:0)/PC 16:0/22:5, Cer(dl8:l/18:0)/PC 14:0/22:6,
Cer(dl8:l/24:l)/Cer(dl8:l/24:0), Cer(dl8:l/18:0)/PC 36:6, Cer(dl8:l/16:0)/PC 38:5,
Cer(dl8:l/18:0)/Cer(dl8:l/16:0), Cer(dl8:l/18:0)/PC(16:0/16:0), Cer(dl8:l/18:0)/LPC(15-MHDA),
Cer(dl8:l/18:0)/PC 32:0, Cer(dl8:l/18:0)/PC(16:0/22:5), Cer(dl8:l/18:0)/Cer(dl8:l/24:0),
Cer(dl8:l/18:0)/PC 38:5, Cer(dl8:l/24:l)/PC(16:0/22:5), Cer(d 18: 1/24: 1)/LPC( 15-M H DA),
Cer(dl8:l/16:0)/LPC(15-MHDA), Cer(dl8:l/24:l)/PC 38:5, Cer(dl8:l/16:0)/Cer(dl8:l/24:0),
Cer(dl8:l/24:l)/PC 36:6, Cer(dl8:l/24:l)/PC(14:0/22:6), Cer(dl8:l/16:0)/PC(14:0/22:6),
Cer(dl8:l/16:0)/PC 36:6, Cer(dl8:l/16:0)/PC(16:0/16:0), Cer(dl8:l/24:l)/PC(16:0/16:0),
Cer(dl8:l/16:0)/PC 32:0 and/or Cer(dl8:l/24:l)/PC 32:0.
81. The method of any one of claims 1-6, wherein the method further comprises administering a treatment to the subject, and optionally wherein the subject has been identified as being in need of the treatment according to the methods described in claims 1-6.
82. The method of any one of claims 7-9, 76 or 81, wherein the treatment comprises any therapeutic treatment typically given to a subject suffering from a pulmonary condition and/or needing immediate or intensive care, such as hospitalization, intensive care, life support care, noninvasive or invasive medical care, monitoring and/or supporting bodily functions, administering a drug, administering supplemental oxygen (e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation), extracorporeal membrane oxygenation, a surgical procedure or lung transplantation.
83. The method of claim 82, wherein the drug comprises any pharmaceutical typically given to a subject suffering from a pulmonary condition and/or its symptoms, such as an anti-inflammatory agent, antibiotic, antiviral drug, corticosteroid, analgesic, sedative, antithrombotic, anticoagulant or antiplatelet drug.
84. The method or use of any one of the proceeding claims, wherein the method or use further comprises requesting a test or receiving test results from a laboratory which provides the results of an assay useful for determining the concentration of the at least one biomarker selected from Table 1.
85. The method or use of any one of the proceeding claims, wherein the method or use further comprises adding at least one isotope-labelled biomarker to the sample prior to assaying the concentration of the at least one biomarker selected from Table 1, optionally wherein the at least one isotope-labelled biomarker is 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 11, at least 12 or more isotope-labelled biomarkers, and further optionally wherein the at least one isotope-labelled biomarker is deuterium-labelled, 13C -labelled or 15N -labelled biomarker selected from Table 1.
86. The method or use of claim 85, wherein the at least one isotope-labelled biomarker comprises
Cer(d 18: 1/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(dl6:l/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC
34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC 36:7, PC 38:7, PC
36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC 37:1, PC 38:3, PC
31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC 35:4, PC 38:0, PC
75 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC 35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
87. The method or use of claim 85 or 86, wherein the at least one isotope-labelled biomarker is d7- Cer(d 18: 1/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7-Cer(dl8:l/24:l), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
88. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the concentration of the at least one biomarker is determined by using mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, dual polarisation interferometry, a high performance separation method, such as liquid chromatography, gas chromatography, two-dimensional liquid chromatography, two-dimensional gas chromatography, high performance liquid chromatography, ultra high performance liquid chromatography or ultra performance liquid chromatography, an immunoassay, such as an ELISA, and/or an assay with a binding moiety capable of specifically binding the analyte.
89. The method or use of claim 88, wherein the concentration is determined by using mass spectrometry, and optionally wherein the mass spectrometry is coupled to a direct sample infusion method, such as a robotic nanoflow ion source device, or to a high performance separation method, such as high performance liquid chromatography, ultra high performance liquid chromatography or ultra performance liquid chromatography.
90. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the concentrations of the 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 11, at least 12, or more biomarkers selected from Table 1 are determined by one assay, and optionally the one assay is performed with mass spectrometry.
91. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the pulmonary condition comprises any disorder affecting the lungs and other parts of the respiratory system, and optionally wherein the pulmonary condition is an acute or chronic condition caused by an infection, inflammation, apoptosis, smoking, secondhand smoking, air pollution, exposure to dust, smoke or fumes, and/or genetic factors.
92. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the pulmonary condition comprises a pulmonary infection, pulmonary inflammation, pulmonary damage, injury or distress, pneumonia, acute respiratory distress
76 syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, emphysema, bronchitis, pulmonary edema, asthma, lung cancer, pulmonary embolism and/or pulmonary hypertension.
93. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the pulmonary infection or inflammation is caused by a pathogen, smoking, secondhand smoking, air pollution, exposure to dust, smoke or fumes and/or genetic factors.
94. The method, use, pulmonary marker combination, standard combination, composition or kit of claim 93, wherein the pathogen is an infectious microorganism or agent that causes disease or illness to its host, and optionally wherein the pathogen is a virus, bacterium, protozoan, prion, viroid, fungus or any other eukaryotic or prokaryotic invader of host.
95. The method, use, pulmonary marker combination, standard combination, composition or kit of claim 94, wherein the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D, any subtype or variant of type A, B, C or D influenza virus or any other virus capable of infecting mammalian subjects.
96. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the severe pulmonary condition comprises any clinically severe, critical or life-threatening pulmonary condition which may require immediate enhanced care, hospitalization or care in the intensive care unit, and optionally wherein the severe pulmonary condition comprises pneumonia, pulmonary fibrosis, respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), pulmonary embolism or lung cancer.
97. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the complication comprises any symptom caused by a pulmonary condition, and optionally wherein the complication is a clinically severe, critical or lifethreatening symptom.
98. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the complication comprises cough, sore throat, shortness of breath, difficulty breathing, chest pain, pain when breathing, pain in the airways, chest pressure or tightness, fever, chills, headache, tiredness, nasal congestion, runny nose, muscle and joint pain, rash,
77 nausea, diarrhea, vomiting, loss of taste or smell, eye problems, mucus production, expectoration, wheezing, gastrointestinal symptoms, oxygen deficiency, pneumonia, pulmonary fibrosis, thrombosis, pulmonary embolism, hypertension, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g. acute kidney injury), sepsis, additional viral or bacterial infection, multisystem inflammatory syndrome, chronic fatigue syndrome, acute or chronic cardiovascular damage, such as myocardial injury, myocarditis, arrhythmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure, seizure, clinical deterioration or death.
99. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the serious complication requires immediate enhanced care or treatment, hospitalization or care in the intensive care unit, and optionally wherein the serious complication comprises oxygen deficiency, pneumonia, pulmonary fibrosis, thrombosis, pulmonary embolism, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g. acute kidney injury), sepsis, acute or chronic cardiovascular damage, such as myocardial injury, myocarditis, arrhythmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure, seizure, clinical deterioration or death.
100. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the thrombosis comprises venous thrombosis, arterial thrombosis, pulmonary embolism or deep vein thrombosis.
101. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject is a healthy individual with no previous signs or symptoms of a pulmonary condition, or the subject is suffering or has previously suffered from a pulmonary condition.
102. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject is suspected of suffering from a pulmonary condition, is at high risk of developing a severe pulmonary condition and/or its serious complication or is suffering from a pulmonary condition and is at high risk of progressing to a more severe form of the condition.
103. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject has a high risk of a severe pulmonary condition
78 and/or its serious complication, and optionally wherein the subject has one or more underlying medical condition, such as diabetes, a cardiovascular disease (e.g. heart failure, atherosclerosis, coronary artery disease, cardiomyopathy or hypertension), severe obesity, kidney disease, liver disease, sickle cell disease, weakened immune system, lung disease or neurological condition.
104. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject is or has been hospitalized or under a treatment or medication, or is not or has not been hospitalized or under any previous treatment or medication.
105. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject is infected by a virus, and optionally wherein the virus is SARS-CoV-2, SARS-CoV, MERS-CoV, any other coronavirus or a subtype or variant of a coronavirus, any influenza virus A, B, C or D or any subtype or variant of type A, B, C or D influenza virus.
106. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject is a healthy individual with no previous signs or symptoms of thrombosis, is suffering or has previously suffered from thrombosis, is suspected of suffering from thrombosis or is at high risk of developing thrombosis.
107. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the sample is a biological sample, and optionally wherein the sample is a blood sample, serum sample, plasma sample, saliva sample, urine sample, tissue sample or a fraction thereof, such as a lipoprotein fraction.
108. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the sample is a dried blood spot, and optionally wherein the blood sample is dried on a filter.
109. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the sample is a plasma or serum sample separated from a blood sample and dried on a filter card.
110. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the control is a control sample or a control value, concentration, score, threshold, limit, limit value or set of data previously determined, calculated or extrapolated, or to be determined, calculated or extrapolated, or taken from the literature.
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111. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the control is from a healthy individual, a generalized population of healthy individuals, a pulmonary patient that has remained free of any severe condition or serious complication, or a group of pulmonary patients that have remained free of any severe condition or serious complication.
112. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the control sample is previously obtained from the same subject or the control value is obtained from a previously taken sample from the same subject.
113. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination comprises at least one biomarker ratio or at least two biomarkers selected from Table 1 and at least one biomarker ratio, and optionally wherein the biomarker ratio is selected from Table 3.
114. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination comprises a score, and optionally wherein the score is selected from Tables 4-10.
115. The pulmonary marker combination of any one of claims 25-31, wherein the at least two biomarkers are 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 11, at least 12, or more biomarkers selected from Table 1.
116. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination is a ratio, sum, difference, product, remainder, value, score, calculation, formula, equation, algorithm, or any combination thereof, of the at least two biomarkers selected from Table 1.
117. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination is calculated from the concentrations of the at least two biomarkers selected from Table 1.
118. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination further comprises other biomarkers, such as CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT-proBNP, cystatin C, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof, and optionally wherein the pulmonary marker combination further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data, or any combination thereof.
80
119. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination is constructed by linear combinations, regression models, other unsupervised or supervised frequentist or Bayesian statistical classification or machine learning methods, such as support vector machines, kernel estimations, decision trees or neural networks.
120. The pulmonary marker combination of any one of claims 25-31, wherein the pulmonary marker combination is a continuous or discrete scoring systems based on the biomarkers selected from Table
I, and optionally wherein for discrete scoring, the points are given based on intervals with non-equal probabilities, quantiles or other cut-off values, determined from the same or other populations, of the individual score components.
121. The standard combination of any one of claims 32-40, wherein the standard combination comprises at least one lipid selected from the lipid ratios of Table 3, at least two lipids selected from Table 1 and at least one lipid selected from the lipid ratios of Table 3 or at least one component of the scores selected from Tables 4-10.
122. The standard combination of any one of claims 32-40, wherein the standard combination comprises at least one isotope-labelled lipid, and optionally wherein the isotope of the isotope-labelled lipid is deuterium, 13C or 15N.
123. The standard combination of any one of claims 32-40, wherein the standard combination comprises 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
II, at least 12, or more lipids selected from Table 1.
124. A use of a standard combination in a preparation of a reagent, kit or composition, wherein the standard combination comprises at least two lipids selected from Table 1.
125. The composition or kit of any one of claims 54-63, wherein the composition or kit further comprises other biomarkers, such as CRP, hs-CRP, a cytokine, troponin T, troponin I, BNP, proBNP, NT- proBNP, cystatin C, creatinine, glucose, oxygen saturation, blood oxygen level, any other inflammatory or oxidation marker, or any combination thereof, and optionally wherein the composition or kit further comprises other personal information or health data, such as sex, age, blood pressure, BMI, smoking status, disease history, medication, ethnic background, geographical location, medical imaging data , or any combination thereof.
126. The method of any one of claims 99, 104, 118 or 125, wherein the treatment comprises any therapeutic treatment typically given to a subject suffering from a pulmonary condition and/or needing
81 immediate or intensive care, such as hospitalization, intensive care, life support care, noninvasive or invasive medical care, monitoring and/or supporting bodily functions, administering a drug, administering supplemental oxygen (e.g. oxygen therapy, nasal high-flow oxygen therapy, noninvansive or invasive mechanical ventilation), extracorporeal membrane oxygenation, a surgical procedure or lung transplantation.
127. The use of any one of claims 41-53, wherein the one or more isotope-labelled lipid of the reagent is selected from Cer(dl8:l/18:0), Cer(dl8:l/24:1), Cer(dl8:l/16:0), Cer(dl8:l/24:0), Cer(dl8:l/20:0), Cer(dl8:l/22:0), Cer(dl8:l/26:1), Cer(dl8:l/26:0), Cer(dl8:l/23:0), Cer(dl8:l/14:0), Cer(dl6:l/16:0), Cer(d 16: 1/18:0), Cer(dl6:l/20:0), Cer(dl6:l/24:0), Cer(dl6:l/23:0), Cer(dl6:l/22:0), Cer(dl6:l/26:0), Cer(dl6:l/24:1), Cer(dl8:0/16:0), Cer(dl8:0/24:l), Cer(dl8:0/23:0), Cer(dl8:0/24:0), Cer(dl8:0/22:0), Cer(dl8:2/18:0), Cer(dl8:2/16:0), Cer(dl8:2/20:0), Cer(dl8:2/26:1), Cer(dl8:2/23:0), Cer(dl8:2/26:0), Cer(dl8:2/24:0), Cer(dl8:2/22:0), Cer(dl8:2/24:1), Cer(d20:l/24:l), Cer(d20:l/22:0), Cer(d20:l/24:0), Cer(d20:l/23:0), PC (14:0/22:6), PC (16:0/22:5), PC (16:0/16:0), PC 38:5, PC 36:6, PC 32:0, PC 30:2, PC 32:2, PC 38:6, PC 34:4, PC 28:0, PC 30:1, PC 34:3, PC 34:5, PC 32:3, PC 36:5, PC 35:3, PC 40:8, PC 30:0, PC
36:7, PC 38:7, PC 36:3, PC 37:2, PC 35:2, PC 38:2, PC 36:0, PC 28:1, PC 35:0, PC 31:1, PC 36:2, PC 35:5, PC
37:1, PC 38:3, PC 31:0, PC 36:1, PC 34:0, PC 34:2, PC 33:2, PC 32:1, PC 39:4, PC 40:5, PC 33:3, PC 40:4, PC
35:4, PC 38:0, PC 40:1, PC 37:3, PC 37:4, PC 36:4, PC 33:1, PC 39:6, PC 37:6, PC 39:0, PC 38:1, PC 34:1, PC
35:1, PC 39:5, PC 40:3, PC 40:2, PC 40:6, PC 38:4 and/or PC 39:2.
128. The use of any one of claims 41-53, wherein the one or more isotope-labelled lipid of the reagent is d7-Cer(dl8:l/16:0), d7-Cer(dl8:l/18:0), d7-Cer(dl8:l/24:0), d7-Cer(dl8:l/24:l), d9-PC 16:0/22:5, d9-PC 14:0/22:6 and/or d9-PC 16:0/16:0.
129. The method of claim 7, wherein the method further comprises determining the risk of the subject to develop a severe pulmonary condition according to the methods described herein prior to administering the treatment.
130. The method of claim 8, wherein the method further comprises determining that the subject needs a treatment to prevent the occurrence of a serious complication, or its progression, of the pulmonary condition according to the methods described herein prior to administering the treatment.
131. The method of claim 9, wherein the method further comprises determining the risk of the subject to develop thrombosis according to the methods described herein prior to administering the treatment.
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132. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the pulmonary condition is caused by severe acute respiratory syndrome coronavirus 2 (SARS-Co-2).
133. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the increased concentration of the at least one biomarker is 0.01 - 10 000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers, and optionally wherein the increased concentration of the at least one biomarker is 1 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
134. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the increased concentration of the at least one biomarker is 10 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers, and optionally wherein the increased concentration of the at least one biomarker is 25 - 1000 % higher than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
135. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the decreased concentration of the at least one biomarker is 0.01- 10000% lowerthan a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers, and optionally wherein the decreased concentration of the at least one biomarker is 1 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
136. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the decreased concentration of the at least one biomarker is 10 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers, and optionally wherein the decreased concentration of the at least one biomarker is 25 - 1000 % lower than a control value, such as a concentration, a score, a threshold or a combination value of the concentrations of the biomarkers.
137. A method for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
83 (b) determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
138. A method for obtaining or collecting data for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
139. A method for generating quantitative data for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the method comprises assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1.
140. A use of a pulmonary marker combination for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
141. A use of a standard combination for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine concentrations of the at least two biomarkers selected from Table 1; and
(b) determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains increased and/or decreased concentrations of the at least two biomarkers as indicated in Table 1, when compared to a control.
84
142. A use of one or more reagent in a preparation of a reagent, kit or composition for determining the risk of a subject to develop one or more complication(s) of a pulmonary condition, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the subject has a risk to develop one or more complication(s) of the pulmonary condition, if the sample contains an increased or decreased concentration of the at least one biomarker as indicated in Table 1, when compared to a control.
143. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the one or more complication(s) of a pulmonary condition is a serious complication which requires immediate enhanced care or treatment, hospitalization or care in the intensive care unit, and optionally wherein the serious complication comprises oxygen deficiency, pneumonia, pulmonary fibrosis, thrombosis, pulmonary embolism, pulmonary hypertension, respiratory failure, acute respiratory distress syndrome (ARDS), lung cancer, damage of the internal organs (e.g. acute kidney injury), sepsis, acute or chronic cardiovascular damage, such as myocardial injury, myocarditis, arrhythmias, cardiomyopathy, acute coronary syndrome, myocardial infarction, stroke, transient ischemic attacks or heart failure, seizure, clinical deterioration or death.
144. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the cytokine comprises one or more of any interleukin (IL), interferon (INF), tumor negrosis factor (TNF), transforming growth factor (TGF), chemokine, lymphokine and/or monokine.
145. The method, use, pulmonary marker combination, standard combination, composition or kit of claim 144, wherein the interleukin comprises one or more of IL-1, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL- 14, IL-15, IL-16, IL-17, IL-18 or any IL from IL19 to IL36, the interferon comprises one or more of INF type I (e.g. INF-a or INF-P), INF type II (e.g. INF-y) or INF type III, and/or the tumor negrosis factor comprises one or more of TNF-a orTNF-p.
146. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the subject is suffering from coronavirus disease (COVID-19) or the subject has previously suffered from COVID-19.
147. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the control is obtained from a symptomless pulmonary
85 patient or a pulmonary patient who has mild symptoms, or a group of symptomless pulmonary patients or a group of pulmonary patients who have mild symptoms.
148. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the control is obtained from a symptomless COVID-19 patient or a COVID-19 patient who has mild symptoms, or a group of symptomless COVID-19 patients or a group of COVID-19 patients who have mild symptoms.
149. A method for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more its complications in a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
150. A method for obtaining or collecting data for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more its complications in a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
151. A method for generating quantitative data for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more its complications in a subject, wherein the method comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
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152. A use of a pulmonary marker combination for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the pulmonary marker combination comprises at least two biomarkers selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
153. A use of a standard combination for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the standard combination comprises at least two lipids selected from Table 1, and wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
154. A use of one or more reagent in a preparation of a reagent, kit or composition for evaluating the effectiveness of a treatment, or the response to a treatment, of a pulmonary condition and/or one or more of its complications in a subject, wherein the use comprises
(a) assaying a sample from the subject to determine a concentration of at least one biomarker selected from Table 1; and
(b) determining that the treatment is effective, or the subject is responding to the treatment, if the sample contains an increased or decreased concentration of the at least one biomarker in an opposite direction of change to the direction of change indicated in Table 1, when compared to a control.
155. The method or use of any one of claims 149-154, wherein the method or use further comprises after the determining step, changing, supplementing, or keeping the same an already administered treatment in the subject based on the concentrations of the at least one biomarker obtained in the determining step.
87
156. The method, use, pulmonary marker combination, standard combination, composition or kit of any one of the proceeding claims, wherein the drug comprises one or more of a functional inhibitor of acid sphingomyelinase (FIASMA), molnupiravir, dexamethasone, a ceramide synthase or an inhibitor of a ceramide synthase.
88
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