WO2011012553A1 - Method for predicting the likelihood of an onset of an inflammation associated organ failure - Google Patents

Method for predicting the likelihood of an onset of an inflammation associated organ failure Download PDF

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Publication number
WO2011012553A1
WO2011012553A1 PCT/EP2010/060745 EP2010060745W WO2011012553A1 WO 2011012553 A1 WO2011012553 A1 WO 2011012553A1 EP 2010060745 W EP2010060745 W EP 2010060745W WO 2011012553 A1 WO2011012553 A1 WO 2011012553A1
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cer
double bonds
organ failure
acid
acyl
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PCT/EP2010/060745
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French (fr)
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Hans-Peter Deigner
Matthias Kohl
David Enot
Therese Koal
Matthias Keller
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Biocrates Life Sciences Ag
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Priority to AU2010277664A priority Critical patent/AU2010277664A1/en
Priority to CA2767763A priority patent/CA2767763A1/en
Priority to JP2012522121A priority patent/JP2013501215A/en
Priority to CN2010800341097A priority patent/CN102472756A/en
Priority to EP10737041A priority patent/EP2460014A1/en
Priority to US13/387,572 priority patent/US20120202240A1/en
Publication of WO2011012553A1 publication Critical patent/WO2011012553A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical 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
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20Supervised data analysis
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/26Infectious diseases, e.g. generalised sepsis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/17Nitrogen containing
    • Y10T436/173845Amine and quaternary ammonium

Definitions

  • the present invention relates to a method for predicting the likelihood of an onset of an inflammation or infection associated organ failure from a biological sample of a mammalian subject in vitro in accordance with claim 1.
  • the invention generally relates to biomarkers for organ failure as tools in clinical diagnosis for early detection of organ failure, therapy monitoring and methods based on the same biomarkers.
  • Organ failure in acute pancreatitis was predicted by using a combination of plasma interleukin 10 and serum calcium measurements (Early Prediction of Organ Failure by Combined Markers in Patients With Acute Pancreatitis Mentula P, Kylanpaa M-L, Kemppainen E, Br J Surg, 92, 68 - 75, 2005).
  • interleukin 6 and interleukin 10 were used for multiple OF prediction (Lausevic Z, Lausevic M, Trbojevic-Stankovic J, Krstic S, Stojimirovic, Predicting multiple organ failure in patients with severe trauma B Can J Surg. 2008, 51 , 97-102).
  • Severe sepsis also includes OF and occurs when one or more vital organs are compromised. It can lead to septic shock, which is marked by low blood pressure that does not respond to standard treatment, problems in vital organs, and oxygen
  • CRP C-reactive protein
  • PCT procalcitonin
  • Sepsis by definition comprises systemic inflammatory response syndrome (SIRS) and infection with pathogens.
  • SIRS systemic inflammatory response syndrome
  • SIRS Systemic inflammatory response syndrome
  • the quantitative metabolomics profile of the endogenous organ failure predictive target metabolites can be combined with any of the above classical clinical laboratory parameters.
  • Organ failure includes a systemic inflammatory response syndrome (SIRS) together with an infection.
  • Sepsis commonly called a "blood stream infection” denotes the presence of bacteria (bacteremia) or other infectious organisms or their toxins in the blood (septicemia) or in other tissue of the body and the immune response of the host.
  • Organ failure due to sepsis is currently thought to start with the interaction between the host response and the presence of micro-organisms and/or their toxins within the body.
  • the observed host responses include immune, coagulation, pro and anti-inflammatory responses.
  • Septic organ failure thus comprises a systemic response to infection, defined as hypothermia or hyperthermia, tachycardia, tachypnea, a clinically evident focus of infection or positive blood cultures, one or more end organs with either dysfunction or inadequate perfusion, cerebral dysfunction, hypoxaemia, increased plasma lactate or unexplained metabolic acidosis, and oliguria.
  • WBC white blood cell
  • SBP systolic blood pressure
  • MAP mean arterial blood pressure
  • SvO2 mixed venous oxygen saturation
  • INR international normalized ratio
  • aPTT activated partial thromboplastin time
  • tachycardia may be absent in hypothermic patients
  • at least one of the following indications of altered organ function altered mental status, hypoxemia, increased serum lactate level.
  • Hypotension is defined by a systolic arterial pressure below 90 mm Hg, a MAP ⁇ 70 mmHg, or a reduction in systolic blood pressure of >40 mm Hg from baseline, despite adequate volume resuscitation, in the absence of other causes for hypotension.
  • US 2009/0104596 A1 discloses methods and kits for diagnosing a disease state of cachexia by measuring biomarker profiles.
  • the biomarkers concerned are those known from the energy metabolism, namely lactate, citrate, formate, acetoacetate, 3-hydroxy butyrate and some amino acids.
  • Organ failure of any kind is not addressed.
  • Freund et al., Ann. Surg. (1979), 190, 571 -576 desclose the use of a plasma amino acid pattern as predictors of the severity and outcome of sepsis for discriminating between septic encephalopathy and no encephalopathy, wherein the degree of encephalopathy of a patient is considered an expression for the severity of the septic process.
  • the medical practitioner uses a number of diagnostic tools for diagnosing a patient suffering from a certain disease.
  • diagnostic tools for diagnosing a patient suffering from a certain disease.
  • measurement of a series of single routine parameters, e.g. in a blood sample is a common diagnostic laboratory approach.
  • These single parameters comprise for example enzyme activities and enzyme concentration and/or detection.
  • the present invention provides a solution to these problems based on the application of a new technology in this context and on an unknown list of endogenous metabolites as diagnostic marker. Since metabolite concentration differences in biological fluids and tissues provide links to the various phenotypical responses, metabolites are suitable biomarker candidates.
  • the present invention allows for accurate, rapid, and sensitive prediction and diagnosis of OF through a measurement of a plurality of endogenous metabolic biomarker (metabolites) taken from a biological sample at a single point in time. This is accomplished by obtaining a biomarker panel at a single point in time from an individual, particularly an individual at risk of developing OF, having OF, or suspected of having OF, and comparing the biomarker profile from the individual to reference biomarker values or scores.
  • the reference biomarker values may be obtained from a population of individuals (a "reference population") who are, for example, afflicted with OF or who are suffering from either the onset of OF or a particular stage in the progression of OF.
  • biomarker panel values or score from the individual contains appropriately characteristic features of the biomarker values or scores from the reference population, then the individual is diagnosed as having a more likely chance of getting OF, as being afflicted with OF or as being at the particular stage in the progression of OF as the reference population.
  • the present invention provides, inter alia, methods of predicting the likelihood of an onset of OF in an individual.
  • the methods comprise obtaining a biomarker score at a single point in time from the individual and comparing the individual's biomarker profile to a reference biomarker profile. Comparison of the biomarker profiles can predict the onset of OF in the individual preferably with an accuracy of at least about . This method may be repeated again at any time prior to the onset of OF.
  • the present invention further provides a method of determining the progression (i.e., the stage) of sepsis in an individual towards OF.
  • This method comprises obtaining a biomarker profile at a single point in time from the individual and comparing the individual's biomarker profile to a reference biomarker score. Comparison of the biomarker scores can determine the progression of sepsis in the individual preferably with an accuracy of at least about 90 %. This method may also be repeated on the individual at any time.
  • the present invention provides a method of diagnosing OF in an individual having or suspected of having OF. This method comprises obtaining a biomarker score at a single point in time from the individual and comparing the individual's biomarker score to a reference biomarker score. Comparison of the biomarker profiles can diagnose OF in the individual with an accuracy of at least about 90 %. This method may also be repeated on the individual at any time.
  • the invention provides, inter alia, a method of determining the status of OF or diagnosing OF in an individual comprising applying a decision rule.
  • the decision rule comprises comparing (i) a biomarker score generated from a biological sample taken from the individual at a single point in time with (ii) a biomarker score generated from a reference population.
  • Application of the decision rule determines the status of sepsis or diagnoses OF in the individual.
  • the method may be repeated on the individual at one or more separate, single points in time.
  • the present invention further provides, inter alia, a method of determining the status of OF or diagnosing OF in an individual comprising obtaining a biomarker score from a biological sample taken from the individual and comparing the individual's biomarker score to a reference biomarker score. A single such comparison is capable of classifying the individual as having membership in the reference population. Comparison of the biomarker scores determines the status of OF or diagnoses OF in the individual.
  • the present invention provides, inter alia, a method of determining the status of OF or diagnosing OF in an individual.
  • the method comprises comparing a measurable characteristic of at least one biomarker between a biomarker panel or biomarker score composed by (processed or unprocessed) values of this panel obtained from a biological sample from the individual and a biomarker score obtained from biological samples from a reference population. Based on this comparison, the individual is classified as belonging to or not belonging to the reference population. The comparison, therefore, determines the likelihood of OF or diagnoses OF in the individual.
  • the biomarkers in one embodiment, are selected from the group of biomarkers shown in any one of TABLES 1 to 3.
  • the present invention provides methods for predicting organ failure, which is clinically deary to be distinguished from methods of diagnosing sepsis, SIRS, and the like.
  • Such methods comprise the steps of: analyzing a biological sample from a subject to determine the level(s) of a purality of biomarkers for organ failure in the sample, where the plurality of biomarkers are selected from Table 1 and comparing the level(s) of the the plurality of biomarkers - respectively a composed value / score generated by subjecting the concentrations of individual biomarkers in the sample to a classification method such as affording an equation processing single concentration values - to obtain a separation between both (diseased and healthy) groups or comparing the level(s) of the the plurality of biomarkers in the sample to organ failure positive or organ failure negative reference levels of the the plurality of biomarkers in order to determine at a very early state whether the subject is developing organ failure or not, so that suitable therapeutic measures can be started.
  • the present invention provides a solution to the problem described above, and generally relates to the use of metabolomics data, generated by quantitation of endogenous metabolites by but not limited to mass spectrometry (MS), in particular MS- technologies such as MALDI, ESI, atmospheric pressure pressure chemical ionization (APCI), and other methods, determination of metabolite concentrations by use of MS- technologies or alternative methods coupled to separation (LC-MS, GC-MS, CE-MS), subsequent feature selection and /or the combination of features to classifiers including molecular data of at least two molecules.
  • MS mass spectrometry
  • MS- technologies such as MALDI, ESI, atmospheric pressure pressure chemical ionization (APCI), and other methods
  • determination of metabolite concentrations by use of MS- technologies or alternative methods coupled to separation (LC-MS, GC-MS, CE-MS), subsequent feature selection and /or the combination of features to classifiers including molecular data of at least two molecules.
  • concentrations of the individual markers, analytes, metabolites thus are measured and compared to reference values or data combined and processed to scores, classifiers and compared to reference values thus indicating diseased states etc. with superior sensitivities and specificities compared to known procedures, clinical parameters and biomarkers.
  • the present invention provides a method of diagnosing organ failure and/or duration/severity comprising: detecting the presence or absence of a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, etc. measured together in a multiplex or panel format) of organ failure specific metabolites in a sample
  • organ failure based on the presence of the organ failure specific metabolite.
  • the present invention further provides a method of screening compounds, comprising: contacting an animal, a tissue, a cell containing a organ failure-specific metabolite with a test compound; and detecting the level of the organ failure specific metabolite.
  • the method further comprises the step of comparing the level of the organ failure specific metabolite in the presence of the test compound or therapeutic intervention to the level of the organ failure specific metabolite in the absence of the organ failure specific metabolite.
  • the cell is in vitro, in a non- human mammal, or ex vivo.
  • the test compound is a small molecule or a nucleic acid (e.g., antisense nucleic acid, a siRNA, or a miRNA) or oxygen/xenon or any neuroprotective drug that inhibits the expression of an enzyme involved in the synthesis or breakdown of an organ failure specific metabolite.
  • the method is a high throughput method.
  • the present invention relates to:
  • a method for predicting the likelihood of onset of an inflammation associated organ failure from a biological sample of a mammalian subject in vitro wherein a. the subject's quantitative metabolomics profile comprising a plurality of endogenous metabolites, is detected in the biological sample by means of quantitative metabolomics analysis, and b. the quantitative metabolomics profile of the subject's sample is compared with a quantitative reference metabolomics profile of a plurality of endogenous organ failure predictive target metabolites in order to predict whether the subject is likely or unlikely to develop an organ failure; and c.
  • said endogenous organ failure predictive target metabolites have a molecular mass less than 1500 Da and are selected from the group consisting of: Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid (glutamate), glutamine, leucine, isoleucine, histidine, ornithine, proline, phenylalanine, serine, tryptophane, tyrosine, valine, kynurenine; phenylthio carbamyl amino acids (PTC-amino acids), in particular, PCT-arginine, PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-tyrosine, PTC-valine; dimethylarginine, in particular N,N-dimethyl-L-arginine; carboxylic acids
  • Ceramides with an N-acyl residue having from 2 to 30 Carbon atoms in the acyl residue and having from 0 to 5 double bonds and having from 0 to 5 hydroxy groups; carnitine; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue and having 1 to 4 double bonds in the acyl residue; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue and having from 1 to 3 OH-groups in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue with 1 to 4 double bonds and 1 to 3 OH- groups in the acyl residue; phospholipides, in particular lysophosphatidylcholines (monoacylphospha-tidylcholines) having from 1 to 30 carbon atoms in the acyl residue; lysophosphatidylcho
  • prostaglandines namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane
  • oxysterols namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20 ⁇ - hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3 ⁇ ,5 ⁇ ,6 ⁇ - trihydroxycholesterol, 7 ⁇ - hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5 ⁇ ,6 ⁇ - epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycolithocholic acid sulfate, glycoursodeoxycholic acid, lithocholic acid
  • inflammation associated organ failure comprises “infection associated organ failure” and/or “sepsis associated organ failure”.
  • a preferred method is one, wherein the biological sample is selected from the group consisting of stool; body fluids, in particular blood, liquor, cerebrospinal fluid, urine, ascitic fluid, seminal fluid, saliva, puncture fluid, cell content, tissue samples, in particular liver biopsy material; or a mixture thereof.
  • a preferred embodiment of the method according to the present invention is one, wherein said quantitative metabolomics profile is achieved by a quantitative metabolomics profile analysis method comprising the generation of intensity data for the quantitation of endogenous metabolites by mass spectrometry (MS), in particular, by high- throughput mass spectrometry, preferably by MS-technologies such as Matrix Assisted Laser Desorption/lonisation (MALDI), Electro Spray Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), 1 H-, 13 C- and/or 31 P- Nuclear
  • MS-technologies such as Matrix Assisted Laser Desorption/lonisation (MALDI), Electro Spray Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), 1 H-, 13 C- and/or 31 P- Nuclear
  • Magnetic Resonance spectroscopy optionally coupled to MS, determination of metabolite concentrations by use of MS-technologies and/or methods coupled to separation, in particular Liquid Chromatography (LC-MS), Gas Chromatography (GC- MS), or Capillary Electrophoresis (CE-MS).
  • MS-technologies and/or methods coupled to separation in particular Liquid Chromatography (LC-MS), Gas Chromatography (GC- MS), or Capillary Electrophoresis (CE-MS).
  • intensity data of said metabolomics profile are normalized with a set of endogenous housekeeper metabolites by relating detected intensities of the selected endogenous organ failure predictive target metabolites to intensities of said endogenous housekeeper metabolites.
  • a particularly preferred method according to the present invention is one, wherein said endogenous housekeeper metabolites are selected from the group consisting of such endogeneous metabolites which show stability in accordance with statistical stability measures being selected from the group consisting of coefficient of variation (CV) of raw intensity data, standard deviation (SD) of logarithmic intensity data, stability measure (M) of geNorm - algorithm or stability measure value (rho) of NormFinder- algorithm.
  • CV coefficient of variation
  • SD standard deviation
  • M stability measure
  • rho stability measure value
  • said quantitative metabolomics profile comprises the results of measuring at least one of the parameters selected from the group consisting of:
  • a panel of reference endogenous organ failure predictive target metabolites or derivatives thereof is established by:
  • step a) said classifier algorithms of step b) being trained on at least one training data set containing preprocessed data from subjects being divided into classes according to their likelihood to develop an organ failure, in order to select a classifier function to map said preprocessed data to said likelihood; d) applying said trained classifier algorithms of step c) to a preprocesse
  • the endogenous organ failure predictive target metabolites for easier and/or more sensitive detection are preferably detected by means of chemically modified derivatives thereof, such as phenylisothiocyanates for amino acids.
  • said endogenous organ failure predictive target metabolites are selected from the group consisting of:
  • phosphatidylcholines (diacylphosphatidylcholines, PC aa chain length:total number of double bonds or PC ae) in particular, PC aa C28:1 , PC aa C38:0, PC aa C42:0, PC aa C42:1 , PC ae C40:1 , PC ae C40:2, PC ae C40:6, PC ae C42:2, PC ae C42:3, PC ae C42:4, PC ae C44:5, PC ae C44:6, PC aa C36:4, PC aa C38:1 , PC aa C38:2, PC aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:5, PC aa C40:6, PC aa C40:7, PC aa C40:8, PC ae C36:4, PC ae C36:5, PC ae C38:4,
  • lysophosphatidylcholines (monoacylphosphatidylcholines, PC a chain length:total number of double bonds), in particular, PC a C18:2, PC a C20:4, PC a C20:3, PC a C26:0;
  • oxycholesterols in particular, 3 ⁇ ,5 ⁇ ,6 ⁇ -trihydroxycholestan, 7-ketocholesterol, 5 ⁇ ,6 ⁇ - epoxycholesterol;
  • lysophosphatidylethanolamins (monoacylphosphatidylcholins, PE a chain length:total number of double bonds), in particular, PE a C18:1 , PE a C18:2, PE a C20:4, PE a C22:5, PE a C22:6;
  • phosphatidylethanolamins (diacylphosphatidylcholins, PE aa chain length:total number of double bonds), in particular, PE aa C38:0, PE aa C38:2;
  • ceramids (N-chain length:total number of double bonds), in particular, N-C2:0-Cer, N- C7:0-Cer, N-C9:3-Cer, N-C17:1 -Cer, N-C22:1 -Cer, N-C25:0-Cer, N-C27:1 -Cer, N-C5:1 - Cer(2H), N-C7:1 -Cer(2H), N-C8:1 -Cer(2H), N-C1 1 :1 -Cer(2H), N-C20:0-Cer(2H), N- C21 :0-Cer(2H), N-C22:1 -Cer(2H), N-C25:1 -Cer(2H), N-C26:1 -Cer(2H), N-C24:0(OH)- Cer, N-C26:0(OH)-Cer, N-C6:0(OH)-Cer, N
  • said plurality of endogenous organ failure predictive target metabolites or derivatives thereof comprises 2 to 80, in particular 2 to 60, preferably 2 to 50, preferred 2 to 30, more preferred 2 to 20, particularly preferred 2 to 10 endogenous metabolites
  • a particular embodiment of the present invention is the use of a plurality of endogenous metabolites for predicting of an onset of an infection associated organ failure from a biological sample of a mammalian subject in vitro, wherein the metabolites are selected from the group consisting of : Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid (glutamate), glutamine, leucine, isoleucine, histidine, ornithine, proline, phenylalanine, serine, tryptophane, tyrosine, valine, kynurenine; phenylthio carbamyl amino acids (PTC-amino acids), in particular, PCT-arginine, PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-ty
  • hydroxysphingoyelines having a total number of carbon atoms in the acyl residues from 10 to 30 and 1 to 5 double bonds; prostaglandines, namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane B2; putrescine; oxysterols, namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20 ⁇ - hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3 ⁇ ,5 ⁇ ,6 ⁇ - trihydroxycholesterol, 7 ⁇ - hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5 ⁇ ,6 ⁇ - epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely
  • Particularly preferred endogenous organ failure predictive target metabolites are selected from the group consisting of:
  • Carnitin acylcarnitines (C chain length :total number of double bonds), in particular, C12-DC, C14:1 , C14:1 -OH, C14:2, C14:2-OH, C18, C6:1 ;
  • sphingomyelins (SM chain length :total number of double bonds), in particular, SM C16:0, SM C17:0, SM C18:0, SM C19:0, SM C21 :1 , SM C21 :3, SM C22:2, SM C23:0, SM C23:1 , SM C23:2, SM C23:3, SM C24:0, SM C24:1 , SM C24:2, SM C24:3, SM C24:4, SM C26:4, SM C3:0, SM (OH) C22:1 , SM (OH) C22:2, SM (OH) C24:1 , SM C26:0, SM C26:1 ;
  • phosphatidylcholines (diacylphosphatidylcholines, PC aa chain length:total number of double bonds or PC ae) in particular, PC aa C28:1 , PC aa C38:0, PC aa C42:0, PC aa C42:1 , PC ae C40:1 , PC ae C40:2, PC ae C40:6, PC ae C42:2, PC ae C42:3, PC ae C42:4, PC ae C44:5, PC ae C44:6, PC aa C36:4, PC aa C38:1 , PC aa C38:2, PC aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:5, PC aa C40:6, PC aa C40:7, PC aa C40:8, PC ae C36:4, PC ae C36:5, PC ae C38:4,
  • oxycholesterols in particular, 3 ⁇ ,5 ⁇ ,6 ⁇ -trihydroxycholestan, 7-ketocholesterol, 5 ⁇ ,6 ⁇ - epoxycholesterol;
  • lysophosphatidylethanolamins (monoacylphosphatidylcholins, PE a chain length:total number of double bonds), in particular, PE a C18:1 , PE a C18:2, PE a C20:4, PE a C22:5, PE a C22:6;
  • phosphatidylethanolamins (diacylphosphatidylcholins, PE aa chain length:total number of double bonds), in particular, PE aa C38:0, PE aa C38:2;
  • ceramids (N-chain length:total number of double bonds), in particular, N-C2:0-Cer, N- C7:0-Cer, N-C9:3-Cer, N-C17:1 -Cer, N-C22:1 -Cer, N-C25:0-Cer, N-C27:1 -Cer, N-C5:1 - Cer(2H), N-C7:1 -Cer(2H), N-C8:1 -Cer(2H), N-C1 1 :1 -Cer(2H), N-C20:0-Cer(2H), N-
  • the present invention includes a kit for carrying out a method for predicting the likelihood of an onset of an infection associated organ failure from a biological sample of a mammalian subject in vitro, in a biological sample, comprising: a) calibration agents for the quantitative detection of endogenous organ failure predictive target metabolites, wherein said metabolites are selected from the group consisting of: Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid
  • PTC-amino acids phenylthio carbamyl amino acids
  • PCT-arginine PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-tyrosine, PTC-valine
  • dimethylarginine in particular N,N-dimethyl-L-arginine
  • carboxylic acids namely 15(S)-hydroxy-5Z,8Z,1 1 Z,13E-eicosatetraenoic acid [(5Z,8Z,1 1 Z,13E,15S)-15-Hydroxyicosa-5
  • Ceramides with an N-acyl residue having from 2 to 30 Carbon atoms in the acyl residue and having from 0 to 5 double bonds and having from 0 to 5 hydroxy groups; carnitine; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue and having 1 to 4 double bonds in the acyl residue; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue and having from 1 to 3 OH-groups in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue with 1 to 4 double bonds and 1 to 3 OH- groups in the acyl residue; phospholipides, in particular lysophosphatidylcholines (monoacylphospha-tidylcholines) having from 1 to 30 carbon atoms in the acyl residue; lysophosphatidylcho
  • prostaglandines namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane
  • oxysterols namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20 ⁇ - hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3 ⁇ ,5 ⁇ ,6 ⁇ - trihydroxycholesterol, 7 ⁇ - hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5 ⁇ ,6 ⁇ - epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycolithocholic acid sulfate, glycoursodeoxycholic acid, lithocholic acid
  • step c) classification software for generating the quantitative metabolomics profiles achieved with said calibration agents of step a) and classifying the results based on the processed data of step b).
  • Classifiers are typically deterministic functions that map a multi-dimensional vector of biological measurements to a binary (or n-ary) outcome variable that encodes the absence or existence of a clinically-relevant class, phenotype, distinct physiological state or distinct state of disease.
  • classification methods such as, but not limited to, logistic regression, (diagonal) linear or quadratic discriminant analysis (LDA, QDA, DLDA, DQDA), perceptron, shrunken centroids regularized discriminant analysis (RDA), random forests (RF), neural networks (NN), Bayesian networks, hidden Markov models, support vector machines (SVM), generalized partial least squares (GPLS), partitioning around medoids (PAM), inductive logic programming (ILP), generalized additive models, gaussian processes, regularized least square regression, self organizing maps (SOM), recursive partitioning and regression trees, K-nearest neighbor classifiers (K-NN), fuzzy classifiers, bagging, boosting, and na ⁇ ve Bayes and many more can be used.
  • logistic regression logistic regression
  • QDA linear or quadratic discriminant analysis
  • DLDA linear or quadratic discriminant analysis
  • DQDA perceptron
  • RDA shrunken centroids regularized discriminant analysis
  • RDA random forests
  • RF neural
  • Fig.1 is a Venn diagram showing the agreement between adjusted p value
  • Fig. 2 is a graph showing classifier accuracy for support vector machines (SVM) with linear kernel, diagonal linear discriminant analysis (DLDA) and k nearest neighbors (KNN) with k equal to one where the features are selected using a ranker which ranks the metabolites combining adjusted p value, fold change and AUC;
  • SVM support vector machines
  • DLDA diagonal linear discriminant analysis
  • KNN k nearest neighbors
  • Fig. 3 is a graph showing classifier accuracy for support vector machines (SVM) with linear kernel, diagonal linear discriminant analysis (DLDA) and k nearest neighbors (KNN) with k equal to one where the features are selected by a so-called wrapper using boosted regression trees;
  • SVM support vector machines
  • DLDA diagonal linear discriminant analysis
  • KNN k nearest neighbors
  • Fig. 4 is a Venn diagram showing the agreement between adjusted p value
  • Sese sepsis refers to sepsis associated with organ dysfunction, hypoperfusion abnormalities, or sepsis-induced hypotension. Hypoperfusion abnormalities include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status.
  • Stress refers to sepsis-induced hypotension that is not responsive to adequate intravenous fluid challenge and with manifestations of peripheral hypoperfusion.
  • a "converter patient” refers to a SIRS-positive patient who progresses to clinical suspicion of sepsis during the period the patient is monitored, typically during an ICU stay.
  • non- converter patient refers to a SIRS-positive patient who does not progress to clinical suspicion of sepsis during the period the patient is monitored, typically during an ICU stay.
  • a patient with OF has a clinical presentation that is classified as OF, as defined above, but is not clinically deemed to have OF.
  • Individuals who are at risk of developing OF include patients in an ICU and those who have otherwise suffered from a physiological trauma, such as a burn or other insult.
  • organ failure includes all stages of OF including, but not limited to, the onset of OF and multi organ failure (MOD), e.g. associated with the end stages of sepsis.
  • Sepsis refers to a SIRS-positive condition that is associated with a confirmed infectious process. Clinical suspicion of sepsis arises from the suspicion that the SIRS- positive condition of a SIRS patient is a result of an infectious process.
  • the "onset of OF” refers to an early stage of OF, i.e., prior to a stage when the clinical manifestations are sufficient to support a clinical suspicion of OF. Because the methods of the present invention are used to detect OF prior to a time that OF would be suspected using conventional techniques, the patient's disease status at early OF can only be confirmed retrospectively, when the manifestation of OF is more clinically obvious. The exact mechanism by which a patient acquires OF is not a critical aspect of the invention. The methods of the present invention can detect changes in the biomarker score independent of the origin of the OF. Regardless of how OF arises, the methods of the present invention allow for determining the status of a patient having, or suspected of having, OF, as classified by previously used criteria.
  • organ failure specific metabolite refers to a metabolite that is differentially present or differentially concentrated in septic organisms compared to non- septic organisms.
  • organ failure specific metabolites are present in septic tissues but not in non- in septic tissues.
  • organ failure-specific metabolites are absent in septic tissues but present in non-septic cells, tissues, body liquids. In still further embodiments, organ failure specific metabolites are present at different levels (e.g., higher or lower) in septic tissue/cells as compared to non-septic cells.
  • an organ failure specific metabolite may be differentially present at any level, but is generally present at a level that is increased by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more; or is generally present at a level that is decreased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 40%
  • An organ failure-specific metabolite is preferably differentially present at a level that is statistically significant (e.g., an adjusted p-value less than 0.05 as determined using either Analysis of Variance, Welch's t-test or its non parametric equivalent versions). Exemplary organ failure-specific metabolites are described in the detailed description and experimental sections below.
  • sample in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture. On the other hand, it is meant to include both biological and environmental samples.
  • a sample may include a specimen of synthetic origin.
  • Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, such biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, rodents, etc.
  • a biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material from a subject.
  • the sample can be isolated from any suitable biological tissue or fluid such as, for example, tissue, blood, blood plasma, urine, or cerebral spinal fluid (CSF).
  • CSF cerebral spinal fluid
  • a “reference level” of a metabolite means a level of the metabolite that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof.
  • a "positive" reference level of a metabolite means a level that is indicative of a particular disease state or phenotype.
  • a “negative” reference level of a metabolite means a level that is indicative of a lack of a particular disease state or phenotype.
  • a "organ failure-positive reference level" of a metabolite means a level of a metabolite that is indicative of a positive diagnosis of organ failure in a subject
  • an "organ failure-negative reference level" of a metabolite means a level of a metabolite that is indicative of a negative diagnosis of organ failure in a subject.
  • a “reference level" of a metabolite may be an absolute or relative amount or concentration of the metabolite, a presence or absence of the metabolite, a range of amount or concentration of the metabolite, a minimum and/or maximum amount or concentration of the metabolite, a mean amount or concentration of the metabolite, and/or a median amount or concentration of the metabolite; and, in addition, “reference levels” of combinations of metabolites may also be ratios of absolute or relative amounts or concentrations of two or more metabolites with respect to each other or a composed value / score obtained by classification.
  • Appropriate positive and negative reference levels of metabolites for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired metabolites in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age- matched so that comparisons may be made between metabolite levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of metabolites in biological samples (e.g., LC-MS, GC-MS, etc.), where the levels of metabolites may differ based on the specific technique that is used.
  • cell refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells
  • processor refers to a device that performs a set of steps according to a program (e.g., a digital computer).
  • processors for example, include Central Processing Units ("CPUs"), electronic devices, or systems for receiving, transmitting, storing and/or manipulating data under programmed control.
  • CPUs Central Processing Units
  • electronic devices or systems for receiving, transmitting, storing and/or manipulating data under programmed control.
  • memory device refers to any data storage device that is readable by a computer, including, but not limited to, random access memory, hard disks, magnetic (floppy) disks, compact discs, DVDs, magnetic tape, flash memory, and the like.
  • Mass Spectrometry is a technique for measuring and analyzing molecules that involves fragmenting a target molecule, then analyzing the fragments, based on their mass/charge ratios, to produce a mass spectrum that serves as a "molecular fingerprint". Determining the mass/charge ratio of an object is done through means of determining the wavelengths at which electromagnetic energy is absorbed by that object. There are several commonly used methods to determine the mass to charge ration of an ion, some measuring the interaction of the ion trajectory with electromagnetic waves, others measuring the time an ion takes to travel a given distance, or a combination of both. The data from these fragment mass measurements can be searched against databases to obtain definitive identifications of target molecules. Mass spectrometry is also widely used in other areas of chemistry, like petrochemistry or pharmaceutical quality control, among many others.
  • the term "metabolite” denotes endogenous organic compounds of a cell, an organism, a tissue or being present in body liquids and in extracts obtained from the aforementioned sources with a molecular weight typically below 1500 Dalton.
  • Typical examples of metabolites are carbohydrates, lipids, phospholipids, sphingolipids and sphingophospholipids, amino acids, cholesterol, steroid hormones and oxidized sterols and other compounds such as collected in the Human Metabolite database [Wishart DS et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan ;35 (Database issue) :D521-6(see http://www.hmdb.ca/) / and other databases and literature. This includes any substance produced by metabolism or by a metabolic process and any substance involved in metabolism.
  • Methodomics designates the comprehensive quantitative measurement of several (2-thousands) metabolites by, but not limited to, methods such as mass spectroscopy, coupling of liquid chromatography, gas chromatography and other separation methods chromatography with mass spectroscopy.
  • separation refers to separating a complex mixture into its component proteins or metabolites. Common laboratory separation techniques include gel electrophoresis and chromatography.
  • capillary electrophoresis refers to an automated analytical technique that separates molecules in a solution by applying voltage across buffer-filled capillaries.
  • Capillary electrophoresis is generally used for separating ions, which move at different speeds when the voltage is applied, depending upon the size and charge of the ions.
  • the solutes (ions) are seen as peaks as they pass through a detector and the area of each peak is proportional to the concentration of ions in the solute, which allows quantitative determinations of the ions.
  • Chromatographic refers to a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Chromatographic output data may be used for manipulation by the present invention.
  • An “ion” is a charged object formed by adding electrons to or removing electrons from an atom.
  • a “mass spectrum” is a plot of data produced by a mass spectrometer, typically containing m/z values on x-axis and intensity values on y-axis.
  • a “peak” is a point on a mass spectrum with a relatively high y-value.
  • m/z refers to the dimensionless quantity formed by dividing the mass number of an ion by its charge number. It has long been called the "mass-to-charge” ratio.
  • metabolism refers to the chemical changes that occur within the tissues of an organism, including “anabolism” and “catabolism”. Anabolism refers to biosynthesis or the buildup of molecules and catabolism refers to the breakdown of molecules.
  • post-surgical tissue refers to tissue that has been removed from a subject during a surgical procedure. Examples include, but are not limited to, biopsy samples, excised organs, and excised portions of organs.
  • detect may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition.
  • clinical failure refers to a negative outcome following organ failure treatment.
  • a biomarker in this context is a characteristic, comprising data of at least one metabolite that is measured and evaluated as an indicator of biologic processes, pathogenic processes, or responses to a therapeutic intervention associated with organ failure or related to organ failure treatment.
  • a combined biomarker as used here may be selected from at least two small endogenous molecules and metabolites.
  • the present invention relates to markers of Organ failure and its duration/severity as well of the effect of therapeutic interventions.
  • the present invention provides metabolites that are differentially present in Organ failure.
  • Experiments conducted during the course of development of embodiments of the present invention identified a series of metabolites as being differentially present in
  • Tables 2 and 3 provide additional metabolites present in plasma serum or other body liquids.
  • the disclosed markers find use as diagnostic and therapeutic targets.
  • the present invention provides methods and compositions for diagnosing organ failure, including but not limited to, characterizing risk of organ failure, stage of organ failure, duration and severity etc. based on the presence of organ failure specific metabolites or their derivatives, precursors, metabolites, etc. Exemplary diagnostic methods are described below.
  • a method of diagnosing (or aiding in diagnosing) whether a subject has organ failure comprises (1 ) detecting the presence or absence or a differential level of a plurality of organ failure specific metabolites selected from tables 1 *** and b) diagnosing organ failure based on the presence, absence or differential level of the organ failure specific metabolite.
  • the results of the method may be used along with other methods (or the results thereof) useful in the clinical determination of whether a subject has organ failure.
  • the sample may be tissue (e.g., a biopsy sample or post-surgical tissue), blood, urine, or a fraction thereof (e.g., plasma, serum, urine supernatant, urine cell pellet).
  • tissue e.g., a biopsy sample or post-surgical tissue
  • blood e.g., blood, urine, or a fraction thereof (e.g., plasma, serum, urine supernatant, urine cell pellet).
  • the patient sample undergoes preliminary processing designed to isolate or enrich the sample for organ failure specific metabolites or cells that contain organ failure specific metabolites.
  • preliminary processing designed to isolate or enrich the sample for organ failure specific metabolites or cells that contain organ failure specific metabolites.
  • a variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited: centrifugation; immunocapture; and cell lysis.
  • Metabolites may be detected using any suitable method including, but not limited to, liquid and gas phase chromatography, alone or coupled to mass spectrometry (See e.g., experimental section below), NMR, immunoassays, chemical assays, spectroscopy and the like. In some embodiments, commercial systems for chromatography and NMR analysis are utilized.
  • metabolites are detected using optical imaging techniques such as magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), CAT scans, ultra sound, MS-based tissue imaging or X-ray detection methods (e.g., energy dispersive x-ray fluorescence detection).
  • optical imaging techniques such as magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), CAT scans, ultra sound, MS-based tissue imaging or X-ray detection methods (e.g., energy dispersive x-ray fluorescence detection).
  • any suitable method may be used to analyze the biological sample in order to determine the presence, absence or level(s) of the the plurality of metabolites in the sample. Suitable methods include chromatography (e.g., HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g., MS, MS-MS), enzyme-linked immunosorbent assay (ELISA), antibody linkage, other immunochemical techniques, biochemical or enzymatic reactions or assays, and combinations thereof. Further, the level(s) of the the plurality of metabolites may be measured indirectly, for example, by using an assay that measures the level of a compound (or compounds) that correlates with the level of the biomarker(s) that are desired to be measured.
  • chromatography e.g., HPLC, gas chromatography, liquid chromatography
  • mass spectrometry e.g., MS, MS-MS
  • ELISA enzyme-linked immunosorbent assay
  • antibody linkage other immunochemical techniques, biochemical or enzymatic reactions or as
  • the levels of the plurality of the recited metabolites may be determined in the methods of the present invention. For example, the level(s) of one metabolites, two or more metabolites, three or more metabolites, four or more metabolites, five or more metabolites, six or more metabolites, seven or more metabolites, eight or more metabolites, nine or more metabolites, ten or more metabolites, etc., including a combination of some or all of the metabolites including, but not limited to those listed in table 2, may be determined and used in such methods.
  • Determining levels of combinations of the metabolites may allow greater sensitivity and specificity in the methods, such as diagnosing organ failure and aiding in the diagnosis of organ failure, and may allow better differentiation or characterization of organ failure from other disorders or other organ failure that may have similar or overlapping metabolites to organ failure (as compared to a subject not having organ failure). For example, ratios of the levels of certain metabolites in biological samples may allow greater sensitivity and specificity in diagnosing organ failure and aiding in the diagnosis of organ failure and allow better differentiation or characterization of organ failure from other organ failure or other disorders of the that may have similar or overlapping metabolites to organ failure (as compared to a subject not having organ failure).
  • a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of an organ failure specific metabolite) into data of predictive value for a clinician.
  • the clinician can access the predictive data using any suitable means.
  • the present invention provides the further benefit that the clinician, who is not likely to be trained in metabolite analysis, need not understand the raw data.
  • the data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.
  • the present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects.
  • a sample e.g., a biopsy or a blood, urine or serum sample
  • a profiling service e.g., clinical lab at a medical facility, etc.
  • any part of the world e.g., in a country different than the country where the subject resides or where the information is ultimately used
  • the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a plasma sample) and directly send it to a profiling center.
  • the sample comprises previously determined biological information
  • the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems).
  • the profiling service Once received by the profiling service, the sample is processed and a profile is produced (i.e., metabolic profile), specific for the diagnostic or prognostic information desired for the subject.
  • the profile data is then prepared in a format suitable for interpretation by a treating clinician.
  • the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of organ failure being present) for the subject, along with recommendations for particular treatment options.
  • the data may be displayed to the clinician by any suitable method.
  • the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.
  • the information is first analyzed at the point of care or at a regional facility.
  • the raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient.
  • the central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis.
  • the central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.
  • the subject is able to directly access the data using the electronic communication system.
  • the subject may chose further intervention or counseling based on the results.
  • the data is used for research use.
  • the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease.
  • the amount(s) or level(s) of the plurality of metabolites in the sample may be compared to organ failure metabolite- reference levels, such as -organ failure-positive and/or organ failure-negative reference levels to aid in diagnosing or to diagnose whether the subject has organ failure.
  • organ failure metabolite- reference levels such as -organ failure-positive and/or organ failure-negative reference levels to aid in diagnosing or to diagnose whether the subject has organ failure.
  • Levels of the plurality of metabolites in a sample corresponding to the organ failure-positive reference levels are indicative of a diagnosis of organ failure in the subject.
  • Levels of the plurality of metabolites in a sample corresponding to the organ failure-negative reference levels are indicative of a diagnosis of no organ failure in the subject.
  • levels of the plurality of metabolites that are differentially present (especially at a level that is statistically significant) in the sample as compared to organ failure-negative reference levels are indicative of a diagnosis of organ failure in the subject.
  • Levels of the plurality of metabolites that are differentially present (especially at a level that is statistically significant) in the sample as compared to organ failure-positive reference levels are indicative of a diagnosis of no organ failure in the subject.
  • the level(s) of the plurality of metabolites may be compared to organ failure-positive and/or organ failure-negative reference levels using various techniques, including a simple comparison (e.g., a manual comparison) of the level(s) of the plurality of metabolites in the biological sample to organ failure-positive and/or organ failure- negative reference levels.
  • the level(s) of the plurality of metabolites in the biological sample may also be compared to organ failure-positive and/or organ failure-negative reference levels using one or more statistical analyses (e.g., t-test, Welch's t-test, Wilcoxon's rank sum test, random forests, support vector machines, linear discriminant analysis, k nearest neighbours).
  • compositions for use include reagents for detecting the presence or absence of organ failure specific metabolites. Any of these compositions, alone or in combination with other compositions of the present invention, may be provided in the form of a kit. Kits may further comprise appropriate controls and/or detection reagents.
  • Embodiments of the present invention provide for multiplex or panel assays that simultaneously detect a plurality of the markers of the present invention depicted in tables 1 to 3, alone or in combination with additional organ failure markers known in the art.
  • panel or combination assays are provided that detected 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more, 30 or more, 40 or more markers in a single assay.
  • assays are automated or high throughput.
  • a preferred embodiment of the present invention is the use of markers listed in tables 2 and 3 for prediction/diagnosis of organ failure and its duration/severity where said mammalian subject is a human being, said biological sample blood and/or blood cells.
  • additional organ failure markers are included in multiplex or panel assays. Markers are selected for their predictive value alone or in combination with the metabolic markers described herein.
  • the present invention provides therapeutic methods (e.g., that target the organ failure specific metabolites described herein).
  • the therapeutic methods target enzymes or pathway components of the organ failure specific metabolites described herein.
  • the present invention provides compounds that target the organ failure specific metabolites of the present invention.
  • the compounds may decrease the level of organ failure specific metabolite by, for example, interfering with synthesis of the organ failure specific metabolite (e.g., by blocking transcription or translation of an enzyme involved in the synthesis of a metabolite, by inactivating an enzyme involved in the synthesis of a metabolite (e.g., by post translational modification or binding to an irreversible inhibitor), or by otherwise inhibiting the activity of an enzyme involved in the synthesis of a metabolite) or a precursor or metabolite thereof, by binding to and inhibiting the function of the organ failure specific metabolite, by binding to the target of the organ failure specific metabolite (e.g., competitive or non competitive inhibitor), or by increasing the rate of break down or clearance of the metabolite.
  • interfering with synthesis of the organ failure specific metabolite e.g., by blocking transcription or translation of an enzyme involved in the synthesis of a
  • the compounds may increase the level of organ failure specific metabolite by, for example, inhibiting the break down or clearance of the organ failure specific metabolite (e.g., by inhibiting an enzyme involved in the breakdown of the metabolite), by increasing the level of a precursor of the organ failure specific metabolite, or by increasing the affinity of the metabolite for its target.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates.
  • the present invention provides drug screening assays (e.g., to screen for anti - organ failure drugs).
  • the screening methods of the present invention utilize organ failure specific metabolites described herein.
  • test compounds are small molecules, nucleic acids, or antibodies.
  • test compounds target organ failure specific metabolites directly. In other embodiments, they target enzymes involved in metabolic pathways of organ failure specific metabolites.
  • Plasma samples were prepared by standard procedures and stored at (-70 0 C). To enable analysis of all samples simultaneously within one batch, samples were thawed on ice (1 h) on the day of analysis and centrifuged at 18000 g at 2°C for 5 min. All tubes were prepared with 0.001 % BHT (butylated hydroxytoluene; Sigma-Aldrich, Vienna, Austria) to prevent artificial formation of prostaglandins caused by autooxidation .
  • BHT butylated hydroxytoluene
  • Liver tissue samples were homogenized using a Precellys® 24 homogenizer with Cryolys cooling module before analysis. Typically 50 mg of tissue were homogenized in ethanol : phosphate buffer 9:1 (v/v) for 30 min and unsolved material and beads for tissue desintegration removed by 5 min centrifugation at 10 000g.
  • VLM nitrogen evaporator
  • Mass spectrometric analysis was performed on an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies) equipped with an electro-spray ionization (ESI)-source using the analysis acquisition method as provided in the Absolute/DQ kit.
  • the standard FIA-MS/MS method was applied for all measurements with two subsequent 20 ⁇ l_ injections (one for positive and one for negative mode analysis).
  • Multiple reaction monitoring (MRM) detection was used for quantification applying the spectra parsing algorithm integrated into the MetlQ software (Biocrates Life Sciences AG). Concentration values for 148 metabolites (all analytes determined with the metabolomics kit besides of the amino acids, which were determined by a different method) obtained by internal calibration were exported for comprehensive statistical analysis.
  • Amino acids and biogenic amines were quantitatively analyzed by reversed phase LC- MS/MS to obtain chromatographic separation of isobaric (same MRM ion pairs) metabolites for individual quantitation performed by external calibration and by use of internal standards.
  • 10 ⁇ L sample volume (plasma, brain homogenate) is required for the analysis using the following sample preparation procedure. Samples were added on filter spots placed in a 96- solvinert well plate (internal standards were placed and dried down under nitrogen before), fixed above a 96 deep well plate (capture plate). 20 ⁇ L of 5% phenyl-isothiocyanate derivatization reagent was added.
  • the derivatized samples were extracted after incubation by aqueous methanol into the capture plate.
  • Sample extracts were analyzed by LC-ESI-MS/MS in positive MRM detection mode with an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies).
  • the analyzed individual metabolite concentrations (Analyst 1.4.2 software, Applied Biosystems) were exported for comprehensive statistical analysis.
  • Bile acids (LC-MS/MS) A highly selective reversed phase LC-MS/MS analysis method in negative MRM detection mode was applied to determine the concentration of bile acids in plasma samples. Samples were extracted via dried filter spot technique in 96 well plate format, which is well suitable for high throughput analysis. For highly accurate quantitation internal standards and external calibration were applied. In brief, internal standards and 20 ⁇ l_ sample volume placed onto filter spots were extracted and simultaneously protein precipitated with aqueous methanol. These sample extracts were measured by LC-ESI- MS/MS with an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies). Data of bile acids were quantified with Analyst 1.4.2 software (Applied Biosystems) and finally exported for comprehensive statistical analysis.
  • Prostanoids - a term summarizing prostaglandins (PG), thromboxanes (TX) and prostacylines - and oxidised fatty acid metabolites were analyzed in plasma extracts by
  • Oxysterols are determined after extraction and saponification by HPLC-Tandem mass spectrometer (HPLC-API-MS/MS) in positive detection mode using Multiple Reaction Mode (MRM).
  • HPLC-Tandem mass spectrometer HPLC-API-MS/MS
  • MRM Multiple Reaction Mode
  • Chromatographic separation and detection is performed by using a Zorbax Eclipse XDB C18, 150 x 2.0 mm, 3.5 ⁇ m HPLC-Column at a flow rate of 0.3 mL/min followed by electrospray ionization on the API4000/QTRAP4000 tandem mass spectrometer.
  • the Analyst Quantitation software from Applied Bioystems was used.
  • hdyrophilic interaction liquid chromatography HILIC-ESI-MS/MS method in highly selective negative MRM detection mode was used.
  • the MRM detection was performed using an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies). 20 ⁇ l_ sample volume (plasma, brain homogenate) was protein precipitated and extracted simultaneously with aqueous methanol in a 96 well plate format. Internal standards (ratio external to internal standard) and external calibration were used for highly accurate quantitation. Data were quantified with Analyst 1.4.2 software (Applied Biosystems) and finally exported for statistical analysis.
  • ADMA B.Am total DMA B.Am.
  • Table 1 summarizes analyzed metabolites and respective abbreviations; Glycero-phospholipids are further differentiated with respect to the presence of ester (a) and ether (e) bonds in the glycerol moiety, where two letters (aa, ea, or ee) denote that the first and the second position of the glycerol scaffold are bound to a fatty acid residue, whereas a single letter (a or e) indicates a bond with only one fatty acid residue; e.g. PC_ea_33:1 denotes a plasmalogen phosphatidylcholine with 33 carbons in the two fatty acid side chains and a single double bond in one of them.
  • ester (a) and ether (e) bonds in the glycerol moiety where two letters (aa, ea, or ee) denote that the first and the second position of the glycerol scaffold are bound to a fatty acid residue, whereas a single letter (a or e) indicates
  • missing metabolite concentrations are replaced by the average value of the 6 closest samples to the one where the measurement is missing (impute: Imputation for microarray data, Hastie T., Tibshirani R., Narasimhan B. and Chu G., R package version 1.14.0).
  • Imputation for microarray data Hastie T., Tibshirani R., Narasimhan B. and Chu G., R package version 1.14.0.
  • FC fold change
  • the ImFit function in the package limma (Limma: linear models for microarray data, Smyth G. K. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer, New York, pp 397-420, R package version 2.16.5) is used to compute the moderated statistics between measurements from septic patients samples and samples from patient developing organ failure. Resulting p values are adjusted by the method described in Benjamini and Hochberg (Benjamini Y. and Hochberg Y., Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society Series B, 1995, 57, 289-300) leading to so-called q values.
  • Sensitivity/specificity properties of a classifier comprising one analyte or a combination of analytes are summarised in terms of Area Under the Receiver Operating Characteristic Curve (AUC).
  • AUC Area Under the Receiver Operating Characteristic Curve
  • the function colAUC (caTools: Tools: moving window statistics, GIF, Base64, ROC AUC, etc., Tuszynski J., 2008, R package version 1.9) is used to compute and plot ROC curves. From the three univariate statistics (adjusted p value (q value), fold change and AUC), features are ranked according to a 2 step strategy: 1 ) the 3 measures are first used as input to the multiple objective algorithm described by Chen et al.
  • gbm function from gbm R package (gbm: Generalized Boosted Regression Models, Ridgeway G., 2007, R package version 1.6-3) was used to perform tree based gradient boosting specifying a gaussian loss function, a shrinkage parameter of 0.05 and allowing trees with up to 3 trees splits.
  • feature relevance score is presented as the average rank calculated by leaving one set out on the training set.
  • Performance of single markers as well as of combinations of markers is assessed by three classification algorithms that rely on different mechanisms to ensure that the outcome is not dependent on the modelling technique: support vector machine (SVM) with linear kernel using the R function svm in package e1071 (e1071 : Misc Functions of the Department of Statistics (e1071 ), Dimitriadou E., Hornik k., Leisch F., Meyer D.
  • SVM support vector machine
  • Table 2 depicts the ranks of the individual analytes and metabolites in terms of discriminatory power for detecting the onset of infection associated organ failure. Ranking was performed using a ranker combining adjusted p values, fold changes and AUCs as well as using a multivariate wrapper which is based on boosted regression trees as described above. For additional information see Fig 1-3.
  • the effective dosis of the extract (to induce either sepsis or organ failure) has to be pre- determined for each batch (of stool from one individual human subject).
  • sepsis can be induced within 24 h with a complete recovery of the animals > 48 h or septic organ failure can be induced by applying a higher dosage; for instance sepsis can be induced by injection of 0.5 ml of extract and organ failure by injection of 1.0 ml intraperitoneal ⁇ . All samples of liver tissue were drawn 24 h after intraperitoneal injection of the extract.
  • missing metabolite concentrations are replaced by the average value of the 6 closest samples to the one where the measurement is missing (impute: Imputation for microarray data, Hastie T., Tibshirani R., Narasimhan B. and Chu G., R package version 1.14.0).
  • Imputation for microarray data Hastie T., Tibshirani R., Narasimhan B. and Chu G., R package version 1.14.0.
  • FC fold change
  • the ImFit function in the package limma (Limma: linear models for microarray data, Smyth G. K. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer, New York, pp 397-420, R package version 2.16.5) is used to compute the moderated statistics between measurements from septic patients samples and samples from patient developing organ failure. Resulting p values are adjusted by the method described in Benjamini and Hochberg (Benjamini Y. and Hochberg Y., Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society Series B, 1995, 57, 289-300) leading to so-called q values.
  • Sensitivity/specificity properties of a classifier comprising one analyte or a combination of analytes are summarised in terms of Area Under the Receiver Operating Characteristic Curve (AUC).
  • AUC Area Under the Receiver Operating Characteristic Curve
  • the function colAUC (caTools: Tools: moving window statistics, GIF, Base64, ROC AUC, etc., Tuszynski J., 2008, R package version 1.9) is used to compute and plot ROC curves. From the three univariate statistics (adjusted p value (q value), fold change and AUC), features are ranked according to a 2 step strategy: 1 ) the 3 measures are first used as input to the multiple objective algorithm described by Chen et al.
  • Desmosterol 30 5.26E-002 -68,61 0,80
  • Table 3 depicts the ranks of the individual analytes and metabolites in terms of discriminatory power for detecting the onset of infection associated organ failure. Ranking was performed using a univariate ranker which combines adjusted p values, fold changes and AUCs. For additional information see Fig 4.
  • Table 4 shows the endogenous organ failure predictive targe metabolites as used in the present invention with their abbreviations and chemical names

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Abstract

The present invention relates to a reliable and statistically significant method for predicting the likelihood of an onset of an inflammation associated organ failure from a biological sample of a mammalian subject in vitro, by means of a subject's quantitative metabolomics profile comprising a plurality of endogenous metabolites, and comparing it with a quantitative reference metabolomics profile of a plurality of endogenous organ failure predictive target metabolites in order to predict whether the subject is likely or unlikely to develop an organ failure. Furthermore, the invention relates to the usefulness of endogenous organ failure predictive target metabolites in such a method.

Description

Method for Predicting the likelihood of an Onset of an Inflammation Associated Organ Failure The present invention relates to a method for predicting the likelihood of an onset of an inflammation or infection associated organ failure from a biological sample of a mammalian subject in vitro in accordance with claim 1.
The invention generally relates to biomarkers for organ failure as tools in clinical diagnosis for early detection of organ failure, therapy monitoring and methods based on the same biomarkers.
BACKGROUND of the Invention Organ failure (OF) strikes an estimated 200 000 people in the U.S.
annually and kills 60% of them. While organ failure may arise from an infection and hospitals are seeing more cases in part due to increasing numbers of
immunosuppressed cancer and transplant patients, an increasing number of hospital patients are at risk.
The mortality of multiorgan dysfinction syndrome (MODS) in hospitals is around 50%. The main etiological factors for MODS still are severe infection, major operations, trauma and severe pancreatitis. (Zhang SW, Wang C, Yin CH, Wang H, Wang BE, Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2004, 16, 328-32. Multi-center clinical study on the diagnostic criteria for multiple organ dysfunction syndrome with illness severity score system).
Diagnostics of OF and MODS so far relies on clinical criteria and scores such as the Atlanta criteria and Sepsis-Related Organ Failure Assessment (SOFA)-score as well as on the use of few unreliable protein marker. For instance, severe acute pancreatitis with systemic organ dysfunctions develops in about 25% of patients with acute pancreatitis. Biochemical parameters are limited to protein markers such as procalcitonin (PCT), C reactive protein (CRP) and interleukins (Beger HG, Rau BM, Severe acute pancreatitis: Clinical course and management World J Gastroenterol. 2007, 13, 5043-51 ). Organ failure in acute pancreatitis was predicted by using a combination of plasma interleukin 10 and serum calcium measurements (Early Prediction of Organ Failure by Combined Markers in Patients With Acute Pancreatitis Mentula P, Kylanpaa M-L, Kemppainen E, Br J Surg, 92, 68 - 75, 2005). In trauma patients, interleukin 6 and interleukin 10 were used for multiple OF prediction (Lausevic Z, Lausevic M, Trbojevic-Stankovic J, Krstic S, Stojimirovic, Predicting multiple organ failure in patients with severe trauma B Can J Surg. 2008, 51 , 97-102).
Severe sepsis also includes OF and occurs when one or more vital organs are compromised. It can lead to septic shock, which is marked by low blood pressure that does not respond to standard treatment, problems in vital organs, and oxygen
deprivation. About half of patients who suffer septic shock die.
Early diagnosis of beginning OF, however, is difficult because its clinical signs can mimic other conditions. The complexity of the host's response during the systemic inflammatory response has complicated efforts towards understanding disease pathogenesis (Reviewed in Healy, Annul. Pharmacother. 36: 648-54 (2002).). Early diagnosis, however, is the key to saving more lives, but available diagnostics so far do not indicate beginning organ failure. Consequently, some labs have started to offer faster tests for OF markers to speed diagnosis.
Besides critical care medicine therapy such as antibiotics therapy and symptomatic therapy, the treatment of organ failure is still limited to preventive measures and symptomatic supportive strategies.
Current diagnostics in clinical routine is limited to a) clinical information b) use of basic biochemical clinical parameters as outlined below in the definitions
Or unspecific biomarkers like C-reactive protein (CRP) or procalcitonin (PCT) with low sensitivities and specificities (Critical Care Medicine 2006; 34:1996-2003, Archives of Surgery 2007; 142:134-142).
Sepsis by definition comprises systemic inflammatory response syndrome (SIRS) and infection with pathogens. Systemic inflammatory response syndrome (SIRS) is considered to be present when two or more of the following clinical findings are present:
1. Body temperature >38°C or <36°C;
2. Heart rate >90 min"1 ;
-1
3. Hyperventilation evidenced by a respiratory rate of >20 min or a PaCO2 of <32 mm Hg; and
-1 -1
4. White blood cell count of >12,000 cells μl_ or <4,000 μl_
The quantitative metabolomics profile of the endogenous organ failure predictive target metabolites can be combined with any of the above classical clinical laboratory parameters.
Organ failure includes a systemic inflammatory response syndrome (SIRS) together with an infection. Sepsis (commonly called a "blood stream infection") denotes the presence of bacteria (bacteremia) or other infectious organisms or their toxins in the blood (septicemia) or in other tissue of the body and the immune response of the host. Organ failure due to sepsis is currently thought to start with the interaction between the host response and the presence of micro-organisms and/or their toxins within the body. The observed host responses include immune, coagulation, pro and anti-inflammatory responses. Septic organ failure thus comprises a systemic response to infection, defined as hypothermia or hyperthermia, tachycardia, tachypnea, a clinically evident focus of infection or positive blood cultures, one or more end organs with either dysfunction or inadequate perfusion, cerebral dysfunction, hypoxaemia, increased plasma lactate or unexplained metabolic acidosis, and oliguria.
While usually related to infection, it can also be associated with noninfectious insults such as trauma, burns, and pancreatitis. It is one of the most common causes of adult respiratory distress syndrome.
A precise definition of the term sepsis has been introduced by the ACCP/SCCM Consensus Conference Committee (1992): Definition for sepsis and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 20(6):864-874. The 2001 International Organ failure Definitions Conference attempted to improve the above definition with the aim of increasing the accuracy of the diagnosis of sepsis Levy M, Fink M, Mitchell P, Marshall JC, Abraham E, et al. for the International Sepsis Definitions Conference. 2001 SCCM/ESICM/ACCP/ATS/SIS. The statement suggested that although the SIRS concept was valid, in the future if supported by further epidemiologic data, it may be possible to use purely biochemical and/or immunologic, rather than clinical criteria to identify the inflammatory response. It also defined infection as a pathologic process induced by a micro-organism, and that organ failure should be defined as a patient with documented or suspected infection' exhibiting some of the following variables:
1. General variables
o Fever (core temperature >38.3 °C)
o Hypothermia (core temperature <36°C)
o Heart rate >90 min"1 or >2 SD above the normal value for age
o Tachypnea
o Altered mental status
o Significant oedema or positive fluid balance (>20 mL/kg over 24 hrs) o Hyperglycemia (plasma glucose >7.7 mmol/L) in the absence of diabetes
2. Inflammatory variables
o Leukocytosis - WBC count >12,000 μL"1
o Leukopaenia - WBC count <4000 μL"1
o Normal WBC count with >10% immature forms
o Plasma C-reactive protein >2 SD above the normal value
o Plasma procalcitonin >2 SD above the normal value
3. Hemodynamic variables
o Arterial hypotension (SBP <90 mmHg, MAP <70 mmHg, or an SBP decrease >40 mmHg in adults)
o SvO2a >70%
o Cardiac index > 3.5 Lmin"1 M"2
4. Organ dysfunction variables
o Arterial hypoxemia (PaO2/FIO2 <300)
o Acute oliguria (urine output <0.5 mLkg"1 hr"1 for at least 2hrs) o Creatinine increase >0.5 mg/dL
o Coagulation abnormalities (INR >1.5 or aPTT >60 sees)
o Ileus (absent bowel sounds)
o Thrombocytopenia (platelet count <1 OOμL)
o Hyperbilirubinemia (plasma total bilirubin>4 mg/dL or 70 mmol/L)
5. Tissue perfusion variables
o Hyperlactatemia (>1 mmol/L)
o Decreased capillary refill or mottling (WBC, white blood cell; SBP, systolic blood pressure; MAP, mean arterial blood pressure; SvO2, mixed venous oxygen saturation; INR, international normalized ratio; aPTT, activated partial thromboplastin time; tachycardia (may be absent in hypothermic patients), and at least one of the following indications of altered organ function: altered mental status, hypoxemia, increased serum lactate level.
The definition of severe sepsis remained unchanged and refers to sepsis complicated by organ dysfunction. Organ dysfunction is defined using Multiple Organ Dysfunction score Marshall JC, Cook DJ, Christou NV, et al. Multiple organ dysfunction score: A reliable descriptor of a complex clinical outcome. Crit Care Med 1995; 23: 1638-1652 or the definitions used for the Sequential Organ Failure Assessment (SOFA) score Ferreira FL, Bota DP, Bross A, et al. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA 2002; 286: 1754-1758. Septic shock in adults refers to a state of acute circulatory failure characterized by persistent arterial hypotension unexplained by other causes. Hypotension is defined by a systolic arterial pressure below 90 mm Hg, a MAP <70 mmHg, or a reduction in systolic blood pressure of >40 mm Hg from baseline, despite adequate volume resuscitation, in the absence of other causes for hypotension.
The mortality rate associated with organ failure, severe sepsis and septic shock are high and reported as 25 to 30% and 40 to 70% respectively. Bernard GR, Vincent JL,
Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001 ; 344: 699-709. Annane D, Aegerter P, Jars- Guincestre MC, Guidet B. Current epidemiology of septic shock: the CUB-Rea Network. Am J Respir Crit Care Med 2003; 168: 165-72.
A number of other prognositic approaches appear in the scientific community, a selection is shown below. However, all these approaches do not address the problem of predicting the likelihood of an onset of an inflammation associated organ failure:
Xu et al., J. Infection (2008) 56, 471 -481 describes a metabonomic approach to early prognostic evaluation of experimental sepsis in rats by using linolenic acid, linoleic acid, oleic acid, stearic acid, docosahexanoic acid and docosapentaenoic acid as biomarkers to discriminate surving, non-surving and sham-operated groups of animals. Nowhere in this paper, organ failure is mentioned, let alone addressed by specifically disclosed biomarkers. Bradford et al., Toxicology and Applied Pharmacology 232 (2008), 236-243 describes metabolomic profiling of a modified alcohol liquid diet model for liver injury in the mouse using amino acids. However, a prdeiction of an inflammation associated organ failure is not mentioned. US 2009/0104596 A1 discloses methods and kits for diagnosing a disease state of cachexia by measuring biomarker profiles. The biomarkers concerned are those known from the energy metabolism, namely lactate, citrate, formate, acetoacetate, 3-hydroxy butyrate and some amino acids. Organ failure of any kind is not addressed. Freund et al., Ann. Surg. (1979), 190, 571 -576 desclose the use of a plasma amino acid pattern as predictors of the severity and outcome of sepsis for discriminating between septic encephalopathy and no encephalopathy, wherein the degree of encephalopathy of a patient is considered an expression for the severity of the septic process. Additionally, this document discriminates between survivors and non-survivors of a sepsis. Predictors of organ failure are not mentioned. Munoz et al., Transplantation Proceedings (1993), 25, 1779-1782 desclose serum amino acids as an indicator of hepatic graft functional status following orthotopic liver transplantation. Furthermore, WO 2006/071583 A2 relates to method and compositions for determining treatment regimens in SIRS. Although, multiple organ dysfunftion syndrome (MODS) is mentioned, this document does not provide any information which biomarkers could be used for a prognosis of MODS, let alone which biomarkers could be used for a prediction of of the likelihood of an onset of inflammation associated organ failure.
Moyer et al., The Journal of Trauma (1981 ), 21 , 862-869 discloses death predictors in the trauma-septic state by means of an amino acid pattern, however, no predictors for the likelihood of an onset of an inflammation associated organ failure is mentioned. Finally, background information on HPLC analysis of amino acids in physiological samples is described in Fekkes, D., Journal of Chromatography B (1996), 682, 3-22, and the identification of phenylthiocarbamyl amino acids for compositional analysis by thermospray LC/MS is disclosed in Pramanik et al., Analyt. Biochem. (1989), 176, 269- 277.
Despite some advances in the management of severe sepsis and septic shock, problems remain regarding the usefulness of the currently used definitions and the often encountered delays in diagnosis. The reliable diagnosis of organ failure still remains a challenge.
The identification, let alone the quantification of pathogens or of nucleic acids from these pathogens in an ill subject is far from being reliable, validated or sufficient for diagnosis, a large body of scientific evidence supports diagnostics based on the molecular response and immune response of the host, actually reflecting the individual clinical state of the subject, regardless of the nature or quantities of the underlying pathogens, respectively fragments of these organisms.
In classical patient screening and diagnosis, the medical practitioner uses a number of diagnostic tools for diagnosing a patient suffering from a certain disease. Among these tools, measurement of a series of single routine parameters, e.g. in a blood sample, is a common diagnostic laboratory approach. These single parameters comprise for example enzyme activities and enzyme concentration and/or detection.
As far as such diseases are concerned which easily and unambiguously can be correlated with one single parameter or a few number of parameters achieved by clinical chemistry, these parameters have proved to be indispensable tools in modern laboratory medicine and diagnosis. However, in pathophysiological conditions, such as cancer or demyelinating diseases such as multiple sclerosis which share a lack of an unambiguously assignable single parameter or marker, differential diagnosis from blood or tissue samples is currently difficult to impossible.
Although RNA-based diagnosis of organ failure from blood cells has been explored recently, these approaches, however, suffer from several serious limitations:
The required sample size of usually several ml of blood is a problem for continuous monitoring of a critically ill subject; alternatives applying amplification of transcripts are lengthy and prone to error. The whole procedure affords numerous steps and due to laborious sample preparation and RNA isolation, transcription and array or PCR analysis still takes at least several hours and a large technological effort.
Currently used diagnostic methods thus require time and appropriate equipment with high costs and frequently unsatisfying sensitivities. However this used diagnostic means have major limitations either to reduced area under the curve (AUC) and/or delay of diagnosis or increased costs due to equipment required. Accordingly these procedures do not allow a timely assessment of an acute and rapidly evolving disease and overall the situation is far from satisfying and from providing a rapid and reliable diagnosis of severe sepsis and organ failure.
Therefore, there is still an urgent need for an early, rapid and reliable diagnosis of organ failure or any other state of health providing the unspecific clinical symptoms, ideally requiring only minute amounts of blood; there is an urgent need for timely treatment and early diagnosis of organ failure as well as, an urgent need for therapy monitoring.
Further, there is an urgent need for early organ failure biomarkers enabling early and reliable diagnosis.
These needs are met by a method for in vitro predicting the likelihood of an onset of organ failure in accordance with claim 1. In particular, the present invention provides a solution to these problems based on the application of a new technology in this context and on an unknown list of endogenous metabolites as diagnostic marker. Since metabolite concentration differences in biological fluids and tissues provide links to the various phenotypical responses, metabolites are suitable biomarker candidates.
The present invention allows for accurate, rapid, and sensitive prediction and diagnosis of OF through a measurement of a plurality of endogenous metabolic biomarker (metabolites) taken from a biological sample at a single point in time. This is accomplished by obtaining a biomarker panel at a single point in time from an individual, particularly an individual at risk of developing OF, having OF, or suspected of having OF, and comparing the biomarker profile from the individual to reference biomarker values or scores. The reference biomarker values may be obtained from a population of individuals (a "reference population") who are, for example, afflicted with OF or who are suffering from either the onset of OF or a particular stage in the progression of OF. If the biomarker panel values or score from the individual contains appropriately characteristic features of the biomarker values or scores from the reference population, then the individual is diagnosed as having a more likely chance of getting OF, as being afflicted with OF or as being at the particular stage in the progression of OF as the reference population.
Accordingly, the present invention provides, inter alia, methods of predicting the likelihood of an onset of OF in an individual. The methods comprise obtaining a biomarker score at a single point in time from the individual and comparing the individual's biomarker profile to a reference biomarker profile. Comparison of the biomarker profiles can predict the onset of OF in the individual preferably with an accuracy of at least about . This method may be repeated again at any time prior to the onset of OF.
The present invention further provides a method of determining the progression (i.e., the stage) of sepsis in an individual towards OF. This method comprises obtaining a biomarker profile at a single point in time from the individual and comparing the individual's biomarker profile to a reference biomarker score. Comparison of the biomarker scores can determine the progression of sepsis in the individual preferably with an accuracy of at least about 90 %. This method may also be repeated on the individual at any time. Additionally, the present invention provides a method of diagnosing OF in an individual having or suspected of having OF. This method comprises obtaining a biomarker score at a single point in time from the individual and comparing the individual's biomarker score to a reference biomarker score. Comparison of the biomarker profiles can diagnose OF in the individual with an accuracy of at least about 90 %. This method may also be repeated on the individual at any time.
In another embodiment, the invention provides, inter alia, a method of determining the status of OF or diagnosing OF in an individual comprising applying a decision rule. The decision rule comprises comparing (i) a biomarker score generated from a biological sample taken from the individual at a single point in time with (ii) a biomarker score generated from a reference population. Application of the decision rule determines the status of sepsis or diagnoses OF in the individual. The method may be repeated on the individual at one or more separate, single points in time.
The present invention further provides, inter alia, a method of determining the status of OF or diagnosing OF in an individual comprising obtaining a biomarker score from a biological sample taken from the individual and comparing the individual's biomarker score to a reference biomarker score. A single such comparison is capable of classifying the individual as having membership in the reference population. Comparison of the biomarker scores determines the status of OF or diagnoses OF in the individual.
In yet another embodiment, the present invention provides, inter alia, a method of determining the status of OF or diagnosing OF in an individual. The method comprises comparing a measurable characteristic of at least one biomarker between a biomarker panel or biomarker score composed by (processed or unprocessed) values of this panel obtained from a biological sample from the individual and a biomarker score obtained from biological samples from a reference population. Based on this comparison, the individual is classified as belonging to or not belonging to the reference population. The comparison, therefore, determines the likelihood of OF or diagnoses OF in the individual. The biomarkers, in one embodiment, are selected from the group of biomarkers shown in any one of TABLES 1 to 3.
The present invention provides methods for predicting organ failure, which is clinically deary to be distinguished from methods of diagnosing sepsis, SIRS, and the like. Such methods comprise the steps of: analyzing a biological sample from a subject to determine the level(s) of a purality of biomarkers for organ failure in the sample, where the plurality of biomarkers are selected from Table 1 and comparing the level(s) of the the plurality of biomarkers - respectively a composed value / score generated by subjecting the concentrations of individual biomarkers in the sample to a classification method such as affording an equation processing single concentration values - to obtain a separation between both (diseased and healthy) groups or comparing the level(s) of the the plurality of biomarkers in the sample to organ failure positive or organ failure negative reference levels of the the plurality of biomarkers in order to determine at a very early state whether the subject is developing organ failure or not, so that suitable therapeutic measures can be started.
The present invention provides a solution to the problem described above, and generally relates to the use of metabolomics data, generated by quantitation of endogenous metabolites by but not limited to mass spectrometry (MS), in particular MS- technologies such as MALDI, ESI, atmospheric pressure pressure chemical ionization (APCI), and other methods, determination of metabolite concentrations by use of MS- technologies or alternative methods coupled to separation (LC-MS, GC-MS, CE-MS), subsequent feature selection and /or the combination of features to classifiers including molecular data of at least two molecules.
The concentrations of the individual markers, analytes, metabolites thus are measured and compared to reference values or data combined and processed to scores, classifiers and compared to reference values thus indicating diseased states etc. with superior sensitivities and specificities compared to known procedures, clinical parameters and biomarkers.
Those skilled in the art will understand that for the quantitation of certain metabolites, also chemically modified metabolites may be used. For example, it is a well established practice to use the phenylisothiocyanates of amino acids for a more sensitive (sensitivity enhancement up to 100 fold) and preciser quantification, as one gets a better separation on the column material used prior to the MS-technologies.
Furthermore, in some embodiments, the present invention provides a method of diagnosing organ failure and/or duration/severity comprising: detecting the presence or absence of a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, etc. measured together in a multiplex or panel format) of organ failure specific metabolites in a sample
(e.g., a tissue (e.g., biopsy) sample, a blood sample, a serum sample, or a urine sample) from a subject; and diagnosing organ failure based on the presence of the organ failure specific metabolite.
The present invention further provides a method of screening compounds, comprising: contacting an animal, a tissue, a cell containing a organ failure-specific metabolite with a test compound; and detecting the level of the organ failure specific metabolite. In some embodiments, the method further comprises the step of comparing the level of the organ failure specific metabolite in the presence of the test compound or therapeutic intervention to the level of the organ failure specific metabolite in the absence of the organ failure specific metabolite. In some embodiments, the cell is in vitro, in a non- human mammal, or ex vivo. In some embodiments, the test compound is a small molecule or a nucleic acid (e.g., antisense nucleic acid, a siRNA, or a miRNA) or oxygen/xenon or any neuroprotective drug that inhibits the expression of an enzyme involved in the synthesis or breakdown of an organ failure specific metabolite. In some embodiments, the organ failure specific metabolite groups given in Tables 2 and 3. In some embodiments, the method is a high throughput method.
In particular, the present invention relates to:
A method for predicting the likelihood of onset of an inflammation associated organ failure from a biological sample of a mammalian subject in vitro, wherein a. the subject's quantitative metabolomics profile comprising a plurality of endogenous metabolites, is detected in the biological sample by means of quantitative metabolomics analysis, and b. the quantitative metabolomics profile of the subject's sample is compared with a quantitative reference metabolomics profile of a plurality of endogenous organ failure predictive target metabolites in order to predict whether the subject is likely or unlikely to develop an organ failure; and c. wherein said endogenous organ failure predictive target metabolites have a molecular mass less than 1500 Da and are selected from the group consisting of: Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid (glutamate), glutamine, leucine, isoleucine, histidine, ornithine, proline, phenylalanine, serine, tryptophane, tyrosine, valine, kynurenine; phenylthio carbamyl amino acids (PTC-amino acids), in particular, PCT-arginine, PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-tyrosine, PTC-valine; dimethylarginine, in particular N,N-dimethyl-L-arginine; carboxylic acids, namely 15(S)-hydroxy-5Z,8Z,1 1 Z,13E-eicosatetraenoic acid [(5Z,8Z,1 1 Z,13E,15S)-15-Hydroxyicosa-5,8,11 ,13-tetraenoic acid], succinic acid (succinate);
Ceramides, with an N-acyl residue having from 2 to 30 Carbon atoms in the acyl residue and having from 0 to 5 double bonds and having from 0 to 5 hydroxy groups; carnitine; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue and having 1 to 4 double bonds in the acyl residue; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue and having from 1 to 3 OH-groups in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue with 1 to 4 double bonds and 1 to 3 OH- groups in the acyl residue; phospholipides, in particular lysophosphatidylcholines (monoacylphospha-tidylcholines) having from 1 to 30 carbon atoms in the acyl residue; lysophosphatidylcholines having from 3 to 30 carbon atoms in the acyl residue and having 1 to 6 double bonds in the acyl residue; phosphatidylcholines (diacylphosphatidylcholines) having a total of from 1 to 50 carbon atoms in the acyl residues; phosphatidylcholines having a total from 3 to 50 carbon atoms in the acyl residues and having a total of 1 to 8 double bonds in the acyl residues; sphingolipids, in particular sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30; sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30 and 1 to 5 double bonds; hydroxysphinogomyelines having a total number of carbon atoms in the acyl residues from 10 to 30;
hydroxysphingoyelines having a total number of carbon atoms in the acyl residues from
10 to 30 and 1 to 5 double bonds; prostaglandines, namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane
B2; putrescine; oxysterols, namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20α- hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3β,5α,6β- trihydroxycholesterol, 7α- hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5α,6α- epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycolithocholic acid sulfate, glycoursodeoxycholic acid, lithocholic acid, taurocholic acid, taurochenodeoxycholic acid taurodeoxycholic acid, taurolithocholic acid, taurolithocholic acid sulfate, tauroursodeoxycholic acid, ursodeoxycholic acid; biogenic amines, namely histamine, serotonine, palmitoyl ethanolamine.
According to the present invention, the term "inflammation associated organ failure" comprises "infection associated organ failure" and/or "sepsis associated organ failure".
A preferred method is one, wherein the biological sample is selected from the group consisting of stool; body fluids, in particular blood, liquor, cerebrospinal fluid, urine, ascitic fluid, seminal fluid, saliva, puncture fluid, cell content, tissue samples, in particular liver biopsy material; or a mixture thereof.
Advantageously, a preferred embodiment of the method according to the present invention is one, wherein said quantitative metabolomics profile is achieved by a quantitative metabolomics profile analysis method comprising the generation of intensity data for the quantitation of endogenous metabolites by mass spectrometry (MS), in particular, by high- throughput mass spectrometry, preferably by MS-technologies such as Matrix Assisted Laser Desorption/lonisation (MALDI), Electro Spray Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), 1H-, 13C- and/or 31 P- Nuclear
Magnetic Resonance spectroscopy (NMR), optionally coupled to MS, determination of metabolite concentrations by use of MS-technologies and/or methods coupled to separation, in particular Liquid Chromatography (LC-MS), Gas Chromatography (GC- MS), or Capillary Electrophoresis (CE-MS).
Furthermore, preferably, intensity data of said metabolomics profile are normalized with a set of endogenous housekeeper metabolites by relating detected intensities of the selected endogenous organ failure predictive target metabolites to intensities of said endogenous housekeeper metabolites.
A particularly preferred method according to the present invention is one, wherein said endogenous housekeeper metabolites are selected from the group consisting of such endogeneous metabolites which show stability in accordance with statistical stability measures being selected from the group consisting of coefficient of variation (CV) of raw intensity data, standard deviation (SD) of logarithmic intensity data, stability measure (M) of geNorm - algorithm or stability measure value (rho) of NormFinder- algorithm.
Additionally, said quantitative metabolomics profile comprises the results of measuring at least one of the parameters selected from the group consisting of:
concentration, level or amount of each individual endogenous metabolite of said plurality of endogenous metabolites in said sample, qualitative and/or quantitative molecular pattern and/or molecular signature; and using and storing the obtained set of values in a database.
A panel of reference endogenous organ failure predictive target metabolites or derivatives thereof is established by:
a) mathematically preprocessing intensity values obtained for generating the metabolomics profiles in order to reduce technical errors being inherent to the
measuring procedures used to generate the metabolomics profiles;
b) selecting at least one suitable classifying algorithm from the group consisting of logistic regression, (diagonal) linear or quadratic discriminant analysis (LDA, QDA,
DLDA, DQDA), perceptron, shrunken centroids regularized discriminant analysis (RDA), random forests (RF), neural networks (NN), Bayesian networks, hidden Markov models, support vector machines (SVM), generalized partial least squares (GPLS), partitioning around medoids (PAM), inductive logic programming (ILP), generalized additive models, gaussian processes, regularized least square regression, self organizing maps (SOM), recursive partitioning and regression trees, K-nearest neighbour classifiers (K-NN), fuzzy classifiers, bagging, boosting, and naϊve Bayes; and applying said selected classifier algorithm to said preprocessed data of step a); c) said classifier algorithms of step b) being trained on at least one training data set containing preprocessed data from subjects being divided into classes according to their likelihood to develop an organ failure, in order to select a classifier function to map said preprocessed data to said likelihood; d) applying said trained classifier algorithms of step c) to a preprocessed data set of a subject with unknown organ failure likelihood, and using the trained classifier algorithms to predict the class label of said data set in order to predict the likelihood for a subject to develop an organ failure.
The endogenous organ failure predictive target metabolites for easier and/or more sensitive detection are preferably detected by means of chemically modified derivatives thereof, such as phenylisothiocyanates for amino acids.
In a preferred embodiment of the present invention, said endogenous organ failure predictive target metabolites are selected from the group consisting of:
Carnitin, acylcarnitines (C chain length :total number of double bonds), in particular, C12-DC, C14:1 , C14:1 -OH, C14:2, C14:2-OH, C18, C6:1 ; sphingomyelins (SM chain length :total number of double bonds), in particular, SM C16:0, SM C17:0, SM C18:0, SM C19:0, SM C21 :1 , SM C21 :3, SM C22:2, SM C23:0, SM C23:1 , SM C23:2, SM C23:3, SM C24:0, SM C24:1 , SM C24:2, SM C24:3, SM C24:4, SM C26:4, SM C3:0, SM (OH) C22:1 , SM (OH) C22:2, SM (OH) C24:1 , SM C26:0, SM C26:1 ;
phosphatidylcholines, (diacylphosphatidylcholines, PC aa chain length:total number of double bonds or PC ae) in particular, PC aa C28:1 , PC aa C38:0, PC aa C42:0, PC aa C42:1 , PC ae C40:1 , PC ae C40:2, PC ae C40:6, PC ae C42:2, PC ae C42:3, PC ae C42:4, PC ae C44:5, PC ae C44:6, PC aa C36:4, PC aa C38:1 , PC aa C38:2, PC aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:5, PC aa C40:6, PC aa C40:7, PC aa C40:8, PC ae C36:4, PC ae C36:5, PC ae C38:4, PC ae C38:6;
lysophosphatidylcholines (monoacylphosphatidylcholines, PC a chain length:total number of double bonds), in particular, PC a C18:2, PC a C20:4, PC a C20:3, PC a C26:0;
Phe; oxycholesterols, in particular, 3β,5α,6β-trihydroxycholestan, 7-ketocholesterol, 5α,6α- epoxycholesterol;
lysophosphatidylethanolamins (monoacylphosphatidylcholins, PE a chain length:total number of double bonds), in particular, PE a C18:1 , PE a C18:2, PE a C20:4, PE a C22:5, PE a C22:6;
phosphatidylethanolamins, (diacylphosphatidylcholins, PE aa chain length:total number of double bonds), in particular, PE aa C38:0, PE aa C38:2;
ceramids, (N-chain length:total number of double bonds), in particular, N-C2:0-Cer, N- C7:0-Cer, N-C9:3-Cer, N-C17:1 -Cer, N-C22:1 -Cer, N-C25:0-Cer, N-C27:1 -Cer, N-C5:1 - Cer(2H), N-C7:1 -Cer(2H), N-C8:1 -Cer(2H), N-C1 1 :1 -Cer(2H), N-C20:0-Cer(2H), N- C21 :0-Cer(2H), N-C22:1 -Cer(2H), N-C25:1 -Cer(2H), N-C26:1 -Cer(2H), N-C24:0(OH)- Cer, N-C26:0(OH)-Cer, N-C6:0(OH)-Cer, N-C8:0(OH)-Cer(2H), N-C10:0(OH)-Cer(2H), N-C25:0(OH)-Cer(2H), N-C26:0(OH)-Cer(2H), N-C27:0(OH)-Cer(2H), N-C28:0(OH)- Cer(2H).
For generating a metabolomics analysis profile, said plurality of endogenous organ failure predictive target metabolites or derivatives thereof comprises 2 to 80, in particular 2 to 60, preferably 2 to 50, preferred 2 to 30, more preferred 2 to 20, particularly preferred 2 to 10 endogenous metabolites
A particular embodiment of the present invention is the use of a plurality of endogenous metabolites for predicting of an onset of an infection associated organ failure from a biological sample of a mammalian subject in vitro, wherein the metabolites are selected from the group consisting of : Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid (glutamate), glutamine, leucine, isoleucine, histidine, ornithine, proline, phenylalanine, serine, tryptophane, tyrosine, valine, kynurenine; phenylthio carbamyl amino acids (PTC-amino acids), in particular, PCT-arginine, PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-tyrosine, PTC-valine; dimethylarginine, in particular N,N-dimethyl-L-arginine; carboxylic acids, namely 15(S)-hydroxy-5Z,8Z,1 1 Z,13E-eicosatetraenoic acid [(5Z,8Z,1 1 Z,13E,15S)-15-Hydroxyicosa-5,8,11 ,13-tetraenoic acid], succinic acid (succinate); Ceramides, with an N-acyl residue having from 2 to 30 Carbon atoms in the acyl residue and having from 0 to 5 double bonds and having from 0 to 5 hydroxy groups; carnitine; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue and having 1 to 4 double bonds in the acyl residue; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue and having from 1 to 3 OH-groups in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue with 1 to 4 double bonds and 1 to 3 OH- groups in the acyl residue; phospholipides, in particular lysophosphatidylcholines (monoacylphospha-tidylcholines) having from 1 to 30 carbon atoms in the acyl residue; lysophosphatidylcholines having from 3 to 30 carbon atoms in the acyl residue and having 1 to 6 double bonds in the acyl residue; phosphatidylcholines (diacylphosphatidylcholines) having a total of from 1 to 50 carbon atoms in the acyl residues; phosphatidylcholines having a total from 3 to 50 carbon atoms in the acyl residues and having a total of 1 to 8 double bonds in the acyl residues; sphingolipids, in particular sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30; sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30 and 1 to 5 double bonds; hydroxysphinogomyelines having a total number of carbon atoms in the acyl residues from 10 to 30;
hydroxysphingoyelines having a total number of carbon atoms in the acyl residues from 10 to 30 and 1 to 5 double bonds; prostaglandines, namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane B2; putrescine; oxysterols, namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20α- hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3β,5α,6β- trihydroxycholesterol, 7α- hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5α,6α- epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycolithocholic acid sulfate, glycoursodeoxycholic acid, lithocholic acid, taurocholic acid, taurochenodeoxycholic acid taurodeoxycholic acid, taurolithocholic acid, taurolithocholic acid sulfate, tauroursodeoxycholic acid, ursodeoxycholic acid; biogenic amines, namely histamine, serotonine, palmitoyl ethanolamine.
It is emphasized that every of the above mentioned groups of chemical compounds, such as e.g. "amino acids", "bile acids", "oxysterols", and the like, per se can be used as organ failure predictive target metabolites (OF predictors) within the frame of the present invention.
Particularly preferred endogenous organ failure predictive target metabolites are selected from the group consisting of:
Carnitin, acylcarnitines (C chain length :total number of double bonds), in particular, C12-DC, C14:1 , C14:1 -OH, C14:2, C14:2-OH, C18, C6:1 ;
sphingomyelins (SM chain length :total number of double bonds), in particular, SM C16:0, SM C17:0, SM C18:0, SM C19:0, SM C21 :1 , SM C21 :3, SM C22:2, SM C23:0, SM C23:1 , SM C23:2, SM C23:3, SM C24:0, SM C24:1 , SM C24:2, SM C24:3, SM C24:4, SM C26:4, SM C3:0, SM (OH) C22:1 , SM (OH) C22:2, SM (OH) C24:1 , SM C26:0, SM C26:1 ;
phosphatidylcholines, (diacylphosphatidylcholines, PC aa chain length:total number of double bonds or PC ae) in particular, PC aa C28:1 , PC aa C38:0, PC aa C42:0, PC aa C42:1 , PC ae C40:1 , PC ae C40:2, PC ae C40:6, PC ae C42:2, PC ae C42:3, PC ae C42:4, PC ae C44:5, PC ae C44:6, PC aa C36:4, PC aa C38:1 , PC aa C38:2, PC aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:5, PC aa C40:6, PC aa C40:7, PC aa C40:8, PC ae C36:4, PC ae C36:5, PC ae C38:4, PC ae C38:6; lysophosphatidylcholines (monoacylphosphatidylcholines, PC a chain length:total number of double bonds), in particular, PC a C18:2, PC a C20:4, PC a C20:3, PC a C26:0;
Phe;
oxycholesterols, in particular, 3β,5α,6β-trihydroxycholestan, 7-ketocholesterol, 5α,6α- epoxycholesterol;
lysophosphatidylethanolamins (monoacylphosphatidylcholins, PE a chain length:total number of double bonds), in particular, PE a C18:1 , PE a C18:2, PE a C20:4, PE a C22:5, PE a C22:6;
phosphatidylethanolamins, (diacylphosphatidylcholins, PE aa chain length:total number of double bonds), in particular, PE aa C38:0, PE aa C38:2;
ceramids, (N-chain length:total number of double bonds), in particular, N-C2:0-Cer, N- C7:0-Cer, N-C9:3-Cer, N-C17:1 -Cer, N-C22:1 -Cer, N-C25:0-Cer, N-C27:1 -Cer, N-C5:1 - Cer(2H), N-C7:1 -Cer(2H), N-C8:1 -Cer(2H), N-C1 1 :1 -Cer(2H), N-C20:0-Cer(2H), N-
C21 :0-Cer(2H), N-C22:1 -Cer(2H), N-C25:1 -Cer(2H), N-C26:1 -Cer(2H), N-C24:0(OH)- Cer, N-C26:0(OH)-Cer, N-C6:0(OH)-Cer, N-C8:0(OH)-Cer(2H), N-C10:0(OH)-Cer(2H), N-C25:0(OH)-Cer(2H), N-C26:0(OH)-Cer(2H), N-C27:0(OH)-Cer(2H), N-C28:0(OH)- Cer(2H).
Furthermore, the present invention includes a kit for carrying out a method for predicting the likelihood of an onset of an infection associated organ failure from a biological sample of a mammalian subject in vitro, in a biological sample, comprising: a) calibration agents for the quantitative detection of endogenous organ failure predictive target metabolites, wherein said metabolites are selected from the group consisting of: Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid
(glutamate), glutamine, leucine, isoleucine, histidine, ornithine, proline, phenylalanine, serine, tryptophane, tyrosine, valine, kynurenine; phenylthio carbamyl amino acids (PTC-amino acids), in particular, PCT-arginine, PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-tyrosine, PTC-valine; dimethylarginine, in particular N,N-dimethyl-L-arginine; carboxylic acids, namely 15(S)-hydroxy-5Z,8Z,1 1 Z,13E-eicosatetraenoic acid [(5Z,8Z,1 1 Z,13E,15S)-15-Hydroxyicosa-5,8,11 ,13-tetraenoic acid], succinic acid (succinate);
Ceramides, with an N-acyl residue having from 2 to 30 Carbon atoms in the acyl residue and having from 0 to 5 double bonds and having from 0 to 5 hydroxy groups; carnitine; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue and having 1 to 4 double bonds in the acyl residue; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue and having from 1 to 3 OH-groups in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue with 1 to 4 double bonds and 1 to 3 OH- groups in the acyl residue; phospholipides, in particular lysophosphatidylcholines (monoacylphospha-tidylcholines) having from 1 to 30 carbon atoms in the acyl residue; lysophosphatidylcholines having from 3 to 30 carbon atoms in the acyl residue and having 1 to 6 double bonds in the acyl residue; phosphatidylcholines (diacylphosphatidylcholines) having a total of from 1 to 50 carbon atoms in the acyl residues; phosphatidylcholines having a total from 3 to 50 carbon atoms in the acyl residues and having a total of 1 to 8 double bonds in the acyl residues; sphingolipids, in particular sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30; sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30 and 1 to 5 double bonds; hydroxysphinogomyelines having a total number of carbon atoms in the acyl residues from 10 to 30;
hydroxysphingoyelines having a total number of carbon atoms in the acyl residues from
10 to 30 and 1 to 5 double bonds; prostaglandines, namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane
B2; putrescine; oxysterols, namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20α- hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3β,5α,6β- trihydroxycholesterol, 7α- hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5α,6α- epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycolithocholic acid sulfate, glycoursodeoxycholic acid, lithocholic acid, taurocholic acid, taurochenodeoxycholic acid taurodeoxycholic acid, taurolithocholic acid, taurolithocholic acid sulfate, tauroursodeoxycholic acid, ursodeoxycholic acid; biogenic amines, namely histamine, serotonine, palmitoyl ethanolamine; b) data base with processed data from healthy patients and patients who developed an infection associated organ failure;
c) classification software for generating the quantitative metabolomics profiles achieved with said calibration agents of step a) and classifying the results based on the processed data of step b).
Data classification is the categorization of data for its most effective and efficient use. Classifiers are typically deterministic functions that map a multi-dimensional vector of biological measurements to a binary (or n-ary) outcome variable that encodes the absence or existence of a clinically-relevant class, phenotype, distinct physiological state or distinct state of disease. To achieve this various classification methods such as, but not limited to, logistic regression, (diagonal) linear or quadratic discriminant analysis (LDA, QDA, DLDA, DQDA), perceptron, shrunken centroids regularized discriminant analysis (RDA), random forests (RF), neural networks (NN), Bayesian networks, hidden Markov models, support vector machines (SVM), generalized partial least squares (GPLS), partitioning around medoids (PAM), inductive logic programming (ILP), generalized additive models, gaussian processes, regularized least square regression, self organizing maps (SOM), recursive partitioning and regression trees, K-nearest neighbor classifiers (K-NN), fuzzy classifiers, bagging, boosting, and naϊve Bayes and many more can be used.
Further aspects, advantages and embodiments of the present invention will become evident by the description of examples, from the experimental sections below and by means of the drawings.
Fig.1 is a Venn diagram showing the agreement between adjusted p value
(P.adj), fold change and area under the receiver operating characteristic curve (AUC) for the comparison between septic patients and septic patients developing an organ failure where those metabolites with adjusted p value < 0.01 , absolute fold change > 50% and AUC > 0.80 were selected;
Fig. 2 is a graph showing classifier accuracy for support vector machines (SVM) with linear kernel, diagonal linear discriminant analysis (DLDA) and k nearest neighbors (KNN) with k equal to one where the features are selected using a ranker which ranks the metabolites combining adjusted p value, fold change and AUC;
Fig. 3 is a graph showing classifier accuracy for support vector machines (SVM) with linear kernel, diagonal linear discriminant analysis (DLDA) and k nearest neighbors (KNN) with k equal to one where the features are selected by a so-called wrapper using boosted regression trees;
Fig. 4 is a Venn diagram showing the agreement between adjusted p value
(P.adj), fold change and area under the receiver operating characteristic curve (AUC) for the comparison between septic mice and septic mice developing liver failure where those metabolites with adjusted p value < 0.05, absolute fold change > 50% and AUC > 0.8 were selected; "Organ failure" (OF) in this context relates to any diseased state, however, particularly addresses an infection associated organ failure.
"Severe sepsis" refers to sepsis associated with organ dysfunction, hypoperfusion abnormalities, or sepsis-induced hypotension. Hypoperfusion abnormalities include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status. "Septic shock" refers to sepsis-induced hypotension that is not responsive to adequate intravenous fluid challenge and with manifestations of peripheral hypoperfusion. A "converter patient" refers to a SIRS-positive patient who progresses to clinical suspicion of sepsis during the period the patient is monitored, typically during an ICU stay. A "non- converter patient" refers to a SIRS-positive patient who does not progress to clinical suspicion of sepsis during the period the patient is monitored, typically during an ICU stay. A patient with OF has a clinical presentation that is classified as OF, as defined above, but is not clinically deemed to have OF. Individuals who are at risk of developing OF include patients in an ICU and those who have otherwise suffered from a physiological trauma, such as a burn or other insult.
As used herein, "organ failure" (OF) includes all stages of OF including, but not limited to, the onset of OF and multi organ failure (MOD), e.g. associated with the end stages of sepsis.
"Sepsis" refers to a SIRS-positive condition that is associated with a confirmed infectious process. Clinical suspicion of sepsis arises from the suspicion that the SIRS- positive condition of a SIRS patient is a result of an infectious process.
The "onset of OF" refers to an early stage of OF, i.e., prior to a stage when the clinical manifestations are sufficient to support a clinical suspicion of OF. Because the methods of the present invention are used to detect OF prior to a time that OF would be suspected using conventional techniques, the patient's disease status at early OF can only be confirmed retrospectively, when the manifestation of OF is more clinically obvious. The exact mechanism by which a patient acquires OF is not a critical aspect of the invention. The methods of the present invention can detect changes in the biomarker score independent of the origin of the OF. Regardless of how OF arises, the methods of the present invention allow for determining the status of a patient having, or suspected of having, OF, as classified by previously used criteria.
As used herein, the term "organ failure specific metabolite" refers to a metabolite that is differentially present or differentially concentrated in septic organisms compared to non- septic organisms. For example, in some embodiments, organ failure specific metabolites are present in septic tissues but not in non- in septic tissues.
In other embodiments, organ failure-specific metabolites are absent in septic tissues but present in non-septic cells, tissues, body liquids. In still further embodiments, organ failure specific metabolites are present at different levels (e.g., higher or lower) in septic tissue/cells as compared to non-septic cells. For example, an organ failure specific metabolite may be differentially present at any level, but is generally present at a level that is increased by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 1 10%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more; or is generally present at a level that is decreased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent).
An organ failure-specific metabolite is preferably differentially present at a level that is statistically significant (e.g., an adjusted p-value less than 0.05 as determined using either Analysis of Variance, Welch's t-test or its non parametric equivalent versions). Exemplary organ failure-specific metabolites are described in the detailed description and experimental sections below.
The term "sample" in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture. On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin.
Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, such biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, rodents, etc. A biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material from a subject. The sample can be isolated from any suitable biological tissue or fluid such as, for example, tissue, blood, blood plasma, urine, or cerebral spinal fluid (CSF).
A "reference level" of a metabolite means a level of the metabolite that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof. A "positive" reference level of a metabolite means a level that is indicative of a particular disease state or phenotype. A "negative" reference level of a metabolite means a level that is indicative of a lack of a particular disease state or phenotype. For example, a "organ failure-positive reference level" of a metabolite means a level of a metabolite that is indicative of a positive diagnosis of organ failure in a subject, and an "organ failure-negative reference level" of a metabolite means a level of a metabolite that is indicative of a negative diagnosis of organ failure in a subject. A "reference level" of a metabolite may be an absolute or relative amount or concentration of the metabolite, a presence or absence of the metabolite, a range of amount or concentration of the metabolite, a minimum and/or maximum amount or concentration of the metabolite, a mean amount or concentration of the metabolite, and/or a median amount or concentration of the metabolite; and, in addition, "reference levels" of combinations of metabolites may also be ratios of absolute or relative amounts or concentrations of two or more metabolites with respect to each other or a composed value / score obtained by classification.
Appropriate positive and negative reference levels of metabolites for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired metabolites in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age- matched so that comparisons may be made between metabolite levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of metabolites in biological samples (e.g., LC-MS, GC-MS, etc.), where the levels of metabolites may differ based on the specific technique that is used.
As used herein, the term "cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
As used herein, the term "processor" refers to a device that performs a set of steps according to a program (e.g., a digital computer). Processors, for example, include Central Processing Units ("CPUs"), electronic devices, or systems for receiving, transmitting, storing and/or manipulating data under programmed control.
As used herein, the term "memory device," or "computer memory" refers to any data storage device that is readable by a computer, including, but not limited to, random access memory, hard disks, magnetic (floppy) disks, compact discs, DVDs, magnetic tape, flash memory, and the like.
"Mass Spectrometry" (MS) is a technique for measuring and analyzing molecules that involves fragmenting a target molecule, then analyzing the fragments, based on their mass/charge ratios, to produce a mass spectrum that serves as a "molecular fingerprint". Determining the mass/charge ratio of an object is done through means of determining the wavelengths at which electromagnetic energy is absorbed by that object. There are several commonly used methods to determine the mass to charge ration of an ion, some measuring the interaction of the ion trajectory with electromagnetic waves, others measuring the time an ion takes to travel a given distance, or a combination of both. The data from these fragment mass measurements can be searched against databases to obtain definitive identifications of target molecules. Mass spectrometry is also widely used in other areas of chemistry, like petrochemistry or pharmaceutical quality control, among many others.
As used here, the term "metabolite" denotes endogenous organic compounds of a cell, an organism, a tissue or being present in body liquids and in extracts obtained from the aforementioned sources with a molecular weight typically below 1500 Dalton. Typical examples of metabolites are carbohydrates, lipids, phospholipids, sphingolipids and sphingophospholipids, amino acids, cholesterol, steroid hormones and oxidized sterols and other compounds such as collected in the Human Metabolite database [Wishart DS et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan ;35 (Database issue) :D521-6(see http://www.hmdb.ca/) / and other databases and literature. This includes any substance produced by metabolism or by a metabolic process and any substance involved in metabolism.
"Metabolomics" as understood within the scope of the present invention designates the comprehensive quantitative measurement of several (2-thousands) metabolites by, but not limited to, methods such as mass spectroscopy, coupling of liquid chromatography, gas chromatography and other separation methods chromatography with mass spectroscopy. The term "separation" refers to separating a complex mixture into its component proteins or metabolites. Common laboratory separation techniques include gel electrophoresis and chromatography.
The term "capillary electrophoresis" refers to an automated analytical technique that separates molecules in a solution by applying voltage across buffer-filled capillaries.
Capillary electrophoresis is generally used for separating ions, which move at different speeds when the voltage is applied, depending upon the size and charge of the ions.
The solutes (ions) are seen as peaks as they pass through a detector and the area of each peak is proportional to the concentration of ions in the solute, which allows quantitative determinations of the ions.
The term "chromatography" refers to a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Chromatographic output data may be used for manipulation by the present invention.
An "ion" is a charged object formed by adding electrons to or removing electrons from an atom. A "mass spectrum" is a plot of data produced by a mass spectrometer, typically containing m/z values on x-axis and intensity values on y-axis. A "peak" is a point on a mass spectrum with a relatively high y-value.
The term "m/z" refers to the dimensionless quantity formed by dividing the mass number of an ion by its charge number. It has long been called the "mass-to-charge" ratio.
The term "metabolism" refers to the chemical changes that occur within the tissues of an organism, including "anabolism" and "catabolism". Anabolism refers to biosynthesis or the buildup of molecules and catabolism refers to the breakdown of molecules.
As used herein, the term "post-surgical tissue" refers to tissue that has been removed from a subject during a surgical procedure. Examples include, but are not limited to, biopsy samples, excised organs, and excised portions of organs.
As used herein, the terms "detect", "detecting", or "detection" may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition.
As used herein, the term "clinical failure" refers to a negative outcome following organ failure treatment.
A biomarker in this context is a characteristic, comprising data of at least one metabolite that is measured and evaluated as an indicator of biologic processes, pathogenic processes, or responses to a therapeutic intervention associated with organ failure or related to organ failure treatment. A combined biomarker as used here may be selected from at least two small endogenous molecules and metabolites.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to markers of Organ failure and its duration/severity as well of the effect of therapeutic interventions. In particular embodiments, the present invention provides metabolites that are differentially present in Organ failure. Experiments conducted during the course of development of embodiments of the present invention identified a series of metabolites as being differentially present in
Tables 2 and 3 provide additional metabolites present in plasma serum or other body liquids. The disclosed markers find use as diagnostic and therapeutic targets.
Diagnostic Applications
In some embodiments, the present invention provides methods and compositions for diagnosing organ failure, including but not limited to, characterizing risk of organ failure, stage of organ failure, duration and severity etc. based on the presence of organ failure specific metabolites or their derivatives, precursors, metabolites, etc. Exemplary diagnostic methods are described below.
Thus, for example, a method of diagnosing (or aiding in diagnosing) whether a subject has organ failure comprises (1 ) detecting the presence or absence or a differential level of a plurality of organ failure specific metabolites selected from tables 1 *** and b) diagnosing organ failure based on the presence, absence or differential level of the organ failure specific metabolite. When such a method is used to aid in the diagnosis of organ failure, the results of the method may be used along with other methods (or the results thereof) useful in the clinical determination of whether a subject has organ failure.
Any mammalian sample suspected of containing organ failure specific metabolites is tested according to the methods described herein. By way of non-limiting examples, the sample may be tissue (e.g., a biopsy sample or post-surgical tissue), blood, urine, or a fraction thereof (e.g., plasma, serum, urine supernatant, urine cell pellet).
In some embodiments, the patient sample undergoes preliminary processing designed to isolate or enrich the sample for organ failure specific metabolites or cells that contain organ failure specific metabolites. A variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited: centrifugation; immunocapture; and cell lysis.
Metabolites may be detected using any suitable method including, but not limited to, liquid and gas phase chromatography, alone or coupled to mass spectrometry (See e.g., experimental section below), NMR, immunoassays, chemical assays, spectroscopy and the like. In some embodiments, commercial systems for chromatography and NMR analysis are utilized.
In other embodiments, metabolites (i.e. biomarkers and derivatives thereof) are detected using optical imaging techniques such as magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), CAT scans, ultra sound, MS-based tissue imaging or X-ray detection methods (e.g., energy dispersive x-ray fluorescence detection).
Any suitable method may be used to analyze the biological sample in order to determine the presence, absence or level(s) of the the plurality of metabolites in the sample. Suitable methods include chromatography (e.g., HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g., MS, MS-MS), enzyme-linked immunosorbent assay (ELISA), antibody linkage, other immunochemical techniques, biochemical or enzymatic reactions or assays, and combinations thereof. Further, the level(s) of the the plurality of metabolites may be measured indirectly, for example, by using an assay that measures the level of a compound (or compounds) that correlates with the level of the biomarker(s) that are desired to be measured.
The levels of the plurality of the recited metabolites may be determined in the methods of the present invention. For example, the level(s) of one metabolites, two or more metabolites, three or more metabolites, four or more metabolites, five or more metabolites, six or more metabolites, seven or more metabolites, eight or more metabolites, nine or more metabolites, ten or more metabolites, etc., including a combination of some or all of the metabolites including, but not limited to those listed in table 2, may be determined and used in such methods.
Determining levels of combinations of the metabolites may allow greater sensitivity and specificity in the methods, such as diagnosing organ failure and aiding in the diagnosis of organ failure, and may allow better differentiation or characterization of organ failure from other disorders or other organ failure that may have similar or overlapping metabolites to organ failure (as compared to a subject not having organ failure). For example, ratios of the levels of certain metabolites in biological samples may allow greater sensitivity and specificity in diagnosing organ failure and aiding in the diagnosis of organ failure and allow better differentiation or characterization of organ failure from other organ failure or other disorders of the that may have similar or overlapping metabolites to organ failure (as compared to a subject not having organ failure).
Data Analysis
In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of an organ failure specific metabolite) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in metabolite analysis, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.
The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy or a blood, urine or serum sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a plasma sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., metabolic profile), specific for the diagnostic or prognostic information desired for the subject.
The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of organ failure being present) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.
In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.
In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease.
When the amount(s) or level(s) of the plurality of metabolites in the sample are determined, the amount(s) or level(s) may be compared to organ failure metabolite- reference levels, such as -organ failure-positive and/or organ failure-negative reference levels to aid in diagnosing or to diagnose whether the subject has organ failure. Levels of the plurality of metabolites in a sample corresponding to the organ failure-positive reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of a diagnosis of organ failure in the subject. Levels of the plurality of metabolites in a sample corresponding to the organ failure-negative reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of a diagnosis of no organ failure in the subject. In addition, levels of the plurality of metabolites that are differentially present (especially at a level that is statistically significant) in the sample as compared to organ failure-negative reference levels are indicative of a diagnosis of organ failure in the subject. Levels of the plurality of metabolites that are differentially present (especially at a level that is statistically significant) in the sample as compared to organ failure-positive reference levels are indicative of a diagnosis of no organ failure in the subject.
The level(s) of the plurality of metabolites may be compared to organ failure-positive and/or organ failure-negative reference levels using various techniques, including a simple comparison (e.g., a manual comparison) of the level(s) of the plurality of metabolites in the biological sample to organ failure-positive and/or organ failure- negative reference levels. The level(s) of the plurality of metabolites in the biological sample may also be compared to organ failure-positive and/or organ failure-negative reference levels using one or more statistical analyses (e.g., t-test, Welch's t-test, Wilcoxon's rank sum test, random forests, support vector machines, linear discriminant analysis, k nearest neighbours).
Compositions for use (e.g., sufficient for, necessary for, or useful for) in the diagnostic methods of some embodiments of the present invention include reagents for detecting the presence or absence of organ failure specific metabolites. Any of these compositions, alone or in combination with other compositions of the present invention, may be provided in the form of a kit. Kits may further comprise appropriate controls and/or detection reagents.
Embodiments of the present invention provide for multiplex or panel assays that simultaneously detect a plurality of the markers of the present invention depicted in tables 1 to 3, alone or in combination with additional organ failure markers known in the art. For example, in some embodiments, panel or combination assays are provided that detected 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more, 30 or more, 40 or more markers in a single assay. In some embodiments, assays are automated or high throughput.
A preferred embodiment of the present invention is the use of markers listed in tables 2 and 3 for prediction/diagnosis of organ failure and its duration/severity where said mammalian subject is a human being, said biological sample blood and/or blood cells.
In some embodiments, additional organ failure markers are included in multiplex or panel assays. Markers are selected for their predictive value alone or in combination with the metabolic markers described herein.
Therapeutic Methods
In some embodiments, the present invention provides therapeutic methods (e.g., that target the organ failure specific metabolites described herein). In some embodiments, the therapeutic methods target enzymes or pathway components of the organ failure specific metabolites described herein.
For example, in some embodiments, the present invention provides compounds that target the organ failure specific metabolites of the present invention. The compounds may decrease the level of organ failure specific metabolite by, for example, interfering with synthesis of the organ failure specific metabolite (e.g., by blocking transcription or translation of an enzyme involved in the synthesis of a metabolite, by inactivating an enzyme involved in the synthesis of a metabolite (e.g., by post translational modification or binding to an irreversible inhibitor), or by otherwise inhibiting the activity of an enzyme involved in the synthesis of a metabolite) or a precursor or metabolite thereof, by binding to and inhibiting the function of the organ failure specific metabolite, by binding to the target of the organ failure specific metabolite (e.g., competitive or non competitive inhibitor), or by increasing the rate of break down or clearance of the metabolite.
The compounds may increase the level of organ failure specific metabolite by, for example, inhibiting the break down or clearance of the organ failure specific metabolite (e.g., by inhibiting an enzyme involved in the breakdown of the metabolite), by increasing the level of a precursor of the organ failure specific metabolite, or by increasing the affinity of the metabolite for its target.
Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates.
In some embodiments, the present invention provides drug screening assays (e.g., to screen for anti - organ failure drugs). The screening methods of the present invention utilize organ failure specific metabolites described herein. As described above, in some embodiments, test compounds are small molecules, nucleic acids, or antibodies. In some embodiments, test compounds target organ failure specific metabolites directly. In other embodiments, they target enzymes involved in metabolic pathways of organ failure specific metabolites.
EXPERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
General Analytics:
Sample preparation and metabolomic analyses were performed at BIOCRATES life sciences AG, Innsbruck, Austria. We used a multi-parametric, highly robust, sensitive and high-throughput targeted metabolomic platform consisting of flow injection analysis (FIA)-MS/MS and LC-MS/MS methods for the simultaneous quantification of a broad range of endogenous intermediates namely from the panel disclosed in table 1. All procedures (sample handling, analytics) were performed by co-workers blinded to the groups.
Plasma homogenization
Plasma samples were prepared by standard procedures and stored at (-700C). To enable analysis of all samples simultaneously within one batch, samples were thawed on ice (1 h) on the day of analysis and centrifuged at 18000 g at 2°C for 5 min. All tubes were prepared with 0.001 % BHT (butylated hydroxytoluene; Sigma-Aldrich, Vienna, Austria) to prevent artificial formation of prostaglandins caused by autooxidation .
Liver tissue samples were homogenized using a Precellys® 24 homogenizer with Cryolys cooling module before analysis. Typically 50 mg of tissue were homogenized in ethanol : phosphate buffer 9:1 (v/v) for 30 min and unsolved material and beads for tissue desintegration removed by 5 min centrifugation at 10 000g.
Acylcarnitines, Sphingomyelins, Hexoses, Glycerophospholipids (FIA-MS/MS)
To determine the concentration of acylcarnitines, sphingomyelins and glycerophospholipids in brain homogenates and in plasma the Absolute/DQ kit p150
(Biocrates Life Sciences AG) was prepared as described in the manufacturer's protocol.
In brief, 10 μL of brain homogenate was added to the center of the filter on the upper
96-well kit plate, and the samples were dried using a nitrogen evaporator (VLM
Laboratories). Subsequently, 20 μL of a 5 % solution of phenyl-isothiocyanate was added for derivatization. After incubation, the filter spots were dried again using an evaporator. The metabolites were extracted using 300 μl_ of a 5 mM ammonium acetate solution in methanol. The extracts were obtained by centrifugation into the lower 96- deep well plate followed by a dilution step with 600 μl_ of kit MS running solvent. Mass spectrometric analysis was performed on an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies) equipped with an electro-spray ionization (ESI)-source using the analysis acquisition method as provided in the Absolute/DQ kit. The standard FIA-MS/MS method was applied for all measurements with two subsequent 20 μl_ injections (one for positive and one for negative mode analysis). Multiple reaction monitoring (MRM) detection was used for quantification applying the spectra parsing algorithm integrated into the MetlQ software (Biocrates Life Sciences AG). Concentration values for 148 metabolites (all analytes determined with the metabolomics kit besides of the amino acids, which were determined by a different method) obtained by internal calibration were exported for comprehensive statistical analysis.
Amino acids, Biogenic amines (LC-MS/MS)
Amino acids and biogenic amines were quantitatively analyzed by reversed phase LC- MS/MS to obtain chromatographic separation of isobaric (same MRM ion pairs) metabolites for individual quantitation performed by external calibration and by use of internal standards. 10 μL sample volume (plasma, brain homogenate) is required for the analysis using the following sample preparation procedure. Samples were added on filter spots placed in a 96- solvinert well plate (internal standards were placed and dried down under nitrogen before), fixed above a 96 deep well plate (capture plate). 20 μL of 5% phenyl-isothiocyanate derivatization reagent was added. The derivatized samples were extracted after incubation by aqueous methanol into the capture plate. Sample extracts were analyzed by LC-ESI-MS/MS in positive MRM detection mode with an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies). The analyzed individual metabolite concentrations (Analyst 1.4.2 software, Applied Biosystems) were exported for comprehensive statistical analysis.
Bile acids (LC-MS/MS) A highly selective reversed phase LC-MS/MS analysis method in negative MRM detection mode was applied to determine the concentration of bile acids in plasma samples. Samples were extracted via dried filter spot technique in 96 well plate format, which is well suitable for high throughput analysis. For highly accurate quantitation internal standards and external calibration were applied. In brief, internal standards and 20 μl_ sample volume placed onto filter spots were extracted and simultaneously protein precipitated with aqueous methanol. These sample extracts were measured by LC-ESI- MS/MS with an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies). Data of bile acids were quantified with Analyst 1.4.2 software (Applied Biosystems) and finally exported for comprehensive statistical analysis.
Prostanoids, oxidized fatty acids (LC-MS/MS)
Prostanoids - a term summarizing prostaglandins (PG), thromboxanes (TX) and prostacylines - and oxidised fatty acid metabolites were analyzed in plasma extracts by
LC-ESI-MS/MS [Unterwurzacher at al. Clin Chem Lab Med 2008; 46 (11 ):1589-1597] and in brain homogenate extracts by online solid phase extraction (SPE)-LC-MS/MS
[Unterwurzacher et al. Rapid Commun Mass Spec submitted] with an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies) in negative MRM detection mode. The sample preparation was the same for both, plasma and brain homogenates. In brief, filter spots in a 96 well plate were spiked with internal standard; 20 μl_ of plasma or tissue homogenates were added and extracted with aqueous methanol, the individual extracts then were analysed. Data of prostanoids and oxidized fatty acids were quantified with Analyst 1.4.2 software (Applied Biosystems) and finally exported for statistical analysis.
Oxysterols
Oxysterols are determined after extraction and saponification by HPLC-Tandem mass spectrometer (HPLC-API-MS/MS) in positive detection mode using Multiple Reaction Mode (MRM).
Samples (20 μl_), calibrators and internal standard mixture were placed into a capture plate and were protein precipitated in the first step by means of addition of 200 μl_ acetonitrile and centrifugation. 180 μl_ of the appropriate supernatants were transferred on a new filter plate with 7 mm filter spots, dried down, hydrolysed with 0.35 M KOH in 95 % Ethanol and after washing steps extracted with 100 μl_ aqueous MeOH. An aliquot of the extracted sample is injected onto the H P LC- MS/MS system. Chromatographic separation and detection is performed by using a Zorbax Eclipse XDB C18, 150 x 2.0 mm, 3.5 μm HPLC-Column at a flow rate of 0.3 mL/min followed by electrospray ionization on the API4000/QTRAP4000 tandem mass spectrometer. For the quantitation the Analyst Quantitation software from Applied Bioystems was used.
Energy metabolism (Organic Acids) (LC-MS/MS)
For the quantitative analysis of energy metabolism intermediates (glycolysis, citrate cycle, pentose phosphate pathway, urea cycle) hdyrophilic interaction liquid chromatography (HILIC)-ESI-MS/MS method in highly selective negative MRM detection mode was used. The MRM detection was performed using an API4000 QTrap® tandem mass spectrometry instrument (Applied Biosystems/MDS Analytical Technologies). 20 μl_ sample volume (plasma, brain homogenate) was protein precipitated and extracted simultaneously with aqueous methanol in a 96 well plate format. Internal standards (ratio external to internal standard) and external calibration were used for highly accurate quantitation. Data were quantified with Analyst 1.4.2 software (Applied Biosystems) and finally exported for statistical analysis.
Lab name Family
CO AcCa.
C10 AcCa.
C10:1 AcCa.
C10:2 AcCa.
C12 AcCa.
C12-DC AcCa.
C12:1 AcCa.
C14 AcCa.
C14:1 AcCa.
C14:1 -OH AcCa.
C14:2 AcCa.
C14:2-OH AcCa.
C16 AcCa.
C16-OH AcCa.
C16:1 AcCa.
C16:1 -OH AcCa.
C16:2 AcCa. C16:2-OH AcCa.
C18 AcCa.
C18:1 AcCa.
C18:1 -OH AcCa.
C18:2 AcCa.
C2 AcCa.
C3 AcCa.
C3-OH AcCa.
C3:1 AcCa.
C4 AcCa.
C4-OH (C3-DC) AcCa.
C4:1 AcCa.
C5 AcCa.
C5-DC (C6-OH) AcCa.
C5-M-DC AcCa.
C5-OH (C3-DC-M) AcCa.
C5:1 AcCa.
C5:1 -DC AcCa.
C6 (C4:1 -DC) AcCa.
C6:1 AcCa.
C7-DC AcCa.
C8 AcCa.
C8:1 AcCa.
C9 AcCa.
H1 Sug.
SM (OH) C14:1 S.L.
SM (OH) CI 6:1 S.L.
SM (OH) C22:1 S.L.
SM (OH) C22:2 S.L.
SM (OH) C24:1 S.L.
SM C26:0 S.L.
SM C26:1 S.L.
PC aa C24:0 GP. L.
PC aa C26:0 GP. L.
PC aa C28:1 GP. L.
PC aa C32:3 GP. L.
PC aa C34:4 GP. L.
PC aa C36:6 GP. L.
PC aa C38:0 GP. L.
PC aa C40:1 GP. L.
PC aa C40:2 GP. L.
PC aa C40:3 GP. L.
PC aa C42:0 GP. L.
PC aa C42:1 GP. L.
PC aa C42:2 GP. L.
PC aa C42:4 GP. L.
PC aa C42:5 GP. L.
PC aa C42:6 GP. L. PC ae C30:0 GP. L.
PC ae C30:1 GP. L.
PC ae C30:2 GP. L.
PC ae C32:2 GP. L.
PC ae C36:0 GP. L.
PC ae C38:0 GP. L.
PC ae C40:0 GP. L.
PC ae C40:1 GP. L.
PC ae C40:2 GP. L.
PC ae C40:3 GP. L.
PC ae C40:4 GP. L.
PC ae C40:6 GP. L.
PC ae C42:0 GP. L.
PC ae C42:1 GP. L.
PC ae C42:2 GP. L.
PC ae C42:3 GP. L.
PC ae C42:4 GP. L.
PC ae C42:5 GP. L.
PC ae C44:3 GP. L.
PC ae C44:4 GP. L.
PC ae C44:5 GP. L.
PC ae C44:6 GP. L. lysoPC a C14:0 GP. L. lysoPC a C16:1 GP. L. lysoPC a C17:0 GP. L. lysoPC a C20:3 GP. L. lysoPC a C24:0 GP. L. lysoPC a C26:0 GP. L. lysoPC a C26:1 GP. L. lysoPC a C28:0 GP. L. lysoPC a C28:1 GP. L. lysoPC a C6:0 GP. L.
GIy Am .Ac.
Ala Am .Ac.
Ser Am .Ac.
Pro Am .Ac.
VaI Am .Ac.
Thr Am .Ac.
XIe Am .Ac.
Leu Am .Ac. lie Am .Ac.
Asn Am .Ac.
Asp Am .Ac.
GIn Am.Ac.
GIu Am.Ac.
Met Am.Ac.
His Am.Ac.
Phe Am.Ac. Arg Am .Ac.
Cit Am .Ac.
Tyr Am .Ac.
Trp Am .Ac.
Om Am .Ac.
Lys Am .Ac.
ADMA B.Am, total DMA B.Am.
Met-SO Am .Ac.
Kyn B.Am.
Putrescine B.Am.
Spermidine B.Am.
Spermine B.Am.
Creatinine B.Am.
9-HODE P. G.
13S-HODE P.G.
12S-HETE P.G.
15S-HETE P.G.
LTB4 P.G.
DHA P.G.
PGE2 P.G.
PGD2 P.G.
AA P.G.
Lac En. Met.
Sue En. Met.
Hex En. Met.
22ROHC Ox.St.
24SOHC Ox.St.
25OHC Ox.St.
27OHC Ox.St.
THC Ox.St.
7aOHC Ox.St.
7KC Ox.St.
5a,6a,EPC Ox.St.
4BOHC Ox.St.
Desmosterol Ox.St.
7DHC Ox.St.
Lanosterol Ox.St.
PEaC16:0 GP. L.
PEaC18:0 GP. L.
PEaC18:1 GP. L.
PEaCI 8:2 GP. L.
PEaC20:4 GP. L.
PEaC22:4 GP. L.
PEaC22:5 GP. L.
PEaC22:6 GP. L.
PEeC18:0 GP. L.
PGeC14:2 GP. L. PEaaC20:0 GP. L. PE aa C22:2 GP. L. PE aa C26:4 GP. L. PE aa C28:4 GP. L. PEaaC28:5 GP. L. PE aa C34:0 GP. L. PEaaC34:1 GP. L. PE aa C34:2 GP. L. PE aa C34:3 GP. L. PEaaC36:0 GP. L. PE aaC36:1 GP. L. PE aa C36:2 GP. L. PE aa C36:3 GP. L. PE aa C36:4 GP. L. PEaaC36:5 GP. L. PE aaC38:0 GP. L. PE aaC38:1 GP. L. PE aa C38:2 GP. L. PE aa C38:3 GP. L. PE aa C38:4 GP. L. PE aaC38:5 GP. L. PE aaC38:6 GP. L. PEaaC38:7 GP. L. PE aa C40:2 GP. L. PE aa C40:3 GP. L. PE aa C40:4 GP. L. PE aa C40:5 GP. L. PE aa C40:6 GP. L. PE aa C40:7 GP. L. PEaaC48:1 GP. L. PE ae C34:1 GP. L. PE ae C34:2 GP. L. PE ae C34:3 GP. L. PEaeC36:1 GP. L. PE ae C36:2 GP. L. PE ae C36:3 GP. L. PE ae C36:4 GP. L. PE ae C36:5 GP. L. PEaeC38:1 GP. L. PE ae C38:2 GP. L. PE ae C38:3 GP. L. PE ae C38:4 GP. L. PE ae C38:5 GP. L. PE ae C38:6 GP. L. PE ae C40:1 GP. L. PE ae C40:2 GP. L. PE ae C40:3 GP. L. PE ae C40:4 GP. L. PE ae C40:5 GP. L. PE ae C40:6 GP. L. PE ae C42:1 GP. L. PE ae C42:2 GP. L. PE ae C46:5 GP. L. PE ae C46:6 GP. L. PG aa C30:0 GP. L. PG aa C32:0 GP. L. PG aa C32:1 GP. L. PG aa C33:6 GP. L. PG aa C34:0 GP. L. PG aa C34:1 GP. L. PG aa C34:2 GP. L. PG aa C34:3 GP. L. PG aa C36:0 GP. L. PG aa C36:1 GP. L. PG aa C36:2 GP. L. PG aa C36:3 GP. L. PG aa C36:4 GP. L. PG aa C38:5 GP. L. PG ae C32:0 GP. L. PG ae C34:0 GP. L. PG ae C34:1 GP. L. PG ae C36:1 GP. L. PS aa C34:1 GP. L. PS aa C34:2 GP. L. PS aa C36:0 GP. L. PS aa C36:1 GP. L. PS aa C36:2 GP. L. PS aa C36:3 GP. L. PS aa C36:4 GP. L. PS aa C38:1 GP. L. PS aa C38:2 GP. L. PS aa C38:3 GP. L. PS aa C38:4 GP. L. PS aa C38:5 GP. L. PS aa C40:1 GP. L. PS aa C40:2 GP. L. PS aa C40:3 GP. L. PS aa C40:4 GP. L. PS aa C40:5 GP. L. PS aa C40:6 GP. L. PS aa C40:7 GP. L. PS aa C42:1 GP. L. PS aa C42:2 GP. L. PS aa C42:4 GP. L. PS aa C42:5 GP. L. PS ae C34:2 GP. L. PSaeC36:1 GP. L.
PSaeC36:2 GP. L.
PSaeC38:4 GP. L.
SMC14:0 S. L.
SMC16:0 S. L.
SMC16:1 S. L.
SMC17:0 S. L.
SMC18:0 S. L.
SMC18:1 S. L.
SMC19:0 S. L.
SMC19:1 S.L.
SMC19:2 S.L.
SM C20:0 S.L.
SMC20:1 S.L.
SM C20:2 S.L.
SMC21:0 S.L.
SMC21:1 S.L.
SMC21:2 S.L.
SMC21:3 S.L.
SM C22:0 S.L.
SM C22:1 S.L.
SM C22:2 S.L.
SM C22:3 S.L.
SM C23:0 S.L.
SM C23:1 S.L.
SM C23:2 S.L.
SM C23:3 S.L.
SM C24:0 S.L.
SM C24:1 S.L.
SM C24:2 S.L.
SM C24:3 S.L.
SM C24:4 S.L.
SM C26:3 S.L.
SM C26:4 S.L.
SM C3:0 S.L. lysoPCaC16:0 GP. L. lysoPCaC18:0 GP. L. lysoPCaC18:1 GP. L. lysoPCaC18:2 GP. L. lysoPC a C20:4 GP. L.
PCeC18:0 GP. L.
PCaaC30:0 GP. L.
PCaaC30:1 GP. L.
PCaaC30:2 GP. L.
PCaaC32:0 GP. L.
PCaaC32:1 GP. L.
PC aa C32:2 GP. L.
PC aa C34:0 GP. L. PC aa C34:1 GP. L. PC aa C34:2 GP. L. PC aa C34:3 GP. L. PC aa C36:0 GP. L. PC aa C36:1 GP. L. PC aa C36:2 GP. L. PC aa C36:3 GP. L. PC aa C36:4 GP. L. PC aa C36:5 GP. L. PC aa C38:1 GP. L. PC aa C38:2 GP. L. PC aa C38:3 GP. L. PC aa C38:4 GP. L. PC aa C38:5 GP. L. PC aa C38:6 GP. L. PC aa C40:4 GP. L. PC aa C40:5 GP. L. PC aa C40:6 GP. L. PC aa C40:7 GP. L. PC aa C40:8 GP. L. PC ae C32:0 GP. L. PC ae C32:1 GP. L. PC ae C32:6 GP. L. PC ae C34:0 GP. L. PC ae C34:1 GP. L. PC ae C34:2 GP. L. PC ae C34:3 GP. L. PC ae C34:6 GP. L. PC ae C36:1 GP. L. PC ae C36:2 GP. L. PC ae C36:3 GP. L. PC ae C36:4 GP. L. PC ae C36:5 GP. L. PC ae C38:1 GP. L. PC ae C38:2 GP. L. PC ae C38:3 GP. L. PC ae C38:4 GP. L. PC ae C38:5 GP. L. PC ae C38:6 GP. L. PC ae C40:5 GP. L. N-C2:0-Cer Cer. N-C3:1 -Cer Cer. N-C3:0-Cerr Cer. N-C4:1 -Cer Cer. N-C4:0-Cer Cer. N-C5:1 -Cer Cer. N-C5:0-Cer Cer. N-C6:1 -Cer Cer. N-C6:0-Cer Cer.
N-C7:1-Cer Cer.
N-C7:0-Cer Cer.
N-C8:1-Cer Cer.
N-C8:0-Cer Cer.
N-C9:3-Cer Cer.
N-C9:1-Cer Cer.
N-C9:0-Cer Cer.
N-C10:1-Cer Cer.
N-C10:0-Cer Cer.
N-C11:1-Cer Cer.
N-C11:0-Cer Cer.
N-C12:1-Cer Cer.
N-C12:0-Cer Cer.
N-(OH)CI 1:0-Cer Cer.
N-C13:1-Cer Cer.
N-C13:0-Cer Cer.
N-C14:1-Cer Cer.
N-C14:0-Cer Cer.
N-C15:1-Cer Cer.
N-C15:0-Cer Cer.
N-C16:1-Cer Cer.
N-C16:0-Cer Cer.
N-C17:1-Cer Cer.
N-C17:0-Cer Cer.
N-(2xOH)C15:0-Cer Cer.
N-C18:1-Cer Cer.
N-C18:0-Cer Cer.
N-C19:1-Cer Cer.
N-C19:0-Cer Cer.
N-C20:1-Cer Cer.
N-C20:0-Cer Cer.
N-C21:1-Cer Cer.
N-C21 :0-Cer Cer.
N-C22:1-Cer Cer.
N-C22:0-Cer Cer.
N-C23:1-Cer Cer.
N-C23:0-Cer Cer.
N-C24:1-Cer Cer.
N-C24:0-Cer Cer.
N-C25:1-Cer Cer.
N-C25:0-Cer Cer.
N-C26:1-Cer Cer.
N-C26:0-Cer Cer.
N-C27:1-Cer Cer.
N-C27:0-Cer Cer.
N-C28:1-Cer Cer.
N-C28:0-Cer Cer. N-C2:0-Cer(2H) Cer.
N-C3:1-Cer(2H) Cer.
N-C3:0-Cer(2H) Cer.
N-C4:1-Cer(2H) Cer.
N-C4:0-Cer(2H) Cer.
N-C5:1-Cer(2H) Cer.
N-C5:0-Cer(2H) Cer.
N-C6:1-Cer(2H) Cer.
N-C6:0-Cer(2H) Cer.
N-C7:1-Cer(2H) Cer.
N-C7:0-Cer(2H) Cer.
N-C8:1-Cer(2H) Cer.
N-C8:0-Cer(2H) Cer.
N-C9:1-Cer(2H) Cer.
N-C9:0-Cer(2H) Cer.
N-C10:1-Cer(2H) Cer.
N-C10:0-Cer(2H) Cer.
N-C11:1-Cer(2H) Cer.
N-C11:0-Cer(2H) Cer.
N-C12:1-Cer(2H) Cer.
N-C12:0-Cer(2H) Cer.
N-C13:1-Cer(2H) Cer.
N-C13:0-Cer(2H) Cer.
N-C14:1-Cer(2H) Cer.
N-C14:0-Cer(2H) Cer.
N-C15:1-Cer(2H) Cer.
N-C15:0-Cer(2H) Cer.
N-C16:1-Cer(2H) Cer.
N-C16:0-Cer(2H) Cer.
N-C17:1-Cer(2H) Cer.
N-C17:0-Cer(2H) Cer.
N-C18:1-Cer(2H) Cer.
N-C18:0-Cer(2H) Cer.
N-C19:1-Cer(2H) Cer.
N-C19:0-Cer(2H) Cer.
N-C18:0-Cer(2H) Cer.
N-C20:0-Cer(2H) Cer.
N-C21:1-Cer(2H) Cer.
N-C21 :0-Cer(2H) Cer.
N-C22:1-Cer(2H) Cer.
N-C22:0-Cer(2H) Cer.
N-C23:1-Cer(2H) Cer.
N-C23:0-Cer(2H) Cer.
N-C24:1-Cer(2H) Cer.
N-C24:0-Cer(2H) Cer.
N-C25:1-Cer(2H) Cer.
N-C25:0-Cer(2H) Cer.
N-C26:1-Cer(2H) Cer. N-C26:0-Cer(2H) Cer.
N-C27:1 -Cer(2H) Cer.
N-C27:0-Cer(2H) Cer.
N-C28:1 -Cer(2H) Cer.
N-C28:0-Cer(2H) Cer.
N-C3:0(OH)-Cer Cer.
N-C4:0(OH)-Cer Cer.
N-(2xOH)C3:0-Cer Cer.
N-C5:0(OH)-Cer Cer.
N-C6:0(OH)-Cer Cer.
N-C7:2(OH)-Cer Cer.
N-C7:1 (OH)-Cer Cer.
N-C7:0(OH)-Cer Cer.
N-C8:0(OH)-Cer Cer.
N-C9:0(OH)-Cer Cer.
N-C10:0(OH)-Cer Cer.
N-C1 1 :1 (OH)-Cer Cer.
N-C1 1 :0(OH)-Cer Cer.
N-C12:0(OH)-Cer Cer.
N-C13:0(OH)-Cer Cer.
N-C14:0(OH)-Cer Cer.
N-C15:0(OH)-Cer Cer.
N-C16:0(OH)-Cer Cer.
N-C17:1 (OH)-Cer Cer.
N-C17:0(OH)-Cer Cer.
N-C18:0(OH)-Cer Cer.
N-C19:0(OH)-Cer Cer.
N-C20:0(OH)-Cer Cer.
N-C19:0(2xOH)-Cer Cer.
N-C21 :0(OH)-Cer Cer.
N-C22:0(OH)-Cer Cer.
N-C23:0(OH)-Cer Cer.
N-C24:0(OH)-Cer Cer.
N-C23:0(2xOH)-Cer Cer.
N-C25:0(OH)-Cer Cer.
N-C26:1 (OH)-Cer Cer.
N-C26:0(OH)-Cer Cer.
N-C27:0(OH)-Cer Cer.
N-C28:0(OH)-Cer Cer.
N-C3:0(OH)-Cer(2H) Cer.
N-C4:0(OH)-Cer(2H) Cer.
N-C5:0(OH)-Cer(2H) Cer.
N-C6:0(OH)-Cer(2H) Cer.
N-C7:0(OH)-Cer(2H) Cer.
N-C8:0(OH)-Cer(2H) Cer.
N-C9:0(OH)-Cer(2H) Cer.
N-C10:0(OH)-Cer(2H) Cer.
N-C1 1 :0(OH)-Cer(2H) Cer. N-C13:0(OH)-Cer(2H) Cer.
N-C14:0(OH)-Cer(2H) Cer.
N-C15:0(OH)-Cer(2H) Cer.
N-C16:0(OH)-Cer(2H) Cer.
N-C17:0(OH)-Cer(2H) Cer.
N-C18:0(OH)-Cer(2H) Cer.
N-C19:0(OH)-Cer(2H) Cer.
N-C20:0(OH)-Cer(2H) Cer.
N-C21 :0(OH)-Cer(2H) Cer.
N-C22:0(OH)-Cer(2H) Cer.
N-C23:0(OH)-Cer(2H) Cer.
N-C24:0(OH)-Cer(2H) Cer.
N-C25:0(OH)-Cer(2H) Cer.
N-C26:0(OH)-Cer(2H) Cer.
N-C27:0(OH)-Cer(2H) Cer.
N-C28:0(OH)-Cer(2H) Cer.
Histamine B.Am.
Serotonin B.Am.
PEA B.Am.
TXB2 P.G.
PGF2a P.G.
24,25,EPC Ox. St.
5B,6B,EPC Ox. St.
24DHLan Ox. St.
GCDCA Bi.Ac.
GLCA Bi.Ac.
TCDCA Bi.Ac.
TLCA Bi.Ac.
GCA Bi.Ac.
CA Bi.Ac.
UDCA Bi.Ac.
CDCA Bi.Ac.
DCA Bi.Ac.
TDCA Bi.Ac.
TLCAS Bi.Ac.
GDCA Bi.Ac.
GUDCA Bi.Ac.
Table 1 summarizes analyzed metabolites and respective abbreviations; Glycero-phospholipids are further differentiated with respect to the presence of ester (a) and ether (e) bonds in the glycerol moiety, where two letters (aa, ea, or ee) denote that the first and the second position of the glycerol scaffold are bound to a fatty acid residue, whereas a single letter (a or e) indicates a bond with only one fatty acid residue; e.g. PC_ea_33:1 denotes a plasmalogen phosphatidylcholine with 33 carbons in the two fatty acid side chains and a single double bond in one of them. Detailed Examples
1. Human
We use data of 29 subjects where data are obtained by 17 patients with mixed sepsis (i.e., sepsis with mixed foci including peritonitis (4), pneumonia (5) and also unidentified foci (12 patients with mixed sepsis) developing a systemic infection (sepsis) associated organ failure. Diagnosis was confirmed diagnosis clinical criteria and microbiological evidence for infection (blood culture, PCR for pathogens).
Statistical Analysis
All statistical calculations have been performed using the statistics software R (R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2009, ISBN 3-900051 -07-0).
Analytes that were detected in at least 15% of the samples were selected for further analyses resulting in a list of 521 unique compounds/metabolites (Table 1 ). The metabolic data is left censored due to thresholding of the mass spectrometer data resulting in non detected peak/signals. By a combination of metabolic pathway dynamism, complex sample molecular interaction and overall efficiency of the analytical protocol, replacement of missing data by means of a multivariate algorithm is preferred to a naive imputation by a pre-specified value like for instance zero. Hence, missing metabolite concentrations are replaced by the average value of the 6 closest samples to the one where the measurement is missing (impute: Imputation for microarray data, Hastie T., Tibshirani R., Narasimhan B. and Chu G., R package version 1.14.0). At the exception of fold change (FC) determination, all statistical analyses are performed on preprocessed - that is, log transformed - data.
The ImFit function in the package limma (Limma: linear models for microarray data, Smyth G. K. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer, New York, pp 397-420, R package version 2.16.5) is used to compute the moderated statistics between measurements from septic patients samples and samples from patient developing organ failure. Resulting p values are adjusted by the method described in Benjamini and Hochberg (Benjamini Y. and Hochberg Y., Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society Series B, 1995, 57, 289-300) leading to so-called q values.
Sensitivity/specificity properties of a classifier comprising one analyte or a combination of analytes are summarised in terms of Area Under the Receiver Operating Characteristic Curve (AUC). The function colAUC (caTools: Tools: moving window statistics, GIF, Base64, ROC AUC, etc., Tuszynski J., 2008, R package version 1.9) is used to compute and plot ROC curves. From the three univariate statistics (adjusted p value (q value), fold change and AUC), features are ranked according to a 2 step strategy: 1 ) the 3 measures are first used as input to the multiple objective algorithm described by Chen et al. (Chen J.J., Tsai C-A., Tzeng S. -Land Chen C-H., Gene selection with multiple ordering criteria, BMC Bioinformatics 2007, 8:74) 2) ties (i.e. metabolites belonging to the same front) are broken according by simple Borda count. The function vennDiagram from the R package limma (Limma: linear models for microarray data, Smyth G. K. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer, New York, pp 397-420, R package version 2.16.5) is employed to display the number of features selected by each ranking technique; confer Figure 1. Numbers in dark (resp. grey) express the count of metabolites that exhibit higher (resp. lower) concentration in the samples of those patients developing organ failure than in the septic patients samples. Following thresholds are used: adjusted p value (q-value) less than 0.01 , absolute fold change higher than 50% and AUC greater than 0.8.
In addition to univariate statistics, additional ranking that take into account multivariate interactions is computing from boosted regression tree models. Similarly to the variable importance measures in Breiman's Random Forests, feature relative influence is determined as the effect of class labels permutation on reducing the loss function
(Friedman J. H., Greedy Function Approximation: A Gradient Boosting Maof Statistics,
2001 , 29(5):1189-1232). gbm function from gbm R package (gbm: Generalized Boosted Regression Models, Ridgeway G., 2007, R package version 1.6-3) was used to perform tree based gradient boosting specifying a gaussian loss function, a shrinkage parameter of 0.05 and allowing trees with up to 3 trees splits. To reduce variance in the ranking, feature relevance score is presented as the average rank calculated by leaving one set out on the training set.
Performance of single markers as well as of combinations of markers is assessed by three classification algorithms that rely on different mechanisms to ensure that the outcome is not dependent on the modelling technique: support vector machine (SVM) with linear kernel using the R function svm in package e1071 (e1071 : Misc Functions of the Department of Statistics (e1071 ), Dimitriadou E., Hornik k., Leisch F., Meyer D. and Weingessel A., R package version 1.5-19); diagonal discriminant analysis (DLDA) using the R function dDa in package sfsmisc (sfsmisc: Utilities from Seminar fuer Statistik ETH Zurich, Maechler M., R package version 1.0-7) and the nearest neighbour algorithm(KNN) with k equal to one using the R function knn in package class (Modern Applied Statistics with S, Venables W.N. And Ripley B. D., Springer, New York, R package version 7.2-47). Predictive abilities of the models are computed using stratified boostrap (B=20), repeated 10 times to obtain a performance estimate and its associated variance (FIEmspro: Flow Injection Electrospray Mass Spectrometry Processing: data processing, classification modelling and variable selection in metabolite fingerprinting, Beckmann M., Enot D. and Lin W., 2007, R package version 1.1 -0).
Based on the accuracy computations for the three classification algorithms SVM, DLDA, and KNN (cf. Figures 2 and 3) we select the top 60 metabolites for the ranker combining adjusted p values, fold change and AUC as well as for the multivariate wrapper which uses boosted regression trees leading to 97 different analytes and metabolites; confer Table 2.
Table 2 depicts the ranks of the individual analytes and metabolites in terms of discriminatory power for detecting the onset of infection associated organ failure. Ranking was performed using a ranker combining adjusted p values, fold changes and AUCs as well as using a multivariate wrapper which is based on boosted regression trees as described above. For additional information see Fig 1-3.
Adjusted p Multivariate
Name Univariate rank value Fold change AUC rank
CO 290 9,85E-OOl 40,23 0,50 27 C 12-DC 386 6,06E-OOl -0,79 0,58 43
C14:l 4 1.64E-003 106,12 0,93 13
C14:1-OH 326 5,75E-OOl 13,33 0,63 56
C14:2 60 2,25E-OOl 90,48 0,82 16
C14:2-OH 214 3,45E-OOl 44,44 0,70 29
C18 200 6,06E-OOl 56,00 0,66 26
C6:l 31 6,06E-OOl -325,41 0,64 124
SM (OH) C22:1 2 4,39E-005 111,63 0,92 39
SM (OH) C22:2 24 1.03E-004 87,48 0,90 254
SM (OH) C24:1 50 1.25E-004 77,60 0,88 38
SM C26:0 57 2.79E-003 89,00 0,83 298
SM C26:1 19 4,44E-005 84,43 0,91 169
PC aa C28:l 256 1,48E-OOl 10,75 0,64 52
PC aa C38:0 27 2,57E-003 103,52 0,85 209
PC aa C42:0 58 1.55E-002 91,30 0,80 154
PC aa C42:l 36 2.73E-003 102,52 0,85 253
PC ae C40:l 33 1.83E-003 96,56 0,88 500
PC ae C40:2 39 2.73E-003 91,53 0,87 455
PC ae C40:6 32 2,22E-004 81,86 0,92 108
PC ae C42:2 10 2,57E-003 147,86 0,84 419
PC ae C42:3 8 2,96E-003 134,67 0,87 331
PC ae C42:4 41 1.37E-002 126,49 0,79 50
PC ae C44:5 42 9,27E-002 182,51 0,74 141
PC ae C44:6 29 1.90E-002 120,88 0,81 61 lysoPC a C20:3 54 4,48E-002 118,52 0,73 93 lysoPC a C26:0 298 4,27E-OOl 18,11 0,56 41
Phe 251 9,40E-OOl -27,92 0,70 60
THC 15 7,04E-002 -380,12 0,80 6
7KC 17 7,04E-002 -437,25 0,76 74
5a,6a,EPC 18 7,04E-002 -224,71 0,75 37
PE a C18:l 53 8,50E-002 144,30 0,74 487
PE a C18:2 30 9,15E-002 248,48 0,75 389
PE a C20:4 49 5,45E-002 122,02 0,77 334
PE a C22:5 47 1,02E-OOl 136,84 0,76 394
PE a C22:6 16 4,74E-002 195,51 0,74 281
PE aa C38:0 119 5,41E-003 52,04 0,85 58
PE aa C38:2 59 7,01E-002 108,83 0,76 395
SM C16:0 46 1.97E-005 60,14 0,93 64
SM C17:0 56 7,25E-005 64,61 0,91 3
SM C18:0 83 2.11E-004 54,73 0,88 40
SM C19:0 52 4,44E-005 48,58 0,94 36
SM C21:1 48 4,44E-005 62,77 0,90 63
SM C21:3 45 6,41E-005 69,05 0,95 20
SM C22:2 28 5,09E-006 58,61 0,96 14
SM C23:0 6 1.56E-005 75,15 0,96 4
SM C23:1 25 6,88E-005 79,68 0,91 161
SM C23:2 26 9,32E-006 70,13 0,94 62
SM C23:3 44 9,97E-005 73,55 0,92 197
SM C24:0 3 3,89E-006 78,55 0,96 42 SM C24:1 20 9,99E-006 77,52 0,95 35
SM C24:2 5 2,71E-006 73,35 0,98 9
SM C24:3 11 2,71E-006 55,12 0,99 21
SM C24:4 38 2,64E-004 85,17 0,86 137
SM C26:4 43 2,l lE-004 83,13 0,89 104
SM C3:0 13 2,08E-003 171,48 0,80 66 lysoPC a C18:2 14 l,06E-002 180,95 0,78 178 lysoPC a C20:4 23 8,22E-003 153,07 0,80 17
PC aa C36:4 35 4,82E-005 64,50 0,95 8
PC aa C38:l 37 l,39E-004 77,32 0,93 267
PC aa C38:2 21 l,39E-004 86,17 0,93 215
PC aa C38:4 79 7,00E-004 60,00 0,90 18
PC aa C38:5 12 4,71E-005 58,58 0,99 15
PC aa C38:6 40 2,10E-003 90,17 0,86 120
PC aa C40:5 68 2,79E-004 73,08 0,90 28
PC aa C40:6 51 l,83E-003 84,16 0,89 55
PC aa C40:7 55 2,22E-004 73,36 0,91 182
PC aa C40:8 9 2,57E-003 119,32 0,86 151
PC ae C36:4 70 l,31E-003 70,81 0,90 30
PC ae C36:5 22 2,91E-004 87,31 0,94 10
PC ae C38:4 7 4,82E-005 79,47 0,94 98
PC ae C38:6 1 4,82E-005 96,66 0,97 59
N-C2:0-Cer 312 8,48E-OOl 20,06 0,65 25
N-C7:0-Cer 209 6,02E-OOl 44,44 0,71 46
N-C9:3-Cer? 144 4,45E-OOl 71,25 0,73 57
N-C17:l-Cer 354 9,99E-OOl -22,50 0,61 49
N-C22:l-Cer 364 9,99E-OOl -27,07 0,51 23
N-C25:0-Cer 34 2,95E-003 88,98 0,91 12
N-C27:l-Cer 253 4,68E-OOl 17,49 0,70 19
N-C5:l-Cer(2H) 178 9,52E-OOl 62,93 0,68 5
N-C7:l-Cer(2H) 289 9,52E-OOl 31,68 0,67 48
N-C8:l-Cer(2H) 254 9,52E-OOl 41,96 0,66 22
N-Cl l:l-Cer(2H) 311 9,99E-OOl 31,82 0,62 53
N-C20:0-Cer(2H) 103 1,29E-OOl 80,11 0,76 33
N-C21:0-Cer(2H) 457 9,99E-OOl 4,89 0,58 24
N-C22:l-Cer(2H) 223 4,45E-OOl 48,84 0,67 54
N-C25:l-Cer(2H) 228 4,45E-OOl 38,12 0,71 11
N-C26:l-Cer(2H) 140 3,45E-OOl 59,22 0,80 31
N-C6:0(OH)-Cer 276 9,99E-OOl 38,31 0,62 51
N-C24:0(OH)-Cer 236 4,45E-OOl 32,03 0,71 1
N-C26:0(OH)-Cer 260 4,45E-OOl -14,17 0,69 45
N-C8:0(OH)-Cer(2H) 415 7,98E-OOl -11,72 0,56 32
N-C10:0(OH)-Cer(2H) 100 6,61E-002 -74,99 0,82 47
N-C25:0(OH)-Cer(2H) 318 9,52E-OOl 20,16 0,65 7
N-C26:0(OH)-Cer(2H) 462 9,99E-OOl 2,38 0,58 34
N-C27:0(OH)-Cer(2H) 493 9,99E-OOl 8,35 0,52 44
N-C28:0(OH)-Cer(2H) 151 4,45E-OOl 28,55 0,82 2 2. Mouse
We use data of 1 1 (BL6) mice obtained from 5 animals with sepsis and induced liver failure and 6 mice with sepsis. Sepsis and organ failure were induced by intraperitoneal injection of an extract of human faeces. Typically 20 g of human stool (weight determined without further treatment) were homogenized in 40 ml of ice-cooled (4 C) sterile phosphate buffered saline (pH 7.4) using a Potter homogenizer or an Ultra Turrax, briefly centrifuged to remove bigger particles and the extract stored as frozen aliquots.
The effective dosis of the extract (to induce either sepsis or organ failure) has to be pre- determined for each batch (of stool from one individual human subject). Depending of the dosage, sepsis can be induced within 24 h with a complete recovery of the animals > 48 h or septic organ failure can be induced by applying a higher dosage; for instance sepsis can be induced by injection of 0.5 ml of extract and organ failure by injection of 1.0 ml intraperitoneal^. All samples of liver tissue were drawn 24 h after intraperitoneal injection of the extract.
Statistical Analysis
All statistical calculations have been performed using the statistics software R (R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2009, ISBN 3-900051 -07-0).
Analytes that were detected in at least 15% of the samples were selected for further analyses resulting in a list of 218 unique compounds/metabolites (Table 1 ). The metabolic data is left censored due to thresholding of the mass spectrometer data resulting in non detected peak/signals. By a combination of metabolic pathway dynamism, complex sample molecular interaction and overall efficiency of the analytical protocol, replacement of missing data by means of a multivariate algorithm is preferred to a naive imputation by a pre-specified value like for instance zero. Hence, missing metabolite concentrations are replaced by the average value of the 6 closest samples to the one where the measurement is missing (impute: Imputation for microarray data, Hastie T., Tibshirani R., Narasimhan B. and Chu G., R package version 1.14.0). At the exception of fold change (FC) determination, all statistical analyses are performed on preprocessed - that is, log transformed - data.
The ImFit function in the package limma (Limma: linear models for microarray data, Smyth G. K. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer, New York, pp 397-420, R package version 2.16.5) is used to compute the moderated statistics between measurements from septic patients samples and samples from patient developing organ failure. Resulting p values are adjusted by the method described in Benjamini and Hochberg (Benjamini Y. and Hochberg Y., Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society Series B, 1995, 57, 289-300) leading to so-called q values.
Sensitivity/specificity properties of a classifier comprising one analyte or a combination of analytes are summarised in terms of Area Under the Receiver Operating Characteristic Curve (AUC). The function colAUC (caTools: Tools: moving window statistics, GIF, Base64, ROC AUC, etc., Tuszynski J., 2008, R package version 1.9) is used to compute and plot ROC curves. From the three univariate statistics (adjusted p value (q value), fold change and AUC), features are ranked according to a 2 step strategy: 1 ) the 3 measures are first used as input to the multiple objective algorithm described by Chen et al. (Chen J.J., Tsai C-A., Tzeng S. -Land Chen C-H., Gene selection with multiple ordering criteria, BMC Bioinformatics 2007, 8:74) 2) ties (i.e. metabolites belonging to the same front) are broken according by simple Borda count. The function vennDiagram from the R package limma (Limma: linear models for microarray data, Smyth G. K. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor, Springer, New York, pp 397-420, R package version 2.16.5) is employed to display the number of features selected by each ranking technique; confer Figure 4. Numbers in dark (resp. grey) express the count of metabolites that exhibit higher (resp. lower) concentration in the samples of those patients developing organ failure than in the septic patients samples. Following thresholds are used: adjusted p value (q-value) less than 0.05, absolute fold change higher than 50% and AUC greater than 0.8.
Due to the relatively small number of samples we performed no multivariate analyses avoiding overfitting. We select the top 60 metabolites for the ranker combining adjusted p values, fold changes and AUCs; confer Table 3.
Name Univariate rank Adjusted p value Fold change AUC
Putrescine 1 6.75E-005 166,67 1,00
Lanosterol 2 3.50E-003 -186,85 0,97
C5-DC (C6-OH) 3 3.39E-002 90,38 1,00
25OHC 4 1.14E-003 122,16 0,87
SMC16:1 5 9.74E-003 47,95 0,98
24SOHC 6 2.07E-004 104,06 0,80
C14 7 3.06E-003 -163,87 0,63
C4-OH (C3-DC) 8 2.65E-002 129,92 0,93
CO 9 2.17E-002 82,21 0,93
C5-M-DC 10 3.49E-002 71,15 0,98
C6(C4:1-DC) 11 2.14E-001 134,29 0,75
PC aa C38:4 12 6.03E-003 14,29 0,87
GLCA 13 6.57E-001 -150,89 0,60
Ala 14 3.91E-001 -144,80 0,50
4BOHC 15 8.26E-002 59,11 0,93
24DHLan 16 1.23E-001 -51,66 0,93
TLCA 17 1.35E-001 87,93 0,87
Serotonin 18 1.48E-001 84,52 0,87
ADMA 19 7.50E-002 -114,30 0,67
PC aa C36:1 20 3.12E-003 -20,78 0,53
SMC16:0 21 3.52E-002 35,88 0,93
C5:1-DC 22 2.90E-001 88,46 0,83
7aOHC 23 1.39E-001 -26,38 0,93
27OHC 24 3.87E-001 -94,61 0,77
Cit 25 3.17E-001 -126,99 0,50
lysoPC a C20:4 26 2.90E-001 59,50 0,87
GCA 27 3.00E-001 98,25 0,67
lysoPCaCi6:0 28 1.59E-001 51,93 0,90
He 29 5.49E-002 42,99 0,87
Desmosterol 30 5.26E-002 -68,61 0,80
PEA 31 5.06E-001 -112,16 0,60
total DMA 32 2.50E-002 -35,97 0,53
Trp 33 7.03E-002 28,10 0,90
C3:1 34 8.68E-001 50,00 0,90
lysoPCaCi8:0 35 2.76E-001 50,86 0,87
VaI 36 3.40E-001 38,05 0,90
PC ae C38:0 37 6.05E-002 -50,52 0,67
PGF2a 38 5.38E-001 -96,77 0,60
SM(OH)C14:1 39 2.68E-001 35,29 0,90
lysoPCaCi8:2 40 3.57E-001 39,10 0,87
THC 41 3.15E-001 26,62 0,90
PC ae C40:4 42 1.17E-001 12,60 0,87 24,25,EPC 43 1 ,71 E-001 -84,00 0,53
PC ae C36:5 44 2.10E-001 24,65 0,90
PG D2 45 4.49E-001 56,29 0,80
GIy 46 2.00E-001 45,29 0,83
5B,6B,EPC 47 1 .30E-001 -16,12 0,80
PC ae C40:0 48 9,41 E-002 -24,60 0,67
PC ae C36:1 49 1 ,21 E-001 -37,70 0,53
C18 50 2.07E-001 44,24 0,73
C16:2 51 4.96E-001 55,26 0,75
PC aa C36:5 52 1 ,41 E-001 -36,1 1 0,63
PC aa C38:5 53 1 .46E-001 -27,05 0,67
PC aa C30:2 54 5,91 E-001 57,78 0,73
13S-HODE 55 5.25E-001 -72,09 0,57
C9 56 4,81 E-001 16,22 0,87
15S-H ETE 57 4.58E-001 -66,46 0,53
SM C22:3 58 1 .80E-001 -36,27 0,53
C5:1 59 4.16E-001 32,69 0,83
lysoPC a C17:0 60 6.28E-001 36,24 0,80
Table 3 depicts the ranks of the individual analytes and metabolites in terms of discriminatory power for detecting the onset of infection associated organ failure. Ranking was performed using a univariate ranker which combines adjusted p values, fold changes and AUCs. For additional information see Fig 4.
These 60 metabolites comprise a preferred embodyment of the present invention, as claimed in dependent claim 12.
Table 4 shows the endogenous organ failure predictive targe metabolites as used in the present invention with their abbreviations and chemical names
Table 4
Common Name
No. Name
1 CO Carnitine (free)
2 ClO Decanoylcarnitine [Caprylcarnitine] (Fumarylcarnitine) 3 C10:l Decenoylcarnitine
4 C10:2 Decadienoylcarnitine
5 C 12 Dodecanoylcarnitine [Laurylcarnitine]
6 C 12-DC Dodecanedioylcarnitine
7 C12:1 Dodecenoylcarnitine
8 C 14 Tetradecanoylcarnitine
9 C14:l Tetradecenoylcarnitine [Myristoleylcarnitine]
10 C14:1-OH 3-Hydroxytetradecenoylcarnitine [3-Hydroxymyristoleylcarnitine]
11 C14:2 Tetradecadienoylcarnitine
12 C14:2-OH 3-Hydroxytetradecadienoylcarnitine
13 C16 Hexadecanoylcarnitine [Palmitoylcarnitine]
14 C16-OH 3-Hydroxyhexadecanolycarnitine [3-Hydroxypalmitoylcarnitine]
15 C16:l Hexadecenoylcarnitine [Palmitoleylcarnitine]
3-Hydroxyhexadecenoylcarnitine [3-Hydroxypalmitoleylcarnitine] 16 C16:1-OH
17 C16:2 Hexadecadienoylcarnitine C16:2-OH 3-Hydroxyhexadecadienoylcarnitine
Cl 8 Octadecanoylcarnitine [Stearylcarnitine] C 18 : 1 Octadecenoylcarnitine [Oleylcarnitine] C 18 : 1 -OH S-Hydroxyoctadecenoylcarnitine [3-Hydroxyoleylcarnitine] Cl 8:2 Octadecadienoylcarnitine [Linoleylcarnitine]
C2 Acetylcarnitine
C3 Propionylcarnitine
C3-OH Hydroxypropionylcarnitine
C3:1 Propenoylcarnitine
C4 Butyrylcarnitine / Isobutyrylcarnitine
C4-OH (C3-DC) 3-Hydroxybutyrylcarnitine / Malonylcarnitine
C4:1 Butenoylcarnitine
C5 Isovalerylcarnitine / 2-Methylbutyrylcarnitine / Valerylcarnitine C5-DC (C6-OH) Glutarylcarnitine / Hydroxycaproylcarnitine
C5-M-DC Methylglutarylcarnitine
C5-OH (C3-DC- M) 3-Hydroxyisovalerylcarnitine / 3-Hydroxy-2-methylbutyryl C5: 1 Tiglylcarnitine / 3-Methyl-crotonylcarnitine
C5:1-DC Tiglylcarnitine / 3-Methyl-crotonylcarnitine
C6 (C4:1-DC) Hexanoylcarnitine [Caproylcarnitine]
C6:1 Hexenoylcarnitine
C7-DC Pimelylcarnitine
C8 Octanoylcarnitine [Caprylylcarnitine] C8:l Octenoylcarnitine
C9 Nonoylcarnitine [Pelargonylcarnitine]
Hl Hexose pool
SM (OH) C14: 1 Sphingomyelin with acyl residue sum (OH) C14: 1
SM (OH) C 16: 1 Sphingomyelin with acyl residue sum (OH) C 16: 1
SM (OH) C22: 1 Sphingomyelin with acyl residue sum (OH) C22: 1
SM (OH) C22:2 Sphingomyelin with acyl residue sum (OH) C22:2
SM (OH) C24: 1 Sphingomyelin with acyl residue sum (OH) C24: 1
SM C26:0 Sphingomyelin with acyl residue sum C26:0 SM C26:1 Sphingomyelin with acyl residue sum C26:l
PC aa C24:0 Phosphatidylcholine with diacyl residue sum C24:0PC aa C26:0 Phosphatidylcholine with diacyl residue sum C26:0PC aa C28:l Phosphatidylcholine with diacyl residue sum C28:l PC aa C32:3 Phosphatidylcholine with diacyl residue sum C32:3 PC aa C34:4 Phosphatidylcholine with diacyl residue sum C34:4 PC aa C36:6 Phosphatidylcholine with diacyl residue sum C36:6 PC aa C38:0 Phosphatidylcholine with diacyl residue sum C38:0PC aa C40:l Phosphatidylcholine with diacyl residue sum C40:l PC aa C40:2 Phosphatidylcholine with diacyl residue sum C40:2 PC aa C40:3 Phosphatidylcholine with diacyl residue sum C40:3 PC aa C42:0 Phosphatidylcholine with diacyl residue sum C42:0PC aa C42:l Phosphatidylcholine with diacyl residue sum C42: 1 PC aa C42:2 Phosphatidylcholine with diacyl residue sum C42:2 PC aa C42:4 Phosphatidylcholine with diacyl residue sum C42:4 PC aa C42:5 Phosphatidylcholine with diacyl residue sum C42:5 PC aa C42:6 Phosphatidylcholine with diacyl residue sum C42:6 PC ae C30:0 Phosphatidylcholine with acyl-alkyl residue sum C30:0PC ae C30:l Phosphatidylcholine with acyl-alkyl residue sum C30:l PC ae C30:2 Phosphatidylcholine with acyl-alkyl residue sum C30:2 PC ae C32:2 Phosphatidylcholine with acyl-alkyl residue sum C32:2 PC ae C36:0 Phosphatidylcholine with acyl-alkyl residue sum C36:0PC ae C38:0 Phosphatidylcholine with acyl-alkyl residue sum C38:0 PC ae C40:0 Phosphatidylcholine with acyl-alkyl residue sum C40:0PC ae C40:l Phosphatidylcholine with acyl-alkyl residue sum C40:l PC ae C40:2 Phosphatidylcholine with acyl-alkyl residue sum C40:2 PC ae C40:3 Phosphatidylcholine with acyl-alkyl residue sum C40:3 PC ae C40:4 Phosphatidylcholine with acyl-alkyl residue sum C40:4 PC ae C40:6 Phosphatidylcholine with acyl-alkyl residue sum C40:6 PC ae C42:0 Phosphatidylcholine with acyl-alkyl residue sum C42:0PC ae C42:l Phosphatidylcholine with acyl-alkyl residue sum C42: 1 PC ae C42:2 Phosphatidylcholine with acyl-alkyl residue sum C42:2PC ae C42:3 Phosphatidylcholine with acyl-alkyl residue sum C42:3 PC ae C42:4 Phosphatidylcholine with acyl-alkyl residue sum C42:4 PC ae C42:5 Phosphatidylcholine with acyl-alkyl residue sum C42:5 PC ae C44:3 Phosphatidylcholine with acyl-alkyl residue sum C44:3 PC ae C44:4 Phosphatidylcholine with acyl-alkyl residue sum C44:4 PC ae C44:5 Phosphatidylcholine with acyl-alkyl residue sum C44:5 PC ae C44:6 Phosphatidylcholine with acyl-alkyl residue sum C44:6 lysoPC a C14:0 Ly s opho sphatidylcholine with acyl residue sum C 14:01ysoPC a C16:l Ly s opho sphatidylcholine with acyl residue sum C 16:1 lysoPC a C17:0 Ly s opho sphatidylcholine with acyl residue sum C 17:01ysoPC a C20:3 Ly s opho sphatidylcholine with acyl residue sum C20:3 lysoPC a C24:0 Ly s opho sphatidylcholine with acyl residue sum C24:0 lysoPC a C26:0 Ly s opho sphatidylcholine with acyl residue sum C26:01ysoPC a C26:l Ly s opho sphatidylcholine with acyl residue sum C26:l lysoPC a C28:0 Ly s opho sphatidylcholine with acyl residue sum C28:01ysoPC a C28:l Ly s opho sphatidylcholine with acyl residue sum C28:l lysoPC a C6:0 Ly s opho sphatidylcholine with acyl residue sum C6:0 98 GIy Glycine
99 AIa Alanine
100 Ser Serine
101 Pro Proline
102 VaI Valine
103 Thr Threonine
104 XIe Leucine + Isoleucine
105 Leu Leucine
106 Ee Isoleucine
107 Asn Asparagine
108 Asp Aspartate
109 GIn Glutamine
110 GIu Glutamate
111 Met Methionine
112 His Histidine
113 Phe Phenylalanine
114 Arg Arginine
115 Cit Citrulline
116 Tyr Tyrosine
117 Trp Tryptophan
118 Orn Ornithine
119 Lys Lysine
120 ADMA asymmetrical Dimethylarginin
121 total DMA Total dimethylarginine: sum ADMA + SDMA
122 Met-SO Methionine-Sulfoxide
123 Kyn Kynurenine
124 Putrescine Putrescine
125 Spermidine Spermidine
126 Spermine Spermine
127 Creatinine Creatinine
128 9-HODE (±)9-hydroxy-10E,12Z-octadecadienoic acid
129 13S-HODE 13(S)-hydroxy-9Z,l lE-octadecadienoic acid
130 12S-HETE 12(S)-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid
131 15S-HETE 15(S)-hydroxy-5Z,8Z,l lZ,13E-eicosatetraenoic acid
132 LTB4 Leukotriene B4
133 DHA Docosahexaenoic acid
134 PGE2 Prostaglandin E2
135 PGD2 Prostaglandin D2
136 AA Arachidonic acid
137 Lac Lactate
138 Sue Succinic acid (succite)
139 Hex Hexose pool
140 22ROHC 22-R-Hydroxycholesterol
141 24SOHC 24- S -Hydroxycholesterol
142 25OHC 25-Hydroxycholesterol
143 27OHC 27-Hydroxycholesterol
144 THC 3β,5α,6β-Trihydroxycholestan
145 7aOHC 7α-Hydroxycholesterol
146 7KC 7 - Ketocholesterol 147 5a,6a,EPC 5α,6α-Epoxycholesterol
148 4BOHC 4β-Hydroxycholesterol
149 Desmosterol Desmosterol
150 7DHC 7-Dehydrocholesterol (Vitamin D3)
151 Lanosterol Lanosterol
152 PE a C16:0 Ly s opho sphatidylethanolamine with acyl residue sum C 16:0 153 PE a C18:0 Ly s opho sphatidylethanolamine with acyl residue sum Cl 8:0 154 PE a C18:l Ly s opho sphatidylethanolamine with acyl residue sum Cl 8:1 155 PE a C18:2 Ly s opho sphatidylethanolamine with acyl residue sum Cl 8:2
156 PE a C20:4 Ly s opho sphatidylethanolamine with acyl residue sum C20:4
157 PE a C22:4 Ly s opho sphatidylethanolamine with acyl residue sum C22:4
158 PE a C22:5 Ly s opho sphatidylethanolamine with acyl residue sum C22:5
159 PE a C22:6 Ly s opho sphatidylethanolamine with acyl residue sum C22:6 160 PE e C18:0 Ly s opho sphatidylethanolamine with alkyl residue sum Cl 8:0 161 PG e C14:2 Lysophosphatidylglycerol with alkyl residue sum C 14:2
162 PE aa C20:0 Pho sphatidylethanolamine with diacyl residue sum C20:0
163 PE aa C22:2 Pho sphatidylethanolamine with diacyl residue sum C22:2
164 PE aa C26:4 Pho sphatidylethanolamine with diacyl residue sum C26:4
165 PE aa C28:4 Pho sphatidylethanolamine with diacyl residue sum C28:4
166 PE aa C28:5 Pho sphatidylethanolamine with diacyl residue sum C28:5
167 PE aa C34:0 Pho sphatidylethanolamine with diacyl residue sum C34:0 168 PE aa C34:l Pho sphatidylethanolamine with diacyl residue sum C34: 1
169 PE aa C34:2 Pho sphatidylethanolamine with diacyl residue sum C34:2
170 PE aa C34:3 Pho sphatidylethanolamine with diacyl residue sum C34:3 171 PE aa C36:0 Pho sphatidylethanolamine with diacyl residue sum C36:0 172 PE aa C36:l Pho sphatidylethanolamine with diacyl residue sum C36:l
173 PE aa C36:2 Pho sphatidylethanolamine with diacyl residue sum C36:2
174 PE aa C36:3 Pho sphatidylethanolamine with diacyl residue sum C36:3
175 PE aa C36:4 Pho sphatidylethanolamine with diacyl residue sum C36:4
176 PE aa C36:5 Pho sphatidylethanolamine with diacyl residue sum C36:5
177 PE aa C38:0 Pho sphatidylethanolamine with diacyl residue sum C38:0 178 PE aa C38:l Pho sphatidylethanolamine with diacyl residue sum C38:l 179 PE aa C38:2 Pho sphatidylethanolamine with diacyl residue sum C38:2 180 PE aa C38:3 Pho sphatidylethanolamine with diacyl residue sum C38:3 181 PE aa C38:4 Pho sphatidylethanolamine with diacyl residue sum C38:4 182 PE aa C38:5 Pho sphatidylethanolamine with diacyl residue sum C38:5 183 PE aa C38:6 Pho sphatidylethanolamine with diacyl residue sum C38:6 184 PE aa C38:7 Pho sphatidylethanolamine with diacyl residue sum C38:7 185 PE aa C40:2 Pho sphatidylethanolamine with diacyl residue sum C40:2 186 PE aa C40:3 Pho sphatidylethanolamine with diacyl residue sum C40:3 187 PE aa C40:4 Pho sphatidylethanolamine with diacyl residue sum C40:4 188 PE aa C40:5 Pho sphatidylethanolamine with diacyl residue sum C40:5 189 PE aa C40:6 Pho sphatidylethanolamine with diacyl residue sum C40:6 190 PE aa C40:7 Pho sphatidylethanolamine with diacyl residue sum C40:7 191 PE aa C48:l Pho sphatidylethanolamine with diacyl residue sum C48:l 192 PE ae C34:l Pho sphatidylethanolamine with acyl-alkyl residue sum C34: 1
193 PE ae C34:2 Pho sphatidylethanolamine with acyl-alkyl residue sum C34:2
194 PE ae C34:3 Pho sphatidylethanolamine with acyl-alkyl residue sum C34:3 195 PE ae C36:l Pho sphatidylethanolamine with acyl-alkyl residue sum C36:l 196 PE ae C36:2 Phosphatidylethanolamine with acyl-alkyl residue sum C36:2
197 PE ae C36:3 Phosphatidylethanolamine with acyl-alkyl residue sum C36:3
198 PE ae C36:4 Phosphatidylethanolamine with acyl-alkyl residue sum C36:4
199 PE ae C36:5 Phosphatidylethanolamine with acyl-alkyl residue sum C36:5
200 PE ae C38:l Phosphatidylethanolamine with acyl-alkyl residue sum C38:l
201 PE ae C38:2 Phosphatidylethanolamine with acyl-alkyl residue sum C38:2
202 PE ae C38:3 Phosphatidylethanolamine with acyl-alkyl residue sum C38:3
203 PE ae C38:4 Phosphatidylethanolamine with acyl-alkyl residue sum C38:4
204 PE ae C38:5 Phosphatidylethanolamine with acyl-alkyl residue sum C38:5
205 PE ae C38:6 Phosphatidylethanolamine with acyl-alkyl residue sum C38:6
206 PE ae C40:l Phosphatidylethanolamine with acyl-alkyl residue sum C40:l
207 PE ae C40:2 Phosphatidylethanolamine with acyl-alkyl residue sum C40:2
208 PE ae C40:3 Phosphatidylethanolamine with acyl-alkyl residue sum C40:3
209 PE ae C40:4 Phosphatidylethanolamine with acyl-alkyl residue sum C40:4
210 PE ae C40:5 Phosphatidylethanolamine with acyl-alkyl residue sum C40:5
211 PE ae C40:6 Phosphatidylethanolamine with acyl-alkyl residue sum C40:6
212 PE ae C42:l Phosphatidylethanolamine with acyl-alkyl residue sum C42:l
213 PE ae C42:2 Phosphatidylethanolamine with acyl-alkyl residue sum C42:2
214 PE ae C46:5 Phosphatidylethanolamine with acyl-alkyl residue sum C46:5
215 PE ae C46:6 Phosphatidylethanolamine with acyl-alkyl residue sum C46:6
216 PG aa C30:0 Phosphatidylglycerol with diacyl residue sum C30:0
217 PG aa C32:0 Phosphatidylglycerol with diacyl residue sum C32:0
218 PG aa C32: 1 Phosphatidylglycerol with diacyl residue sum C32: 1
219 PG aa C33:6 Phosphatidylglycerol with diacyl residue sum C33:6
220 PG aa C34:0 Phosphatidylglycerol with diacyl residue sum C34:0
221 PG aa C34:l Phosphatidylglycerol with diacyl residue sum C34:l
222 PG aa C34:2 Phosphatidylglycerol with diacyl residue sum C34:2
223 PG aa C34:3 Phosphatidylglycerol with diacyl residue sum C34:3
224 PG aa C36:0 Phosphatidylglycerol with diacyl residue sum C36:0
225 PG aa C36: 1 Phosphatidylglycerol with diacyl residue sum C36: 1
226 PG aa C36:2 Phosphatidylglycerol with diacyl residue sum C36:2
227 PG aa C36:3 Phosphatidylglycerol with diacyl residue sum C36:3
228 PG aa C36:4 Phosphatidylglycerol with diacyl residue sum C36:4
229 PG aa C38:5 Phosphatidylglycerol with diacyl residue sum C38:5
230 PG ae C32:0 Phosphatidylglycerol with acyl-alkyl residue sum C32:0
231 PG ae C34:0 Phosphatidylglycerol with acyl-alkyl residue sum C34:0
232 PG ae C34: 1 Phosphatidylglycerol with acyl-alkyl residue sum C34: 1 233 PG ae C36:l Phosphatidylglycerol with acyl-alkyl residue sum C36:l
234 PS aa C34: 1 Phosphatidylserine with diacyl residue sum C34: 1
235 PS aa C34:2 Phosphatidylserine with diacyl residue sum C34:2
236 PS aa C36:0 Phosphatidylserine with diacyl residue sum C36:0
237 PS aa C36:l Phosphatidylserine with diacyl residue sum C36:l
238 PS aa C36:2 Phosphatidylserine with diacyl residue sum C36:2
239 PS aa C36:3 Phosphatidylserine with diacyl residue sum C36:3
240 PS aa C36:4 Phosphatidylserine with diacyl residue sum C36:4
241 PS aa C38:l Phosphatidylserine with diacyl residue sum C38:l
242 PS aa C38:2 Phosphatidylserine with diacyl residue sum C38:2
243 PS aa C38:3 Phosphatidylserine with diacyl residue sum C38:3
244 PS aa C38:4 Phosphatidylserine with diacyl residue sum C38:4 245 PS aa C38:5 Phosphatidylserine with diacyl residue sum C38:5
246 PS aa C40:l Phosphatidylserine with diacyl residue sum C40:l
247 PS aa C40:2 Phosphatidylserine with diacyl residue sum C40:2
248 PS aa C40:3 Phosphatidylserine with diacyl residue sum C40:3
249 PS aa C40:4 Phosphatidylserine with diacyl residue sum C40:4
250 PS aa C40:5 Phosphatidylserine with diacyl residue sum C40:5
251 PS aa C40:6 Phosphatidylserine with diacyl residue sum C40:6
252 PS aa C40:7 Phosphatidylserine with diacyl residue sum C40:7
253 PS aa C42: 1 Phosphatidylserine with diacyl residue sum C42: 1
254 PS aa C42:2 Phosphatidylserine with diacyl residue sum C42:2
255 PS aa C42:4 Phosphatidylserine with diacyl residue sum C42:4
256 PS aa C42:5 Phosphatidylserine with diacyl residue sum C42:5
257 PS ae C34:2 Phosphatidylserine with acyl-alkyl residue sum C34:2 258 PS ae C36:l Phosphatidylserine with acyl-alkyl residue sum C36:l
259 PS ae C36:2 Phosphatidylserine with acyl-alkyl residue sum C36:2
260 PS ae C38:4 Phosphatidylserine with acyl-alkyl residue sum C38:4
261 SM C14:0 Sphingomyelin with acyl residue sum C14:0
262 SM C16:0 Sphingomyelin with acyl residue sum C16:0
263 SM C16:l Sphingomyelin with acyl residue sum C16:l
264 SM C17:0 Sphingomyelin with acyl residue sum C17:0
265 SM C 18:0 Sphingomyelin with acyl residue sum Cl 8:0
266 SM C 18:1 Sphingomyelin with acyl residue sum Cl 8:1
267 SM C 19:0 Sphingomyelin with acyl residue sum C 19:0
268 SM C19:l Sphingomyelin with acyl residue sum C19:l
269 SM C19:2 Sphingomyelin with acyl residue sum C19:2
270 SM C20:0 Sphingomyelin with acyl residue sum C20:0
271 SM C20:l Sphingomyelin with acyl residue sum C20:l
272 SM C20:2 Sphingomyelin with acyl residue sum C20:2
273 SM C21:0 Sphingomyelin with acyl residue sum C21:0
274 SM C21 : 1 Sphingomyelin with acyl residue sum C21 : 1
275 SM C21:2 Sphingomyelin with acyl residue sum C21:2
276 SM C21:3 Sphingomyelin with acyl residue sum C21:3
277 SM C22:0 Sphingomyelin with acyl residue sum C22:0
278 SM C22: 1 Sphingomyelin with acyl residue sum C22: 1
279 SM C22:2 Sphingomyelin with acyl residue sum C22:2
280 SM C22:3 Sphingomyelin with acyl residue sum C22:3
281 SM C23:0 Sphingomyelin with acyl residue sum C23:0
282 SM C23: 1 Sphingomyelin with acyl residue sum C23: 1
283 SM C23:2 Sphingomyelin with acyl residue sum C23:2
284 SM C23:3 Sphingomyelin with acyl residue sum C23:3
285 SM C24:0 Sphingomyelin with acyl residue sum C24:0
286 SM C24: 1 Sphingomyelin with acyl residue sum C24: 1
287 SM C24:2 Sphingomyelin with acyl residue sum C24:2
288 SM C24:3 Sphingomyelin with acyl residue sum C24:3
289 SM C24:4 Sphingomyelin with acyl residue sum C24:4
290 SM C26:3 Sphingomyelin with acyl residue sum C26:3
291 SM C26:4 Sphingomyelin with acyl residue sum C26:4
292 SM C3:0 Sphingomyelin with acyl residue sum C3:0
293 lysoPC a C16:0 Lysophosphatidylcholine with acyl residue sum C16:0 294 1ysoPC a C18:0 Ly s opho sphatidylcholine with acyl residue sum C 18:0
295 1ysoPC a C18:l Ly s opho sphatidylcholine with acyl residue sum C 18:1
296 1ysoPC a C18:2 Ly s opho sphatidylcholine with acyl residue sum C 18:2
297 lysoPC a C20:4 Ly s opho sphatidylcholine with acyl residue sum C20:4
298 PC e C18:0 Ly s opho sphatidylcholine with alkyl residue sum C 18:0
299 PC aa C30:0 Phosphatidylcholine with diacyl residue sum C30:0
300 PC aa C30:l Phosphatidylcholine with diacyl residue sum C30:l
301 PC aa C30:2 Phosphatidylcholine with diacyl residue sum C30:2
302 PC aa C32:0 Phosphatidylcholine with diacyl residue sum C32:0
303 PC aa C32:l Phosphatidylcholine with diacyl residue sum C32: 1
304 PC aa C32:2 Phosphatidylcholine with diacyl residue sum C32:2
305 PC aa C34:0 Phosphatidylcholine with diacyl residue sum C34:0 306 PC aa C34:l Phosphatidylcholine with diacyl residue sum C34: 1
307 PC aa C34:2 Phosphatidylcholine with diacyl residue sum C34:2
308 PC aa C34:3 Phosphatidylcholine with diacyl residue sum C34:3
309 PC aa C36:0 Phosphatidylcholine with diacyl residue sum C36:0 310 PC aa C36:l Phosphatidylcholine with diacyl residue sum C36:l 311 PC aa C36:2 Phosphatidylcholine with diacyl residue sum C36:2 312 PC aa C36:3 Phosphatidylcholine with diacyl residue sum C36:3 313 PC aa C36:4 Phosphatidylcholine with diacyl residue sum C36:4 314 PC aa C36:5 Phosphatidylcholine with diacyl residue sum C36:5 315 PC aa C38:l Phosphatidylcholine with diacyl residue sum C38:l 316 PC aa C38:2 Phosphatidylcholine with diacyl residue sum C38:2 317 PC aa C38:3 Phosphatidylcholine with diacyl residue sum C38:3 318 PC aa C38:4 Phosphatidylcholine with diacyl residue sum C38:4 319 PC aa C38:5 Phosphatidylcholine with diacyl residue sum C38:5
320 PC aa C38:6 Phosphatidylcholine with diacyl residue sum C38:6
321 PC aa C40:4 Phosphatidylcholine with diacyl residue sum C40:4
322 PC aa C40:5 Phosphatidylcholine with diacyl residue sum C40:5
323 PC aa C40:6 Phosphatidylcholine with diacyl residue sum C40:6
324 PC aa C40:7 Phosphatidylcholine with diacyl residue sum C40:7
325 PC aa C40:8 Phosphatidylcholine with diacyl residue sum C40:8
326 PC ae C32:0 Phosphatidylcholine with acyl-alkyl residue sum C32:0 327 PC ae C32:l Phosphatidylcholine with acyl-alkyl residue sum C32: 1
328 PC ae C32:6 Phosphatidylcholine with acyl-alkyl residue sum C32:6
329 PC ae C34:0 Phosphatidylcholine with acyl-alkyl residue sum C34:0 330 PC ae C34:l Phosphatidylcholine with acyl-alkyl residue sum C34: 1 331 PC ae C34:2 Phosphatidylcholine with acyl-alkyl residue sum C34:2
332 PC ae C34:3 Phosphatidylcholine with acyl-alkyl residue sum C34:3
333 PC ae C34:6 Phosphatidylcholine with acyl-alkyl residue sum C34:6 334 PC ae C36:l Phosphatidylcholine with acyl-alkyl residue sum C36:l
335 PC ae C36:2 Phosphatidylcholine with acyl-alkyl residue sum C36:2
336 PC ae C36:3 Phosphatidylcholine with acyl-alkyl residue sum C36:3
337 PC ae C36:4 Phosphatidylcholine with acyl-alkyl residue sum C36:4
338 PC ae C36:5 Phosphatidylcholine with acyl-alkyl residue sum C36:5 339 PC ae C38:l Phosphatidylcholine with acyl-alkyl residue sum C38:l
340 PC ae C38:2 Phosphatidylcholine with acyl-alkyl residue sum C38:2
341 PC ae C38:3 Phosphatidylcholine with acyl-alkyl residue sum C38:3
342 PC ae C38:4 Phosphatidylcholine with acyl-alkyl residue sum C38:4 343 PC ae C38:5 Phosphatidylcholine with acyl-alkyl residue sum C38:5
344 PC ae C38:6 Phosphatidylcholine with acyl-alkyl residue sum C38:6
345 PC ae C40:5 Phosphatidylcholine with acyl-alkyl residue sum C40:5
Ceramide: chain length and number of double bonds is
346 N-C2:0-Cer determined by the measured mass C2:0
Ceramide: chain length and number of double bonds is
347 N-C3: 1-Cer determined by the measured mass C3: 1
Ceramide: chain length and number of double bonds is
348 N-C3:0-Cerr determined by the measured mass C3:0
Ceramide: chain length and number of double bonds is
349 N-C4: 1-Cer determined by the measured mass C4: 1
Ceramide: chain length and number of double bonds is
350 N-C4:0-Cer determined by the measured mass C4:0
Ceramide: chain length and number of double bonds is
351 N-C5 : 1 -Cer determined by the measured mass C5 : 1
Ceramide: chain length and number of double bonds is
352 N-C5:0-Cer determined by the measured mass C5:0
Ceramide: chain length and number of double bonds is
353 N-C6: 1-Cer determined by the measured mass C6: 1
Ceramide: chain length and number of double bonds is
354 N-C6:0-Cer determined by the measured mass C6:0
Ceramide: chain length and number of double bonds is
355 N-C7: 1-Cer determined by the measured mass C7: 1
Ceramide: chain length and number of double bonds is
356 N-C7:0-Cer determined by the measured mass C7:0
Ceramide: chain length and number of double bonds is
357 N-C8: 1-Cer determined by the measured mass C8: 1
Ceramide: chain length and number of double bonds is
358 N-C8:0-Cer determined by the measured mass C8:0
Ceramide: chain length and number of double bonds is
359 N-C9:3-Cer determined by the measured mass C9:3
Ceramide: chain length and number of double bonds is
360 N-C9: 1-Cer determined by the measured mass C9: 1
Ceramide: chain length and number of double bonds is
361 N-C9:0-Cer determined by the measured mass C9:0
Ceramide: chain length and number of double bonds is
362 N-ClO: 1-Cer determined by the measured mass ClO: 1
Ceramide: chain length and number of double bonds is
363 N-C10:0-Cer determined by the measured mass C10:0
Ceramide: chain length and number of double bonds is
364 N-C 11:1 -Cer determined by the measured mass C 11 : 1
Ceramide: chain length and number of double bonds is
365 N-Cl l:0-Cer determined by the measured mass Cl 1:0
Ceramide: chain length and number of double bonds is
366 N-C12: 1-Cer determined by the measured mass C12: 1
Ceramide: chain length and number of double bonds is
367 N-C12:0-Cer determined by the measured mass C12:0
Ceramide: chain length and number of double bonds is
368 N-(OH)Cl l:0-Cer determined by the measured mass (OH)Cl 1:0 Ceramide: chain length and number of double bonds is
369 N-C 13:1 -Cer determined by the measured mass C 13 : 1
Ceramide: chain length and number of double bonds is
370 N-C13:0-Cer determined by the measured mass C13:0
Ceramide: chain length and number of double bonds is
371 N-C14:l-Cer determined by the measured mass C14:l
Ceramide: chain length and number of double bonds is
372 N-C14:0-Cer determined by the measured mass C14:0
Ceramide: chain length and number of double bonds is
373 N-C 15:1 -Cer determined by the measured mass C 15 : 1
Ceramide: chain length and number of double bonds is
374 N-C15:0-Cer determined by the measured mass C15:0
Ceramide: chain length and number of double bonds is
375 N-C16: 1-Cer determined by the measured mass C16: 1
Ceramide: chain length and number of double bonds is
376 N-C16:0-Cer determined by the measured mass C16:0
Ceramide: chain length and number of double bonds is
377 N-C17: 1-Cer determined by the measured mass C17: 1
Ceramide: chain length and number of double bonds is
378 N-C17:0-Cer determined by the measured mass C17:0
N-(2xOH)C15:0- Ceramide: chain length and number of double bonds is
379 Cer determined by the measured mass (2xOH)C15:0
Ceramide: chain length and number of double bonds is
380 N-C18: 1-Cer determined by the measured mass C18: 1
Ceramide: chain length and number of double bonds is
381 N-C18:0-Cer determined by the measured mass C18:0
Ceramide: chain length and number of double bonds is
382 N-C19: 1-Cer determined by the measured mass C19: 1
Ceramide: chain length and number of double bonds is
383 N-C19:0-Cer determined by the measured mass C19:0
Ceramide: chain length and number of double bonds is
384 N-C20: 1-Cer determined by the measured mass C20: 1
Ceramide: chain length and number of double bonds is
385 N-C20:0-Cer determined by the measured mass C20:0
Ceramide: chain length and number of double bonds is
386 N-C21 : 1-Cer determined by the measured mass C21 : 1
Ceramide: chain length and number of double bonds is
387 N-C21 :0-Cer determined by the measured mass C21 :0
Ceramide: chain length and number of double bonds is
388 N-C22: 1-Cer determined by the measured mass C22: 1
Ceramide: chain length and number of double bonds is
389 N-C22:0-Cer determined by the measured mass C22:0
Ceramide: chain length and number of double bonds is
390 N-C23: 1-Cer determined by the measured mass C23: 1
Ceramide: chain length and number of double bonds is
391 N-C23:0-Cer determined by the measured mass C23:0
Ceramide: chain length and number of double bonds is
392 N-C24: 1-Cer determined by the measured mass C24: 1
393 N-C24:0-Cer Ceramide: chain length and number of double bonds is determined by the measured mass C24:0
Ceramide: chain length and number of double bonds is
394 N-C25:l-Cer determined by the measured mass C25: 1
Ceramide: chain length and number of double bonds is 395 N-C25:0-Cer determined by the measured mass C25:0
Ceramide: chain length and number of double bonds is 396 N-C26:l-Cer determined by the measured mass C26: 1
Ceramide: chain length and number of double bonds is 397 N-C26:0-Cer determined by the measured mass C26:0
Ceramide: chain length and number of double bonds is 398 N-C27:l-Cer determined by the measured mass C27: 1
Ceramide: chain length and number of double bonds is 399 N-C27:0-Cer determined by the measured mass C27:0
Ceramide: chain length and number of double bonds is 400 N-C28:l-Cer determined by the measured mass C28: 1
Ceramide: chain length and number of double bonds is
401 N-C28:0-Cer determined by the measured mass C28:0
Dihydroceramide: chain length and number of double bonds is
402 N-C2:0-Cer(2H) determined by the measured mass C2:0
Dihydroceramide: chain length and number of double bonds is 403 N-C3:l-Cer(2H) determined by the measured mass C3: 1
Dihydroceramide: chain length and number of double bonds is 404 N-C3:0-Cer(2H) determined by the measured mass C3:0
Dihydroceramide: chain length and number of double bonds is 405 N-C4:l-Cer(2H) determined by the measured mass C4: 1
Dihydroceramide: chain length and number of double bonds is 406 N-C4:0-Cer(2H) determined by the measured mass C4:0
Dihydroceramide: chain length and number of double bonds is 407 N-C5:l-Cer(2H) determined by the measured mass C5: 1
Dihydroceramide: chain length and number of double bonds is 408 N-C5:0-Cer(2H) determined by the measured mass C5:0
Dihydroceramide: chain length and number of double bonds is 409 N-C6:l-Cer(2H) determined by the measured mass C6: 1
Dihydroceramide: chain length and number of double bonds is 410 N-C6:0-Cer(2H) determined by the measured mass C6:0
Dihydroceramide: chain length and number of double bonds is 411 N-C7:l-Cer(2H) determined by the measured mass C7: 1
Dihydroceramide: chain length and number of double bonds is 412 N-C7:0-Cer(2H) determined by the measured mass C7:0
Dihydroceramide: chain length and number of double bonds is 413 N-C8:l-Cer(2H) determined by the measured mass C8: 1
Dihydroceramide: chain length and number of double bonds is 414 N-C8:0-Cer(2H) determined by the measured mass C8:0
Dihydroceramide: chain length and number of double bonds is
415 N-C9:l-Cer(2H) determined by the measured mass C9: 1
N-C9:0-Cer(2H) Dihydroceramide: chain length and number of double bonds is 416 Q3+NL cor determined by the measured mass C9:0
Dihydroceramide: chain length and number of double bonds is
417 N-ClO: l-Cer(2H) determined by the measured mass ClO: 1 Dihydroceramide: chain length and number of double bonds is
418 N-C10:0-Cer(2H) determined by the measured mass C10:0
Dihydroceramide: chain length and number of double bonds is
419 N-Cl l:l-Cer(2H) determined by the measured mass C 11 : 1
Dihydroceramide: chain length and number of double bonds is
420 N-Cll:0-Cer(2H) determined by the measured mass Cl 1:0
Dihydroceramide: chain length and number of double bonds is 421N-C12:l-Cer(2H) determined by the measured mass C12:l
Dihydroceramide: chain length and number of double bonds is 422 N-C12:0-Cer(2H) determined by the measured mass C 12:0
Dihydroceramide: chain length and number of double bonds is 423N-C13:l-Cer(2H) determined by the measured mass C 13:1
Dihydroceramide: chain length and number of double bonds is 424 N-C13:0-Cer(2H) determined by the measured mass C13:0
Dihydroceramide: chain length and number of double bonds is 425N-C14:l-Cer(2H) determined by the measured mass C14:l
Dihydroceramide: chain length and number of double bonds is 426 N-C14:0-Cer(2H) determined by the measured mass C 14:0
Dihydroceramide: chain length and number of double bonds is 427N-C15:l-Cer(2H) determined by the measured mass C 15:1
Dihydroceramide: chain length and number of double bonds is 428 N-C15:0-Cer(2H) determined by the measured mass C15:0
Dihydroceramide: chain length and number of double bonds is 429N-C16:l-Cer(2H) determined by the measured mass C16:l
Dihydroceramide: chain length and number of double bonds is 430 N-C16:0-Cer(2H) determined by the measured mass C16:0
Dihydroceramide: chain length and number of double bonds is 431N-C17:l-Cer(2H) determined by the measured mass C17:l
Dihydroceramide: chain length and number of double bonds is 432 N-C17:0-Cer(2H) determined by the measured mass C17:0
Dihydroceramide: chain length and number of double bonds is 433N-C18:l-Cer(2H) determined by the measured mass C 18:1
Dihydroceramide: chain length and number of double bonds is 434N-C18:0-Cer(2H) determined by the measured mass C18:0
Dihydroceramide: chain length and number of double bonds is 435N-C19:l-Cer(2H) determined by the measured mass C19:l
Dihydroceramide: chain length and number of double bonds is
436 N-C19:0-Cer(2H) determined by the measured mass C19:0
Dihydroceramide: chain length and number of double bonds is
437 N-C18:0-Cer(2H) determined by the measured mass C18:0
Dihydroceramide: chain length and number of double bonds is
438 N-C20:0-Cer(2H) determined by the measured mass C20:0
Dihydroceramide: chain length and number of double bonds is 439N-C21:l-Cer(2H) determined by the measured mass C21:l
Dihydroceramide: chain length and number of double bonds is 440N-C21:0-Cer(2H) determined by the measured mass C21:0
Dihydroceramide: chain length and number of double bonds is
441N-C22:l-Cer(2H) determined by the measured mass C22: 1
442 N-C22:0-Cer(2H) Dihydroceramide: chain length and number of double bonds is determined by the measured mass C22:0
Dihydroceramide: chain length and number of double bonds is
443N-C23:l-Cer(2H) determined by the measured mass C23: 1
Dihydroceramide: chain length and number of double bonds is 444 N-C23:0-Cer(2H) determined by the measured mass C23:0
Dihydroceramide: chain length and number of double bonds is 445N-C24:l-Cer(2H) determined by the measured mass C24: 1
Dihydroceramide: chain length and number of double bonds is 446 N-C24:0-Cer(2H) determined by the measured mass C24:0
Dihydroceramide: chain length and number of double bonds is 447N-C25:l-Cer(2H) determined by the measured mass C25: 1
Dihydroceramide: chain length and number of double bonds is 448 N-C25:0-Cer(2H) determined by the measured mass C25:0
Dihydroceramide: chain length and number of double bonds is 449N-C26:l-Cer(2H) determined by the measured mass C26: 1
Dihydroceramide: chain length and number of double bonds is 450 N-C26:0-Cer(2H) determined by the measured mass C26:0
Dihydroceramide: chain length and number of double bonds is 451N-C27:l-Cer(2H) determined by the measured mass C27: 1
Dihydroceramide: chain length and number of double bonds is 452 N-C27:0-Cer(2H) determined by the measured mass C27:0
Dihydroceramide: chain length and number of double bonds is 453N-C28:l-Cer(2H) determined by the measured mass C28: 1
Dihydroceramide: chain length and number of double bonds is
454 N-C28:0-Cer(2H) determined by the measured mass C28:0
Ceramide: chain length and number of double bonds is
455 N-C3:0(OH)-Cer determined by the measured mass C3:0(OH)
Ceramide: chain length and number of double bonds is
456 N-C4:0(OH)-Cer determined by the measured mass C4:0(OH)
Ceramide: chain length and number of double bonds is
457 N-(2xOH)C3:0-Cer determined by the measured mass (2xOH)C3:0
Ceramide: chain length and number of double bonds is
458 N-C5:0(OH)-Cer determined by the measured mass C5:0(OH)
Ceramide: chain length and number of double bonds is
459 N-C6:0(OH)-Cer determined by the measured mass C6:0(OH)
Ceramide: chain length and number of double bonds is
460 N-C7:2(OH)-Cer determined by the measured mass C7:2(OH)
Ceramide: chain length and number of double bonds is 461N-C7:1(OH)-Cer determined by the measured mass C7:1(OH)
Ceramide: chain length and number of double bonds is
462 N-C7:0(OH)-Cer determined by the measured mass C7:0(OH)
Ceramide: chain length and number of double bonds is
463 N-C8:0(OH)-Cer determined by the measured mass C8:0(OH)
Ceramide: chain length and number of double bonds is
464 N-C9:0(OH)-Cer determined by the measured mass C9:0(OH)
Ceramide: chain length and number of double bonds is
465 N-C10:0(OH)-Cer determined by the measured mass C10:0(OH)
Ceramide: chain length and number of double bonds is
466 N-Cl1:1(OH)-Cer determined by the measured mass C 11 : 1 (OH) Ceramide: chain length and number of double bonds is
467 N-CI l :0(OH)-Cer determined by the measured mass Cl 1:0(OH)
Ceramide: chain length and number of double bonds is
468 N-C12:0(OH)-Cer determined by the measured mass C12:0(OH)
Ceramide: chain length and number of double bonds is
469 N-C13:0(OH)-Cer determined by the measured mass C13:0(OH)
Ceramide: chain length and number of double bonds is
470 N-C14:0(OH)-Cer determined by the measured mass C14:0(OH)
Ceramide: chain length and number of double bonds is
471 N-C15:0(OH)-Cer determined by the measured mass C15:0(OH)
Ceramide: chain length and number of double bonds is
472 N-C16:0(OH)-Cer determined by the measured mass C16:0(OH)
Ceramide: chain length and number of double bonds is 473 N-C17:1(OH)-Cer determined by the measured mass C17:1(OH)
Ceramide: chain length and number of double bonds is
474 N-C17:0(OH)-Cer determined by the measured mass C17:0(OH)
Ceramide: chain length and number of double bonds is
475 N-C18:0(OH)-Cer determined by the measured mass C18:0(OH)
Ceramide: chain length and number of double bonds is
476 N-C19:0(OH)-Cer determined by the measured mass C19:0(OH)
Ceramide: chain length and number of double bonds is
477 N-C20:0(OH)-Cer determined by the measured mass C20:0(OH)
N-C19:0(2xOH)- Ceramide: chain length and number of double bonds is
478 Cer determined by the measured mass C19:0(2xOH)
Ceramide: chain length and number of double bonds is
479 N-C21:0(OH)-Cer determined by the measured mass C21:0(OH)
Ceramide: chain length and number of double bonds is
480 N-C22:0(OH)-Cer determined by the measured mass C22:0(OH)
Ceramide: chain length and number of double bonds is
481 N-C23:0(OH)-Cer determined by the measured mass C23:0(OH)
Ceramide: chain length and number of double bonds is
482 N-C24:0(OH)-Cer determined by the measured mass C24:0(OH)
N-C23:0(2xOH)- Ceramide: chain length and number of double bonds is
483 Cer determined by the measured mass C23:0(2xOH)
Ceramide: chain length and number of double bonds is
484 N-C25:0(OH)-Cer determined by the measured mass C25:0(OH)
Ceramide: chain length and number of double bonds is 485 N-C26:1(OH)-Cer determined by the measured mass C26:1(OH)
Ceramide: chain length and number of double bonds is
486 N-C26:0(OH)-Cer determined by the measured mass C26:0(OH)
Ceramide: chain length and number of double bonds is
487 N-C27:0(OH)-Cer determined by the measured mass C27:0(OH)
Ceramide: chain length and number of double bonds is
488 N-C28:0(OH)-Cer determined by the measured mass C28:0(OH)
N-C3:0(OH)- Dihydroceramide: chain length and number of double bonds is
489 Cer(2H) determined by the measured mass C3:0(OH)
N-C4:0(OH)- Dihydroceramide: chain length and number of double bonds is
490 Cer(2H) determined by the measured mass C4:0(OH)
491 N-C5:0(OH)- Dihydroceramide: chain length and number of double bonds is Cer(2H) determined by the measured mass C5:0(OH) N-C6:0(OH)- Dihydroceramide: chain length and number of double bonds is
492 Cer(2H) determined by the measured mass C6:0(OH)
N-C7:0(OH)- Dihydroceramide: chain length and number of double bonds is
493 Cer(2H) determined by the measured mass C7:0(OH)
N-C8:0(OH)- Dihydroceramide: chain length and number of double bonds is
494 Cer(2H) determined by the measured mass C8:0(OH)
N-C9:0(OH)- Dihydroceramide: chain length and number of double bonds is
495 Cer(2H) determined by the measured mass C9:0(OH)
N-ClOiO(OH)- Dihydroceramide: chain length and number of double bonds is
496 Cer(2H) determined by the measured mass C10:0(OH)
N-Cl IiO(OH)- Dihydroceramide: chain length and number of double bonds is
497 Cer(2H) determined by the measured mass C11:0(OH)
N-C13:0(OH)- Dihydroceramide: chain length and number of double bonds is
498 Cer(2H) determined by the measured mass C13:0(OH)
N-C14:0(OH)- Dihydroceramide: chain length and number of double bonds is
499 Cer(2H) determined by the measured mass C14:0(OH)
N-C15:0(OH)- Dihydroceramide: chain length and number of double bonds is
500 Cer(2H) determined by the measured mass C15:0(OH)
N-C16:0(OH)- Dihydroceramide: chain length and number of double bonds is
501 Cer(2H) determined by the measured mass C16:0(OH)
N-C17:0(OH)- Dihydroceramide: chain length and number of double bonds is
502 Cer(2H) determined by the measured mass C17:0(OH)
N-C18:0(OH)- Dihydroceramide: chain length and number of double bonds is
503 Cer(2H) determined by the measured mass C18:0(OH)
N-C19:0(OH)- Dihydroceramide: chain length and number of double bonds is
504 Cer(2H) determined by the measured mass C19:0(OH)
N-C20:0(OH)- Dihydroceramide: chain length and number of double bonds is
505 Cer(2H) determined by the measured mass C20:0(OH)
N-C21:0(OH)- Dihydroceramide: chain length and number of double bonds is
506 Cer(2H) determined by the measured mass C21:0(OH)
N-C22:0(OH)- Dihydroceramide: chain length and number of double bonds is
507 Cer(2H) determined by the measured mass C22:0(OH)
N-C23:0(OH)- Dihydroceramide: chain length and number of double bonds is
508 Cer(2H) determined by the measured mass C23:0(OH)
N-C24:0(OH)- Dihydroceramide: chain length and number of double bonds is
509 Cer(2H) determined by the measured mass C24:0(OH)
N-C25:0(OH)- Dihydroceramide: chain length and number of double bonds is
510 Cer(2H) determined by the measured mass C25:0(OH)
N-C26:0(OH)- Dihydroceramide: chain length and number of double bonds is 511 Cer(2H) determined by the measured mass C26:0(OH)
N-C27:0(OH)- Dihydroceramide: chain length and number of double bonds is 512 Cer(2H) determined by the measured mass C27:0(OH)
N-C28:0(OH)- Dihydroceramide: chain length and number of double bonds is 513 Cer(2H) determined by the measured mass C28:0(OH)
514 Histamine Histamine
515 Serotonin Serotonin
516 PEA Phenylethylamine
517 TXB2 Tromboxane B2 518 PGF2a Prostaglandin F2alpha
519 24,25,EPC 24,25-Epoxycholesterol
520 5B,6B,EPC 5B,6B-Epoxycholesterol
521 24DHLan 24-Dihydrolanosterol
522 GCDCA Glycochenodeoxycholic Acid
523 GLCA Glycolithocholic Acid
524 TCDCA Taurochenodeoxycholic Acid
525 TLCA Taurolithocholic Acid
526 GCA Glycocholic Acid
527 CA Cholic Acid
528 UDCA Ursodeoxycholic Acid
529 CDCA Chenodeoxycholic Acid
530 DCA Deoxycholic Acid
531 TDCA Taurodeoxycholic Acid
532 TLCAS Taurolithocholic Acid sulfate
533 GDCA Glycodeoxycholic Acid
534 GUDCA Glycoursodeoxycholic Acid

Claims

Claims
1. Method for predicting the likelihood of onset of an inflammation
associated organ failure from a biological sample of a mammalian subject in vitro, wherein a. the subject's quantitative metabolomics profile comprising a plurality of endogenous metabolites, is detected in the biological sample by means of quantitative metabolomics analysis, and b. the quantitative metabolomics profile of the subject's sample is compared with a quantitative reference metabolomics profile of a plurality of endogenous organ failure predictive target metabolites in order to predict whether the subject is likely or unlikely to develop an organ failure; and c. wherein said endogenous organ failure predictive target metabolites have a molecular mass less than 1500 Da and are selected from the group consisting of: Amino acids, in particular, arginine, aspartic acid, citrulline, glutamic acid (glutamate), glutamine, leucine, isoleucine, histidine, ornithine, proline, phenylalanine, serine, tryptophane, tyrosine, valine, kynurenine; phenylthio carbamyl amino acids (PTC-amino acids), in particular, PCT-arginine, PTC- glutamine, PTC-histidine, PTC-methionine, PTC-ornithine, PTC-phenylalanine, PTC- proline, PTC-serine, PTC-tryptophane, PTC-tyrosine, PTC-valine; dimethylarginine, in particular N,N-dimethyl-L-arginine; carboxylic acids, namely 15(S)-hydroxy-5Z,8Z,1 1 Z,13E-eicosatetraenoic acid [(5Z,8Z,1 1 Z,13E,15S)-15-Hydroxyicosa-5,8,11 ,13-tetraenoic acid], succinic acid (succinate); Ceramides, with an N-acyl residue having from 2 to 30 Carbon atoms in the acyl residue and having from 0 to 5 double bonds and having from 0 to 5 hydroxy groups; carnitine; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue and having 1 to 4 double bonds in the acyl residue; acylcarnitines having from 1 to 20 carbon atoms in the acyl residue and having from 1 to 3 OH-groups in the acyl residue; acylcarnitines having from 3 to 20 carbon atoms in the acyl residue with 1 to 4 double bonds and 1 to 3 OH- groups in the acyl residue; phospholipides, in particular lysophosphatidylcholines (monoacylphospha-tidylcholines) having from 1 to 30 carbon atoms in the acyl residue; lysophosphatidylcholines having from 3 to 30 carbon atoms in the acyl residue and having 1 to 6 double bonds in the acyl residue; phosphatidylcholines (diacylphosphatidylcholines) having a total of from 1 to 50 carbon atoms in the acyl residues; phosphatidylcholines having a total from 3 to 50 carbon atoms in the acyl residues and having a total of 1 to 8 double bonds in the acyl residues; sphingolipids, in particular sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30; sphingomyelines having a total number of carbon atoms in the acyl chains from 10 to 30 and 1 to 5 double bonds; hydroxysphinogomyelines having a total number of carbon atoms in the acyl residues from 10 to 30;
hydroxysphingoyelines having a total number of carbon atoms in the acyl residues from 10 to 30 and 1 to 5 double bonds; prostaglandines, namely 6-keto-prostaglandin F1 alpha, prostaglandin D2, thromboxane B2; putrescine; oxysterols, namely 22-R-hydroxycholesterol, 24-S-hydroxycholesterol, 25- hydroxycholesterol, 27- hydroxycholesterol, 20α- hydroxycholesterol, 22-S- hydroxycholesterol, 24,25- epoxycholesterol,3β,5α,6β- trihydroxycholesterol, 7α- hydroxycholesterol, 7-Ketocholesterol, 53,63- epoxycholesterol, 5α,6α- epoxycholesterol, 43- hydroxycholesterol, desmosterol (vitamin D3), 7- dehydrocholesterol, cholestenone, lanosterol, 24-dehydrolanosterol; bile acids, namely cholic acid, chenodeoxycholic acid, deoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, glycolithocholic acid, glycolithocholic acid sulfate, glycoursodeoxycholic acid, lithocholic acid, taurocholic acid, taurochenodeoxycholic acid taurodeoxycholic acid, taurolithocholic acid, taurolithocholic acid sulfate, tauroursodeoxycholic acid, ursodeoxycholic acid; biogenic amines, namely histamine, serotonine, palmitoyl ethanolamine.
2. Method according to claim 1 , wherein inflammation associated organ failure comprises infection associated organ failure and/or sepsis associated organ failure.
3. Method according to claimi or 2, wherein the biological sample is selected from the group consisting of stool; body fluids, in particular blood, liquor, cerebrospinal fluid, urine, ascitic fluid, seminal fluid, saliva, puncture fluid, cell content, tissue samples, in particular liver biopsy material; or a mixture thereof.
4. Method according to anyone of claims 1 to 3, wherein said quantitative metabolomics profile is achieved by a quantitative metabolomics profile analysis method comprising the generation of intensity data for the quantitation of endogenous
metabolites by mass spectrometry (MS), in particular, by high- throughput mass spectrometry, preferably by MS-technologies such as Matrix Assisted Laser
Desorption/lonisation (MALDI), Electro Spray Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI), 1H-, 13C- and/or 31 P- Nuclear Magnetic Resonance spectroscopy (NMR), optionally coupled to MS, determination of metabolite
concentrations by use of MS-technologies and/or methods coupled to separation, in particular Liquid Chromatography (LC-MS), Gas Chromatography (GC-MS), or Capillary Electrophoresis (CE-MS).
5. Method according to anyone of claims 1 to 4, wherein intensity data of said metabolomics profile are normalized with a set of endogenous housekeeper metabolites by relating detected intensities of the selected endogenous organ failure predictive target metabolites to intensities of said endogenous housekeeper metabolites.
6. Method according to claim 5, wherein said endogenous housekeeper metabolites are selected from the group consisting of such endogeneous metabolites which show stability in accordance with statistical stability measures being selected from the group consisting of coefficient of variation (CV) of raw intensity data, standard deviation (SD) of logarithmic intensity data, stability measure (M) of geNorm - algorithm or stability measure value (rho) of NormFinder-algorithm.
7. Method according to anyone of claims 1 to 6, wherein said quantitative metabolomics profile comprises the results of measuring at least one of the parameters selected from the group consisting of: concentration, level or amount of each individual endogenous metabolite of said plurality of endogenous metabolites in said sample, qualitative and/or quantitative molecular pattern and/or molecular signature; and using and storing the obtained set of values in a database.
8. Method according to anyone of claims 1 to 7, wherein a panel of reference endogenous organ failure predictive target metabolites or derivatives thereof is established by: a) mathematically preprocessing intensity values obtained for generating the metabolomics profiles in order to reduce technical errors being inherent to the
measuring procedures used to generate the metabolomics profiles;
b) selecting at least one suitable classifying algorithm from the group consisting of logistic regression, (diagonal) linear or quadratic discriminant analysis (LDA, QDA, DLDA, DQDA), perceptron, shrunken centroids regularized discriminant analysis (RDA), random forests (RF), neural networks (NN), Bayesian networks, hidden Markov models, support vector machines (SVM), generalized partial least squares (GPLS), partitioning around medoids (PAM), inductive logic programming (ILP), generalized additive models, gaussian processes, regularized least square regression, self organizing maps (SOM), recursive partitioning and regression trees, K-nearest neighbour classifiers (K-NN), fuzzy classifiers, bagging, boosting, and naϊve Bayes; and applying said selected classifier algorithm to said preprocessed data of step a); c) said classifier algorithms of step b) being trained on at least one training data set containing preprocessed data from subjects being divided into classes according to their likelihood to develop an organ failure, in order to select a classifier function to map said preprocessed data to said likelihood; d) applying said trained classifier algorithms of step c) to a preprocessed data set of a subject with unknown organ failure likelihood, and using the trained classifier algorithms to predict the class label of said data set in order to predict the likelihood for a subject to develop an organ failure.
9. Method according to anyone of claims 1 to 8, wherein said endogenous organ failure predictive target metabolites for easier and/or more sensitive detection are detected by means of chemically modified derivatives thereof, such as
phenyisothiocyanates for amino acids.
10. Method according to anyone of claims 1 to 9, wherein said endogenous organ failure predictive target metabolites are selected from the group consisting of:
Carnitin, acylcarnitines (C chain length :total number of double bonds), in particular, C12-DC, C14:1 , C14:1 -OH, C14:2, C14:2-OH, C18, C6:1 ; sphingomyelins (SM chain length :total number of double bonds), in particular, SM C16:0, SM C17:0, SM C18:0, SM C19:0, SM C21 :1 , SM C21 :3, SM C22:2, SM C23:0, SM C23:1 , SM C23:2, SM C23:3, SM C24:0, SM C24:1 , SM C24:2, SM C24:3, SM C24:4, SM C26:4, SM C3:0, SM (OH) C22:1 , SM (OH) C22:2, SM (OH) C24:1 , SM C26:0, SM C26:1 ;
phosphatidylcholines, (diacylphosphatidylcholines, PC aa chain length:total number of double bonds or PC ae) in particular, PC aa C28:1 , PC aa C38:0, PC aa C42:0, PC aa C42:1 , PC ae C40:1 , PC ae C40:2, PC ae C40:6, PC ae C42:2, PC ae C42:3, PC ae C42:4, PC ae C44:5, PC ae C44:6, PC aa C36:4, PC aa C38:1 , PC aa C38:2, PC aa C38:4, PC aa C38:5, PC aa C38:6, PC aa C40:5, PC aa C40:6, PC aa C40:7, PC aa C40:8, PC ae C36:4, PC ae C36:5, PC ae C38:4, PC ae C38:6;
lysophosphatidylcholines (monoacylphosphatidylcholines, PC a chain length:total number of double bonds), in particular, PC a C18:2, PC a C20:4, PC a C20:3, PC a C26:0;
Phe;
oxycholesterols, in particular, 3β,5α,6β-trihydroxycholestan, 7-ketocholesterol, 5α,6α- epoxycholesterol;
lysophosphatidylethanolamins (monoacylphosphatidylcholins, PE a chain length:total number of double bonds), in particular, PE a C18:1 , PE a C18:2, PE a C20:4, PE a C22:5, PE a C22:6;
phosphatidylethanolamins, (diacylphosphatidylcholins, PE aa chain length:total number of double bonds), in particular, PE aa C38:0, PE aa C38:2;
ceramids, (N-chain length:total number of double bonds), in particular, N-C2:0-Cer, N- C7:0-Cer, N-C9:3-Cer, N-C17:1 -Cer, N-C22:1 -Cer, N-C25:0-Cer, N-C27:1 -Cer, N-C5:1 - Cer(2H), N-C7:1 -Cer(2H), N-C8:1 -Cer(2H), N-C1 1 :1 -Cer(2H), N-C20:0-Cer(2H), N- C21 :0-Cer(2H), N-C22:1 -Cer(2H), N-C25:1 -Cer(2H), N-C26:1 -Cer(2H), N-C24:0(OH)- Cer, N-C26:0(OH)-Cer, N-C6:0(OH)-Cer, N-C8:0(OH)-Cer(2H), N-C10:0(OH)-Cer(2H), N-C25:0(OH)-Cer(2H), N-C26:0(OH)-Cer(2H), N-C27:0(OH)-Cer(2H), N-C28:0(OH)- Cer(2H).
1 1. Method according to anyone of claims 1 to 10, wherein said plurality of endogenous organ failure predictive target metabolites or derivatives thereof comprises 2 to 80, in particular 2 to 60, preferably 2 to 50, preferred 2 to 30, more preferred 2 to 20, particularly preferred 2 to 10 endogenous metabolites.
12. Method according to anyone of claims 1 to 11 , wherein said plurality of endogenous organ failure predictive target metabolites is selected from the group consisting of:
Putrescine
Lanosterol
C5-DC (C6-OH)
25OHC, SM C16:1
24SOHC
C14
C4-OH (C3-DC)
CO C5-M-DC
C6(C4:1-DC)
PC aa C38:4
GLCA
Ala
4BOHC
24DHLan
TLCA
Serotonin ADMA
PC aa C36:1
SMC16:0
C5:1-DC
7aOHC 27OHC
Cit lysoPC a C20:4
GCA
lysoPCaC16:0 lie
Desmosterol
PEA
total DMA
Trp
C3:1 lysoPCaC18:0
VaI
PC ae C38:0
PGF2a SM(OH)C14:1 lysoPCaC18:2
THC
PC ae C40:4
24,25,EPC PC ae C36:5
PG D2
GIy
5B,6B,EPC
PC ae C40:0 PC ae C36:1
C18
C16:2
PC aa C36:5
PC aa C38:5 PC aa C30:2
13S-HODE
C9
15S-H ETE
SM C22:3 C5:1 lysoPCaC17:0
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