Patent Publication Number: US-2020284805-A1

Title: Biomarkers related to organ function

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This is a continuation of U.S. application Ser. No. 15/024,297, filed Mar. 23, 2016, which is the § 371 U.S. National Stage of International Application No. PCT/US2014/057049, filed Sep. 23, 2014, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 61/881,333, filed Sep. 23, 2013, which is incorporated by reference herein it its entirety. 
    
    
     FIELD 
     This disclosure relates to biomarkers related to organ or tissue function, particularly methods of identifying such biomarkers utilizing an ex vivo perfusion system and methods of predicting organ or tissue function by determining one or more biomarkers. 
     BACKGROUND 
     There are a large and increasing number of individuals in need of organ transplantation. Transplant candidates&#39; waiting times have continued to grow around the world, imposing further morbidity and mortality for this population. Meanwhile, the discard rates of human organs have continued to increase in spite of the high mortality rate on the transplant waiting lists (18 patients/day) across the country. 
     Even when an appropriate transplant organ is obtained, failure rates of transplanted organs range from 5-25%. Furthermore, organ dysfunction (such as liver or kidney dysfunction) is becoming increasingly common in the general population. Thus, there is a need to identify biomarkers for organ dysfunction, for both organs for transplantation and in individuals who have or are at risk for organ dysfunction. 
     SUMMARY 
     Disclosed herein are methods of identifying biomarkers (such as nucleic acids (e.g., DNA, RNA or mRNA), proteins, and/or small molecules) that can be used to predict organ or tissue function or dysfunction. In some embodiments, the methods include ex vivo perfusion of the organ or tissue, collection of samples from the organ or tissue (for example, perfusate, fluids produced by the organ (such as bile or urine) or tissue biopsies) and measuring the level of one or more biomarkers in the sample. In some embodiments, the organ is perfused with a hemoglobin-based oxygen carrier (HBOC) solution. The organ function (or dysfunction) is analyzed and biomarkers associated with organ function or dysfunction are identified. In some examples, one or more biomarkers that increase or decrease in organs with good function (for example compared with a reference or control) are identified as predictors of organ function. In other examples, one or more biomarkers that increase or decrease in organs with poor function (for example, compared with a reference or control) are identified as predictors of organ dysfunction. 
     It is also disclosed herein that an analysis of biomarkers (such as nucleic acids (e.g., DNA, RNA or mRNA), proteins, and/or small molecules) present in a biological sample from an organ, tissue, or subject can be used to identify whether the organ, tissue, or subject is at risk for (or has) organ dysfunction or organ failure. In some embodiments, the methods utilize analyzing gene expression profiles, protein profiles, and/or small molecule profiles of metabolites. In other embodiments, the methods utilize analyzing the presence of one or more specific genes, proteins, and/or metabolites. These methods can be used, for example, to identify individuals at risk of (or having) organ dysfunction or failure or identify organs that are suitable or unsuitable for organ transplantation. In particular disclosed examples, the methods determine biomarkers associated with liver dysfunction (such as liver failure) and can be used to assist in the identification of persons with liver disease, to assess the severity of liver disease and the necessity of liver transplantation, and to help identify or rank donated livers in terms of their suitability for transplantation and/or likelihood of long-term organ survival following transplantation. 
     The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram showing an exemplary methodology for biomarker analysis in an ex vivo perfusion model. 
         FIGS. 2A-2G  are a series of graphs showing post-operative levels of lactate ( FIG. 2A ), albumin ( FIG. 2B ), AST ( FIG. 2C ), ALT ( FIG. 2D ), BUN ( FIG. 2E ), creatinine ( FIG. 2F ), and peripheral blood pH ( FIG. 2G ) in the control and study group animals. 
         FIG. 3  is a graph showing histological analysis of ischemia-reperfusion (IR). IR scores (Suzuki modified) were determined by serial analysis of inflammatory changes within the portal tracts and the hepatic lobules. The graphic shows a longitudinal comparison of average IR scores for cold static preservation (CSP—(b)) and machine perfusion (MP—(a)). The IR scores at necropsy were significantly lower in the MP group (p=0.01), showing the benefits of effective ex-vivo oxygenation on liver tissue viability after transplantation. 
         FIGS. 4A-4C  are a series of graphs showing mitochondrial function in control and study groups.  FIG. 4A  shows respiratory control ratio,  FIG. 4B  shows ATP production, and  FIG. 4C  shows reactive oxygen species generation (H 2 O 2 ). 
         FIG. 5  is a graphic representation of the transcriptomic analysis (microarray) of 20,000 genes obtained from pig liver samples after machine perfusion showing the top 100 genes with increased expression after 8 hours sustained ex vivo oxygenation before liver allograft implantation. 
         FIG. 6  is a graphic representation of the transcriptomic analysis (microarray) of 20,000 genes obtained from pig liver samples showing the genes with the greatest differences in gene expression immediately following liver allograft reperfusion compared to the fifth post-operative day (end-study necropsy). 
         FIG. 7  shows pathway analysis (Ingenuity®) of genes with altered expression in the machine perfusion group (top) or cold ischemia group (bottom) at the fifth post-operative day (end-study necropsy) from microarray analysis of 20,000 genes. The machine perfusion group showed that genes associated with liver damage were significantly down-regulated, while the cold ischemia group showed significant up-regulation of genes associated with liver pathology. 
         FIG. 8  is a graphic representation of the transcriptomic analysis (microarray) of 20,000 genes obtained from pig liver samples showing the enrichment by biological process networks following 8 hours of machine perfusion (Ingenuity®). 
         FIG. 9A  is a graphic representation of the transcriptomic analysis (microarray) of 20,000 genes obtained from pig liver samples showing the metabolic network analysis (Ingenuity®) following 8 hours of machine perfusion. Effective ex-vivo oxygenation enhanced significantly (p=0.01) the genes regulating drug, amino acid, free radical scavenging, vitamin, mineral and carbohydrate metabolism while enhancing energy production. 
         FIG. 9B  is a graphic representation of the transcriptomic analysis (microarray) of 20,000 genes obtained from pig liver samples showing the metabolic network analysis (Ingenuity®) following 8 hours of machine perfusion. Effective ex-vivo oxygenation enhanced significantly (p=0.01) the genes regulating biological processes involved in hepatic system development and function, cellular growth and proliferation, cellular morphology, cellular development, cellular signaling, cellular assembly and organization, cell-to-cell signaling and interaction, tissue morphology and cellular function and maintenance. Increased expression of several genes associated with entry of hepatocytes into G1 phase was observed. 
         FIG. 10  is a graphic representation of the transcriptomic analysis (microarray) of 20,000 genes obtained from pig liver samples showing the metabolic network analysis (Ingenuity®) following 8 hours of machine perfusion. Effective ex-vivo oxygenation enhanced significantly (p=0.01) the genes expression of signaling pathways that are critical for liver growth. NF-κB can inhibit the TNF-α induced apoptotic pathway and increase the expression of survival gene products. 
         FIG. 11  is a graph showing interferon-α levels in control (cold ischemia) and experimental (machine perfusion) samples. Overall two-way ANOVA p=0.001. 
         FIG. 12  is a graph showing tumor necrosis factor-α levels in control (cold ischemia) and experimental (machine perfusion) samples. Overall two-way ANOVA p=0.032. 
         FIG. 13  is a graph showing interferon-γ levels in control (cold ischemia) and experimental (machine perfusion) samples. Overall two-way ANOVA p=0.022. 
         FIG. 14  is a graph showing interleukin-4 levels in control (cold ischemia) and experimental (machine perfusion) samples. Overall two-way ANOVA p=0.021. 
         FIG. 15  is a graph showing interleukin-10 levels in control (cold ischemia) and experimental (machine perfusion) samples. Overall two-way ANOVA p=0.001. 
         FIG. 16  is a graph showing interleukin-12/interleukin-23 (p40) levels in control (cold ischemia) and experimental (machine perfusion) samples. Overall two-way ANOVA p=0.001. 
         FIGS. 17A-17C  are a series of graphs showing branched chain amino acids in machine perfused livers and cold ischemia livers over the course of the experiment (hours).  FIG. 17A  shows valine,  FIG. 17B  shows leucine, and  FIG. 17C  shows isoleucine. The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIGS. 18A-18B  are a series of graphs showing Krebs pathway byproducts in machine perfused livers and cold ischemia livers over the course of the experiment (hours).  FIG. 18A  shows alpha-ketoglutarate,  FIG. 18B  shows citrate. The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIGS. 19A-19W  are a series of panels of graphs showing different metabolites in machine perfused livers and cold ischemia livers over the course of the experiment (hours). The upper line in each plot is the machine perfused liver, except for 3-hydroxypropanoate, beta-alanine, butylcarnitine, C-glycosyltryptophan, creatinine, eiconsenoate (20:1n9 or 11), ethanolamine, galactose, GABA, gluconate, glutathione, oxidized (GSSG), glycerol 2-phosphate, glycerol 3-phosphate (G3P), glycerophosphorylcholine, glycohenodeoxycholate, glycocholate, glycohyodeoxycholic acid, glycolithocholate, guanosine, hippurate, hypotaurine, hypoxanthine, inosine, ketamine, ophthalmate, ribose 1-phosphate, ribose 5-phosphate, S-methylglutathione, spermine, sucrose, succinate, taurine, taurocholate, uridine, and verbascose, where the lower line is the machine perfused liver. The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIGS. 20A-20E  are a series of graphs showing compounds of the gluconeogenesis pathway in machine perfused livers and cold storage livers over the course of the experiment (hours).  FIG. 20A  shows glucose,  FIG. 20B  shows lactate,  FIG. 20C  shows pyruvate,  FIG. 20D  shows fructose 6-phosphate, and  FIG. 20E  shows glucose 6-phosphate. The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIGS. 21A and 21B  are a pair of graphs showing alpha-ketoglutarate ( FIG. 21A ) and glutamate production ( FIG. 21B ) in machine perfused and cold storage liver perfusate over the course of the experiment (hours). The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIG. 22  is a graph showing ascorbate in in machine perfused and cold storage liver perfusate over the course of the experiment (hours). The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIGS. 23A and 23B  are a pair of graphs showing 15-HETE ( FIG. 23A ) and 12-HETE ( FIG. 23B ) in machine perfused and cold storage liver perfusate over the course of the experiment (hours). The X-axis shows concentration in I/U. The Y-axis shows 3 time points (0=3 hours, 5=6 hours and 10=9 hours) when samples were obtained. 
         FIG. 24  is a graph showing bile production in the post-operative period by machine perfused (study) and cold storage (control) animals following allograft reperfusion. 
         FIG. 25  is a graph showing principal component analysis (PCA) carried out on the metabolomic profile of perfusate at three time points (3, 6 and 9 hours). Variables are ordered by the sum of their contribution to all components, with contributions to individual components represented by different colored sections of the bars. In MP livers, variables representing carbohydrate metabolism (ribulose, ribose, glycolate) and antioxidant defenses (oxidized homo-glutathione—GSSG) were principal drivers of metabolic changes. 
         FIG. 26  is a graph showing PCA carried out on the metabolomic profile of perfusate at three time points (3, 6 and 9 hours). Variables are ordered by the sum of their contribution to all components, with contributions to individual components represented by different colored sections of the bars. In CSP livers, PCA showed ethanolamine to be the principal driver of metabolic changes, suggesting a role for fatty acid metabolism. 
         FIG. 27  is a representation of the method Dynamic Bayesian Networks (DBN) utilized to establish the role of different cytokines while interacting in response to an initial inflammatory event (e.g. ischemia-reperfusion acquired during liver preservation). 
         FIG. 28  shows DBN analysis suggesting two different pathways for cytokine regulation in livers being perfused by different techniques (Control with cold static preservation—CSP and Study with machine perfusion in combination with the hemoglobin-based-oxygen carrier solution—MP/HBOC). 
     
    
    
     DETAILED DESCRIPTION 
     Disclosed herein is a new discovery platform for biomarkers related to organ or tissue function, particularly methods of identifying such biomarkers utilizing an ex vivo perfusion system where organs can be perfused outside of the body for several hours by a system combining machine perfusion with a cell free oxygen carrier solution at variable temperatures. This system providing effective oxygenation through ex vivo perfusion for several hours can be utilized to assess tissue and organ viability (for both acute and long term features) while determining the role of biochemical components (e.g., transcriptomics, cytokines, chemokines, damage-associated molecular pattern molecules (DAMPs), toll-like receptors (TLRs) and/or metabolomics) in predicting subsequent organ function. 
     This system can also map out biological features related to a previously known disease (diabetes, hypertension, steatosis, acute kidney injury, etc.) experienced by a subject, whose organs can be further studied post-mortem by this new ex vivo perfusion environment. This should create a new platform to define cardinal events for acute (e.g., initial markers involved in acute organ failure) and chronic diseases (e.g., initial cell signaling for the development of fibrosis) that are currently limited by the subject&#39;s survival. Finally, this technology represents a new model system, for testing new therapies and diagnostics for acute diseases of solid organs. 
     The current platforms utilized for the discovery of clinically-relevant biomarkers related to organ function and additional medical conditions are primarily based on biological samples (e.g., blood, urine, bile, saliva, etc.) obtained from live individuals. Biomarkers can be divided in pharmacodiagnostic, pharmacological, and disease related categories. These biochemicals are essential tools in preventive and personalized medicine, modern drug development, and in outcomes prediction for medical treatment and/or diseases. 
     Predictive biomarkers are the building blocks for personalized medicine when capable to enhance the evidence-based environment needed for subsequent diagnostic and therapeutic decisions. These new biomarkers should reflect the heterogeneity of human diseases while stratifying patients and biological pathways within their predictable outcomes. The early discovery and exploratory phases involving the inception of new biomarkers are rather lengthy and expensive, since the initial data collection relies primarily on live patients and their body fluids in a rather diverse geographic and clinical environment. 
     This disclosure describes a new platform for the development of biomarkers that is primarily centered on organs and tissues being perfused by machine perfusion technology in association with a recently developed cell-free oxygen carrier solution (discussed below). This ex vivo discovery platform for biomarkers can also be used to predict organ function prior to organ transplantation. Finally, developing new therapies for acute diseases has been challenging because existing model systems involving animals (even transgenic animals) and cell cultures often do not yield results that translate into humans. The platform disclosed herein offers a unique solution to this problem by using human organs and subjecting them to a variety of acute pathologies including infection, trauma and ischemia in order to study treatments and diagnostics. 
     In some embodiments, biomarkers can be identified for pharmacodiagnostic applications (e.g., treatment eligibility, treatment response prediction, drug safety, and/or assessing the efficacy of a given therapy), pharmacological applications (e.g., pharmacodynamics markers, pharmacokinetic markers, and/or outliners of intrinsic mechanisms of action), disease and medical conditions (e.g., screening for a given condition, early prognostic feature, early detection, monitoring tool to detect clinical evolution and recurrence of a given disease), and/or organ transplantation (e.g., predictive marker for organ function, predictive marker for ischemia-reperfusion injuries, predictive factor for immune compatibility, predictive of vascular integrity, and/or predictive of subsequent fibrosis development within the allograft). 
     I. Terms 
     In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided: 
     Biomarker: An organic biomolecule, such as a small molecule, amino acid, sugar, carbon (energy) source, carbohydrate, nucleic acid (such as DNA, RNA, or mRNA, referred to in some examples herein as “genes”) or a polypeptide or protein, which is differentially present in a biological sample. In one example, the biomarker is present in a sample taken from an organ or tissue or a subject who is, or may be at risk for, or has, organ dysfunction. A biomarker can be differentially present in samples from a normal (e.g., healthy or functional) organ, tissue, or subject and samples from an organ, tissue, or subject having or at-risk for organ dysfunction, if it is present at an elevated level or a decreased level in the latter samples as compared to normal samples. 
     Hemoglobin-based oxygen carrier (HBOC): Molecules or compositions with oxygen carrying capabilities derived from the presence of hemoglobin. In some examples, HBOCs include isolated or purified hemoglobin (sometimes referred to as “acellular” HBOCs). Exemplary acellular HBOCs contain polymerized hemoglobin (for example, bovine or human hemoglobin), for example HBOC-201 (HEMOPURE, OPK Biotech, Cambridge, Mass.), HEMOLINK (Hemosol, Inc., Toronto, Canada), and POLYHEME (Northfield Laboratories, Evanston, Ill.) or encapsulated hemoglobin (such as liposome- or polymersome-encapsulated hemoglobin). In other examples, HBOCs include red blood cells. 
     Metabolome: All of the small molecules present in a given sample, tissue, organ, or subject. The metabolome includes both metabolites as well as products of catabolism. In one embodiment, the disclosure encompasses a small molecule profile of the entire (or substantially entire) metabolome of a sample. In other embodiments, the disclosure encompasses a profile of one or more molecules of the metabolome of a sample. Generally the metabolome or small molecule profile includes those molecules with a molecular weight of less than 2,000 Daltons Small molecules do not include large macromolecules, such as proteins (for example, proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Daltons), large nucleic acids (such as nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Daltons), or large polysaccharides (such as polysaccharides with a molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 Daltons). The molecules shown in  FIG. 1 -A-19W and Tables 8 and 9 are non-limiting examples of small molecules of the metabolome. 
     Organ: A part of the body, tissue, or portion thereof. In some examples, organs include those that can be transplanted or preserved ex vivo. Organs include, but are not limited to liver, kidney, heart, lung, pancreas, small intestine, and limb (such as arm or leg, or portion thereof), or extremity (such as hand, foot, finger, toe, or a portion thereof). As used herein, “organ” also includes other tissues, such as tissue grafts, such as composite tissue allografts. 
     Organ dysfunction: Organ dysfunction is a biological and dynamic condition where an organ does not perform its expected function (e.g., has impaired function). Organ dysfunction can migrate towards organ failure if remained untreated. Organ dysfunction can lead into the deregulation of the body homeostasis when metabolically active and filtering organs like liver and kidneys are affected. One example of kidney dysfunction is in drug toxicity leading into renal failure (e.g., excessive use of NSAIDS). In some examples, kidney dysfunction includes decreased glomerular filtration rate due to progressive vasospasm of afferent arterioles within the nephron. An example of liver dysfunction is in metabolic syndrome following morbid obesity. In some examples, liver dysfunction includes enhanced metabolic pathways to gluconeogenesis, followed by fat deposition within hepatocyte cytoplasm, leading to hepatic steatosis. 
     Perfusion: Circulation of a fluid (also referred to as a perfusion solution or perfusate) through an organ to supply the needs of the organ to retain its viability (for example, in an ex vivo system). In some examples, the perfusion solution includes an oxygen carrier (for example, a hemoglobin-based oxygen carrier). Machine perfusion refers to introduction and removal of a perfusion solution to an organ by a mechanical device. Such devices may include one or more chambers for holding an organ and a perfusion solution, one or more pumps for delivery of the perfusion solution to the organ, one or more means to regulate temperature of the perfusion solution, and one or more means to oxygenate the perfusion solution. In some examples, machine perfusion includes introduction of an oxygen carrying fluid into an organ and removal of oxygen depleted fluid from the organ by circulation of the oxygen carrying fluid through the organ. 
     Sample: A specimen containing genomic DNA, RNA (including mRNA), protein, small molecules or combinations thereof, obtained from an organ, tissue, or subject. In some examples, a sample is from an ex vivo tissue or organ, such as perfusate from an ex vivo perfused tissue or organ, fluids produced by an organ or tissue (such as bile, urine, or tissue exudate), or a biopsy from an organ or tissue. Additional examples include, but are not limited to, peripheral blood, bile, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material from a subject. 
     Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as veterinary subjects. 
     II. Methods of Identifying Biomarkers of Organ or Tissue Function 
     Disclosed herein are methods of identifying biomarkers (such as genes (e.g., RNA or mRNA), proteins, and/or small molecules) that can be used to predict organ or tissue function or dysfunction. An advantage of the methods disclosed herein is that samples are obtained from ex vivo tissues or organs, which do not have interference from blood products or cells and does not implicate further allosensitization of the tissue or organ under perfusion. An additional advantage of the methods disclosed herein is that organs that are not suitable for transplantation and would otherwise be discarded can be utilized to identify biomarkers of organ dysfunction. In particular embodiments described herein, the organ is a liver; however, any perfusable organ or tissue (such as liver, kidney, lung, heart, pancreas, small intestine, limbs (for example, arm or leg), extremities (for example, hand, foot, finger, toe, or face)), or a portion thereof can be utilized in the disclosed methods. 
     In some embodiments, the methods include ex vivo perfusion of the organ or tissue, collection of samples from the organ or tissue (for example, perfusate, fluids produced by the organ (such as bile or urine), or tissue biopsies) and measuring the level of one or more biomarkers in the sample. In some embodiments, the organ is perfused with a hemoglobin-based oxygen carrier (HBOC) solution. The organ function (or dysfunction) is analyzed and biomarkers associated with organ function or dysfunction are identified. In some examples, one or more biomarkers that increase or decrease in organs with good function (for example compared with a reference or control) are identified as predictors of organ function. In other examples, one or more biomarkers that increase or decrease in organs with poor function (for example, compared with a reference or control) are identified as predictors of organ dysfunction. 
     In particular embodiments, the methods include determining presence and/or amount of one or more biomarkers, such as one or more biomarkers from a genome, transcriptome, proteome, and/or metabolome of a tissue, organ, or subject, for example as shown schematically in  FIG. 1 . The function (or dysfunction) of the tissue or organ or the health status of the subject from which the sample was obtained is determined or monitored. Biomarkers that increase or decrease in a tissue, organ, or subject with good function, health, or a positive outcome are identified as predictors of organ or tissue function. Biomarkers that increase or decrease in a tissue organ, or subject with poor or decreased function, health, or a poor outcome are identified as predictors of organ or tissue dysfunction. 
     In particular examples, the methods include obtaining samples from an ex vivo perfused organ or tissue. The samples include one or more of perfusate, fluid produced by the organ or tissue (such as bile, urine, or tissue exudate), or tissue biopsy samples. The organ or tissue can be machine perfused by any method known to one of ordinary skill in the art. In some examples, the machine perfusion is carried out with a perfusion solution that includes an oxygen carrier, such as a hemoglobin-based oxygen carrier (HBOC). In one particular example, the machine perfusion is carried out with a perfusion solution that includes a modified HBOC solution comprising a 1:3 mixture of HEMOPURE (OPK Biotech, Cambridge, Mass.) and Belzer machine perfusion solution (BMPS) (e.g., Muhlbacher et al.,  Transplant. Proc.  31:2069, 1999; Kwiatkowski et al.,  Transplant. Proc.  33:913, 2001; Stubenitsky et al.,  Transplant . Int. 68:1469, 1999). The modified HBOC solution is described in detail in U.S. Prov. Pat. Appl. No. 61/713,284, filed Oct. 12, 2012, and International Pat. Publ. No. WO 2014/059316, both of which are incorporated herein by reference in their entirety. However, other machine perfusion solutions known to one of ordinary skill in the art can also be utilized. 
     In some examples, the methods can also include obtaining samples from an ex vivo organ or tissue that is not machine perfused, for example a tissue or organ that is treated by cold storage in a preservation solution. In one example, the cold storage preservation (CSP) solution is UW solution, which is the current standard of care for preservation of organs for transplantation. Organs preserved by CSP in UW solution frequently have poor function (dysfunction), as described in Example 1, below. In some examples, samples from CSP organs are controls for comparison with samples from machine perfused organs, which generally have better function and outcome (see Example 1, below). 
     The samples obtained from the tissue or organ are analyzed for presence and/or amount of one or more biomarkers. In some examples, the genome or transcriptome is analyzed to determine gene expression biomarkers, for example, samples are analyzed for presence and/or amount of one or more nucleic acids (such as DNA, RNA, mRNA, or miRNA). In particular examples, the nucleic acids analyzed are related to cell proliferation or cell differentiation. 
     In other examples, the samples are analyzed for presence and/or amount of one or more proteins or polypeptides, such as analysis of the proteome of the sample. In particular examples, the cytokine and/or chemokine profile of the sample is analyzed (for example, one or more of interferon-α, interferon-γ, interleukin-10, interleukin-12/23 (p40), interleukin-1b, interleukin-4, interleukin-6, interleukin-8, and/or tumor necrosis factor-α); however, any protein of interest can be analyzed. In additional examples, the proteins analyzed include hormones, clotting factors, paracrine factors, and/or growth factors. Exocrine secretions may also be analyzed, for example, exocrine secretions produced by the pancreas or the intestines. In one non-limiting example, hepatocyte growth factor is analyzed. In other examples, one or more of VEGF, TGF-β, ERK/MAPK, ErbB, FAK, HGF, p53, insulin receptor, PI3K/AKT, PDGF, FGF, EGF, and NF-κB are analyzed. 
     In particular examples, nucleic acid or protein biomarkers are analyzed in a tissue sample (such as a biopsy) from an organ, tissue, or subject. However, in some instances, nucleic acids or proteins can also be analyzed in perfusate from a machine perfused organ or tissue or in a fluid from an organ (such as bile or urine). 
     In further examples, the samples are analyzed for presence and/or amount of one or more small molecules, for example, analysis of the metabolome of the sample. In particular non-limiting examples, the small molecule profile is analyzed in perfusate from an ex vivo perfused organ, bile, or urine. 
     Methods of detecting presence and/or amount of nucleic acids, proteins, and small molecules are described in Section IV, below. 
     In some examples, the amount of the one or more biomarkers is compared with the amount of one or more biomarkers in a control or reference sample. In some examples, a “control” refers to a sample or standard used for comparison with an experimental sample, such as a sample or standard from one or more organs with known function or dysfunction. In some embodiments, the control is a sample obtained from a healthy organ. In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of samples that represent baseline or normal values, such as the level of one or more biomarkers in a healthy organ). In other embodiments, the control is a sample obtained from a dysfunctional organ. In some embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of samples that represent the level of one or more biomarkers in an organ with dysfunction). 
     One of skill in the art can identify healthy and/or dysfunctional organs. In some examples, organs (such as human organs) can be thoroughly examined for one or more metabolic features while under ex vivo machine perfusion in order to determine parameters of healthy or dysfunctional organs. The energy production pathways (e.g., glycolytic or glyconeogenic pathways) can by elucidated while outlining additional biological features on carbohydrate, lipid, amino acids, vitamin and minerals metabolism, as well as their free radical scavenging abilities. In one particular example, levels of lactate dehydrogenase, glutathione-S-transferase and aspartate transaminase are correlated with delay graft function of cadaveric kidney allografts (Bhangoo et al.,  Nephrol Dial Transplant  27:3305, 2012). 
     One of skill in the art can readily identify statistical methods and computer programs that can be used to identify an increase or a decrease (such as a statistically significant increase or decrease) in one or more biomarkers, including differences in molecule profiles. Methods of analysis that can be used include linear discriminant analysis and Random Forest analysis. Additional methods of analysis include Principal Component Analysis (PCA) and Dynamic Bayesian Networks (DBN). In some examples, these methods can be used to identify a principal component itself or variations as an increase or a decrease (such as a statistically significant increase or decrease) in one or more biomarkers. One of ordinary skill in the art can identify additional suitable methods of analysis to identify increases or decreases in biomarkers. 
     III. Methods of Predicting Organ Function or Dysfunction 
     Disclosed herein are methods for predicting organ function or dysfunction and methods of treating an organ or subject with predicted dysfunction. The methods include an analysis of biomarkers (such as genes (e.g., RNA or mRNA), proteins, and/or small molecules) present in a biological sample from an organ, tissue, or subject to identify whether the organ, tissue, or subject is at risk for (or has) organ dysfunction or organ failure. In some embodiments, the methods utilize analyzing gene expression profiles, protein profiles, and/or small molecule profiles of metabolites. In some examples a gene expression profile, protein profile, or small molecule profile includes 5 or more (such as 10, 15, 20, 25, 50, 100, 200, 500, 1000, or more) genes, proteins, or small molecule metabolites, respectively. In other embodiments, the methods utilize analyzing the presence of one or more (such as 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more) specific genes, proteins, and/or metabolites. In particular embodiments, the profiles or particular biomarkers include one or more of the genes, proteins, and/or small molecules listed in any one of  FIGS. 5-7, 10-23, 25, 26, and 28  and Tables 8 and 9, or any combination thereof. Exemplary methods of analyzing biomarkers from a sample are discussed in Section IV, below. 
     In some embodiments, the methods include determining the level of one or more (such as 2, 3, 4, 5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or more) small molecules or metabolites in a sample, for example one or more of those listed in Tables 8 and 9 and  FIG. 19A-W , or any combination thereof. In particular examples, the methods include determining the level of one or more markers of glycolysis or gluconeogenesis (such as glucose, lactate, pyruvate, fructose 6-phosphate, and/or glucose 6-phosphate), branched chain amino acids (such as valine, lysine, and/or leucine) or metabolites or side-products of branched chain amino acid synthesis (such as alpha-ketoglutarate, glutamine, and/or glutamate), oxidative stress (such as ascorbate), or lipoxygenase activity (such as 12-HETE and/or 15-HETE) from a sample from an organ (such as a liver) or from a subject. In other examples, the methods include determining the level of one or more of ribose, ribulose, glycolate, oxidized homo-glutathione (GSSG), and/or ethanolamine from a sample from an organ (such as a liver) or from a subject. It is then assessed as to whether the level of the one or more biomarker differs from a control sample or a reference value. A change in the amount of one or more biomarkers in the sample, as compared to control or reference value indicates that the organ or subject is at risk for (or has) organ dysfunction. In some examples, a decrease in the levels of biomarkers of glycolysis, branched chain amino acids or branched chain amino acid synthesis, or lipoxygenase activity compared to a healthy control or reference indicates that the organ or subject is at risk for (or has) organ dysfunction (for example, liver dysfunction). In other examples, an increase in the levels of biomarkers of oxidative stress or lipoxygenase activity compared to a healthy control or reference value indicates that the organ or subject is at risk for (or has) organ dysfunction (for example, liver dysfunction). 
     In particular examples, a decrease in one or more of glucose, lactate, pyruvate, fructose 6-phosphate, glucose 6-phosphate, valine, isoleucine, leucine, alpha-ketoglutarate, glutamate, or 15-HETE as compared to a healthy control or reference value indicates that the organ or subject is at risk for (or has) organ dysfunction. In other particular examples, an increase in ascorbate or 12-HETE as compared to a healthy control or reference value indicates that the organ or subject is at risk for (or has) organ dysfunction. Additional small molecules associated with organ function (particularly liver function) are shown in  FIG. 19A-19W . These molecules may be particularly suitable for identifying subjects at risk for (or having) liver dysfunction and/or determining the severity of the liver dysfunction and/or the necessity of imminent organ transplantation. 
     In other examples, the samples are analyzed for one or more of ribulose, ribose, oxidized homo-glutathione (GSSG), glycolate (hydroxyacetate), xylonate, and/or ethanolamine. For example, a decrease in ribulose, ribose, and/or glycolate and/or an increase in GSSG and/or ethanolamine compared to a control (for example a healthy organ) indicates that an organ has or is predicted to have poor function. 
     In other embodiments, the methods include determining the level of gene expression of one or more genes in the sample (such as 2, 3, 4, 5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or more), for example, one or more of those genes listed in  FIGS. 5-7 and 10 , or any combination thereof. In some examples, the methods include determining the level of one or more genes associated with cell proliferation (such as Jun, NFκB, Gadd45(3, and/or Gadd45a), general metabolic function and free-radical defenses (such as superoxide dismutase 1 and/or acyl coenzyme A synthetase), and/or differentiation (such as albumin, apolipoproteins, and/or cytochrome P450). These genes may be particularly suitable for identifying subjects at risk for (or having) liver dysfunction and/or determining the severity of the liver dysfunction and/or the necessity of imminent organ transplantation. One of ordinary skill in the art can identify genes associated with cell proliferation and/or differentiation in other tissues or organs. 
     In particular examples, a decrease in one or more of Jun, NFκB, apolipoprotein A-II, superoxide dismutase 1, acyl coenzyme A synthetase, thrombospondin 1, prothymosin, alpha, cytochrome c oxidase subunit II, and/or alpha-2-macroglobulin as compared to a healthy control or reference value indicates that the organ or subject is at risk for (or has) organ dysfunction. Additional genes associated with organ function (particularly liver function) are shown in  FIGS. 5-7 and 10 . 
     In other embodiments, the methods include determining the level of one or more proteins in the sample (such as 2, 3, 4, 5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, or more), for example, one or more proteins encoded by the genes shown in  FIGS. 5-7 and 10 , or any combination thereof. In other examples, the methods include determining the level of cytokines and/or chemokines (for example, one or more of interferon-α, interferon-γ, interleukin-10, interleukin-12/23 (p40), interleukin-1b, interleukin-4, interleukin-6, interleukin-8, and/or tumor necrosis factor-α). In particular examples, an increase in one or more of IFN-α, TNF-α, IFN-γ, IL-4, IL-1β, and/or IL-12/IL-23(p40) for example as compared to a healthy control or reference value indicates that the organ or subject is at risk for (or has) organ dysfunction. In other examples, one or more of VEGF, TGF-β, ERK/MAPK, ErbB, FAK, HGF, p53, insulin receptor, PI3K/AKT, PDGF, FGF, EGF, and NF-κB are analyzed. For example, an increase in one or more of VEGF, TGF-β, ERK/MAPK, ErbB, FAK, HGF, p53, insulin receptor, PI3K/AKT, PDGF, FGF, EGF, and NF-κB gene or protein expression (for example compared to a control, such as a dysfunctional organ) indicates good organ function. These proteins may be particularly suitable for identifying subjects at risk for (or having) liver dysfunction and/or determining the severity of the liver dysfunction and/or the necessity of imminent organ transplantation. 
     In still further embodiments, organ function can be measured by production of a fluid, such as bile or urine. In some examples, a decrease in bile production by a liver indicates that the liver is at risk for, or has, dysfunction. In other examples, a decrease in urine production by a kidney indicates that the kidney is at risk for, or has, dysfunction. In further examples, organ function can be measured by the presence or amount of one or more components in a fluid, such as bile or urine. In some examples, production of hydrophilic bile (e.g., decreased levels of taurodeoxycholate) indicates that the liver is predicted to have, or has, good function, while production of hydrophobic bile (e.g., increased levels of glycocholenate sulfate) indicates that the liver is at risk for, or has, dysfunction. In other examples, an increase in hydrophobic bile salts (e.g., glycochenodeoxycholate) indicates that the liver is at risk for, or has, dysfunction, while production of hydrophilic bile salts (e.g., ursodoxycholic acid) indicates that the liver is predicted to have, or has, good function. 
     In some embodiments, the methods include selecting an organ for transplantation, for example, an organ that is predicted to have good function (or an organ that is not predicted to be at risk for or have dysfunction). In one example, an organ that is a candidate for transplantation is machine perfused and samples are collected. The samples are rapidly analyzed (for example in a few hours) and if the organ is predicted to be at risk for or have organ dysfunction, the organ is not utilized for transplantation. If the organ is predicted to have good function, the organ is utilized for transplantation into a transplant recipient. 
     In some embodiments, the methods include administering a treatment to a subject identified as being at risk for or having organ dysfunction. One of ordinary skill in the art, such as a clinician, can identify an appropriate treatment for the subject based on the organ, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the subject undergoing therapy. In one example, liver dysfunction includes enhanced metabolic pathway to gluconeogenesis leading to steatosis, and therapies administered to a subject with liver dysfunction could include intervention in the metabolic pathway (e.g., diet and medication) to reverse the dysfunction. In another example, kidney dysfunction includes decreased glomerular filtration rate due to progressive vasospasm of the afferent arterioles within the nephron and therapies could include identification of vasogenic factors (vasospasm) and amelioration of this condition as precursor of progressive hypertension 
     IV. Methods of Determining Presence or Amount of Biomarkers in a Sample 
     A. Nucleic Acids 
     In some embodiments, the methods disclosed herein include detecting presence and/or amount of one or more nucleic acids (such as DNA, RNA, mRNA, or miRNA) in a sample from a tissue, organ, organ perfusate, fluid, or subject. In some examples, nucleic acids are isolated from the sample. Methods of isolating nucleic acids are known to one of skill in the art. For instance, rapid nucleic acid preparation can be performed using a commercially available kit (such as kits and/or instruments from Qiagen (such as DNEasy®, RNEasy®, or miRNEasy® kits), Life Technologies (such as ChargeSwitch® gDNA, ChargeSwitch® RNA, or mirVana™ kits) Roche Applied Science (such as MagNA Pure kits and instruments), Thermo Scientific (KingFisher mL), bioMérieux (Nuclisens® NASBA Diagnostics), or Epicentre (Masterpure™ kits)). In other examples, the nucleic acids may be extracted using guanidinium isothiocyanate, such as single-step isolation by acid guanidinium isothiocyanate-phenol-chloroform extraction (Chomczynski et al.  Anal. Biochem.  162:156-159, 1987). The sample can be used directly or can be processed, such as by adding solvents, preservatives, buffers, or other compounds or substances. In addition, the nucleic acids may be processed further to produce a nucleic acid suitable for various assays, for example, reverse transcribing mRNA to cDNA. One of skill in the art can identify additional reagents and methods that can be used for nucleic acid purification or preparation for use in the methods disclosed herein. 
     Methods for analyzing nucleic acids in a sample (for example, detecting amount and/or changes in gene expression) are known to one of skill in the art and include, but are not limited to, Southern blotting, Northern blotting, in situ hybridization, RNase protection, subtractive hybridization, differential display, antibody-based methods (such as use of antibodies that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes), microarray-based methods, amplification-based methods, and sequencing-based methods. One of skill in the art can identify additional techniques that can be used to analyze gene expression, and it is to be understood that gene expression detection methods for use in the present disclosure include those developed in the future. 
     In some examples, gene expression (such as presence and/or amount of RNA, mRNA, and/or miRNA) is identified or confirmed using microarray techniques. Thus, expression of one or more genes (or an expression profile) can be measured using microarray technology. In this method, nucleic acids of interest (including for example, cDNAs and/or oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed nucleic acids are then hybridized with isolated nucleic acids (such as cDNA or mRNA) prepared or isolated from the sample. Hybridization of the isolated nucleic acids with the arrayed nucleic acids is detected, for example, based on identification of a label associated with a nucleic acid that is detected at an addressable location non the array. Microarray analysis can be performed by commercially available equipment, following the manufacturer&#39;s protocols, such as are supplied with Affymetrix GeneChip® technology (Affymetrix, Santa Clara, Calif.), or Agilent&#39;s microarray technology (Agilent Technologies, Santa Clara, Calif.). 
     In further examples, nucleic acids are analyzed by amplification techniques including, polymerase chain reaction (PCR), quantitative real-time PCR, reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), digital PCR, strand displacement amplification (see U.S. Pat. No. 5,744,311); transcription-free isothermal amplification (see U.S. Pat. No. 6,033,881); transcription-mediated amplification (TMA); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Pat. No. 6,025,134). 
     In other examples, gene expression is detected using sequencing techniques. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE; Velculescu et al.,  Science  270:484-487, 1995), 454 pyrosequencing (Tones et al.,  Genome Res.  18:172-177, 2008), RNA-seq (Wang et al.,  Nat Rev. Genet.  10:57-63. 2009), and gene expression analysis by massively parallel signature sequencing (MPSS; Brenner et al.,  Nat. Biotechnol.  18:630-634, 2000). 
     B. Proteins 
     In some embodiments, the methods disclosed herein include detecting presence and/or amount of one or more proteins or fragments thereof in a sample from a tissue, organ, organ perfusate, fluid, or subject. In some examples, the samples are used without processing. In other examples, the samples are processed prior to protein analysis, for example by adding one or more components (such as detergents, buffers, or salts), lysis (if cells are present), and/or fractionation. 
     Any standard immunoassay format (such as ELISA, Western blot, or radioimmunoassay) can be used to measure protein levels. Immunohistochemical techniques can also be utilized for protein detection and quantification. General guidance regarding such techniques can be found in Bancroft and Stevens ( Theory and Practice of Histological Techniques , Churchill Livingstone, 1982) and Ausubel et al. ( Current Protocols in Molecular Biology , John Wiley &amp; Sons, New York, 1998). 
     In some examples, protein (or fragments thereof) are detected in a sample using mass spectrometry methods, such as mass spectrometry (MS), tandem MS (MS/MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, or electrospray ionization (ESI)-MS. In some examples, the sample may be subjected to a separation technique, such as liquid chromatography or 2-dimensional electrophoresis prior to MS analysis. Semi-quantitative or quantitative MS methods are also available. (See, e.g., Gygi et al.,  Nat. Biotechnol.  17:994-999, 1999; Aebersold,  J. Inf. Dis.  187:S315-S320, 2003.) In other examples, proteins or fragments thereof are identified or confirmed using microarray techniques. In this method, protein of interest or fragments thereof are plated, or arrayed, on a microchip substrate. The arrayed proteins are then contacted with proteins prepared or isolated from the sample. Binding of the proteins from the sample with the arrayed proteins is detected, for example, based on contacting with a label and localization to an addressable location on the array. Microarray analysis can be performed by commercially available equipment, following the manufacturer&#39;s protocols, such as are supplied with ProtoArray® protein microarray (Life Technologies) or HuProt™ arrays (Cambridge Protein Arrays). 
     In other examples, a bead-based assay is used to measure proteins in the sample. Such assays typically include beads coated with a capture antibody for a specific analyte. The beads are incubated with proteins prepared or isolated from a sample and binding of a protein to a bead is detected, for example using flow cytometry. Bead-based assays can be multiplexed, for example by including beads with different fluorescence intensities, each coated with an antibody specific for a specific protein. The presence and identity of different proteins can thus be detected, based on the fluorescence of the particular bead bound by a protein. Bead-based assays are commercially available (such as the Luminex xMAP® technology or the BD Biosciences Cytometric Bead Array). 
     One of skill in the art can identify additional techniques that can be used to analyze proteins in a sample, and it is to be understood that protein detection methods for use in the present disclosure include those developed in the future. 
     C. Small Molecules/Metabolites 
     The small molecule profile of a sample can be obtained through, for example, a single technique or a combination of techniques for separating and/or identifying small molecules known in the art. Examples of separation and analytical techniques which can be used to separate and identify the compounds of the small molecule profiles include: HPLC, TLC, electrochemical analysis, mass spectroscopy (for example, GC/MS or LC/MS/MS), refractive index spectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent analysis, radiochemical analysis, Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonance spectroscopy (NMR), Light Scattering analysis (LS) and other methods known in the art. One of skill in the art can identify additional techniques that can be used to analyze small molecules, and it is to be understood that detection methods for use in the present disclosure include those developed in the future. 
     The methods can be used to detect electrically neutral as well as electrochemically active compounds. Detection and analytical techniques can be arranged in parallel to optimize the number of molecules identified. In some examples, analysis of the small molecule profile of a sample includes analysis by a commercial provider, such as Metabolon (Durham, N.C.). In particular non-limiting examples, the small molecule profile is analyzed in perfusate from an ex vivo perfused organ, bile, or urine. 
     The present disclosure is illustrated by the following non-limiting Examples. 
     Example 1 
     Experimental Model 
     This example describes the liver transplantation model used for sample collection for further analysis. The machine perfusion system and oxygen carrier perfusion solution are described in related U.S. Provisional Patent Application No. 61/713,284, filed Oct. 12, 2012, and International Pat. Publ. No. WO 2014/059316, both of which are incorporated herein by reference in their entirety. 
     Two groups of 6 swine underwent orthotopic liver transplantation after a period of 9 hours of preservation (cold ischemia time (CIT)=9 hours). Both groups had a 5 day follow up while receiving Tacrolimus (0.3 mg/kg) as their primary immunosuppressive therapy. All surviving animals underwent an end-study necropsy on the 5 th  post-operative day. This challenging swine model (CIT=9 hours) has been consistently demonstrated as having a 70-100% mortality in 5-7 days. 
     The control group had their liver allografts preserved with CSP (University of Wisconsin solution (UW) at 4° C. under anoxic conditions). The study group had their liver allografts preserved with machine perfusion (MP; Organ Assist, Groningen, Netherlands) and a newly developed hemoglobin-based oxygen carrier (HBOC) solution (HEMOPURE (OPK Biotech, Cambridge, Mass.) mixed with Belzer Machine Perfusion Solution (BMPS) at 1:3). The MP livers underwent continuous perfusion under dual pressure (continuous at the portal vein and pulsatile at the hepatic artery) at 21° C. (subnormothermic conditions) and with a FiO 2  of 60%. Both groups had their liver allografts biopsied at 3, 6 and 9 hours while under preservation. Additional biopsies were taken after organ reperfusion and 5 days after the initial procedure (end-study necropsy). The schedule for sample collection is shown in Table 1. All laboratory and clinical parameters were assessed. Repeated measurements were compared between study and control groups using a mixed model with fixed effect (animal group) analysis. Continuous data were compared using either t testing or the nonparametric Wilcoxon two-sample test when appropriate. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sample collection schedule 
               
            
           
           
               
               
               
            
               
                   
                   
                 Time frame 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Post- 
                   
               
               
                   
                   
                   
                   
                   
                   
                 reper- 
                   
               
               
                 Group 
                 Specimen 
                 0 
                 3 
                 6 
                 9 
                 fusion 
                 Final 
               
               
                   
               
               
                 Control 
                 Liver  
                 X 
                 X 
                 X 
                 X 
                 X 
                 necropsy 
               
               
                 (CSP) 
                 biopsy 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Perfusate 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Bile †  
                 No 
                 No 
                 No 
                 No 
                 X 
                 X 
               
               
                 Study 
                 Liver  
                 X 
                 X 
                 X 
                 X 
                 X 
                 necropsy 
               
               
                 (MP) 
                 biopsy 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Perfusate 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 Bile 
                 X 
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                   
               
               
                 † liver allografts do not make bile during CSP 
               
            
           
         
       
     
     The study group (MP) had 100% survival and the control group (CSP) had a 33% survival in the 5 day period (p&lt;0.05). The MP group had none to mild signs of reperfusion syndrome (RS) after liver allograft implantation and the CSP group had moderate to severe signs of RS. The CSP group received a significantly higher (150%, p&lt;0.05) amount on intravenous fluids and a higher amount of vasopressors after liver allograft reperfusion while experiencing major vasodilatation as a result of the moderate to severe RS. 
     The CSP had a higher index of ischemia/reperfusion (IR) tissue injuries revealed by serial histological analysis during liver allograft preservation. IR injuries were considered none to mild in the MP group and moderate to severe in the CSP group (blind analysis by a selected group of transplant pathologists). All the animals that expired in the CSP experienced progressive liver allograft dysfunction leading to irreversible liver failure. The surviving CSP animals were clinically ill (renal failure, coagulopathy, progressive lactic acidosis and decreased mental status) and more ill than the MP group, who had uneventful post-operative courses. 
     All liver allografts were able to make bile, produce glucose and clear lactate under an extended period of time (CIT=9 hours) while under MP. Gasometric analysis of the perfusate showed high (p02&gt;400 mm Hg) levels of oxygenation on both arterial and venous ports, low levels of CO 2  (pCO 2 &lt;18 mm Hg) and sustained pH throughout the entire perfusion protocol. Serial liver biopsies (3, 6, 9 hours) showed normal cytoarchitecture features of the hepatic parenchyma during the ex vivo MP stage. Over 16 physiological parameters were compared over the five day post-operative period and all 16 showed one-directional effect (statistically significant or not) superior to the study group compared to control. Exemplary results are shown in  FIGS. 2A-2G . MP had a statistically significant (p&lt;0.05) beneficial effect in liver allograft preservation when compared to CSP. In assumption that the MP/HBOC does not affect animal physiology post-operatively, the probability of such results is 0.5 in power 16 or p&lt;0.004. 
     Tissue samples were prospectively collected for histological analysis and scoring of ischemia/reperfusion (IR) injury as shown in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Tissue sample collection for IR analysis 
               
            
           
           
               
               
               
            
               
                 Times 
                 Samples 
                 Notes 
               
               
                   
               
               
                 T0 
                 donor baseline 
                 Obtained prior to organ procurement 
               
               
                 T1 
                 back table  
                 Obtained immediately after organ 
               
               
                   
                 (after flush) 
                 recovery 
               
               
                 T2 
                 3h of preservation 
                 Obtained in both groups (CSP and MP) 
               
               
                 T3 
                 6h of preservation 
                 Obtained in both groups (CSP and MP) 
               
               
                 T4 
                 9h of preservation 
                 Obtained in both groups (CSP and MP) 
               
               
                 TRP 
                 post reperfusion 
                 Obtained after liver implantation 
               
               
                 Necropsy 
                 5th post-operative  
                 Obtained at the end-study necropsy 
               
               
                   
                 day 
               
               
                   
               
            
           
         
       
     
     All tissue samples were processed immediately after collection. The samples were initially divided in 2 pieces, for fresh frozen sections initially and for subsequent sections in paraffin afterwards. All tissues were processed and stained (H&amp;E and immunohistochemistry) at the Division of Transplant Pathology, Department of Pathology, UPMC. Tissue samples for the assessment of hepatocellular injury were collected before, during, and after preservation, post-reperfusion and at end study necropsy. All liver samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned (5 μm) and stained with hematoxylin and eosin for histological analyses. The severity of liver IR injuries was blindly graded by transplant pathologists using initially the International Banff Criteria ( Hepatology  3:658-663, 1997). A modified Suzuki&#39;s criteria (Suzuki et al.,  Transplantation  55:1265-1272, 1993) was subsequently applied to quantify the IR injuries and correlate the clinical with the histopathological findings. Complete histological features were assessed both in the portal tracts and the hepatic lobules. The scored number was further weighted (none=0, mild 1-25%=1; moderate 25-50%=2 and significant &gt;50%=3); re-scored based on its contribution to the injury while grouped into four categories and subsequently expressed as mean±standard deviation. The IR scores between the two groups were compared chronologically during liver preservation and after transplantation. 
     The entire histological analysis was combined within a single graphic displaying all the scores for all the samples ( FIG. 3 ). An IR score was calculated for each time point (mean±SD) and compared longitudinally with all values obtained from the different time points. The control group (CSP) histological analysis (H&amp;E) revealed the presence of moderate to severe IR injuries throughout the entire experiment. There was no improvement of the IR injuries after liver implantation and 67% of the animals expired within hours and/or days from liver allograft failure due to the severity of the IR injuries. The surviving animals (33%) revealed significant (p=0.01) allograft damage at the time of the elective end-study necropsy. 
     The study group (MP) presented none to mild IR injuries during preservation and after liver allograft implantation (TRP—reperfusion samples). The IR scores showed a progressive resolution of this transient inflammatory process over the next 5 days and the animals had 100% survival with good liver allograft function. There was a significant (p&lt;0.05) difference between the two groups when comparing the tissue samples analyzed after the end-study necropsy. The magnitude of the IR injuries seen at the control group (CSP) were significantly higher and led to irreversible liver allograft failure and death (only 33% survival, p&lt;0.05). 
     Mitochondrial isolation and respiration: Fresh tissue samples (liver biopsies from the allografts) were obtained from both groups and sent to a mitochondrial functional analysis in an oxygraph chamber. Liver mitochondria were isolated by differential centrifugation in a buffer (250 mM sucrose, 10 mM Tris, 1 mM EGTA, pH 7.4) at 4° C., as previously described. To measure respiration of isolated mitochondria, 1 mg/ml of protein was suspended in respiration buffer (120 mM KCl, 25 mM sucrose, 10 mM HEPES, 1 mM EGTA, 1 mM KH 2 PO 4 , 5 mM MgCl 2 ) in a stirred, sealed chamber fit with a Clark-type oxygen electrode (Instech Laboratories) connected to a data recording device (DATAQ Systems). 
     Mitochondrial function was sustained throughout the entire MP protocol with pulsatile pressures (MAP=20 mmHg) at 21° C. The oxygen delivery was estimated to be around 0.013 mlO 2 /g/min and the oxygen consumption was estimated around 0.0016 mlO 2 /g/min. Both the respiratory control ratio (RCR) ( FIG. 4A ) and ATP assays ( FIG. 4B ) showed uninterrupted and efficient mitochondrial function within the hepatic parenchyma while under machine perfusion at 21° C. The ROS production after liver allograft reperfusion was 2 fold higher in the CSP group ( FIG. 4C ). 
     Example 2 
     Microarray Analysis 
     This example describes microarray analysis of hepatic gene expression in the control and study transplantation groups. 
     Methods 
     Microarray analysis: Microarray analysis was performed using Affymetrix GeneChip® Porcine Genome Array (Affymetrix, Santa Clara, Calif., USA). Liver tissue samples from both groups (control=CSP and study=MP) were obtained before and after liver preservation and at the time of the end-study necropsy. Total RNA (10 μg) or mRNA (0.2 μg) was first reverse transcribed in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling. The biotinylated cRNA targets were fragmented in 1× fragmentation buffer solution provided with the GeneChip sample cleanup module (Affymetrix) at 94° C. for 35 min. A total of 10 μg of fragmented biotin-labeled cRNA per replicate in hybridization mixture then was hybridized to Porcine Genome Array from Affymetrix GeneChips™ and incubated overnight at 45° C. in Affymetrix GeneChip Hybridization Oven 640, all according to the manufacturer&#39;s instructions. 
     The mixture was removed 16 hours after hybridization in several cycles; the chips were washed with non-stringent buffer and stained with streptavidin-phycoerythrin antibody solution (Affymetrix) on an automated Affymetrix GeneChip Fluidic Station 450 station. The data were collected using an Affymetrix GeneChip scanner 3000. Microarray images quantified using Affymetrix GeneChip Operating Software. 
     Microarray data analysis: Normalization and pre-processing of data were performed using dChip software. Expression intensities were log transformed, and genes with less than 80% present calls, expression level lower than 7, or SD smaller than 0.5 were filtered out. Individual expression points of the top 100 genes that were found to be differentially regulated were fitted by statistics and the clustering pattern plotted in Microsoft Excel. The threshold line corresponds to a p value of 0.05 as calculated by the Fischer&#39;s test. 
     The CEL data generated by the microarray were converted using GCOS1.4 software (Affymetrix). The data generated by the Affymetrix platform contain all information required by MIAME protocols to allow the data to be submitted as needed. Details of compliance met by the CGOS1.4 software and all other programs used to convert CEL files to Excel microarray data are provided at ncbi.nlm.nih.gov/geo/info/MIAME.html. 
     Gene ontology analysis: The web tools DAVID (Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists) were used to identify enriched functionally related gene groups following machine perfusion. 
     Pathway analysis: Genes whose expression value was &gt;2 or &lt;2-fold compared to the control group were analyzed for identification of key canonical pathways associated with liver growth and pathology using Ingenuity Software®. The pathways that were found to be most significantly up-regulated were plotted. The biological processes that were found to be significantly affected were displayed along the y-axis. The x-axis displays the-log of p-value and was calculated by Fisher&#39;s exact test right-tailed. 
     Results 
     Hepatic gene expression was analyzed before and after preservation and at necropsy. There was a striking increase in proliferation-associated genes (Jun, Fos, ATP synthase F0 subunit 8, Apolipoprotein A-II, Metallothionein isoform, Acyl coenzyme A synthetase, Syndecan 2, Collagen Alpha 2, Prothymosin alpha) in the MP group. Many hepatocyte-differentiation genes were also upregulated, including albumin, apolipoproteins, and several cytochrome P450 (CYP) members. 
     The top 100 most affected genes in the MP group showed over-expression associated with general metabolic, anti-inflammatory, and regenerative functions, as well as protective mechanisms against free radicals ( FIG. 5 ). MP also resulted in increased expression of several genes associated with entry of hepatocytes into the G1 cell cycle phase ( FIG. 6 ). Genes associated with hepatocyte differentiation were also upregulated ( FIG. 7 ). 
     Enrichment by biological process networks following MP suggested a marked up-regulation of genes related to metabolic process (34%), cellular process (25%), cell communication (17%), as well as additional effects on system process (9%), cell cycle (7%), and cell adhesion (5%) ( FIG. 8 ). Metabolic Network Analysis (Ingenuity Software©) showed significant up-regulation of genes related to drug, amino acid, vitamin, mineral, and carbohydrate metabolism, free radical scavenging, and energy production in the MP group ( FIG. 9A ). Additional Biological Process Network Analysis (Ingenuity Software©) showed a significant (p&lt;0.05) difference in gene expression related to hepatic system development and function, cellular growth and proliferation, cellular development, and cell cycle when MP was compared to CSP ( FIG. 9B ). Metabolic Network Analysis (Ingenuity Software©) showed significant up-regulation of genes related to drug, amino acid, vitamin, mineral, and carbohydrate metabolism, free radical scavenging, and energy production in the MP group. Thus, MP with full oxygenation enhances signaling pathways for HGF, EGF, TGF-β, ErbB and PI3K/AKT among others, and triggers proliferative and regenerative transcriptional pathways when compared to CSP ( FIG. 10 ). 
     Example 3 
     Cytokine Profiling 
     This example describes the cytokine profile of tissue samples from control and study group animals. 
     Additional tissue assays were performed with Affymetrix pig 9 plex Luminex analysis. Approximately 50 mg of the tissue was transferred to a 2.0 ml microcentrifuge tube containing 1 ml of lxBioSource tissue extraction reagent (San Diego, Calif.) (Catalog Number FNN0071) supplemented with 10 ml of 100 mM phenylmethanesulfonyl fluoride in ethanol as a protease inhibitor. The tissue was homogenized for 15-30 sec until the sample was in a consistent solution. The sample was placed on ice, if processing multiple samples, and was then centrifuged at 4° C. for 10 min at 10,000×g. After centrifugation, the supernatant was collected and placed in a new microcentrifuge tube, placed on ice, and assayed for protein content using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, Ill.) using the manufacturer&#39;s protocol. Depending on tissue type, a 1:5 or 1:10 dilution was necessary before addition of samples to the BCA assay. 
     Pig cytokines (INF-α, IFN-γ, IL-10, IL-12/IL-23 (p40), IL-1β, IL-4, IL-6, IL-8 and TNF-α) were detected using a Luminex™ 100 IS apparatus using the BioSource International Pig 9 plex LUMINEX beadset. Cytokine levels are presented as mean±SEM. Differences between the levels of a given cytokine measured in flash-frozen tissue vs. RNALATER™-preserved tissue were assessed by Student&#39;s t-test analysis using SigmaStat™ software (SPSS, Chicago, Ill.). Cytokine levels are shown in  FIGS. 11-16 . Multiple protein-level inflammatory mediators in both tissue and perfusate were significantly (p&lt;0.05) different between groups. MP was associated with downregulation of both type I (IFN-α) and type II (IFN-γ) interferons, consistent with prior studies that showed elevated inflammation and apoptosis subsequent to IR due to the IRF-1 pathway in CSP. The suggestion of a protective mechanism provided by sustainable oxygenation ex vivo was further reinforced by the significant difference in IL-4 levels in the tissues of the MP group. MP was associated with downregulated TNF-α levels in liver tissue when compared to CSP. This detrimental TNF-α activation pathway seen in the CSP group was further corroborated by higher levels of additional Kupffer cells mediators IL-1(3 and IL-12/IL-23 p40 found on liver tissues. MP down-regulated IL-2 expression progressively during preservation when compared to CSP, which might contribute to lower T cell activation after organ implantation. 
     Example 4 
     Metabolomic Analysis 
     This example describes the metabolomics analysis of samples from control and study group animals. 
     Metabolomic analysis was performed on 27 perfusate and 31 bile samples (Tables 3 and 4, respectively) by Metabolon (Durham, N.C.). Following receipt, samples were inventoried, and immediately stored at −80° C. At the time of analysis, samples were extracted and prepared for analysis using Metabolon&#39;s standard solvent extraction method. The extracted samples were split into equal parts for analysis on the GC/MS and LC/MS/MS platforms. Also included were several technical replicate samples created from a homogeneous pool containing a small amount of all study samples. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Liver Perfusate samples 
               
            
           
           
               
               
               
            
               
                   
                   
                 Time Point 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Treatment 
                 0 h 
                 6 h 
                 9 h 
               
               
                   
                   
               
               
                   
                 Cold static perfusion in UW 
                 n = 2 
                 n = 4 
                 n = 4 
               
               
                   
                 buffer (UW/CSP) 
                   
                   
                   
               
               
                   
                 Machine perfusion in HBOC 
                 n = 6 
                 n = 6 
                 n = 5 
               
               
                   
                 buffer (HBOC/MP) 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Bile samples 
               
            
           
           
               
               
            
               
                   
                 Time Point 
               
            
           
           
               
               
               
               
            
               
                 Treatment 
                 0-4 h 
                 16-24 h 
                 64-72 h 
               
               
                   
               
               
                 Cold static perfusion in UW 
                 n = 4 
                 n = 5 
                 n = 4 
               
               
                 buffer (UW/CSP) 
                   
                   
                   
               
               
                 Machine perfusion in HBOC 
                 n = 6 
                 n = 6 
                 n = 6 
               
               
                 buffer (HBOC/MP) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Data Quality: Instrument and Process Variability 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Liver Perfusate 
                 Bile 
               
               
                   
                 QC Sample 
                 Measurement 
                 Median RSD 
                 Median RSD 
               
               
                   
                   
               
               
                   
                 Internal  
                 Instrument  
                  6% 
                  8% 
               
               
                   
                 Standards 
                 Variability 
                   
                   
               
               
                   
                 Endogenous  
                 Total Process  
                 10% 
                 11% 
               
               
                   
                 Biochemicals 
                 Variability 
               
               
                   
                   
               
            
           
         
       
     
     Instrument variability was determined by calculating the median relative standard deviation (RSD) for the internal standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites (non-instrument standards) present in 100% of the Client Matrix samples, which are technical replicates of pooled client samples. Values for instrument and process variability met Metabolon&#39;s acceptance criteria as shown in the Table 5, above. There were 223 compounds of known identity (named biochemicals) in liver perfusate and 377 named biochemicals in bile. Following log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch&#39;s two-sample t-tests were used to identify biochemicals that differed significantly between experimental groups. A summary of the numbers of biochemicals that achieved statistical significance (p&lt;0.05), as well as those approaching significance (0.05&lt;p&lt;0.10), is shown below (Tables 6 and 7). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Summary of biochemicals that differed  
               
               
                 in liver perfusate samples between groups 
               
               
                 Statistical Comparisons (Perfusate) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Total 
                   
               
               
                   
                 Total 
                   
                 bio- 
                   
               
               
                   
                 bio- 
                 Bio- 
                 chemicals 
                 Bio- 
               
               
                 Welch′s Two- 
                 chemicals 
                 chemicals 
                 0.05 &lt;  
                 chemicals 
               
               
                 Sample t-Test 
                 p ≤ 0.05 
                 (↑↓) 
                 p &lt; 0.10 
                 (↑↓) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HBOC/MP 0 h 
                 49 
                 24 | 25 
                 19 
                  4 | 15 
               
               
                 UW/CSP 0 h 
                   
                   
                   
                   
               
               
                 HBOC/MP 6 h 
                 140 
                 89 | 51 
                 15 
                 9 | 6 
               
               
                 UW/CSP 6 h 
                   
                   
                   
                   
               
               
                 HBOC/MP 9 h 
                 121 
                 81 | 40 
                 25 
                 11 | 14 
               
               
                 UW/CSP 9 h 
                   
                   
                   
                   
               
               
                 UW/CSP 6 h 
                 17 
                 16 | 1  
                 18 
                 17 | 1  
               
               
                 UW/CSP 0 h 
                   
                   
                   
                   
               
               
                 UW/CSP 9 h 
                 22 
                 22 | 0  
                 14 
                 12 | 2  
               
               
                 UW/CSP 0 h 
                   
                   
                   
                   
               
               
                 UW/CSP 9 h 
                 2 
                 0 | 2 
                  4 
                 1 | 3 
               
               
                 UW/CSP 6 h 
                   
                   
                   
                   
               
               
                 HBOC/MP 6 h 
                 171 
                 153 | 18  
                 12 
                 11 | 1  
               
               
                 HBOC/MP 0 h 
                   
                   
                   
                   
               
               
                 HBOC/MP 9 h 
                 154 
                 137 | 17  
                 14 
                 14 | 0  
               
               
                 HBOC/MP 0 h 
                   
                   
                   
                   
               
               
                 HBOC/MP 9 h 
                 17 
                 9 | 8 
                 10 
                 5 | 5 
               
               
                 HBOC/MP 6 h 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Summary of biochemicals that differed  
               
               
                 in bile samples between groups 
               
               
                 Statistical Comparisons (Bile) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Total 
                   
               
               
                   
                 Total 
                   
                 bio- 
                   
               
               
                   
                 bio- 
                 Bio- 
                 chemicals 
                 Bio- 
               
               
                 Welch′s Two- 
                 chemicals 
                 chemicals 
                 0.05 &lt;  
                 chemicals 
               
               
                 Sample t-Test 
                 p ≤ 0.05 
                 (↑↓) 
                 p &lt; 0.10 
                 (↑↓) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HBOC/MP 0-4 
                 68 
                 35 | 33 
                 26 
                 10 | 16 
               
               
                 UW/CSP 0-4 
                   
                   
                   
                   
               
               
                 HBOC/MP 16- 
                 68 
                 14 | 54 
                 37 
                 15 | 22 
               
               
                 24 
                   
                   
                   
                   
               
               
                 UW/CSP 16-24 
                   
                   
                   
                   
               
               
                 HBOC/MP 64- 
                 39 
                 35 | 4  
                 31 
                 18 | 13 
               
               
                 72 
                   
                   
                   
                   
               
               
                 UW/CSP 64-72 
                   
                   
                   
                   
               
               
                 UW/CSP 16-24 
                 88 
                 58 | 30 
                 32 
                 17 | 15 
               
               
                 UW/CSP 0-4 
                   
                   
                   
                   
               
               
                 UW/CSP 64-72 
                 82 
                  7 | 75 
                 33 
                  7 | 26 
               
               
                 UW/CSP 0-4 
                   
                   
                   
                   
               
               
                 UW/CSP 64-72 
                 63 
                  2 | 61 
                 40 
                  1 | 39 
               
               
                 UW/CSP 16-24 
                   
                   
                   
                   
               
               
                 HBOC/MP 16- 
                 134 
                 58 | 76 
                 26 
                 10 | 16 
               
               
                 24 
                   
                   
                   
                   
               
               
                 HBOC/MP 0-4 
                   
                   
                   
                   
               
               
                 HBOC/MP 64- 
                 176 
                  35 | 141 
                 28 
                  7 | 21 
               
               
                 72 
                   
                   
                   
                   
               
               
                 HBOC/MP 0-4 
                   
                   
                   
                   
               
               
                 HBOC/MP 64- 
                 68 
                  3 | 65 
                 32 
                  4 | 28 
               
               
                 72 
                   
                   
                   
                   
               
               
                 HBOC/MP 16- 
                   
                   
                   
                   
               
               
                 24 
               
               
                   
               
            
           
         
       
     
     Perfusate Results MP with HBOC had a bigger impact on the metabolic profile over time than cold static perfusion (CSP). Perfusate profiling revealed differences in stress responses over time between the two preservation conditions. Biochemicals that provide insight into oxidative, inflammatory, and energy stress were among those showing a strong separation between perfusate profiles from the two preservation conditions. Possible signs of energy stress and purine nucleotide breakdown were noted in the UW/CSP-preserved livers as indicated by the significantly higher levels of AMP, nucleosides adenosine, guanosine, and inosine, as well as the inosine deamination product hypoxanthine. Evidence of lipoxygenase activity was observed and differed by preservation method. Altogether this suggested that inflammation was greater in the UW/CSP than HBOC/MP samples. The glucose-amino acid cycle and branched-chain amino acid mobilization were markedly elevated in HBOC/MP samples. Branched-chain amino acid (BCAA) oxidation showed a strong differential increase in HBOC/MP perfusates at the 6 and 9 h time points ( FIG. 17A-17C ). In addition, byproducts of the Krebs cycle showed significant differences between the groups ( FIGS. 18A-18B ). These could potentially be used as a marker for adequate aerobic metabolism in tissues experiencing previous ischemic insult. 
     Bile Results Bile acid release during perfusion was suppressed but sterol synthesis after transplant was increased in the HBOC/MP group. Five bile acid conjugates were detected in UW/CSP liver perfusates but were not detected or detected at very low levels in HBOC/MP perfusates, which reinforces the argument towards the inability to sustain effective bile acid conjugation during CSP. MP-treated livers made but did not release glycochenodeoxycholate during perfusion but resumed its secretion almost immediately following liver reperfusion. Although campesterol levels in both treatment groups started out similarly, they remained stable in bile samples collected from MP-preserved livers but rapidly tapered off in bile from UW/CSP-preserved livers. MP-preserved livers were able to adequately support bile-mediated nutrient extraction whereas UW/CSP-preserved livers appeared to be less capable of doing so. 
     After this initial analysis, biomarkers have been grouped by divergent biochemical pathways within known molecular groups (Tables 8 and 9). Analysis of additional markers in control and study group liver perfusate is shown in  FIGS. 19A-19W . 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Biomarkers in perfusate, grouped by divergent pathways within  
               
               
                 known molecular groups/pathways 
               
            
           
           
               
               
               
               
               
            
               
                 Super 
                 Sub  
                   
                 Plat- 
                 Comp  
               
               
                 Pathway 
                 Pathway 
                 Biochemical Name 
                 form 
                 ID 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Amino  
                 Glycine,  
                 glycine 
                 GC/MS 
                 11777 
               
               
                 acid 
                 serine 
                 N-acetylglycine 
                 GC/MS 
                 27710 
               
               
                   
                 and  
                 beta-hydroxypyruvate 
                 GC/MS 
                 15686 
               
               
                   
                 threonine 
                 serine 
                 GC/MS 
                 1648 
               
               
                   
                 metabolism 
                 threonine 
                 GC/MS 
                 1284 
               
               
                   
                   
                 betaine 
                 LC/MS  
                 3141 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Alanine and  
                 aspartate 
                 GC/MS 
                 15996 
               
               
                   
                 aspartate 
                 beta-alanine 
                 GC/MS 
                 55 
               
               
                   
                 metabolism 
                 alanine 
                 GC/MS 
                 1126 
               
               
                   
                 Glutamate  
                 glutamate 
                 GC/MS 
                 57 
               
               
                   
                 metabolism 
                 4-hydroxyglutamate 
                 GC/MS 
                 40499 
               
               
                   
                   
                 glutamine 
                 GC/MS 
                 1647 
               
               
                   
                   
                 pyroglutamine* 
                 LC/MS  
                 32672 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 gamma-aminobutyrate  
                 GC/MS 
                 1416 
               
               
                   
                   
                 (GABA) 
                   
                   
               
               
                   
                 Histidine  
                 histidine 
                 LC/MS  
                 59 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                 Lysine  
                 lysine 
                 GC/MS 
                 1301 
               
               
                   
                 metabolism 
                 2-aminoadipate 
                 GC/MS 
                 6146 
               
               
                   
                 Phenyl-  
                 phenylalanine 
                 LC/MS  
                 64 
               
               
                   
                 alanine &amp;  
                   
                 pos 
                   
               
               
                   
                 tyrosine 
                 tyrosine 
                 LC/MS  
                 1299 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 phenylacetylglycine 
                 LC/MS 
                 33945 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 phenol sulfate 
                 LC/MS  
                 32553 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 5-hydroxymethyl-2- 
                 GC/MS 
                 42040 
               
               
                   
                   
                 furoic acid 
                   
                   
               
               
                   
                 Tryptophan  
                 tryptophan 
                 LC/MS  
                 54 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 C-glycosyltryptophan* 
                 LC/MS  
                 32675 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 3-indoxyl sulfate 
                 LC/MS  
                 27672 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Valine,  
                 3-methyl-2-oxobutyrate 
                 LC/MS  
                 21047 
               
               
                   
                 leucine 
                   
                 neg 
                   
               
               
                   
                 and  
                 3-methyl-2-oxovalerate 
                 LC/MS  
                 15676 
               
               
                   
                 isoleucine 
                   
                 neg 
                   
               
               
                   
                 metabolism 
                 beta-hydroxyisovalerate 
                 GC/MS 
                 12129 
               
               
                   
                   
                 isoleucine 
                 LC/MS  
                 1125 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 leucine 
                 LC/MS  
                 60 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 tigloylglycine 
                 LC/MS  
                 1598 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 valine 
                 LC/MS  
                 1649 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 4-methyl-2- 
                 LC/MS  
                 22116 
               
               
                   
                   
                 oxopentanoate 
                 neg 
                   
               
               
                   
                   
                 isovalerylglycine 
                 LC/MS  
                 35107 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2-methylbutyrylglycine 
                 LC/MS  
                 31928 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 3-methylglutaryl- 
                 LC/MS  
                 37060 
               
               
                   
                   
                 carnitine (C6) 
                 pos 
                   
               
               
                   
                 Cysteine,  
                 cysteine 
                 GC/MS 
                 31453 
               
               
                   
                 methionine, 
                 N-acetylcysteine 
                 LC/MS  
                 1586 
               
               
                   
                 SAM,  
                   
                 pos 
                   
               
               
                   
                 taurine  
                 S-methylcysteine 
                 GC/MS 
                 40262 
               
               
                   
                 metabolism 
                 cystine 
                 GC/MS 
                 39512 
               
               
                   
                   
                 hypotaurine 
                 GC/MS 
                 590 
               
               
                   
                   
                 taurine 
                 GC/MS 
                 2125 
               
               
                   
                   
                 S-adenosylhomo- 
                 LC/MS  
                 15948 
               
               
                   
                   
                 cysteine (SAH) 
                 neg 
                   
               
               
                   
                   
                 methionine 
                 LC/MS  
                 1302 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 2-hydroxybutyrate  
                 GC/MS 
                 21044 
               
               
                   
                   
                 (AHB) 
                   
                   
               
               
                   
                   
                 4-methylthio-2- 
                 LC/MS  
                 40732 
               
               
                   
                   
                 oxobutanoate 
                 neg 
                   
               
               
                   
                 Urea cycle;  
                 ornithine 
                 GC/MS 
                 1493 
               
               
                   
                 arginine-,  
                 urea 
                 GC/MS 
                 1670 
               
               
                   
                 proline-,  
                 proline 
                 LC/MS  
                 1898 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                 Creatine  
                 creatine 
                 LC/MS  
                 27718 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 creatinine 
                 LC/MS  
                 513 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Butanoate  
                 2-aminobutyrate 
                 GC/MS 
                 1577 
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Polyamine  
                 5-methylthioadenosine  
                 LC/MS  
                 1419 
               
               
                   
                 metabolism 
                 (MTA) 
                 pos 
                   
               
               
                   
                   
                 putrescine 
                 GC/MS 
                 1408 
               
               
                   
                   
                 spermidine 
                 GC/MS 
                 485 
               
               
                   
                   
                 spermine 
                 LC/MS  
                 603 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Glutathione  
                 glutathione, reduced  
                 LC/MS  
                 2127 
               
               
                   
                 metabolism 
                 (GSH) 
                 pos 
                   
               
               
                   
                   
                 S-methylglutathione 
                 LC/MS  
                 33944 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 5-oxoproline 
                 LC/MS  
                 1494 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glutathione, oxidized  
                 LC/MS  
                 38783 
               
               
                   
                   
                 (GSSG) 
                 pos 
                   
               
               
                   
                   
                 cysteine-glutathione  
                 LC/MS  
                 35159 
               
               
                   
                   
                 disulfide 
                 pos 
                   
               
               
                   
                   
                 ophthalmate 
                 LC/MS  
                 34592 
               
               
                   
                   
                   
                 pos 
                   
               
               
                 Peptide 
                 Dipeptide 
                 glycylglycine 
                 GC/MS 
                 21030 
               
               
                   
                   
                 cysteinylglycine 
                 GC/MS 
                 35637 
               
               
                   
                 Dipeptide  
                 carnosine 
                 LC/MS  
                 1768 
               
               
                   
                 derivative 
                   
                 neg 
                   
               
               
                   
                 gamma- 
                 gamma- 
                 LC/MS  
                 32393 
               
               
                   
                 glutamyl 
                 glutamylvaline 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 18369 
               
               
                   
                   
                 glutamylleucine 
                 pos 
                   
               
               
                   
                   
                 gamma-glutamyl- 
                 LC/MS  
                 34456 
               
               
                   
                   
                 isoleucine* 
                 pos 
                   
               
               
                   
                   
                 gamma-glutamyl- 
                 LC/MS  
                 37539 
               
               
                   
                   
                 methionine 
                 pos 
                   
               
               
                   
                   
                 gamma-glutamyl- 
                 LC/MS  
                 36738 
               
               
                   
                   
                 glutamate 
                 pos 
                   
               
               
                   
                   
                 gamma-glutamyl- 
                 LC/MS  
                 33422 
               
               
                   
                   
                 phenylalanine 
                 pos 
                   
               
               
                   
                   
                 gamma-glutamyl- 
                 LC/MS  
                 2734 
               
               
                   
                   
                 tyrosine 
                 pos 
                   
               
               
                 Carbo- 
                 Aminosugars  
                 erythronate* 
                 GC/MS 
                 33477 
               
               
                 hydrate 
                 metabolism 
                   
                   
                   
               
               
                   
                 Fructose,  
                 fructose 
                 GC/MS 
                 577 
               
               
                   
                 mannose, 
                 galactitol (dulcitol) 
                 GC/MS 
                 1117 
               
               
                   
                 galactose,  
                 galactose 
                 GC/MS 
                 12055 
               
               
                   
                 starch, 
                 maltose 
                 GC/MS 
                 15806 
               
               
                   
                 and sucrose  
                 mannose 
                 GC/MS 
                 584 
               
               
                   
                 metabolism 
                 mannose-6-phosphate 
                 GC/MS 
                 1469 
               
               
                   
                   
                 Isobar: sorbitol,  
                 LC/MS  
                 33004 
               
               
                   
                   
                 mannitol 
                 pos 
                   
               
               
                   
                   
                 sucrose 
                 LC/MS  
                 1519 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 maltotriose 
                 LC/MS  
                 15913 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 raffinose 
                 LC/MS  
                 586 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 verbascose 
                 LC/MS  
                 37132 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 palatinitol 
                 GC/MS 
                 37469 
               
               
                   
                 Oligo- 
                 lactobionate 
                 GC/MS 
                 20685 
               
               
                   
                 saccharide 
                   
                   
                   
               
               
                   
                 Glycolysis,  
                 glycerate 
                 GC/MS 
                 1572 
               
               
                   
                 gluconeo-  
                 glucose-6-phosphate  
                 GC/MS 
                 31260 
               
               
                   
                 genesis, 
                 (G6P) 
                   
                   
               
               
                   
                 pyruvate  
                 glucose 
                 GC/MS 
                 31263 
               
               
                   
                 metabolism 
                 fructose-6-phosphate 
                 GC/MS 
                 12021 
               
               
                   
                   
                 3-phosphoglycerate 
                 GC/MS 
                 1414 
               
               
                   
                   
                 dihydroxyacetone  
                 GC/MS 
                 15522 
               
               
                   
                   
                 phosphate (DHAP) 
                   
                   
               
               
                   
                   
                 1,3-dihydroxyacetone 
                 GC/MS 
                 35963 
               
               
                   
                   
                 phosphoenolpyruvate  
                 GC/MS 
                 597 
               
               
                   
                   
                 (PEP) 
                   
                   
               
               
                   
                   
                 pyruvate 
                 GC/MS 
                 599 
               
               
                   
                   
                 lactate 
                 GC/MS 
                 527 
               
               
                   
                 Glyoxylate  
                 oxalate (ethanedioate) 
                 GC/MS 
                 20694 
               
               
                   
                 and 
                   
                   
                   
               
               
                   
                 dicarbo-  
                   
                   
                   
               
               
                   
                 xylate 
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Nucleotide  
                 6-phosphogluconate 
                 GC/MS 
                 15442 
               
               
                   
                 sugars,  
                 arabitol 
                 GC/MS 
                 38075 
               
               
                   
                 pentose 
                 ribitol 
                 GC/MS 
                 15772 
               
               
                   
                 metabolism 
                 threitol 
                 GC/MS 
                 35854 
               
               
                   
                   
                 sedoheptulose-7- 
                 GC/MS 
                 35649 
               
               
                   
                   
                 phosphate 
                   
                   
               
               
                   
                   
                 gluconate 
                 GC/MS 
                 587 
               
               
                   
                   
                 ribose 
                 GC/MS 
                 12080 
               
               
                   
                   
                 ribose 5-phosphate 
                 GC/MS 
                 561 
               
               
                   
                   
                 ribose 1-phosphate 
                 GC/MS 
                 1763 
               
               
                   
                   
                 ribulose 
                 GC/MS 
                 35855 
               
               
                   
                   
                 Isobar: ribulose 5- 
                 GC/MS 
                 37288 
               
               
                   
                   
                 phosphate, xylulose  
                   
                   
               
               
                   
                   
                 5-phosphate 
                   
                   
               
               
                   
                   
                 xylitol 
                 GC/MS 
                 4966 
               
               
                   
                   
                 xylose 
                 GC/MS 
                 15835 
               
               
                   
                   
                 xylonate 
                 GC/MS 
                 35638 
               
               
                   
                   
                 xylulose 
                 GC/MS 
                 18344 
               
               
                 Secondary  
                 Advanced  
                 erythrulose 
                 GC/MS 
                 37427 
               
               
                 Metabolism 
                 glycation  
                   
                   
                   
               
               
                   
                 end-product 
                   
                   
                   
               
               
                 Energy 
                 Krebs cycle 
                 citrate 
                 LC/MS  
                 1564 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 alpha-ketoglutarate 
                 GC/MS 
                 33453 
               
               
                   
                   
                 succinate 
                 GC/MS 
                 1437 
               
               
                   
                   
                 fumarate 
                 GC/MS 
                 1643 
               
               
                   
                   
                 malate 
                 GC/MS 
                 1303 
               
               
                   
                 Oxidative  
                 acetylphosphate 
                 GC/MS 
                 15488 
               
               
                   
                 phosphoryl- 
                 phosphate 
                 GC/MS 
                 11438 
               
               
                   
                 ation 
                 pyrophosphate (PPi) 
                 GC/MS 
                 2078 
               
               
                   
                   
                 linoleate (18:2n6) 
                 LC/MS  
                 1105 
               
               
                   
                   
                   
                 neg 
                   
               
               
                 Lipid 
                 Essential  
                 linolenate [alpha or  
                 LC/MS  
                 34035 
               
               
                   
                 fatty acid 
                 gamma; (18:3n3 or 6)] 
                 neg 
                   
               
               
                   
                   
                 dihomo-linolenate  
                 LC/MS  
                 35718 
               
               
                   
                   
                 (20:3n3 or n6) 
                 neg 
                   
               
               
                   
                   
                 eicosapentaenoate  
                 LC/MS  
                 18467 
               
               
                   
                   
                 (EPA; 20:5n3) 
                 neg 
                   
               
               
                   
                   
                 docosapentaenoate  
                 LC/MS  
                 32504 
               
               
                   
                   
                 (n3 DPA; 22:5n3) 
                 neg 
                   
               
               
                   
                   
                 docosahexaenoate  
                 LC/MS  
                 19323 
               
               
                   
                   
                 (DHA; 22:6n3) 
                 neg 
                   
               
               
                   
                 Medium  
                 caproate (6:0) 
                 LC/MS  
                 32489 
               
               
                   
                 chain 
                   
                 neg 
                   
               
               
                   
                 fatty acid 
                 caprylate (8:0) 
                 LC/MS  
                 32492 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2-aminoheptanoic acid 
                 LC/MS  
                 43761 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Long chain  
                 oleate (18:1 n9) 
                 GC/MS 
                 1359 
               
               
                   
                 fatty acid 
                 arachidate (20:0) 
                 LC/MS  
                 44679 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 eicosenoate  
                 LC/MS  
                 33587 
               
               
                   
                   
                 (20:1 n9 or 11) 
                 neg 
                   
               
               
                   
                   
                 dihomo-linoleate  
                 LC/MS  
                 17805 
               
               
                   
                   
                 (20:2n6) 
                 neg 
                   
               
               
                   
                   
                 mead acid (20:3n9) 
                 LC/MS  
                 35174 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 arachidonate (20:4n6) 
                 LC/MS  
                 1110 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Fatty acid,  
                 3-hydroxypropanoate 
                 GC/MS 
                 42103 
               
               
                   
                 mono- 
                 4-hydroxybutyrate (GHB) 
                 GC/MS 
                 34585 
               
               
                   
                 hydroxy 
                 13-HODE + 9-HODE 
                 LC/MS  
                 37752 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Fatty acid,  
                 4-hydroxy-2-oxoglutaric  
                 GC/MS 
                 40062 
               
               
                   
                 dicarbo- 
                 acid 
                   
                   
               
               
                   
                 xylate 
                 hexadecanedioate 
                 LC/MS  
                 35678 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 octadecanedioate 
                 LC/MS  
                 36754 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Eicosanoid 
                 12-HETE 
                 LC/MS  
                 37536 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 15-HETE 
                 LC/MS  
                 37538 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Fatty acid  
                 propionylcarnitine 
                 LC/MS  
                 32452 
               
               
                   
                 metabolism  
                   
                 pos 
                   
               
               
                   
                 (also BCAA  
                 propionylglycine 
                 LC/MS  
                 31932 
               
               
                   
                 metabolism) 
                   
                 neg 
                   
               
               
                   
                   
                 butyrylcarnitine 
                 LC/MS  
                 32412 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Carnitine  
                 carnitine 
                 LC/MS  
                 15500 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 acetylcamitine 
                 LC/MS  
                 32198 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Bile acid  
                 glycocholate 
                 LC/MS  
                 18476 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 taurocholate 
                 LC/MS  
                 18497 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycochenodeoxycholate 
                 LC/MS  
                 32346 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycolithocholate 
                 LC/MS  
                 31912 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 taurolithocholate 
                 LC/MS  
                 31889 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycohyodeoxycholic  
                 LC/MS  
                 43501 
               
               
                   
                   
                 acid 
                 pos 
                   
               
               
                   
                 Glycerolipid  
                 ethanolamine 
                 GC/MS 
                 34285 
               
               
                   
                 metabolism 
                 phosphoethanolamine 
                 GC/MS 
                 12102 
               
               
                   
                   
                 choline 
                 LC/MS  
                 15506 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 glycerol 3-phosphate  
                 GC/MS 
                 15365 
               
               
                   
                   
                 (G3P) 
                   
                   
               
               
                   
                   
                 glycerophosphoryl- 
                 LC/MS  
                 15990 
               
               
                   
                   
                 choline (GPC) 
                 pos 
                   
               
               
                   
                 Inositol  
                 myo-inositol 
                 GC/MS 
                 19934 
               
               
                   
                 metabolism 
                 scyllo-inositol 
                 GC/MS 
                 32379 
               
               
                   
                 Ketone  
                 3-hydroxybutyrate  
                 GC/MS 
                 542 
               
               
                   
                 bodies 
                 (BHBA) 
                   
                   
               
               
                   
                   
                 acetoacetate 
                 GC/MS 
                 33963 
               
               
                   
                   
                 1,2-propanediol 
                 GC/MS 
                 38002 
               
               
                   
                 Lysolipid 
                 1-arachidonoylglycero- 
                 LC/MS  
                 35186 
               
               
                   
                   
                 phosphoethanol amine* 
                 neg 
                   
               
               
                   
                   
                 1-palmitoylglycero- 
                 LC/MS  
                 33955 
               
               
                   
                   
                 phosphocholine (16:0) 
                 neg 
                   
               
               
                   
                   
                 1-oleoylglycerophos- 
                 LC/MS  
                 33960 
               
               
                   
                   
                 phocholine (18:1) 
                 neg 
                   
               
               
                   
                   
                 1-linoleoylglycero- 
                 LC/MS  
                 34419 
               
               
                   
                   
                 phosphocholine (18:2n6) 
                 neg 
                   
               
               
                   
                   
                 2-linoleoylglycero- 
                 LC/MS  
                 38087 
               
               
                   
                   
                 phosphocholine* 
                 neg 
                   
               
               
                   
                   
                 1-arachidonoylglycero- 
                 LC/MS  
                 34061 
               
               
                   
                   
                 phosphocholine (20:4n6)* 
                 neg 
                   
               
               
                   
                 Monoacyl- 
                 1-palmitoylglycerol  
                 GC/MS 
                 21127 
               
               
                   
                 glycerol 
                 (1-monopalmitin) 
                   
                   
               
               
                   
                 Sphingolipid 
                 palmitoyl sphingomyelin 
                 GC/MS 
                 37506 
               
               
                   
                 Sterol/ 
                 pregnanediol-3- 
                 LC/MS  
                 40708 
               
               
                   
                 Steroid 
                 glucuronide 
                 neg 
                   
               
               
                 Nucleotide 
                 Purine  
                 xanthine 
                 LC/MS  
                 3147 
               
               
                   
                 metabolism,  
                   
                 neg 
                   
               
               
                   
                 (hypo) 
                 xanthosine 
                 LC/MS  
                 15136 
               
               
                   
                 xanthine/ 
                   
                 neg 
                   
               
               
                   
                 inosine  
                 hypoxan thine 
                 LC/MS  
                 3127 
               
               
                   
                 containing 
                   
                 neg 
                   
               
               
                   
                   
                 inosine 
                 LC/MS  
                 1123 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2′-deoxyinosine 
                 LC/MS  
                 15076 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Purine  
                 adenine 
                 LC/MS  
                 554 
               
               
                   
                 metabolism,  
                   
                 pos 
                   
               
               
                   
                 adenine  
                 adenosine 
                 LC/MS  
                 555 
               
               
                   
                 containing 
                   
                 pos 
                   
               
               
                   
                   
                 N6-methyladenosine 
                 LC/MS  
                 37114 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 adenosine 5′- 
                 LC/MS  
                 32342 
               
               
                   
                   
                 monophosphate (AMP) 
                 pos 
                   
               
               
                   
                   
                 adenosine-2′,3′-cyclic  
                 LC/MS  
                 37467 
               
               
                   
                   
                 monophosphate 
                 pos 
                   
               
               
                   
                   
                 N6,N6- 
                 LC/MS  
                 42081 
               
               
                   
                   
                 dimethyladenosine 
                 pos 
                   
               
               
                   
                 Purine  
                 guanine 
                 LC/MS  
                 32352 
               
               
                   
                 metabolism,  
                   
                 pos 
                   
               
               
                   
                 guanine  
                 guanosine 
                 LC/MS  
                 1573 
               
               
                   
                 containing 
                   
                 neg 
                   
               
               
                   
                   
                 isoguanine 
                 GC/MS 
                 42958 
               
               
                   
                 Purine  
                 urate 
                 GC/MS 
                 1604 
               
               
                   
                 metabolism,  
                 allantoin 
                 GC/MS 
                 1107 
               
               
                   
                 urate  
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Pyrimidine  
                 cytidine 
                 LC/MS  
                 514 
               
               
                   
                 metabolism, 
                   
                 neg 
                   
               
               
                   
                 cytidine  
                   
                   
                   
               
               
                   
                 containing 
                   
                   
                   
               
               
                   
                 Pyrimidine  
                 3-aminoisobutyrate 
                 GC/MS 
                 1566 
               
               
                   
                 metabolism,  
                   
                   
                   
               
               
                   
                 thymine  
                   
                   
                   
               
               
                   
                 containing;  
                   
                   
                   
               
               
                   
                 Valine,  
                   
                   
                   
               
               
                   
                 leucine and 
                   
                   
                   
               
               
                   
                 isoleucine  
                   
                   
                   
               
               
                   
                 metabolism/ 
                   
                   
                   
               
               
                   
                 Pyrimidine  
                 uracil 
                 GC/MS 
                 605 
               
               
                   
                 metabolism, 
                 uridine 
                 LC/MS  
                 606 
               
               
                   
                 uracil  
                   
                 neg 
                   
               
               
                   
                 containing 
                   
                   
                   
               
               
                   
                 Purine and  
                 methyl phosphate 
                 GC/MS 
                 37070 
               
               
                   
                 pyrimidine 
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                 Cofactors  
                 Ascorbate  
                 ascorbate (Vitamin C) 
                 GC/MS 
                 1640 
               
               
                 and 
                 and aldarate  
                 threonate 
                 GC/MS 
                 27738 
               
               
                 vitamins 
                 metabolism 
                 arabonate 
                 GC/MS 
                 37516 
               
               
                   
                 Hemoglobin  
                 heme 
                 LC/MS  
                 41754 
               
               
                   
                 and  
                   
                 neg 
                   
               
               
                   
                 porphyrin 
                 L-urobilin 
                 LC/MS  
                 40173 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                 Nicotinate  
                 nicotinamide 
                 LC/MS  
                 594 
               
               
                   
                 and 
                   
                 pos 
                   
               
               
                   
                 nicotinamide 
                 N1-Methyl-2-pyridone-  
                 LC/MS  
                 40469 
               
               
                   
                 metabolism 
                 5-carboxamide 
                 pos 
                   
               
               
                   
                 Pantothenate  
                 pantothenate 
                 LC/MS  
                 1508 
               
               
                   
                 and CoA  
                   
                 neg 
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Riboflavin  
                 riboflavin (Vitamin B2) 
                 LC/MS  
                 1827 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                 Xeno- 
                 Benzoate  
                 hippurate 
                 LC/MS  
                 15753 
               
               
                 biotics 
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 2-hydroxyhippurate  
                 LC/MS  
                 18281 
               
               
                   
                   
                 (salicylurate) 
                 neg 
                   
               
               
                   
                 Chemical 
                 glycolate (hydroxyacetate) 
                 GC/MS 
                 15737 
               
               
                   
                   
                 2-hydroxyisobutyrate 
                 GC/MS 
                 22030 
               
               
                   
                   
                 glycerol 2-phosphate 
                 GC/MS 
                 27728 
               
               
                   
                   
                 HEPES 
                 LC/MS  
                 21248 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 trizma acetate 
                 GC/MS 
                 20710 
               
               
                   
                   
                 2-ethylhexanoate (isobar  
                 LC/MS  
                 35490 
               
               
                   
                   
                 with 2-propylpentanoate) 
                 neg 
                   
               
               
                   
                   
                 ricinoleic acid 
                 LC/MS  
                 37464 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Drug 
                 ketamine 
                 LC/MS  
                 35128 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 allopurinol riboside 
                 GC/MS 
                 38321 
               
               
                   
                   
                 vecuronium 
                 LC/MS  
                 42591 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 oxypurinol 
                 GC/MS 
                 41725 
               
               
                   
                   
                 allopurinol 
                 GC/MS 
                 43534 
               
               
                   
                 Food  
                 5-ketogluconate 
                 GC/MS 
                 15687 
               
               
                   
                 component/ 
                 N-glycolylneuraminate 
                 GC/MS 
                 37123 
               
               
                   
                 Plant 
                 stachydrine 
                 LC/MS  
                 34384 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 homostachydrine* 
                 LC/MS  
                 33009 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Sugar, sugar  
                 erythritol 
                 GC/MS 
                 20699 
               
               
                   
                 substitute,  
                   
                   
                   
               
               
                   
                 starch 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Biomarkers in bile, grouped by divergent pathways within  
               
               
                 known molecular groups/pathways 
               
            
           
           
               
               
               
               
               
            
               
                 Super 
                 Sub  
                 Biochemical 
                 Plat- 
                 Comp 
               
               
                 Pathway 
                 Pathway 
                 Name 
                 form 
                 ID 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Amino  
                 Glycine,  
                 glycine 
                 GC/MS 
                 11777 
               
               
                 acid 
                 serine and 
                 dimethylglycine 
                 GC/MS 
                 5086 
               
               
                   
                 threonine  
                 N-acetylglycine 
                 GC/MS 
                 27710 
               
               
                   
                 metabolism 
                 beta-hydroxypyruvate 
                 GC/MS 
                 15686 
               
               
                   
                   
                 serine 
                 GC/MS 
                 1648 
               
               
                   
                   
                 threonine 
                 LC/MS  
                 1284 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N-acetylthreonine 
                 LC/MS  
                 33939 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 betaine 
                 LC/MS  
                 3141 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Alanine  
                 asparagine 
                 GC/MS 
                 34283 
               
               
                   
                 and  
                 beta-alanine 
                 GC/MS 
                 55 
               
               
                   
                 aspartate 
                 3-ureidopropionate 
                 LC/MS  
                 3155 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 N-acetyl-beta-alanine 
                 LC/MS  
                 37432 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 alanine 
                 GC/MS 
                 1126 
               
               
                   
                   
                 N-acetylalanine 
                 LC/MS  
                 1585 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Glutamate  
                 glutamate 
                 LC/MS  
                 57 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 glutamine 
                 LC/MS  
                 53 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Histidine  
                 histidine 
                 LC/MS  
                 59 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 trans-urocanate 
                 LC/MS  
                 607 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 1-methylimidazole- 
                 LC/MS  
                 32350 
               
               
                   
                   
                 acetate 
                 pos 
                   
               
               
                   
                 Lysine  
                 lysine 
                 LC/MS  
                 1301 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 2-aminoadipate 
                 LC/MS  
                 6146 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 pipecolate 
                 GC/MS 
                 1444 
               
               
                   
                   
                 N6-acetyllysine 
                 LC/MS  
                 36752 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Phenyl-  
                 phenyllactate (PLA) 
                 LC/MS  
                 22130 
               
               
                   
                 alanine &amp; 
                   
                 neg 
                   
               
               
                   
                 tyrosine  
                 phenylalanine 
                 LC/MS  
                 64 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 phenylacetate 
                 GC/MS 
                 15958 
               
               
                   
                   
                 p-cresol sulfate 
                 LC/MS  
                 36103 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 m-cresol sulfate 
                 LC/MS  
                 36846 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 tyrosine 
                 LC/MS  
                 1299 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 3-(4-hydroxy- 
                 LC/MS  
                 32197 
               
               
                   
                   
                 phenyl)lactate 
                 neg 
                   
               
               
                   
                   
                 vanillylmandelate  
                 LC/MS  
                 1567 
               
               
                   
                   
                 (VMA) 
                 neg 
                   
               
               
                   
                   
                 4-hydroxyphenyl- 
                 LC/MS  
                 1669 
               
               
                   
                   
                 pyruvate 
                 neg 
                   
               
               
                   
                   
                 4-hydroxyphenyl- 
                 GC/MS 
                 541 
               
               
                   
                   
                 acetate 
                   
                   
               
               
                   
                   
                 3,4-dihydroxy- 
                 LC/MS  
                 18296 
               
               
                   
                   
                 phenylacetate 
                 neg 
                   
               
               
                   
                   
                 phenylacetylglycine 
                 LC/MS  
                 33945 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 phenol sulfate 
                 LC/MS  
                 32553 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 4-hydroxyphenyl- 
                 LC/MS  
                 43525 
               
               
                   
                   
                 acetyl glycine 
                 neg 
                   
               
               
                   
                 Tryptophan  
                 kynurenate 
                 LC/MS  
                 1417 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 kynurenine 
                 LC/MS  
                 15140 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 tryptophan 
                 LC/MS  
                 54 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 indolelactate 
                 GC/MS 
                 18349 
               
               
                   
                   
                 N-acetyl- 
                 LC/MS  
                 33959 
               
               
                   
                   
                 tryptophan 
                 neg 
                   
               
               
                   
                   
                 C-glycosyl- 
                 LC/MS  
                 32675 
               
               
                   
                   
                 tryptophan* 
                 pos 
                   
               
               
                   
                   
                 3-indoxyl  
                 LC/MS  
                 27672 
               
               
                   
                   
                 sulfate 
                 neg 
                   
               
               
                   
                 Valine,  
                 3-methyl-2- 
                 LC/MS  
                 21047 
               
               
                   
                 leucine  
                 oxobutyrate 
                 neg 
                   
               
               
                   
                 and 
                 3-methyl-2- 
                 LC/MS  
                 15676 
               
               
                   
                 isoleucine  
                 oxovalerate 
                 neg 
                   
               
               
                   
                 metabolism 
                 beta-hydroxy- 
                 GC/MS 
                 12129 
               
               
                   
                   
                 isovalerate 
                   
                   
               
               
                   
                   
                 alpha-hydroxy- 
                 GC/MS 
                 22132 
               
               
                   
                   
                 isocaproate 
                   
                   
               
               
                   
                   
                 isoleucine 
                 LC/MS  
                 1125 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 leucine 
                 LC/MS  
                 60 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N-acetylleucine 
                 LC/MS  
                 1587 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N-acetyl- 
                 LC/MS  
                 33967 
               
               
                   
                   
                 isoleucine 
                 pos 
                   
               
               
                   
                   
                 tigloylglycine 
                 LC/MS  
                 1598 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 valine 
                 LC/MS  
                 1649 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 3-hydroxyisobutyrate 
                 GC/MS 
                 1549 
               
               
                   
                   
                 4-methyl-2- 
                 LC/MS  
                 22116 
               
               
                   
                   
                 oxopentanoate 
                 neg 
                   
               
               
                   
                   
                 3-hydroxy-2- 
                 GC/MS 
                 32397 
               
               
                   
                   
                 ethylpropionate 
                   
                   
               
               
                   
                   
                 alpha-hydroxy- 
                 GC/MS 
                 33937 
               
               
                   
                   
                 isovalerate 
                   
                   
               
               
                   
                   
                 isovalerylglycine 
                 LC/MS  
                 35107 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 isobutyrylcarnitine 
                 LC/MS  
                 33441 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 2-methylbutyryl- 
                 LC/MS  
                 35431 
               
               
                   
                   
                 carnitine (C5) 
                 pos 
                   
               
               
                   
                   
                 2-methylbutyryl- 
                 LC/MS  
                 31928 
               
               
                   
                   
                 glycine 
                 pos 
                   
               
               
                   
                   
                 3-methylcrotonyl- 
                 LC/MS  
                 31940 
               
               
                   
                   
                 glycine 
                 pos 
                   
               
               
                   
                   
                 isovalerylcarnitine 
                 LC/MS  
                 34407 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 tiglyl carnitine 
                 LC/MS  
                 35428 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 3-methylglutaryl- 
                 LC/MS  
                 37060 
               
               
                   
                   
                 carnitine (C6) 
                 pos 
                   
               
               
                   
                 Cysteine,  
                 cysteine 
                 GC/MS 
                 31453 
               
               
                   
                 methionine,  
                 S-methylcysteine 
                 LC/MS  
                 39592 
               
               
                   
                 SAM,  
                   
                 pos 
                   
               
               
                   
                 taurine  
                 cystine 
                 GC/MS 
                 31454 
               
               
                   
                 metabolism 
                 S-adenosylhomo- 
                 LC/MS  
                 15948 
               
               
                   
                   
                 cysteine (SAH) 
                 neg 
                   
               
               
                   
                   
                 methionine 
                 LC/MS  
                 1302 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N-acetylmethionine 
                 LC/MS  
                 1589 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2-hydroxybutyrate  
                 GC/MS 
                 21044 
               
               
                   
                   
                 (AHB) 
                   
                   
               
               
                   
                   
                 homocysteine 
                 GC/MS 
                 40266 
               
               
                   
                 Urea  
                 dimethylarginine  
                 LC/MS  
                 36808 
               
               
                   
                 cycle; 
                 (SDMA + ADMA) 
                 pos 
                   
               
               
                   
                 arginine-, 
                 arginine 
                 LC/MS  
                 1638 
               
               
                   
                 proline-,  
                   
                 pos 
                   
               
               
                   
                 metabolism 
                 ornithine 
                 GC/MS 
                 1493 
               
               
                   
                   
                 urea 
                 GC/MS 
                 1670 
               
               
                   
                   
                 proline 
                 LC/MS  
                 1898 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 citrulline 
                 LC/MS  
                 2132 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 trans-4- 
                 LC/MS  
                 32306 
               
               
                   
                   
                 hydroxyproline 
                 pos 
                   
               
               
                   
                   
                 homocitrulline 
                 LC/MS  
                 22138 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N-delta- 
                 LC/MS  
                 43249 
               
               
                   
                   
                 acetylornithine* 
                 pos 
                   
               
               
                   
                   
                 N2,N5- 
                 LC/MS  
                 43591 
               
               
                   
                   
                 diacetylornithine 
                 neg 
                   
               
               
                   
                 Creatine  
                 creatine 
                 LC/MS  
                 27718 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 creatinine 
                 LC/MS  
                 513 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Butanoate  
                 2-aminobutyrate 
                 LC/MS  
                 32348 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                 Polyamine  
                 5-methylthio- 
                 LC/MS  
                 1419 
               
               
                   
                 metabolism 
                 adenosine (MTA) 
                 pos 
                   
               
               
                   
                   
                 acisoga 
                 LC/MS  
                 43258 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Glutathione  
                 S-methyl- 
                 LC/MS  
                 33944 
               
               
                   
                   
                 glutathione 
                 pos 
                   
               
               
                   
                 metabolism 
                 5-oxoproline 
                 LC/MS  
                 1494 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glutathione,  
                 LC/MS  
                 27727 
               
               
                   
                   
                 oxidized (GSSG) 
                 pos 
                   
               
               
                   
                   
                 cysteine-glutathione  
                 LC/MS  
                 35159 
               
               
                   
                   
                 disulfide 
                 pos 
                   
               
               
                   
                   
                 ophthalmate 
                 LC/MS  
                 34592 
               
               
                   
                   
                   
                 pos 
                   
               
               
                 Peptide 
                 Dipeptide 
                 glycylproline 
                 LC/MS  
                 22171 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 leucylleucine 
                 LC/MS  
                 36756 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 pro-hydroxy-pro 
                 LC/MS  
                 35127 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 cysteinylglycine 
                 GC/MS 
                 35637 
               
               
                   
                   
                 valylalanine 
                 LC/MS  
                 41518 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 aspartylleucine 
                 LC/MS  
                 40068 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 isoleucylalanine 
                 LC/MS  
                 40046 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 leucylalanine 
                 LC/MS  
                 40010 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 leucylglutamate 
                 LC/MS  
                 40021 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 leucylphenylalanine 
                 LC/MS  
                 40026 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 leucylserine 
                 LC/MS  
                 40048 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 serylleucine 
                 LC/MS  
                 40066 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 threonylleucine 
                 LC/MS  
                 40051 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 tyrosylleucine 
                 LC/MS  
                 40031 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Dipeptide  
                 carnosine 
                 LC/MS  
                 1768 
               
               
                   
                 derivative 
                   
                 neg 
                   
               
               
                   
                   
                 anserine 
                 LC/MS  
                 15747 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 cys-gly, oxidized 
                 LC/MS  
                 18368 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 N-acetylcarnosine 
                 LC/MS  
                 43488 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 gamma- 
                 gamma-glutamylvaline 
                 LC/MS  
                 32393 
               
               
                   
                 glutamyl 
                   
                 pos 
                   
               
               
                   
                   
                 gamma-glutamyl- 
                 LC/MS  
                 37092 
               
               
                   
                   
                 2-aminobutyrate 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 18369 
               
               
                   
                   
                 glutamylleucine 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 34456 
               
               
                   
                   
                 glutamylisoleucine* 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 33949 
               
               
                   
                   
                 glutamylglycine 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 37539 
               
               
                   
                   
                 glutamylmethionine 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 33422 
               
               
                   
                   
                 glutamylphenylalanine 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 2734 
               
               
                   
                   
                 glutamyltyrosine 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 33364 
               
               
                   
                   
                 glutamylthreonine* 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 33947 
               
               
                   
                   
                 glutamyltryptophan 
                 pos 
                   
               
               
                   
                   
                 gamma- 
                 LC/MS  
                 37063 
               
               
                   
                   
                 glutamylalanine 
                 pos 
                   
               
               
                 Carbo- 
                 Aminosugars  
                 erythronate* 
                 GC/MS 
                 33477 
               
               
                 hydrate 
                 metabolism 
                 fucose 
                 GC/MS 
                 15821 
               
               
                   
                   
                 glucuronate 
                 GC/MS 
                 15443 
               
               
                   
                 Fructose,  
                 fructose 
                 GC/MS 
                 577 
               
               
                   
                 mannose,  
                 galactose 
                 GC/MS 
                 12055 
               
               
                   
                 galactose,  
                 mannitol 
                 GC/MS 
                 15335 
               
               
                   
                 starch, and  
                 mannose 
                 GC/MS 
                 584 
               
               
                   
                 sucrose  
                 sorbitol 
                 GC/MS 
                 15053 
               
               
                   
                 metabolism 
                 sucrose 
                 LC/MS  
                 1519 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 raffinose 
                 LC/MS  
                 586 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Oligo- 
                 lactobionate 
                 GC/MS 
                 20685 
               
               
                   
                 saccharide 
                   
                   
                   
               
               
                   
                 Glycolysis,  
                 glycerate 
                 GC/MS 
                 1572 
               
               
                   
                 gluconeo- 
                 glucose 1-phosphate 
                 GC/MS 
                 33755 
               
               
                   
                 genesis,  
                 glucose 
                 GC/MS 
                 20488 
               
               
                   
                 pyruvate  
                 1,6-anhydroglucose 
                 GC/MS 
                 21049 
               
               
                   
                 metabolism 
                 pyruvate 
                 GC/MS 
                 599 
               
               
                   
                   
                 lactate 
                 GC/MS 
                 527 
               
               
                   
                   
                 Isobar:  
                 LC/MS  
                 33001 
               
               
                   
                   
                 glucuronate,  
                 neg 
                   
               
               
                   
                   
                 galacturonate,  
                   
                   
               
               
                   
                   
                 5-keto-gluconate 
                   
                   
               
               
                   
                 Glyoxylate  
                 oxalate  
                 LC/MS  
                 20694 
               
               
                   
                 and  
                 (ethanedioate) 
                 neg 
                   
               
               
                   
                 dicarboxylate  
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Nucleotide  
                 arabitol 
                 GC/MS 
                 38075 
               
               
                   
                 sugars,  
                 ribitol 
                 GC/MS 
                 15772 
               
               
                   
                 pentose  
                 threitol 
                 GC/MS 
                 35854 
               
               
                   
                 metabolism 
                 gluconate 
                 GC/MS 
                 587 
               
               
                   
                   
                 ribose 
                 GC/MS 
                 12080 
               
               
                   
                   
                 ribonate 
                 GC/MS 
                 38818 
               
               
                   
                   
                 ribulose 
                 GC/MS 
                 35855 
               
               
                   
                   
                 xylitol 
                 GC/MS 
                 41319 
               
               
                   
                   
                 arabinose 
                 GC/MS 
                 575 
               
               
                   
                   
                 xylose 
                 GC/MS 
                 15836 
               
               
                   
                   
                 xylonate 
                 GC/MS 
                 35638 
               
               
                   
                   
                 xylulose 
                 GC/MS 
                 18344 
               
               
                 Energy 
                 Krebs  
                 citrate 
                 GC/MS 
                 1564 
               
               
                   
                 cycle 
                 cis-aconitate 
                 LC/MS  
                 12025 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 alpha-ketoglutarate 
                 GC/MS 
                 33453 
               
               
                   
                   
                 succinate 
                 LC/MS  
                 1437 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 fumarate 
                 GC/MS 
                 1643 
               
               
                   
                   
                 malate 
                 GC/MS 
                 1303 
               
               
                   
                 Oxidative  
                 acetylphosphate 
                 GC/MS 
                 15488 
               
               
                   
                 phos- 
                 phosphate 
                 GC/MS 
                 11438 
               
               
                   
                 phorylation 
                   
                   
                   
               
               
                 Lipid 
                 Essential  
                 linoleate (18:2n6) 
                 LC/MS  
                 1105 
               
               
                   
                 fatty acid 
                   
                 neg 
                   
               
               
                   
                   
                 linolenate [alpha  
                 LC/MS  
                 34035 
               
               
                   
                   
                 or gamma; 
                 neg 
                   
               
               
                   
                   
                 (18:3n3 or 6)] 
                   
                   
               
               
                   
                   
                 dihomo-linolenate  
                 LC/MS  
                 35718 
               
               
                   
                   
                 (20:3n3 or n6) 
                 neg 
                   
               
               
                   
                   
                 eicosapentaenoate  
                 LC/MS  
                 18467 
               
               
                   
                   
                 (EPA; 20:5n3) 
                 neg 
                   
               
               
                   
                   
                 docosapentaenoate  
                 LC/MS  
                 32504 
               
               
                   
                   
                 (n3 DPA; 22:5n3) 
                 neg 
                   
               
               
                   
                   
                 docosapentaenoate  
                 LC/MS  
                 37478 
               
               
                   
                   
                 (n6 DPA; 22:5n6) 
                 neg 
                   
               
               
                   
                   
                 docosahexaenoate  
                 LC/MS  
                 19323 
               
               
                   
                   
                 (DHA; 22:6n3) 
                 neg 
                   
               
               
                   
                 Medium  
                 caproate (6:0) 
                 LC/MS  
                 32489 
               
               
                   
                 chain  
                   
                 neg 
                   
               
               
                   
                 fatty  
                 heptanoate (7:0) 
                 LC/MS  
                 1644 
               
               
                   
                 acid 
                   
                 neg 
                   
               
               
                   
                   
                 caprylate (8:0) 
                 LC/MS  
                 32492 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 pelargonate (9:0) 
                 GC/MS 
                 12035 
               
               
                   
                   
                 laurate (12:0) 
                 GC/MS 
                 1645 
               
               
                   
                   
                 2-aminoheptanoic  
                 LC/MS  
                 43761 
               
               
                   
                   
                 acid 
                 pos 
                   
               
               
                   
                 Long  
                 myristate (14:0) 
                 LC/MS  
                 1365 
               
               
                   
                 chain  
                   
                 neg 
                   
               
               
                   
                 fatty  
                 pentadecanoate  
                 LC/MS  
                 1361 
               
               
                   
                 acid 
                 (15:0) 
                 neg 
                   
               
               
                   
                   
                 palmitate (16:0) 
                 LC/MS  
                 1336 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 palmitoleate  
                 LC/MS  
                 33447 
               
               
                   
                   
                 (16:1n7) 
                 neg 
                   
               
               
                   
                   
                 margarate (17:0) 
                 LC/MS  
                 1121 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 10-heptadecenoate  
                 LC/MS  
                 33971 
               
               
                   
                   
                 (17:1n7) 
                 neg 
                   
               
               
                   
                   
                 stearate (18:0) 
                 LC/MS  
                 1358 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 oleate (18:1n9) 
                 GC/MS 
                 1359 
               
               
                   
                   
                 cis-vaccenate  
                 GC/MS 
                 33970 
               
               
                   
                   
                 (18:1n7) 
                   
                   
               
               
                   
                   
                 nonadecanoate  
                 LC/MS  
                 1356 
               
               
                   
                   
                 (19:0) 
                 neg 
                   
               
               
                   
                   
                 10-nonadecenoate  
                 LC/MS  
                 33972 
               
               
                   
                   
                 (19:1n9) 
                 neg 
                   
               
               
                   
                   
                 arachidate (20:0) 
                 LC/MS  
                 44679 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 eicosenoate  
                 LC/MS  
                 33587 
               
               
                   
                   
                 (20:1n9 or 11) 
                 neg 
                   
               
               
                   
                   
                 dihomo-linoleate  
                 LC/MS  
                 17805 
               
               
                   
                   
                 (20:2n6) 
                 neg 
                   
               
               
                   
                   
                 mead acid (20:3n9) 
                 LC/MS  
                 35174 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 arachidonate  
                 LC/MS  
                 1110 
               
               
                   
                   
                 (20:4n6) 
                 neg 
                   
               
               
                   
                   
                 docosadienoate  
                 LC/MS  
                 32415 
               
               
                   
                   
                 (22:2n6) 
                 neg 
                   
               
               
                   
                   
                 adrenate (22:4n6) 
                 LC/MS  
                 32980 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Fatty  
                 9,10-epoxystearate 
                 LC/MS  
                 39627 
               
               
                   
                 acid,  
                   
                 neg 
                   
               
               
                   
                 oxidized 
                   
                   
                   
               
               
                   
                 Fatty  
                 myristate, methyl ester 
                 GC/MS 
                 12289 
               
               
                   
                 acid,  
                 pentadecanoate,  
                 GC/MS 
                 12288 
               
               
                   
                 methyl  
                 methyl ester 
                   
                   
               
               
                   
                 ester 
                 palmitate, methyl ester 
                 GC/MS 
                 12091 
               
               
                   
                   
                 margarate, methyl ester 
                 GC/MS 
                 11984 
               
               
                   
                   
                 stearate, methyl ester 
                 GC/MS 
                 6097 
               
               
                   
                   
                 oleate, methyl ester 
                 GC/MS 
                 36796 
               
               
                   
                   
                 linoleate, methyl ester 
                 GC/MS 
                 36801 
               
               
                   
                 Fatty  
                 3-hydroxypropanoate 
                 GC/MS 
                 42103 
               
               
                   
                 acid,  
                 2-hydroxystearate 
                 LC/MS  
                 17945 
               
               
                   
                 mono- 
                   
                 neg 
                   
               
               
                   
                 hydroxy 
                 2-hydroxypalmitate 
                 LC/MS  
                 35675 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Fatty  
                 2-hydroxyglutarate 
                 GC/MS 
                 37253 
               
               
                   
                 acid,  
                 sebacate  
                 LC/MS  
                 32398 
               
               
                   
                 dicarb- 
                 (decanedioate) 
                 neg 
                   
               
               
                   
                 oxylate 
                 azelate  
                 LC/MS  
                 18362 
               
               
                   
                   
                 (nonanedioate) 
                 neg 
                   
               
               
                   
                 Fatty acid,  
                 stearamide 
                 GC/MS 
                 37487 
               
               
                   
                 amide 
                   
                   
                   
               
               
                   
                 Fatty acid,  
                 suberylglycine 
                 LC/MS  
                 35419 
               
               
                   
                 beta- 
                   
                 neg 
                   
               
               
                   
                 oxidation 
                   
                   
                   
               
               
                   
                 Fatty acid,  
                 13-methylmyristic  
                 LC/MS  
                 38293 
               
               
                   
                 branched 
                 acid 
                 neg 
                   
               
               
                   
                   
                 15-methylpalmitate  
                 LC/MS  
                 38768 
               
               
                   
                   
                 (isobar with 2-  
                 neg 
                   
               
               
                   
                   
                 methylpalmitate) 
                   
                   
               
               
                   
                   
                 17-methylstearate 
                 LC/MS  
                 38296 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Fatty acid  
                 propionylcarnitine 
                 LC/MS  
                 32452 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                 (also BCAA 
                 propionylglycine 
                 LC/MS  
                 31932 
               
               
                   
                 metabolism) 
                   
                 neg 
                   
               
               
                   
                   
                 butyrylcarnitine 
                 LC/MS  
                 32412 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Fatty acid  
                 isovalerate 
                 LC/MS  
                 34732 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 hexanoylglycine 
                 LC/MS  
                 35436 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Carnitine  
                 deoxycarnitine 
                 LC/MS  
                 36747 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 carnitine 
                 LC/MS  
                 15500 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 3-dehydrocarnitine* 
                 LC/MS  
                 32654 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 acetylcarnitine 
                 LC/MS  
                 32198 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 hexanoylcarnitine 
                 LC/MS  
                 32328 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 octanoylcarnitine 
                 LC/MS  
                 33936 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 laurylcarnitine 
                 LC/MS  
                 34534 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 palmitoylcarnitine 
                 LC/MS  
                 22189 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 stearoylcarnitine 
                 LC/MS  
                 34409 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 oleoylcarnitine 
                 LC/MS  
                 35160 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Bile acid  
                 glycocholate 
                 LC/MS  
                 18476 
               
               
                   
                 metabolism 
                   
                 pos 
                   
               
               
                   
                   
                 glycohyocholate 
                 LC/MS  
                 42574 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 taurohyocholate 
                 LC/MS  
                 42603 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 taurochenode- 
                 LC/MS  
                 18494 
               
               
                   
                   
                 oxycholate 
                 neg 
                   
               
               
                   
                   
                 taurodeoxycholate 
                 LC/MS  
                 12261 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycodeoxycholate 
                 LC/MS  
                 18477 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycochenode- 
                 LC/MS  
                 32346 
               
               
                   
                   
                 oxycholate 
                 neg 
                   
               
               
                   
                   
                 glycolithocholate 
                 LC/MS  
                 31912 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycolithocholate  
                 LC/MS  
                 32620 
               
               
                   
                   
                 sulfate* 
                 neg 
                   
               
               
                   
                   
                 taurolithocholate 
                 LC/MS  
                 31889 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycocholenate  
                 LC/MS  
                 32599 
               
               
                   
                   
                 sulfate* 
                 neg 
                   
               
               
                   
                   
                 taurocholenate sulfate* 
                 LC/MS  
                 32807 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 glycohyodeoxycholic  
                 LC/MS  
                 43501 
               
               
                   
                   
                 acid 
                 pos 
                   
               
               
                   
                 Glycerolipid  
                 ethanolamine 
                 GC/MS 
                 1497 
               
               
                   
                 metabolism 
                 glycerol 
                 GC/MS 
                 15122 
               
               
                   
                   
                 choline 
                 LC/MS  
                 15506 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 glycerol 3-phosphate  
                 GC/MS 
                 15365 
               
               
                   
                   
                 (G3P) 
                   
                   
               
               
                   
                   
                 glycerophosphoryl- 
                 LC/MS  
                 15990 
               
               
                   
                   
                 choline (GPC) 
                 pos 
                   
               
               
                   
                 Inositol  
                 myo-inositol 
                 GC/MS 
                 19934 
               
               
                   
                 metabolism 
                 chiro-inositol 
                 GC/MS 
                 37112 
               
               
                   
                   
                 pinitol 
                 GC/MS 
                 37086 
               
               
                   
                   
                 inositol  
                 GC/MS 
                 1481 
               
               
                   
                   
                 1-phosphate (I1P) 
                   
                   
               
               
                   
                 Ketone  
                 3-hydroxybutyrate  
                 GC/MS 
                 542 
               
               
                   
                 bodies 
                 (BHBA) 
                   
                   
               
               
                   
                   
                 1,2-propanediol 
                 GC/MS 
                 38002 
               
               
                   
                 Lysolipid 
                 1-palmitoylglycero- 
                 LC/MS  
                 35631 
               
               
                   
                   
                 phosphoethanolamine 
                 neg 
                   
               
               
                   
                   
                 2-palmitoylglycero- 
                 LC/MS  
                 35688 
               
               
                   
                   
                 phosphoethanolamine* 
                 neg 
                   
               
               
                   
                   
                 1-stearoylglycero- 
                 LC/MS  
                 34416 
               
               
                   
                   
                 phosphoethanolamine 
                 neg 
                   
               
               
                   
                   
                 1-oleoylglycero- 
                 LC/MS  
                 35628 
               
               
                   
                   
                 phosphoethanolamine 
                 neg 
                   
               
               
                   
                   
                 1-linoleoylglycero- 
                 LC/MS  
                 32635 
               
               
                   
                   
                 phosphoethanolamine* 
                 neg 
                   
               
               
                   
                   
                 2-linoleoylglycero- 
                 LC/MS  
                 36593 
               
               
                   
                   
                 phosphoethanolamine* 
                 neg 
                   
               
               
                   
                   
                 1-arachidonoylglycero- 
                 LC/MS  
                 35186 
               
               
                   
                   
                 phosphoethanolamine* 
                 neg 
                   
               
               
                   
                   
                 2-arachidonoylglycero- 
                 LC/MS  
                 32815 
               
               
                   
                   
                 phosphoethanolamine* 
                 neg 
                   
               
               
                   
                   
                 1-stearoylglycero- 
                 LC/MS  
                 34437 
               
               
                   
                   
                 phosphoglycerol 
                 neg 
                   
               
               
                   
                   
                 2-myristoylglycero- 
                 LC/MS  
                 35626 
               
               
                   
                   
                 phosphocholine* 
                 pos 
                   
               
               
                   
                   
                 1-palmitoylglycero- 
                 LC/MS  
                 33955 
               
               
                   
                   
                 phosphocholine (16:0) 
                 neg 
                   
               
               
                   
                   
                 2-palmitoylglycero- 
                 LC/MS  
                 35253 
               
               
                   
                   
                 phosphocholine* 
                 neg 
                   
               
               
                   
                   
                 1-palmitoleoylglycero- 
                 LC/MS  
                 33230 
               
               
                   
                   
                 phosphocholine (16:1)* 
                 pos 
                   
               
               
                   
                   
                 1-margaroylglycero- 
                 LC/MS  
                 33957 
               
               
                   
                   
                 phosphocholine (17:0) 
                 neg 
                   
               
               
                   
                   
                 1-stearoylglycero- 
                 LC/MS  
                 33961 
               
               
                   
                   
                 phosphocholine (18:0) 
                 pos 
                   
               
               
                   
                   
                 2-stearoylglycero- 
                 LC/MS  
                 35255 
               
               
                   
                   
                 phosphocholine* 
                 pos 
                   
               
               
                   
                   
                 1-oleoylglycerophos- 
                 LC/MS  
                 33960 
               
               
                   
                   
                 phocholine (18:1) 
                 neg 
                   
               
               
                   
                   
                 2-oleoylglycero- 
                 LC/MS  
                 35254 
               
               
                   
                   
                 phosphocholine* 
                 neg 
                   
               
               
                   
                   
                 1-linoleoylglycero- 
                 LC/MS  
                 34419 
               
               
                   
                   
                 phosphocholine (18:2n6) 
                 neg 
                   
               
               
                   
                   
                 2-linoleoylglycero- 
                 LC/MS  
                 38087 
               
               
                   
                   
                 phosphocholine* 
                 neg 
                   
               
               
                   
                   
                 1-dihomo-linoleoylglycero- 
                 LC/MS  
                 33871 
               
               
                   
                   
                 phosphocholine (20:2n6)* 
                 pos 
                   
               
               
                   
                   
                 1-eicosatrienoylglycero- 
                 LC/MS  
                 33821 
               
               
                   
                   
                 phosphocholine (20:3)* 
                 pos 
                   
               
               
                   
                   
                 1-arachidonoylglycero- 
                 LC/MS  
                 33228 
               
               
                   
                   
                 phosphocholine (20:4n6)* 
                 pos 
                   
               
               
                   
                   
                 2-arachidonoylglycero- 
                 LC/MS  
                 35256 
               
               
                   
                   
                 phosphocholine* 
                 pos 
                   
               
               
                   
                   
                 1-docosapentaenoyl- 
                 LC/MS  
                 37231 
               
               
                   
                   
                 glycero- 
                 pos 
                   
               
               
                   
                   
                 phosphocholine (22:5n3)* 
                   
                   
               
               
                   
                   
                 1-docosahexaenoylglycero- 
                 LC/MS  
                 33822 
               
               
                   
                   
                 phosphocholine (22:6n3)* 
                 pos 
                   
               
               
                   
                   
                 2-docosahexaenoyl- 
                 LC/MS  
                 35883 
               
               
                   
                   
                 glycero- 
                 neg 
                   
               
               
                   
                   
                 phosphocholine* 
                   
                   
               
               
                   
                   
                 1-palmitoylplasmenyl- 
                 LC/MS  
                 39270 
               
               
                   
                   
                 ethanolamine* 
                 neg 
                   
               
               
                   
                   
                 1-stearoylplasmenyl- 
                 LC/MS  
                 39271 
               
               
                   
                   
                 ethanolamine* 
                 neg 
                   
               
               
                   
                   
                 1-docosahexaenoyl- 
                 LC/MS  
                 44633 
               
               
                   
                   
                 glycero- 
                 neg 
                   
               
               
                   
                   
                 phosphoethanolamine* 
                   
                   
               
               
                   
                   
                 1-linolenoylglycero- 
                 LC/MS  
                 44562 
               
               
                   
                   
                 phosphocholine (18:3n3)* 
                 pos 
                   
               
               
                   
                   
                 1-eicosapentaenoyl- 
                 LC/MS  
                 44563 
               
               
                   
                   
                 glycero- 
                 pos 
                   
               
               
                   
                   
                 phosphocholine (20:5n3)* 
                   
                   
               
               
                   
                 Mono- 
                 1-palmitoylglycerol  
                 GC/MS 
                 21127 
               
               
                   
                 acyl- 
                 (1-monopalmitin) 
                   
                   
               
               
                   
                 glycerol 
                 1-oleoylglycerol  
                 LC/MS  
                 21184 
               
               
                   
                   
                 (1-monoolein) 
                 pos 
                   
               
               
                   
                   
                 1-linoleoylglycerol  
                 LC/MS  
                 27447 
               
               
                   
                   
                 (1-monolinolein) 
                 neg 
                   
               
               
                   
                 Sphingo- 
                 sphingosine 
                 LC/MS  
                 17747 
               
               
                   
                 lipid 
                   
                 pos 
                   
               
               
                   
                   
                 palmitoyl sphingomyelin 
                 GC/MS 
                 37506 
               
               
                   
                   
                 stearoyl sphingomyelin 
                 GC/MS 
                 19503 
               
               
                   
                 Sterol/ 
                 lathosterol 
                 GC/MS 
                 39864 
               
               
                   
                 Steroid 
                 cholesterol 
                 GC/MS 
                 63 
               
               
                   
                   
                 campesterol 
                 GC/MS 
                 39511 
               
               
                   
                   
                 4-androsten-3beta, 
                 LC/MS  
                 37202 
               
               
                   
                   
                 17beta-diol disultate 1* 
                 neg 
                   
               
               
                   
                   
                 4-androsten-3beta, 
                 LC/MS  
                 37203 
               
               
                   
                   
                 17beta-diol disulfate 2* 
                 neg 
                   
               
               
                   
                   
                 5alpha-androstan-3beta, 
                 LC/MS  
                 37190 
               
               
                   
                   
                 17beta-diol disulfate 
                 neg 
                   
               
               
                   
                   
                 5alpha-pregnan-3beta, 
                 LC/MS  
                 37198 
               
               
                   
                   
                 20alpha-diol disulfate 
                 neg 
                   
               
               
                   
                   
                 pregnen-diol disulfate* 
                 LC/MS  
                 32562 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 21-hydroxy- 
                 LC/MS  
                 37173 
               
               
                   
                   
                 pregnenolone disulfate 
                 neg 
                   
               
               
                 Nucleo- 
                 Purine  
                 xanthine 
                 GC/MS 
                 3147 
               
               
                 tide 
                 metabolism, 
                 xanthosine 
                 LC/MS  
                 15136 
               
               
                   
                 (hypo) 
                   
                 neg 
                   
               
               
                   
                 xanthine/ 
                 hypoxanthine 
                 LC/MS  
                 3127 
               
               
                   
                 inosine  
                   
                 pos 
                   
               
               
                   
                 containing 
                 inosine 
                 LC/MS  
                 1123 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2′-deoxyinosine 
                 LC/MS  
                 15076 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Purine  
                 adenine 
                 LC/MS  
                 554 
               
               
                   
                 metabolism,  
                   
                 pos 
                   
               
               
                   
                 adenine  
                 adenosine 
                 LC/MS  
                 555 
               
               
                   
                 containing 
                   
                 pos 
                   
               
               
                   
                   
                 N1-methyladenosine 
                 LC/MS  
                 15650 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 adenosine-2′,3′-cyclic  
                 LC/MS  
                 37467 
               
               
                   
                   
                 monophosphate 
                 pos 
                   
               
               
                   
                 Purine  
                 guanine 
                 GC/MS 
                 418 
               
               
                   
                 metabolism,  
                 7-methylguanine 
                 LC/MS  
                 35114 
               
               
                   
                 guanine  
                   
                 pos 
                   
               
               
                   
                 containing 
                 guanosine 
                 LC/MS  
                 1573 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2′-deoxy- 
                 LC/MS  
                 1411 
               
               
                   
                   
                 guanosine 
                 neg 
                   
               
               
                   
                   
                 N1-methylguanosine 
                 LC/MS  
                 31609 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N2, N2-dimethyl- 
                 LC/MS  
                 35137 
               
               
                   
                   
                 guanosine 
                 pos 
                   
               
               
                   
                   
                 N6-carbamoylthreonyl- 
                 LC/MS  
                 35157 
               
               
                   
                   
                 adenosine 
                 pos 
                   
               
               
                   
                 Purine  
                 urate 
                 GC/MS 
                 1604 
               
               
                   
                 metabolism,  
                 allantoin 
                 GC/MS 
                 1107 
               
               
                   
                 urate  
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Pyrimidine  
                 cytidine 
                 LC/MS  
                 514 
               
               
                   
                 metabolism,  
                   
                 neg 
                   
               
               
                   
                 cytidine  
                 2′-deoxycytidine 
                 LC/MS  
                 15949 
               
               
                   
                 containing 
                   
                 pos 
                   
               
               
                   
                 Pyrimidine  
                 orotate 
                 GC/MS 
                 1505 
               
               
                   
                 metabolism,  
                   
                   
                   
               
               
                   
                 orotate  
                   
                   
                   
               
               
                   
                 containing 
                   
                   
                   
               
               
                   
                 Pyrimidine  
                 thymidine 
                 LC/MS  
                 2183 
               
               
                   
                 metabolism,  
                   
                 pos 
                   
               
               
                   
                 thymine  
                   
                   
                   
               
               
                   
                 containing 
                   
                   
                   
               
               
                   
                 Pyrimidine  
                 uridine 
                 LC/MS  
                 606 
               
               
                   
                 metabolism,  
                   
                 neg 
                   
               
               
                   
                 uracil  
                 pseudouridine 
                 LC/MS  
                 33442 
               
               
                   
                 containing 
                   
                 pos 
                   
               
               
                   
                 Purine and  
                 methylphosphate 
                 GC/MS 
                 37070 
               
               
                   
                 pyrimidine  
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                 Cofactors  
                 Ascorbate  
                 gulono-1,4-lactone 
                 GC/MS 
                 33454 
               
               
                 and  
                 and  
                 threonate 
                 GC/MS 
                 27738 
               
               
                 vitamins 
                 aldarate 
                 glucurono-6,3-lactone 
                 GC/MS 
                 20680 
               
               
                   
                 metabolism 
                 arabonate 
                 GC/MS 
                 37516 
               
               
                   
                 Pterins 
                 isoxanthopterin 
                 LC/MS  
                 27732 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Hemoglobin  
                 heme 
                 LC/MS  
                 41754 
               
               
                   
                 and  
                   
                 pos 
                   
               
               
                   
                 porphyrin  
                 L-urobilin 
                 LC/MS  
                 40173 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 Coproporphyrin I 
                 LC/MS  
                 39318 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 coproporphyrin III 
                 LC/MS  
                 39317 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 bilirubin (Z,Z) 
                 LC/MS  
                 27716 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 bilirubin (E,E)* 
                 LC/MS  
                 32586 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 biliverdin 
                 LC/MS  
                 2137 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 protoporphyrin  
                 LC/MS  
                 39321 
               
               
                   
                   
                 IX 
                 neg 
                   
               
               
                   
                 Nicotinate  
                 nicotinamide 
                 LC/MS  
                 594 
               
               
                   
                 and  
                   
                 pos 
                   
               
               
                   
                 nicotinamide  
                 quinolinate 
                 GC/MS 
                 1899 
               
               
                   
                 metabolism 
                 N1-Methyl-2-pyridone- 
                 LC/MS  
                 40469 
               
               
                   
                   
                 5-carboxamide 
                 neg 
                   
               
               
                   
                 Pantothenate  
                 pantothenate 
                 LC/MS  
                 1508 
               
               
                   
                 and  
                   
                 neg 
                   
               
               
                   
                 CoA  
                   
                   
                   
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                   
                 Pyridoxal  
                 pyridoxate 
                 LC/MS  
                 31555 
               
               
                   
                 metabolism 
                   
                 neg 
                   
               
               
                   
                 Riboflavin  
                 riboflavin  
                 LC/MS  
                 1827 
               
               
                   
                 metabolism 
                 (Vitamin B2) 
                 pos 
                   
               
               
                   
                 Tocopherol  
                 alpha-tocopherol 
                 GC/MS 
                 1561 
               
               
                   
                 metabolism 
                   
                   
                   
               
               
                 Xeno- 
                 Benzoate  
                 hippurate 
                 LC/MS  
                 15753 
               
               
                 biotics 
                 metabolism 
                   
                 neg 
                   
               
               
                   
                   
                 3-hydroxyhippurate 
                 LC/MS  
                 39600 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 4-hydroxyhippurate 
                 LC/MS  
                 35527 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 3-hydroxymandelate 
                 GC/MS 
                 22112 
               
               
                   
                   
                 4-hydroxymandelate 
                 GC/MS 
                 1568 
               
               
                   
                   
                 benzoate 
                 GC/MS 
                 15778 
               
               
                   
                   
                 p-hydroxybenzaldehyde 
                 GC/MS 
                 17665 
               
               
                   
                 Chemical 
                 glycolate (hydroxyacetate) 
                 GC/MS 
                 15737 
               
               
                   
                   
                 2-hydroxyisobutyrate 
                 GC/MS 
                 22030 
               
               
                   
                   
                 glycerol 2-phosphate 
                 GC/MS 
                 27728 
               
               
                   
                   
                 3-hydroxypyridine 
                 GC/MS 
                 21169 
               
               
                   
                   
                 HEPES 
                 LC/MS  
                 21248 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 2-ethylhexanoate  
                 LC/MS  
                 35490 
               
               
                   
                   
                 (isobar with 2- 
                 neg 
                   
               
               
                   
                   
                 propylpentanoate) 
                   
                   
               
               
                   
                   
                 2-mercaptoethanol* 
                 GC/MS 
                 37225 
               
               
                   
                   
                 2-piperidinone 
                 LC/MS  
                 43400 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 dimethyl sulfone 
                 LC/MS  
                 43424 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                 Drug 
                 ketamine 
                 LC/MS  
                 35128 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 methylprednisolone 
                 LC/MS  
                 42977 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 pantoprazole 
                 LC/MS  
                 38609 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 vecuronium 
                 LC/MS  
                 42591 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 oxypurinol 
                 GC/MS 
                 41725 
               
               
                   
                 Food  
                 5-ketogluconate 
                 GC/MS 
                 15687 
               
               
                   
                 component/ 
                 quinate 
                 GC/MS 
                 18335 
               
               
                   
                 Plant 
                 benzyl alcohol 
                 GC/MS 
                 22294 
               
               
                   
                   
                 ergothioneine 
                 LC/MS  
                 37459 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 N-(2-furoyl)glycine 
                 LC/MS  
                 31536 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 stachydrine 
                 LC/MS  
                 34384 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 homostachydrine* 
                 LC/MS  
                 33009 
               
               
                   
                   
                   
                 pos 
                   
               
               
                   
                   
                 cinnamoylglycine 
                 LC/MS  
                 38637 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 1,1-kestotetraose 
                 LC/MS  
                 39796 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                   
                 equol glucuronide 
                 LC/MS  
                 41948 
               
               
                   
                   
                   
                 neg 
                   
               
               
                   
                 Sugar,  
                 erythritol 
                 GC/MS 
                 20699 
               
               
                   
                 sugar  
                   
                   
                   
               
               
                   
                 substitute,  
                   
                   
                   
               
               
                   
                 starch 
                   
                   
                   
               
               
                   
                 Bacterial 
                 Isobar: tartronate,  
                 GC/MS 
                 42356 
               
               
                   
                   
                 dihydroxyfumarate 
                   
                   
               
               
                   
                 Phthalate 
                 bis(2-ethylhexyl)phthalate 
                 GC/MS 
                 21069 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Biomarkers of Liver Function 
     This example describes the identification of biomarkers of liver function. 
     The glucose-amino acid cycle and branched-chain amino acid mobilization were markedly elevated in livers undergoing machine perfusion as described in Example 1. Glucose was elevated in MP relative to CSP liver perfusates throughout the study and may have been converted to lactate via the glycolysis pathway. In spite a small amount of glucose being initially present at the perfusate in the machine perfusion group, a constant rate of gluconeogenesis can be detected when additional metabolites are analyzed ( FIG. 20A ). Lactate is a predominant source of carbon atoms for glucose synthesis by gluconeogenesis. The livers had an intact aerobic metabolism and were able to convert lactate into pyruvate ( FIGS. 20B and 20C ). The same metabolic pathway can be illustrated by the progressive production of fructose-6-phosphate and glycose-6-phosphate in the livers under machine perfusion ( FIGS. 20D and 20E ). 
     Branched-chain amino acid (BCAA) oxidation showed a strong differential increase in machine perfusion perfusates at the 6 and 9 hour time points as demonstrated by the BCAAs valine, isoleucine, and leucine ( FIG. 17A-17C ) and their respective deamination products 3-methyl-2-oxobutyrate, 3-methyl-2-oxovalerate, and 4-methyl-2-oxopentanoate. Several primary metabolites and side-products of the BCAA pathways were elevated in the machine perfusion group. Deamination by branched-chain aminotransferase (BCAT) is the first (and fully reversible) step in the oxidation of BCAAs and is followed by the irreversible mitochondrial reaction catalyzed by branched-chain ketoacid dehydrogenase. The tricarboxylic acid (TCA) cycle component alpha-ketoglutarate is a co-substrate for BCAT and leads to the co-formation of glutamate. Alpha-ketoglutarate and glutamate were both elevated in the machine perfusion group relative to the cold storage group at 6 and 9 hour as was glutamine, which can be derived from glutamate. It is possible that alpha-ketoglutarate, glutamate, and glutamine were elevated in response to demand for alpha-ketoglutarate that promoted its production by the TCA cycle and its subsequent conversion to glutamate via the action of BCAT ( FIGS. 21A and 21B ). 
     Perfusate profiling revealed differences in stress responses over time between the two preservation conditions. Biochemicals that provide insight into oxidative, inflammatory, and energy stress were among those showing a strong separation between perfusate profiles from the two preservation conditions. For instance, although there were only 2 samples in the perfusate CSP group at the baseline (0 hour) time point, ascorbate was detected in both whereas it was not detected in any of the MP baseline samples. It was detected in all samples from the 6 and 9 hour time points in the CSP samples but was not detected in any of the MP samples at these time points ( FIG. 22 ). Pigs, unlike humans, are capable of synthesizing ascorbate but the expression of the key synthetic enzyme, L-gulono-gamma-lactone oxidase, varies with stress, so ascorbate&#39;s presence in the cold static preservation group samples but absence in the machine perfusion samples could be an indication of different levels of perceived oxidant stress by livers subjected to the different preservation conditions. 
     Evidence of lipoxygenase activity was observed and differed by preservation method. 15-HETE, the product of 15-lipoxygenase, was specifically elevated in machine perfusion perfusates whereas 12-HETE, the product of 12-lipoxygenase, was stably elevated in the cold storage preservation perfusate across all time points but was low in all MP perfusate samples ( FIGS. 23A and 23B ). 15-lipoxygenase is believed to play a role in the selective breakdown and recycling of peroxisomes whereas 12-HETE (and 15-HETE to a lesser extent) has a more traditional role in promoting inflammation. Altogether this suggests that inflammation was greater in the CSP group than MP samples. 
     Finally, bile production was increased within both 24 hours of liver allograft perfusion and over the 5-day post-operative period ( FIG. 24 ). This indicates overall organ function. In addition, biomarkers from bile could be used as indicators of organ function or dysfunction. 
     Example 6 
     Principal Component Analysis of Liver Perfusate 
     This example describes principal component analysis (PCA) of perfusate from MP or CSP livers. 
     PCA is a tool for defining primary characteristics of a highly-dimensional dataset. The analysis achieves dimension reduction by extracting a few (but not all) principal components that describe most of the variation in the original multivariate dataset with the least loss of information. Based on linear transformation and decomposition of a number of correlated variables of a multi-dimensional dataset to a number of uncorrelated components, principal components are identified. The principal components are estimated as the projections of the data set on the eigenvectors of the covariance or correlation matrix of the data set. See, e.g., Janes et al.,  Nat. Rev. Mol. Cell. Biol.  7:820, 2006; Mi et al.,  PLoS ONE  6:19424, 2011. 
     PCA was carried out on the metabolomics profile of liver perfusates from MP or CSP livers at 3, 6, and 9 hour time points. In the MP livers, variables representing carbohydrate metabolism (ribulose, ribose, glycolate) and antioxidant defenses (oxidized homo-glutathione (GSSG)) were principal drivers of metabolic changes ( FIG. 25 ). In the CSP livers, PCA showed ethanolamine to be the principal driver of metabolic changes, suggesting a role for fatty acid metabolism ( FIG. 26 ). 
     Example 7 
     Dynamic Bayesian Network Analysis of Liver Perfusate 
     The example describes dynamic Bayesian network (DBN) analysis of perfusate from MP or CSP livers. 
     DBN analysis infers graphs for each component individually from each liver. If an arrow exists in &gt;50% of the individual networks, it is included in the final consensus network. The thickness of the arrows indicate the percentage of individual networks in which it is present ( FIG. 27 ). 
     DBN was used to determine the role of different cytokines while interacting in response to an initial inflammatory event (e.g., ischemia-reperfusion acquired during liver preservation). The analysis suggests two different pathways for cytokine regulation in livers being perfused by CSP or MP ( FIG. 28 ). 
     In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.