Abstract:
A method for diagnosing heart failure in a subject is provided. The method includes steps of measuring a biological sample of the subject to obtain an amount of at least one biomarker selected from the group consisting of xanthine, spermidine, propionylcarnitine, butyrylcarnitine and P-cresyl sulfate; and comparing the amount of the at least one biomarker to a reference. Moreover, the present invention relates to a method for staging heart failure or evaluating a prognosis of heart failure in a subject.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    This invention relates to a method for diagnosing heart failure or evaluating a prognosis of heart failure in a subject. Moreover, the present invention also relates to a biomarker or kit for diagnosing heart failure or evaluating a prognosis of heart failure in a subject. 
         [0003]    2. Description of Related Art 
         [0004]    Heart failure (HF) is a complex clinical syndrome that represents the end stage of various cardiac diseases. In the past few decades, substantial advances have been made in understanding the underlying pathophysiology and hemodynamics, and in the development of novel pharmaceuticals and interventional therapies. Nevertheless, short- and long-term heart failure-related re-hospitalization and mortality remain high, and demand substantial amounts of healthcare resources. The limited effectiveness of current treatment strategy at the late stage of heart failure necessitates novel interventional measures to cult the maladaptive molecular processes at sub-clinical stage and to avoid the progression of heart failure to advanced stages. 
         [0005]    A variety of biomarkers for heart failure have been identified. B-type natriuretic peptide (BNP) and the N-terminal fragment of the proprotein have emerged as clinically useful markers for diagnosis and prognosis of heart failure. A recent study showed that natriuretic peptides also provide a prognosis for individuals at moderate risk of cardiovascular disease without overt symptoms. Unfortunately, these biomarkers do not provide additional information on molecular targets for therapeutical interventions. Additionally, application of a single biomarker may not be sufficient for evaluating patients with heart failure, and requires compensations through a combination of multiple molecules. 
         [0006]    The etiology of a substantial proportion of heart failure patients remains unexplained according to current knowledge on cardiovascular risk factors. Regardless of the heterogeneous etiologies, the development of heart failure is causally related to the inability of the heart to meet the metabolic demands of the body. The accompanying changes in global metabolism are suggestive of clinical application of heart failure-specific metabolome for diagnostic and prognostic purposes. Current staging on heart failure is based on the consensus developed from American College of Cardiology and the American Heart Association (ACC/AHA), instead of pathogenic mechanism. The ACC/AHA classification of heart failure has four stages. For example, stage A refers to those at risks for heart failure, but who have not yet developed structural heart changes (diabetics, those with coronary disease without prior infarct). Stage B refers to individuals with structural heart disease (i.e. reduced ejection fraction, left ventricular hypertrophy, chamber enlargement), however no symptoms of heart failure have ever developed. Stage C means that patients who have developed clinical heart failure. Stage D is meant to patients with refractory heart failure requiring advanced intervention (biventricular pacemakers, left ventricular assist device, or transplantation). 
         [0007]    In addition to heart failure staging by the definition of ACC/AHA, there is another classification to define the functional status of heart failure, called New York Heart Association functional classification (class I to class IV). This classification relates symptoms to everyday activities and the patient&#39;s quality of life. Class I: No limitation of physical activity. Ordinary physical activity does not cause undue fatigue, palpitation, or dyspnea (shortness of breath). Class II: Slight limitation of physical activity. Comfortable at rest, but ordinary physical activity results in fatigue, palpitation, or dyspnea. Class III: Marked limitation of physical activity. Comfortable at rest, but less than ordinary activity causes fatigue, palpitation, or dyspnea. Class IV: Unable to carry out any physical activity without discomfort; Symptoms of cardiac insufficiency at rest. If any physical activity is undertaken, discomfort is increased. 
         [0008]    Taking advantage of the high throughput and the potential of developing multiple biomarkers, metabolomics is a platform for identification of metabolic signatures associated with pre-heart failure to advanced heart failure subtypes independent of the limitations posed by established traditional risk factors. A thorough understanding of the perturbed metabolism in heart failure, together with advances in nutrigenomic research, potentially moves towards development of personalized preventive measures. 
         [0009]    US patent application publication 2012/0286157 A1 disclosed a method for diagnosing heart failure in a subject, wherein the method comprises determining in a sample of the subject the amount of at least one biomarker, such as mannose, hypoxanthine, glutamate, uric acid, aspartate and etc. In addition, it also disclosed the method for identifying whether a subject is in need for a therapy of heart failure or determining whether a heart failure therapy is successful. 
         [0010]    Although several biomarkers (such as mannose, hypoxanthine, aspartate and etc.) have been used for diagnosing heart failure, there is still a medical need to find more sensitive and specific biomarkers for diagnosing heart failure especially at the early stage of heart failure and evaluating a prognosis of heart failure. 
         [0011]    In the present invention, it was developed to determine the clinical application and significance of metabolomic analysis for diagnosing heart failure and evaluating a prognosis of heart failure, to explore the global sophisticated metabolic perturbation in patients with heart failure, and to provide sensitive evaluation of heart failure at different stages or in regression after therapeutic interventions. 
       SUMMARY OF THE INVENTION 
       [0012]    In view of the prior art&#39;s deficiency, the present invention provides a method for diagnosing heart failure in a subject, comprising steps of: measuring a biological sample of the subject to obtain an amount of at least one biomarker selected from the group consisting of xanthine, spermidine, propionylcarnitine, butyrylcarnitine and P-cresyl sulfate; and comparing the amount of the at least one biomarker to a reference. 
         [0013]    In one embodiment of the present invention, the biological sample is selected from the group consisting of blood, plasma, serum and urine. 
         [0014]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of amino acid; and comparing the amount of the amino acid to a reference of the amino acid. 
         [0015]    In one embodiment of the present invention, the amino acid is selected from the group consisting of glutamine, tyrosine, phenylalanine, histidine, arginine, leucine, tryptophan, threonine, isoleucine, lysine, methionine, valine, and proline. 
         [0016]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of hypoxanthine; and comparing the amount of the hypoxanthine to a reference of the hypoxanthine. 
         [0017]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of phosphatidylcholine; and comparing the amount of the phosphatidylcholine to a reference of the phosphatidylcholine. 
         [0018]    In one embodiment of the present invention, the phosphatidylcholine is selected from the group consisting of phosphatidylcholine diacyl C34:4, phosphatidylcholine acyl-alkyl C36:2, phosphatidylcholine acyl-alkyl C34:2, phosphatidylcholine acyl-alkyl C34:3, phosphatidylcholine diacyl C36:0, phosphatidylcholine diacyl C36:1, phosphatidylcholine diacyl C36:3, phosphatidylcholine diacyl C38:6, phosphatidylcholine diacyl C36:6, phosphatidylcholine diacyl C38:5, phosphatidylcholine diacyl C40:5, phosphatidylcholine diacyl C36:2, phosphatidylcholine acyl-alkyl C36:5, phosphatidylcholine diacyl C38:0, phosphatidylcholine acyl-alkyl C32:3, phosphatidylcholine diacyl C40:4, phosphatidylcholine acyl-alkyl C38:3 and phosphatidylcholine diacyl C42:6. 
         [0019]    In one embodiment of the present invention, the phosphatidylcholine is preferably selected from the group consisting of phosphatidylcholine acyl-alkyl C34:2, phosphatidylcholine acyl-alkyl C34:3 and phosphatidylcholine diacyl C34:4. 
         [0020]    The present invention further provides a method for staging heart failure in a subject, comprising steps of: measuring a biological sample of the subject to obtain an amount of at least one biomarker selected from the group consisting of xanthine, spermidine and propionylcarnitine; and comparing the amount of the at least one biomarker to a reference. 
         [0021]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of amino acid; and comparing the amount of the amino acid to a reference of the amino acid. 
         [0022]    In one embodiment of the present invention, the amino acid is selected from the group consisting of glutamine, tyrosine, phenylalanine, histidine, arginine, leucine, tryptophan, threonine, isoleucine, lysine, methionine, valine, and proline. 
         [0023]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of hypoxanthine; and comparing the amount of the hypoxanthine to a reference of the hypoxanthine. 
         [0024]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of phosphatidylcholine; and comparing the amount of the phosphatidylcholine to a reference of the phosphatidylcholine. 
         [0025]    In one embodiment of the present invention, the phosphatidylcholine is selected from the group consisting of phosphatidylcholine diacyl C34:4, phosphatidylcholine acyl-alkyl C36:2, phosphatidylcholine acyl-alkyl C34:2, phosphatidylcholine acyl-alkyl C34:3, phosphatidylcholine diacyl C36:0, phosphatidylcholine diacyl C36:1, phosphatidylcholine diacyl C36:3, phosphatidylcholine diacyl C38:6, phosphatidylcholine diacyl C36:6, phosphatidylcholine diacyl C38:5, phosphatidylcholine diacyl C40:5, phosphatidylcholine diacyl C36:2, phosphatidylcholine acyl-alkyl C36:5, phosphatidylcholine diacyl C38:0, phosphatidylcholine acyl-alkyl C32:3, phosphatidylcholine diacyl C40:4, phosphatidylcholine acyl-alkyl C38:3 and phosphatidylcholine diacyl C42:6. 
         [0026]    In one embodiment of the present invention, the phosphatidylcholine is preferably selected from the group consisting of phosphatidylcholine acyl-alkyl C34:2, phosphatidylcholine acyl-alkyl C34:3 and phosphatidylcholine diacyl C34:4. 
         [0027]    The present invention further provides a method for evaluating a prognosis of heart failure in a subject, comprising steps of: measuring a biological sample of the subject to obtain an amount of at least one biomarker selected from the group consisting of xanthine, spermidine, butyrylcarnitine and P-cresyl sulfate; and comparing the amount of the at least one biomarker to a reference. 
         [0028]    In one embodiment of the present invention, it further comprises steps of measuring a biological sample of the subject to obtain an amount of amino acid; and comparing the amount of the amino acid to a reference of the amino acid. 
         [0029]    In one embodiment of the present invention, the amino acid is an essential amino acid. 
         [0030]    In one embodiment of the present invention, the essential amino acid is selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. 
         [0031]    In one embodiment of the present invention, the essential amino acid is preferably selected from the group consisting of leucine, threonine and tryptophan. 
         [0032]    In one embodiment of the present invention, it further comprises a step of measuring in the biological sample to obtain dimethylarginine and a ratio of dimethylarginine/arginine. 
         [0033]    In one embodiment of the present invention, it further comprises a step of measuring in the biological sample to obtain symmetric dimethylarginine and a ratio of symmetric dimethylarginine/arginine. 
         [0034]    The present invention further provides a kit for diagnosing heart failure comprising of: a detector for detecting a biomarker selected from the group consisting of xanthine, spermidine, propionylcarnitine, butyrylcarnitine, P-cresyl sulfate and a combination thereof. 
         [0035]    In some embodiments of the present invention, metabonomics/metabolomics technology may use multivariate statistical techniques to analyze the highly complex data sets generated by high-throughput spectroscopy, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). In some aspects of the invention, the combined use of different types of spectroscopic platforms, such as gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS), can take advantage of complementary analytical outcomes and therefore, provide a broadened metabolic “window” for explaining the biological variations associated with pathophysiological conditions. In certain aspects of the invention, identifying metabolites that account for the differences between the metabolic profiles of people with heart failure and healthy counterparts can reveal important underlying molecular mechanisms of the disease. 
         [0036]    In some embodiments of the present invention, profiling methods may include gas chromatography and mass spectrometry. For example, the profiling methods according to an embodiment of the present invention may include gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS). In certain embodiments, more than one profiling method may be used to obtain data about metabolites in a patient sample. In some embodiments, one or more profiling methods may be used together with multivariate statistical techniques to assess a profile of metabolites in a patient sample. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0037]    The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
           [0038]      FIGS. 1A and 1B  show the diagnostic values of global metabolomics for heart failure. The plasma samples from patients at different stages (stages A, stage B, and stage C) of heart failure and normal subjects were collected for determination of global metabolite concentrations by LC-MS/MS. (A) OPLS-DA score plots show the considerable separation between the normal controls, and patients with heart failure at stages A and C. To discriminate the normal controls and patients at stage C by using the whole global metabolomics dataset, a global metabolomics-derived parameter was calculated, called t[1](shown on the x-axis). To discriminate the normal controls and patients at stage A by using the whole global metabolomics dataset, another global metabolomics-derived parameter was calculated, called t[0](shown on the y-axis). On the scale of t[1], the cluster area of patients at stage A is similar to the normal controls, however, shifting up on the scale of t[0] compared to the normal controls. (B) Based on the way how these global metabolomics-derived parameters were calculated, the values of t[1] and t[0] were calculated for patients at stage B. The score plots of patients at stage B span the regions among stage A, stage C and the normal control groups. 
           [0039]      FIG. 2  shows metabolic pathways implicated in the pathogenesis of heart failure (HF). Disturbances in the urea cycle (a), biopterin cycle (b), MTA cycle (c), methionine cycle (d), ornithine-proline-glutamate (e), polyamine synthesis (f), dopamine synthesis (g), methylation (creatinine and phosphatidylcholine) (h), transsulfuration (taurine) (i), p-cresyl sulfate synthesis (j) and purine metabolism (k) pathways were identified in HF patients. Metabolites significantly increased in HF patients (red); metabolites significantly decreased in HF patients (blue); metabolites unchanged in HF patients (black) and metabolites not assayed (grey). 
           [0040]      FIG. 3  shows serial follow-up after acute heart failure. BNP and tPS[1] levels are shown in 32 patients who survived longer than 12 months and improved significantly with New York Heart Association functional class I at the end of 12 months. N, normal controls; M0, M6 and M12, indicate values at pre-discharge, and 6 and 12 months after discharge, respectively. “tPS[ 1 ]”: Based on the combination of four metabolites (Histidine, Phenylalanine, Spermidine and Hypoxanthine), a parameter was produced, called tPS[ 1 ]. 
           [0041]      FIG. 4  shows the diagnostic values of BNP and a few targeted metabolites and targeted metabolites combinations. The ROC curves are shown for the diagnosis of stage C heart failure (versus normal controls) by B-type natriuretic peptide (BNP), t[2], and tPS[2]. “t[2]”: a parameter derived from taking all targeted metabolites into account; “tPS[2]”: Based on the combination of 4 metabolites (histidine, phenylalanine, spermidine, and phosphatidylcholine diacyl C34:4), a parameter was produced, called tPS[2]. 
           [0042]      FIGS. 5A to 5C  show the prognostic value of metabolomics. (A) The ROC curves for comparing the prognostic values of BNP, t[2], tPS[2], and tPS[3]. (B and C) The Kaplan-Meier curves of tPS[3] and BNP, respectively, for predicting a composite event of all-cause death and heart failure-related re-hospitalization. “tPS[3]”: Based on the combination of 4 classes of metabolites (dimethylarginine/Arginine ratio, spermidine, butyrylcarnitine, and total amount of essential amino acid), a parameter was produced, called tPS[3]. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0043]    The following specific examples are used for illustrating the present invention. A person skilled in the art can easily conceive the other advantages and effects of the present invention. The present invention can also be implemented by different specific cases be enacted or application, the details of the instructions can also be based on different perspectives and applications in various modifications and changes do not depart from the spirit of the creation. 
         [0044]    It is further noted that, as used in this specification, the singular forms “a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably with the term “and/or” unless the context clearly indicates otherwise. 
         [0045]    As used herein, the term “subject” or “individual” may be an animal. For example, the subject or individual may be a mammal. Also, the subject or individual may be a human. The subject or individual may be either a male or female. The subject or individual may also be a patient, where a patient is an individual who is under dental or medical care and/or actively seeking medical care for a disorder or disease. 
         [0046]    As used herein, the term “healthy” refers to an individual not having heart failure or other related disorders. 
         [0047]    As used herein, the term “metabolism” refers to the set of chemical reaction that occur in a living organism to maintain life. Metabolism is usually divided into two categories: catabolism and anabolism. Catabolism is a set of chemical reactions that breaks down organic matter (e.g., to harvest energy in cellular respiration). Anabolism is a set of chemical reactions that use energy to construct components of cells (e.g., protein and nucleic acid synthesis). 
         [0048]    As used herein, the term “biomarker” refers to a molecular species which serves as a distinctive biological or biologically derived indicator (such as a biochemical metabolite in the body) of a process, event, or condition (such as aging, disease, or exposure to a toxic substance). 
         [0049]    As used herein, the term “metabolite” is an intermediate or product of metabolism. The term metabolite is generally restricted to small molecules. A “primary metabolite” is a metabolite directly involved in normal growth, development, and reproduction (e.g., alcohol). A “secondary metabolite” is a metabolite not directly involved in those processes, but that usually has an important ecological function (e.g., antibiotics, pigments). Some antibiotics use primary metabolites as precursors, such as actinomycin which is created from the primary metabolite, tryptophan. Rather, for the purposes of the present invention, the term metabolite refers to the small molecules (&lt;1000 Dalton) intermediates and products involved in metabolic pathways such as glycolysis, the citric acid (TCA) cycle, amino acid synthesis and fatty acid metabolism, amongst others. 
         [0050]    As used herein, the term “metabolomics” or “metabonomics” refers to the systematic study of metabolite profiles generated by biological processes in a biological system under a given set of conditions. “Metabolome” refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample (e.g., a biological cell, tissue, organ or organism) that are the end products of cellular processes. Metabolomics is a platform technology to provide top-down, global, and unbiased information. There are two approaches to metabolomics: global metabolic profiling and targeted metabolomics. 
         [0051]    As used herein, the term “metabolite profile” or “metabolite biomarker profile” refers to a panel of metabolites that have been determined to have different levels (e.g., increased or decreased) in healthy subjects as compared to unhealthy subjects (e.g., subject having heart failure) or at different disease states (e.g., different stages of disease). 
         [0052]    As used herein, the term “heart failure (HF)” refers to a condition in which the function of the heart is impaired, such that the heart is unable to pump blood at an adequate rate or in adequate volume. Heart failure can be systolic, such that a significantly reduced ejection fraction of blood from the heart and, thus, a reduced blood flow. Therefore, systolic heart failure is characterized by a significantly reduced left ventricular ejection fraction (LVEF), preferably, an ejection fraction of less than 50%. Alternatively, heart failure can be diastolic, i.e. a failure of the ventricle to properly relax that is usually accompanied by a stiffer ventricular wall. The diastolic heart failure causes inadequate filling of the ventricle, and thus affects the blood flow. Thus, diastolic dysfunction also results in elevated end-diastolic pressures. Heart failure may, thus, affect the right heart (pulmonary circulation), the left heart (body circulation) or both. Techniques for measuring heart failure are well known in the art and include echocardiography, electrophysiology, angiography, and the determination of peptide biomarkers, such as the B-type Natriuretic Peptide (BNP) or the N-terminal fragment of its propeptide, in the blood. It is understood that heart failure can occur permanently or only under certain stress or exercise conditions. Typical symptoms of heart failure include dyspnea, chest pain, dizziness, confusion, pulmonary and/or peripheral edema. Heart failure can be classified as stages A, B, C and D according to American College of Cardiology and the American Heart Association 2001 guidelines. Stage A: patients at high risk for developing heart failure in the future but no functional or structural heart disorder. Stage B: a structural heart disorder but no symptoms at any stage. Stage C: previous or current symptoms of heart failure in the context of an underlying structural heart problem, but managed with medical treatment. Stage D: patients with refractory heart failure requiring advanced intervention. 
         [0053]    As used herein, the term “global metabolites” refers to obtain a global, extensive metabolite profiling which is used for comparison of a large number of analytes in a specific condition, or across several groups of different conditions. It can be achieved by analyzing replicate samples from different treatment conditions (e.g., drug-treated vs. control group) or different pathophysiological states (e.g. diabetic vs. normal group). For this purpose, biospecimens (cells, plasma, urine, saliva, or pathological specimens) are subjected to analysis (by analytical tools such as LC-MS) to generate datasets that are subsequently subjected to univariate or multivariate statistical analysis. Global metabolomics is aimed to identify features that may systemically differentiate a large number of metabolites into groups (classes). 
         [0054]    As used herein, the term “targeted metabolites” refers to the identification and quantification of a defined set of structurally known and annotated metabolites and is based on well established biochemical pathways. 
         [0055]    Many examples have been used to illustrate the present invention. The examples sited below should not be taken as a limit to the scope of the invention. 
       EXAMPLES 
     Materials and Methods for Metabolomics Analysis 
     1. Patients and Study Design: 
       [0056]    During the period from January, 2005 to December, 2009, patients at heart failure stages B and C were enrolled in this study. From May, 2008 to December, 2009, patients at heart failure stage A and normal controls were enrolled. Patients at stage C were those hospitalized due to acute cardiogenic pulmonary edema, and aged 20-85 years. Patients with systolic and diastolic heart failure were included. Patients at stage B were those with post-acute myocardial infarction regardless of their left ventricular ejection fraction (LVEF), those with any severe structure abnormalities or those with a LVEF of &lt;40%. But, patients at stage B are asymptomatic. Patients at stage A were (1) those who have an angiogram-documented coronary artery disease, a LVEF of &gt;50% and are asymptomatic; or (2) those who have risk factors, but are asymptomatic and have no angiogram-documented coronary disease. Normal controls were people who were aged 20-85 years, and had no significant systemic disease, such as hypertension, diabetes mellitus, or coronary artery disease. They were not on any medications, and had a LVEF of &gt;60%. 
         [0057]    Exclusion criteria included (1) the presence of systemic diseases such as hypothyroidism, decompensated liver cirrhosis, and systemic lupus erythematosus; (2) the presence of disorder other than heart failure that might compromise survival within 6 months; (3) patients being bed-ridden for &gt;3 months and/or unable to stand alone; (4) patients with serum creatinine &gt;3 mg/dl; and (5) patients with severe coronary artery disease without complete revascularization therapy. Informed consent was obtained from all patients. The study was designed and carried out in accordance with the principles of the Declaration of Helsinki and with approval from the Ethics Review Board of Chang Gung Memorial Hospital. 
       2. Blood Sampling and Examination 
       [0058]    Blood samples were collected before discharge, and at 6 and 12 months after discharge in EDTA-containing tubes. Plasma was analyzed by metabolomic workflow described in the succeeding section. BNP was measured in triplicate with the Triage BNP Test (Biosite, San Diego, Calif.), which was a fluorescence immunoassay for quantitative determination of plasma BNP. Other measurements, including kidney function, hemoglobin, and C-reactive protein, were conducted in the central core laboratory. 
       3. Disease Management Program 
       [0059]    The patients at stages C were taken care by an HF team consisting of three cardiologists specializing in HF care, one psychologist, one dietary assistant, and two case managers. 
       4. Follow-Up Program 
       [0060]    Follow-up data were prospectively obtained every month from hospital records, personal communication with the patients&#39; physicians, telephone interviews, and patients&#39; regular visits to staff physician outpatient clinics. “Re-hospitalization” was defined as HF-related re-hospitalizations. A committee of 3 cardiologists adjudicated all hospitalizations without knowledge of patients&#39; clinical variables to determine whether the events are related to worsening HF. “All-cause death” was chosen as an endpoint because of the interrelationship of HF with other comorbidities in the patient cohort. The most severe event was considered an endpoint during the follow-up period. Only was the composite event of HF-related re-hospitalization and all-cause death analyzed for the prognostic purpose. 
       5. Plasma Metabolome Analysis 
     (1) Plasma Global Metabolites Analysis by LC-TOFMS 
       [0061]    To 50-μl plasma, 200 μl acetonitrile (ACN) was added. The mixture was vortexed for 30 s, sonicated for 15 min. and centrifuged at 10,000×g for 25 min. The supernatant was collected into a separate glass tube. The pellets were re-extracted with 200 μl 50% methanol. The aqueous methanolic supernatant and acetonitrile supernatant were pooled and dried in a nitrogen evaporator. The residues were saved and stored at −80° C. For metabolomics analysis, the residues were suspended in 100 μl of 95:5 water/acetonitrile and centrifuged at 14,000×g for 5 min. The clear supernatant was collected for LC-MS analysis. 
         [0062]    Liquid chromatographic separation was achieved on a 100 mm×2.1 mm Acquity 1.7-μm C8 column (Waters Corp., Milford, USA) using a ACQUITY™ UPLC system (Waters Corp., Milford, USA). The column was maintained at 45□, and at a flow rate of 0.5 ml/min. Samples were eluted from LC column using a linear gradient: 0-2.5 min: 1-48% B; 2.5-3 min: 48-98% B; 3-4.2 min: 98% B; 4.3-6 min: 1% B for re-equilibration. The mobile phases were 0.1% formic acid (solvent A) in water and 0.1% formic acid in acetonitrile (solvent B). 
         [0063]    The eluent was introduced into the TOF MS system (SYNAPT G1 high-definition mass spectrometer, Waters Corp., Milford, USA) and operated in a ESI-positive ion mode. The conditions were as follows: desolvation gas was set to 700 l/h at a temperature of 300° C., cone gas set to 25 l/h, and source temperature set at 80° C. The capillary voltage and cone voltage were set to 3,000 V and 35 V, respectively. The MCP detector voltage was set to 1,650 V. The data acquisition rate was set at 0.1 s with a 0.02 s interscan delay. The data were collected in centroid mode from 20 to 990 m/z. For accurate mass acquisition, a lock-mass of sulfadimethoxine at a concentration of 60 ng/ml and a flow rate of 60 □l/min (an [M+H] +  ion at 311.0814 Da in ESI-positive mode). 
         [0064]    Raw mass spectrometric data were processed using MassLynx V4.1 and MarkerLynx software (Waters Corp., Milford, USA). The intensity of each mass ion was normalized with respect to the total ion count to generate a data matrix that included the retention time, m/z value, and the normalized peak area. The multivariate data matrix was analyzed by SIMCA-P software (version 13.0, Umetrics AB, Umea, Sweden). OPLS-DA models were carried out prior to the Pareto scaling was applied. SIMCA-P had been used for multivariate data analysis and representation. 
         [0065]    Exact molecular mass data which showed significant differences between two groups, were then submitted for database searching, either in-house or using the online HMDB (http://www.hmdb.ca/) and KEGG (http://www.genome.jp/kegg/) databases. For identification of specific metabolites, standards were subject to UPLC-MS/MS analyses under the conditions identical to those of the profiling experiment. MS/MS spectra were collected at 0.1 spectra per second, with a medium isolation window of ˜4 m/z. The collision energy is set from 5 to 35 V. 
         [0066]    The construction, interaction, and pathway analysis of potential biomarkers was performed with MetaboAnalyst software based on database source including the KEGG and HMDB, to identify the affected metabolic pathways analysis and visualization. The possible biological roles were evaluated by the enrichment analysis. 
       (2) Quantitation of Plasma Targeted Metabolites (Concentrations Determination) 
       [0067]    The targeted metabolite analyses were carried out with the AbsoluteIDQ® p180 Kit (Biocrates Life Science AG Innsbruck, Austria). The kit was used to identify and quantify 184 metabolites covering five metabolite classes, namely 90 glycerophospholipids and 15 sphingolipids (76 phosphatidylcholines, 14 lysophosphatidylcholines and 15 sphingomyelines), 19 biogenic amines, 40 acyl carnitines, 19 amino acids, and hexose. Each 10 μL plasma sample was mixed with isotopically labeled internal standards in a 96-well multititer plate, and dried under a stream of nitrogen. Amino acids and biogenic amines were derivatized with 5% phenylisothiocyanate (PITC) for 20 min, and subsequently dried under nitrogen. Three hundred μL of extraction solution (5 mM ammonium acetate in methanol) was added, and after 30 min incubation, the mixture was centrifuged for 2 min at 100×g. Subsequently, a 150-μL aliquot of filtrate was transferred to a microtiter plate, and diluted with 150 μL of water for analysis of amino acids and biogenic amines by LC-MS/MS. The remaining filtrate was mixed with 400 μl of kit MS running solvent for flow injection analysis coupled with tandem mass spectrometric analysis (FIA-MS/MS). The analysis was performed in positive and negative electrospray ionization modes. Identification and quantification were achieved by multiple reaction monitoring (MRM) and standardized by spiking in of isotopically-labeled standards. In LC-MS analysis, the MS was coupled to an UPLC (Waters Corp, Milford, USA), and the metabolites were separated on a reverse phase column (2.1 mm×50 mm, BEH C18, Waters Corp, Milford, USA). The mobile phases were composed of a gradient mixture of solvent A (0.2% formic acid in water) and solvent B (0.2% formic acid in acetonitrile) (0 min 0% B, 3.5 min 60% B, 3.8 min 0% B, 3.9 min 0% B). Elution was performed at a flow rate of 900 μL/min. The column temperature was maintained at 50° C. For FIA, an isocratic method was used, kit MS running solvent as the mobile phase, with varying flow conditions (0 min, 30 μL/min; 1.6 min 30 μL/min; 2.4 min, 200 μL/min; 2.8 min, 200 μL/min; 3 min 30 μL/min). The corresponding MS settings were as follows: dwell time 0.019-0.025 sec; 3.92 KV voltage for positive mode; 1.5 KV for negative mode; nitrogen as collision gas medium; source temperature 150° C. The parameters for LC-MS were: dwell time 0.006-0.128 s; source temperature 150° C.; 3.20 KV voltage; nitrogen as collision gas medium. Data import and pre-processing steps for targeted MS data analyses were done using TargetLynx (Waters, Mass., USA). The integrated MetIDQ software (Biocrates, Innsbruck, Austria) was applied to streamline data analysis by automated calculation of metabolite concentrations. 
         [0000]    (3) Plasma p-Cresyl Sulfate and Indoxyl Sulfate Quantification 
         [0068]    Plasma samples (10 μL) were prepared by protein precipitation with 500 μL methanol (40 ng/ml d4-indoxyl sulfate as an internal standard) followed by centrifugation at 12,00×g for 10 min at 4° C. The supernatant was collected for p-cresyl sulfate and indoxyl sulfate analysis. LC-MS/MS was carried out on a Xevo TQ MS Acquity UPLC system (Waters Corp., Milford, USA). Separation was achieved on a reversed-phase Acquity UPLC BEH C18 column (1.7□μm, 100 mm×2.1 mm). The column was maintained at 40° C., and at a flow rate of 0.5 ml/min. Samples were eluted from LC column using a linear gradient: 0-0.5 min: 10-20% B; 0.5-3 min: 20-70% B; 3-3.5 min: 70-98% B; 3.5-5 min: 98% B; 5.1-7 min: 10% B for re-equilibration. The mobile phases were water (solvent A) and methanol (solvent B). Mass spectral ionization, fragmentation, and acquisition conditions were optimized on the tandem quadrupole mass spectrometer by using electrospray ionization (ESI) in the negative mode. The conditions were as follows: desolvation gas was set to 1000 l/h at a temperature of 500° C., cone gas set to 30 l/h, and source temperature set at 150° C. The capillary voltage and cone voltage were set to 800 V and 30 V, respectively. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode with dwell and interscan delay times of 0.2 and 0.1 s, respectively. Data were collected and processed by use of Masslynx software (version 4.0). 
       (4) Statistical Analysis 
       [0069]    Results are expressed as the mean±SD for continuous variables and as the number (percentage) for categorical variables. Data were compared by two-sample t-tests, ANOVA and Chi-square, when appropriate. Metabolomics analysis was performed with softwares as specified. To maximize identification of differences in metabolic profiles between groups, the orthogonal projection to latent structure discriminant analysis (OPLS-DA) model was applied, and performed using the SIMCA-P (version 13.0, Umetrics AB, Umea, Sweden). The variable importance in the projection (VIP) value of each variable in the model was calculated to indicate its contribution to the classification. A higher VIP value represents a stronger contribution to the discrimination among groups. The VIP values of those variables greater than 1.0 were considered significantly different. The diagnostic values of metabolomics and BNP for HF were presented by the area under the curve (AUC) of the receiver operating characteristic (ROC) curves. 
         [0070]    Follow-up data were collected as scheduled or at the last available visit. ROC curves and Kaplan-Meier analysis were used to determine the predictors of the first defined events (death, or HF-related re-hospitalization). For Kaplan-Meier analysis, the cutoff value was set at the mean of each variable to get the data of “Log Rank”. The AUC and the value of Log Rank were used to demonstrate the prognosis of metabolomics and BNP in patients with HF. All statistical analyses were 2-sided and performed using SPSS software (version 15.0, SPSS, Chicago, Ill., USA). A p value of &lt;0.05 was considered significant. 
       Example 1 
     Global Metabolomics Analysis for Diagnosing and Staging Heart Failure 
     1. Baseline Characteristics 
       [0071]    A total of 234 subjects were enrolled in this example. This included 51 normal subjects and 183 patients at stages A (n=43), B (n=67), and C (n=73). The baseline characteristics and laboratory data are shown in Table 1. In most of the variables, a significant trend of changes was noted from normal controls to patients at stage A, B, and C. Compared to the normal controls, patients at stage C had remarkably higher BNP levels, wider QRS complex, but lower total cholesterol, low and high density lipoprotein cholesterols, sodium, hemoglobin, albumin, and estimated glomerular filtration rate. In age, although there were no significant differences among the patient groups, they were older than the normal controls. In addition, the percentage of male was also higher in the patient groups. Coronary artery disease was the major etiology of HF patients. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Demographic and laboratory data in the normal controls and patients at stages A, B, and C 
               
             
          
           
               
                   
                 Normal 
                 Stage A 
                 Stage B 
                 Stage C 
                   
               
               
                   
                 n = 51 
                 n = 43 
                 n = 67 
                 n = 73 
                 P value 
               
               
                   
               
             
          
           
               
                 Age (years) 
                 55.2 ± 4.4  
                 60.1 ± 10.8 
                  59.9± 12.8 
                 64.1 ± 12.8 
                 &lt;0.01 
               
               
                 Male (%) 
                   19 (37.3) 
                 35 (81.4) 
                 57 (85.1) 
                 41 (56.2) 
                 &lt;0.01 
               
               
                 LVEF (%) 
                 72.3 ± 8.0  
                 70.1 ± 8.9  
                 50.5 ± 14.1 
                 37.2 ± 15.6 
                 &lt;0.01 
               
               
                 Blood pressure (mm Hg) 
                   
                   
                   
                   
                   
               
               
                 Systolic 
                 125.7 ± 15.7  
                 124.3 ± 16.8  
                 123.4 ± 19.9  
                 124.7 ± 19.7  
                 0.926 
               
               
                 Diastolic 
                 75.6 ± 12.1 
                 76.7 ± 11.8 
                 77.1 ± 12.8 
                 72.0 ± 12.5 
                 0.072 
               
               
                 Heart rate, beats/min 
                 72.3 ± 11.3 
                 76.7 ± 11.3 
                 73.6 ± 9.2  
                 79.5 ± 14.1 
                 0.003 
               
               
                 Co-morbidity 
                   
                   
                   
                   
                   
               
               
                 Diabetes mellitus (%) 
                 0 (0) 
                 18 (41.9) 
                 22 (32.8) 
                 38 (52.1) 
                 &lt;0.01 
               
               
                 Chronic kidney disease (%) 
                 0 (0) 
                 11 (25.6) 
                 16 (23.9) 
                 27 (37)   
                 &lt;0.01 
               
               
                 Hypertension (%) 
                 0 (0) 
                 31 (72.1) 
                 48 (71.6) 
                 54 (74.0) 
                 &lt;0.01 
               
               
                 Atrial fibrillation (%) 
                  0 (0.) 
                 3 (7.0) 
                  7 (10.4) 
                 21 (28.8) 
                 &lt;0.01 
               
               
                 COPD (%) 
                 0 (0) 
                 3 (7.0) 
                 2 (3.0) 
                 12 (16.4) 
                 0.002 
               
               
                 Ischemic (%) 
                 0 (0) 
                 31 (72.1) 
                 59 (8.1)  
                 44 (60.3) 
                 &lt;0.01 
               
               
                 Body mass index (kg/m 2 ) 
                 24.4 ± 3.3  
                 26.1 ± 4.2  
                 25.8 ± 4.3  
                 24.9 ± 4.6  
                 0.136 
               
               
                 Medication 
                   
                   
                   
                   
                   
               
               
                 ACEI or ARB (%) 
                 0 (0) 
                 15 (34.9) 
                 48 (71.6) 
                 62 (84.9) 
                 &lt;0.01 
               
               
                 β-Blocker (%) 
                 0 (0) 
                 18 (41.9) 
                 53 (79.1) 
                 51 (69.9) 
                 &lt;0.01 
               
               
                 Digoxin (%) 
                 0 (0) 
                 0 (0)   
                 5 (7.5) 
                 21 (28.8) 
                 &lt;0.01 
               
               
                 Diuretic (%) 
                 0 (0) 
                 4 (9.3) 
                 15 (22.4) 
                 49 (67.1) 
                 &lt;0.01 
               
               
                 Laboratory data 
                   
                   
                   
                   
                   
               
               
                 B-type natriuretic peptide (pg/ml) 
                 9.2 ± 8.8 
                 43.0 ± 69.4 
                 209.2 ± 359.7 
                 851.6 ± 793.8 
                 &lt;0.01 
               
               
                 Cholesterol (mg/dl) 
                 214.7 ± 35.5  
                 175.1 ± 43.0  
                 182.3 ± 41.9  
                 169.2 ± 58.6  
                 &lt;0.01 
               
               
                 Triglyceride (mg/dl) 
                 99.2 ± 55.9 
                 186.3 ± 110.3 
                 138.4 ± 100.6 
                 120.2 ± 68.6  
                 &lt;0.01 
               
               
                 Low density lipoprotein (mg/dl) 
                 139.2 ± 30.1  
                 104.3 ± 37.8  
                 112.9 ± 39.8  
                 108.1 ± 52.1  
                 &lt;0.01 
               
               
                 High density lipoprotein (mg/dl) 
                 55.3 ± 12.9 
                 39.5 ± 11.3 
                 42.2 ± 13.3 
                 38.6 ± 17.8 
                 &lt;0.01 
               
               
                 Serum sodium (mEq/L) 
                 140.4 ± 1.3  
                 138.5 ± 3.0  
                 139.3 ± 2.2  
                 138.6 ± 4.1  
                 0.003 
               
               
                 Hemoglobin (g/dL) 
                 13.9 ± 1.2  
                 13.9 ± 1.6  
                 13.8 ± 1.9  
                 12.6 ± 2.1  
                 &lt;0.01 
               
               
                 Total bilirubin (mg/dL) 
                 0.8 ± 0.3 
                 0.8 ± 0.3 
                 0.8 ± 0.4 
                 1.1 ± 0.6 
                 &lt;0.01 
               
               
                 Albumin (g/dl) 
                 4.4 ± 0.2 
                 4.1 ± 0.3 
                 3.9 ± 0.3 
                 3.5 ± 0.6 
                 &lt;0.01 
               
               
                 Hemoglobin Alc (%) 
                 5.7 ± 0.3 
                 6.6 ± 1.4 
                 6.5 ± 1.4 
                 6.7 ± 1.5 
                 &lt;0.01 
               
               
                 eGFR (ml/min/1.73 m 2 ) 
                 99.1 ± 19.2 
                 78.6 ± 24.6 
                 79.7 ± 32.2 
                 65.6 ± 27.9 
                 &lt;0.01 
               
               
                 QRS complex, msec 
                 89.6 ± 8.6  
                 94.3 ± 15.6 
                 96.9 ± 19.4 
                 106.3 ± 24.9  
                 &lt;0.01 
               
               
                   
               
               
                 ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; BMI, body mass index; COPD, chronic obstructive pulmonary disease; Chronic kidney disease, estimated glomerular filtration rate (eGFR) &lt;60 ml/min/1.73 m2; LVEF, left ventricular ejection fraction; MDP, multidisciplinary disease management group. 
               
             
          
         
       
     
       2. Global Metabolites Analysis in Heart Failure and Normal Controls 
       [0072]    Global metabolites analysis was performed in this example to separate patients at stages A, B, and C, from the normal controls. 
         [0073]    The OPLS-DA remarkably discriminated the normal controls, and patients at stages A and C in the global metabolites analysis ( FIG. 1A ). In the comparisons among the normal controls, and patients at stages A, B, and C, the metabolites with a VIP score &gt;1.0 are shown in Table 2. To discriminate the normal controls and patients at stage C by using the whole global metabolomics dataset, a global metabolomics-derived parameter was calculated, called t[1](shown on the x-axis). To discriminate the normal controls and patients at stage A by using the whole global metabolomics dataset, another global metabolomics-derived parameter was calculated, called t[0](shown on the y-axis). On the scale of t[1], the score plots cluster area of patients at stage A is similar to the normal controls, however, shifting up on the scale of t[0] compared to the normal controls ( FIG. 1A ). 
         [0074]    Based on the way how these global metabolomics-derived parameters were calculated, the values of t[1] and t[0] were calculated for patients at stage B. The score plots of patients at stage B span the regions among stage A, stage C and the normal control groups ( FIG. 1B ). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Statistical analysis of metabolites (with a VIP of &gt;1.0) between patients at stage C (C) and 
               
               
                 normal controls (N), and between patients at stage A (A) and normal controls (N), and between 
               
               
                 patients at stage B (B) and normal controls (N). 
               
             
          
           
               
                   
                   
                 Fold 
                   
                 Fold 
                   
                 Fold 
                   
               
               
                   
                   
                 Change 
                   
                 Change 
                   
                 Change 
               
               
                 Metabolite ID 
                 M/z 
                 (C/N) 
                 P-value 1   
                 (A/N) 
                 P-value 2   
                 (B/N) 
                 P-value 3   
               
               
                   
               
             
          
           
               
                 Spermine 
                 203.2235 
                 5.9020 
                 &lt;0.0001 
                 1.0588 
                 &lt;0.05 
                 2.0784 
                 &lt;0.0001 
               
               
                 Xanthine 
                 153.0412 
                 4.8854 
                 &lt;0.0001 
                 2.7859 
                 &lt;0.0001 
                 3.7723 
                 &lt;0.0001 
               
               
                 Oleamide 
                 282.2797 
                 4.8534 
                 0.0088 
                 2.7847 
                 &lt;0.0001 
                 3.7255 
                 &lt;0.0001 
               
               
                 Decatrienoylcarnitine 
                 310.2016 
                 4.4185 
                 &lt;0.0001 
                 2.6943 
                 &lt;0.0001 
                 3.5773 
                 &lt;0.0001 
               
               
                 Hypoxanthine 
                 137.0463 
                 3.2584 
                 &lt;0.0001 
                 4.8133 
                 &lt;0.0001 
                 2.9664 
                 &lt;0.0001 
               
               
                 Glutamate 
                 148.0610 
                 2.7740 
                 &lt;0.0001 
                 3.6739 
                 &lt;0.0001 
                 2.8397 
                 &lt;0.0001 
               
               
                 Pyroglutamic acid 
                 130.0504 
                 2.3127 
                 &lt;0.0001 
                 1.3353 
                 &lt;0.001 
                 2.1514 
                 &lt;0.0001 
               
               
                 P-Cresyl sulfate 
                 187.0065 
                 2.2620 
                 0.002 
                 1.3581 
                 0.004 
                 1.7175 
                 0.004 
               
               
                 Octadecadienylcarnitine 
                 424.3427 
                 1.9810 
                 &lt;0.0001 
                 1.5094 
                 &lt;0.0001 
                 1.2264 
                 &lt;0.05 
               
               
                 Butyrylcarnitine 
                 232.1549 
                 1.9418 
                 &lt;0.0001 
                 1.5241 
                 &lt;0.0001 
                 1.1803 
                 &lt;0.05 
               
               
                 Creatinine 
                 114.0667 
                 1.7820 
                 &lt;0.0001 
                 1.3404 
                 &lt;0.0001 
                 1.5646 
                 &lt;0.0001 
               
               
                 Indoxyl Sulfate 
                 212.0017 
                 1.6930 
                 0.008 
                 1.1258 
                 0.02 
                 1.4749 
                 0.003 
               
               
                 Hydroxybutyrylcarnitine 
                 248.1498 
                 1.6330 
                 &lt;0.0001 
                 1.3333 
                 &lt;0.05 
                 1.2000 
                 &lt;0.05 
               
               
                 Theophylline 
                 181.0725 
                 1.5251 
                 0.0126 
                 1.3112 
                 0.0126 
                 1.3722 
                 &lt;0.01 
               
               
                 Butrylcarnitine 
                 230.1392 
                 1.5110 
                 &lt;0.0001 
                 1.2361 
                 &lt;0.0001 
                 1.0667 
                 &lt;0.05 
               
               
                 Octadecenoylcarnitine 
                 426.3583 
                 1.5000 
                 &lt;0.0001 
                 1.2213 
                 &lt;0.0001 
                 1.0246 
                 &lt;0.05 
               
               
                 Ornithine 
                 133.0977 
                 1.4750 
                 &lt;0.0001 
                 0.9511 
                 &lt;0.05 
                 1.1969 
                 &lt;0.01 
               
               
                 Valerycarnitine 
                 246.1705 
                 1.4750 
                 0.002 
                 1.1287 
                 0.002 
                 1.4059 
                 &lt;0.0001 
               
               
                 Propionylcarnitine 
                 218.13868 
                 1.4518 
                 &lt;0.0001 
                 1.4778 
                 &lt;0.0001 
                 1.1373 
                 &lt;0.01 
               
               
                 Spermidine 
                 146.1657 
                 1.4420 
                 &lt;0.0001 
                 0.9496 
                 &lt;0.05 
                 1.0426 
                 &lt;0.05 
               
               
                 Phenylalanine 
                 166.0868 
                 1.3400 
                 &lt;0.0001 
                 1.3523 
                 &lt;0.0001 
                 1.1999 
                 &lt;0.01 
               
               
                 Hexose 
                 179.0555 
                 1.3230 
                 &lt;0.0001 
                 1.2690 
                 &lt;0.0001 
                 1.2261 
                 &lt;0.005 
               
               
                 Tetradecenoylcarnitine 
                 370.2957 
                 1.3100 
                 0.01 
                 1.0238 
                 &lt;0.05 
                 1.0714 
                 0.03 
               
               
                 Taurine 
                 126.0225 
                 1.3050 
                 0.001 
                 0.9082 
                 &lt;0.05 
                 1.1026 
                 0.01 
               
               
                 Proline 
                 116.07061 
                 1.2752 
                 &lt;0.0001 
                 1.3721 
                 &lt;0.0001 
                 1.2105 
                 &lt;0.0001 
               
               
                 Octenoylcarnitine 
                 286.2018 
                 1.2648 
                 0.0002 
                 1.2558 
                 0.0001 
                 1.2015 
                 0.0002 
               
               
                 Prolinebetaine 
                 144.1024 
                 1.2585 
                 0.0740 
                 1.5277 
                 &lt;0.005 
                 1.2003 
                 &lt;0.01 
               
               
                 Choline 
                 105.1153 
                 1.2265 
                 &lt;0.0001 
                 1.5128 
                 &lt;0.0001 
                 1.1944 
                 &lt;0.01 
               
               
                 Phosphatidylcholine acyl-alkyl C44:5 
                 878.7002 
                 1.2140 
                 0.005 
                 1.0607 
                 &lt;0.05 
                 1.0130 
                 0.03 
               
               
                 Hydroxypropionylcarnitine 
                 234.1336 
                 1.1954 
                 &lt;0.0001 
                 1.4776 
                 &lt;0.0001 
                 1.4286 
                 &lt;0.0001 
               
               
                 Tyrosine 
                 182.0817 
                 1.1800 
                 0.001 
                 0.9493 
                 &lt;0.05 
                 1.0567 
                 0.03 
               
               
                 Glycine 
                 76.0398 
                 1.1610 
                 0.005 
                 0.8737 
                 0.002 
                 0.9984 
                 0.04 
               
               
                 Isoleucine 
                 132.1024 
                 1.1610 
                 0.006 
                 1.2120 
                 &lt;0.001 
                 1.2198 
                 0.001 
               
               
                 Phosphatidylcholine diacyl C32:0 
                 734.5700 
                 1.1200 
                 0.007 
                 0.9955 
                 &lt;0.05 
                 0.9571 
                 0.03 
               
               
                 Uric acid 
                 169.0361 
                 1.1176 
                 &lt;0.0001 
                 1.3328 
                 &lt;0.0001 
                 1.2132 
                 &lt;0.0001 
               
               
                 Tiglylcarnitine 
                 244.1543 
                 1.1157 
                 &lt;0.0001 
                 1.5772 
                 &lt;0.0001 
                 1.0000 
                 0.87 
               
               
                 Essential amino acids 
                 — 
                 1.1052 
                 &lt;0.0001 
                 1.6377 
                 &lt;0.0001 
                 1.0900 
                 &lt;0.05 
               
               
                 Methionine 
                 150.0583 
                 0.9256 
                 0.024 
                 0.7983 
                 &lt;0.0001 
                 1.0389 
                 0.04 
               
               
                 Tetradecanoylcarnitine 
                 372.3114 
                 0.9000 
                 0.006 
                 0.9206 
                 &lt;0.05 
                 0.9048 
                 0.01 
               
               
                 Phosphatidylcholinediacyl C30:0 
                 706.5387 
                 0.8940 
                 0.017 
                 0.9555 
                 &lt;0.05 
                 0.8664 
                 0.001 
               
               
                 Phosphatidylcholine acyl-alkyl C38:4 
                 796.6220 
                 0.8890 
                 0.018 
                 0.9614 
                 &lt;0.05 
                 0.8581 
                 0.003 
               
               
                 Phosphatidylcholine acyl-alkyl C38:5 
                 794.6063 
                 0.8880 
                 0.02 
                 0.9050 
                 &lt;0.05 
                 0.8243 
                 0.005 
               
               
                 Phosphatidylcholine acyl-alkyl C38:2 
                 800.6533 
                 0.8740 
                 0.02 
                 0.8588 
                 &lt;0.05 
                 0.8301 
                 0.001 
               
               
                 Phosphatidylcholine diacyl C34:2 
                 758.5700 
                 0.8720 
                 0.005 
                 0.8914 
                 0.003 
                 0.8714 
                 0.02 
               
               
                 Phosphatidylcholine acyl-alkyl C30:1 
                 690.5437 
                 0.8640 
                 0.004 
                 0.8701 
                 0.001 
                 0.8019 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C38:1 
                 802.6689 
                 0.8590 
                 0.005 
                 0.8574 
                 0.001 
                 0.8351 
                 0.001 
               
               
                 L-Acetylcarnitine 
                 204.1236 
                 0.8578 
                 &lt;0.0001 
                 0.8773 
                 &lt;0.0001 
                 0.9336 
                 &lt;0.01 
               
               
                 Arginine 
                 175.1195 
                 0.8560 
                 0.021 
                 0.9121 
                 &lt;0.05 
                 1.0013 
                 0.1 
               
               
                 Phosphatidylcholine diacyl C42:6 
                 862.6326 
                 0.8530 
                 0.002 
                 0.9560 
                 &lt;0.05 
                 0.8881 
                 0.001 
               
               
                 Glutamine 
                 147.0769 
                 0.8510 
                 0.001 
                 0.8009 
                 &lt;0.001 
                 0.8722 
                 0.001 
               
               
                 Phosphatidylcholine acyl-alkyl C32:2 
                 716.5594 
                 0.8500 
                 0.015 
                 0.8234 
                 &lt;0.0001 
                 0.7136 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C40:6 
                 820.6220 
                 0.8470 
                 0.005 
                 0.8139 
                 &lt;0.0001 
                 0.7879 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C30:0 
                 692.5594 
                 0.8460 
                 0.003 
                 0.8824 
                 &lt;0.005 
                 0.7978 
                 &lt;0.0001 
               
               
                 Octanoylcarnitine 
                 288.2175 
                 0.8380 
                 0.014 
                 0.8731 
                 0.03 
                 0.8731 
                 0.001 
               
               
                 Phosphatidylcholine diacyl C38:4 
                 810.6013 
                 0.8350 
                 0.003 
                 1.0890 
                 0.01 
                 0.9346 
                 0.005 
               
               
                 Phosphatidylcholine diacyl C28:1 
                 676.4917 
                 0.8340 
                 0.001 
                 0.9123 
                 0.01 
                 0.8138 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C38:6 
                 806.5700 
                 0.8330 
                 0.013 
                 0.9321 
                 0.02 
                 0.8750 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C36:3 
                 784.5856 
                 0.8310 
                 &lt;0.0001 
                 1.0323 
                 &lt;0.05 
                 0.9398 
                 &lt;0.01 
               
               
                 Phosphatidylcholine acyl-alkyl C36:2 
                 772.6220 
                 0.8270 
                 &lt;0.0001 
                 0.8468 
                 &lt;0.0001 
                 0.7976 
                 &lt;0.0001 
               
               
                 Phosphatidylcholinediacyl C38:0 
                 818.6639 
                 0.8190 
                 0.003 
                 0.8103 
                 &lt;0.0001 
                 0.7697 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C36:4 
                 768.5907 
                 0.8160 
                 0.001 
                 0.8949 
                 0.003 
                 0.7891 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C36:3 
                 770.6063 
                 0.8160 
                 &lt;0.0001 
                 0.8352 
                 &lt;0.0001 
                 0.7576 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C36:5 
                 766.5750 
                 0.8150 
                 0.002 
                 0.8637 
                 0.003 
                 0.7856 
                 &lt;0.0001 
               
               
                 Tryptophan (fragment) 
                 188.0697 
                 0.8139 
                 &lt;0.0001 
                 0.8620 
                 &lt;0.0001 
                 0.8335 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C34:3 
                 756.5543 
                 0.8120 
                 0.003 
                 0.8950 
                 0.002 
                 0.8458 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C42:5 
                 864.6482 
                 0.8090 
                 0.003 
                 0.8723 
                 0.005 
                 0.8262 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C40:1 
                 830.7002 
                 0.8070 
                 0.001 
                 0.8211 
                 &lt;0.0001 
                 0.8218 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C40:6 
                 834.6013 
                 0.8050 
                 0.004 
                 0.8863 
                 0.003 
                 0.8621 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C40:4 
                 838.6326 
                 0.8050 
                 &lt;0.0001 
                 1.0210 
                 &lt;0.05 
                 0.8557 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C38:3 
                 812.6169 
                 0.8020 
                 &lt;0.0001 
                 1.0002 
                 0.04 
                 0.9272 
                 &lt;0.001 
               
               
                 Betaine 
                 118.0868 
                 0.7928 
                 &lt;0.0001 
                 0.7574 
                 &lt;0.0001 
                 0.7279 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C34:2 
                 744.5907 
                 0.7920 
                 &lt;0.0001 
                 0.7779 
                 &lt;0.0001 
                 0.7253 
                 &lt;0.0001 
               
               
                 Carnitine 
                 162.1130 
                 0.7891 
                 &lt;0.0001 
                 1.1229 
                 &lt;0.0001 
                 1.0993 
                 0.04 
               
               
                 Phosphatidylcholine diacyl C36:1 
                 788.6169 
                 0.7890 
                 &lt;0.0001 
                 0.9023 
                 &lt;0.05 
                 0.8522 
                 &lt;0.0001 
               
               
                 Decanoylcarnitine 
                 316.2488 
                 0.7840 
                 0.002 
                 0.8561 
                 &lt;0.0001 
                 0.8201 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C34:3 
                 742.5750 
                 0.7840 
                 0.001 
                 0.7399 
                 &lt;0.0001 
                 0.7068 
                 &lt;0.0001 
               
               
                 Histidine 
                 156.0773 
                 0.7830 
                 &lt;0.0001 
                 0.8303 
                 &lt;0.0001 
                 0.8799 
                 &lt;0.001 
               
               
                 Sphingomyelin C20:2 
                 756.1190 
                 0.7800 
                 &lt;0.0001 
                 0.9223 
                 &lt;0.01 
                 0.8123 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C38:6 
                 792.5907 
                 0.7770 
                 &lt;0.0001 
                 0.7949 
                 &lt;0.0001 
                 0.7334 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C36:0 
                 790.6326 
                 0.7740 
                 &lt;0.0001 
                 0.8538 
                 &lt;0.0001 
                 0.8029 
                 &lt;0.0001 
               
               
                 LysoPhosphatidylcholine acyl C 16:0 
                 496.3403 
                 0.7687 
                 0.0002 
                 1.0143 
                 0.02 
                 1.0327 
                 0.03 
               
               
                 Phosphatidylcholine diacyl C36:2 
                 786.6013 
                 0.7640 
                 &lt;0.0001 
                 0.8470 
                 &lt;0.0001 
                 0.8136 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C38:5 
                 808.5856 
                 0.7610 
                 &lt;0.0001 
                 0.9651 
                 &lt;0.05 
                 0.8378 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C40:5 
                 836.6169 
                 0.7480 
                 &lt;0.0001 
                 0.9421 
                 &lt;0.05 
                 0.8316 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C33:3 
                 742.5387 
                 0.7450 
                 &lt;0.0001 
                 0.7965 
                 &lt;0.0001 
                 0.7532 
                 &lt;0.0001 
               
               
                 Pipecolic acid 
                 130.0868 
                 0.7437 
                 &lt;0.0001 
                 0.7805 
                 &lt;0.0001 
                 0.7587 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C38:0 
                 804.6846 
                 0.7280 
                 0.008 
                 0.7808 
                 &lt;0.0001 
                 0.7692 
                 &lt;0.0001 
               
               
                 Phosphatidylcholinediacyl C36:5 
                 780.5543 
                 0.6740 
                 0.001 
                 0.8210 
                 &lt;0.0001 
                 0.7337 
                 &lt;0.0001 
               
               
                 LysoPhosphatidylcholine acyl C18:0 
                 524.3716 
                 0.6494 
                 &lt;0.0001 
                 0.8996 
                 &lt;0.001 
                 0.9093 
                 &lt;0.001 
               
               
                 LysoPhosphatidylcholine acyl C18:2 
                 520.3403 
                 0.6435 
                 &lt;0.0001 
                 0.9957 
                 &lt;0.01 
                 1.0422 
                 0.04 
               
               
                 Caffeine 
                 195.0882 
                 0.6418 
                 0.0488 
                 0.6822 
                 0.02 
                 0.7174 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C36:6 
                 778.5387 
                 0.6190 
                 &lt;0.0001 
                 0.7841 
                 &lt;0.0001 
                 0.7203 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine diacyl C34:4 
                 754.5387 
                 0.6130 
                 &lt;0.0001 
                 0.9723 
                 &lt;0.01 
                 0.7379 
                 &lt;0.0001 
               
               
                 Phosphatidylcholine acyl-alkyl C38:5 
                 794.6063 
                 0.8880 
                 0.02 
                 0.9050 
                 &lt;0.05 
                 0.8243 
                 0.005 
               
               
                 Phosphatidylcholine diacyl C36:6 
                 778.5387 
                 0.6190 
                 &lt;0.0001 
                 0.7841 
                 &lt;0.0001 
                 0.7203 
                 &lt;0.0001 
               
               
                 Phosphatidylcholin diacyl C34:4 
                 754.5387 
                 0.6130 
                 &lt;0.0001 
                 0.9723 
                 &lt;0.01 
                 0.7379 
                 &lt;0.0001 
               
               
                 M_008 
                 72.0815 
                 0.8319 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_014 
                 82.0149 
                 0.7686 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_018 
                 84.0818 
                 0.7004 
                 0.0007 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_049 
                 104.1077 
                 1.2265 
                 0.0004 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_051 
                 105.0338 
                 1.1593 
                 &lt;0.05 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_052 
                 105.0708 
                 0.8073 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_055 
                 110.0101 
                 0.7677 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_090 
                 126.0927 
                 2.2898 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_095 
                 130.0507 
                 2.6811 
                 0.0032 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_100 
                 130.1597 
                 0.4299 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_102 
                 131.0507 
                 1.1913 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_110 
                 134.0977 
                 0.8814 
                 0.0234 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_113 
                 136.0163 
                 6.7139 
                 0.0398 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_129 
                 143.9976 
                 1.1593 
                 &lt;0.05 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_147 
                 149.024 
                 0.8658 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_157 
                 152.0714 
                 22.8339 
                 0.0163 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_168 
                 158.156 
                 0.8417 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_207 
                 174.0538 
                 2.7941 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_214 
                 176.1189 
                 0.8740 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_227 
                 182.9644 
                 0.6641 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_235 
                 188.0697 
                 0.8139 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_239 
                 191.021 
                 1.2344 
                 0.0052 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_240 
                 191.0416 
                 0.7507 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_269 
                 205.0298 
                 4.8647 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_274 
                 207.0163 
                 0.7653 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_282 
                 209.1192 
                 0.9732 
                 &lt;0.05 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_315 
                 229.1551 
                 3.2227 
                 0.0002 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_382 
                 262.1299 
                 0.6037 
                 0.0005 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_390 
                 265.1193 
                 2.9553 
                 0.0044 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_457 
                 301.1416 
                 0.8767 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_489 
                 316.2487 
                 0.5370 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_526 
                 332.294 
                 0.4792 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_535 
                 337.0646 
                 2.5385 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_570 
                 354.1947 
                 0.7930 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_571 
                 355.0126 
                 0.5224 
                 0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_610 
                 375.0115 
                 1.1735 
                 0.0238 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_618 
                 381.1297 
                 131.8932 
                 &lt;0.05 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_636 
                 390.9849 
                 1.1508 
                 0.0164 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_653 
                 402.1797 
                 430.4213 
                 &lt;0.05 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_664 
                 409.1625 
                 0.8294 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_685 
                 425.1369 
                 0.8488 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_686 
                 426.213 
                 1.7022 
                 &lt;0.05 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_760 
                 520.3401 
                 0.6435 
                 0.0017 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_765 
                 526.2943 
                 0.1465 
                 &lt;0.0001 
                 — 
                 — 
                 — 
                 — 
               
               
                 M_0031 
                 171.1492 
                 0.9832 
                 &lt;0.05 
                 0.9967 
                 0.8737 
                 0.9980 
                 0.9257 
               
               
                 M_0375 
                 104.1072 
                 1.4218 
                 &lt;0.0001 
                 1.1121 
                 0.0578 
                 1.0181 
                 0.7367 
               
               
                 M_0526 
                 260.9942 
                 1.4266 
                 &lt;0.0001 
                 1.6292 
                 0.0002 
                 1.5814 
                 &lt;0.0001 
               
               
                 M_0708 
                 185.9643 
                 0.8677 
                 0.0001 
                 0.9628 
                 0.1338 
                 1.0048 
                 0.8473 
               
               
                 M_0761 
                 189.0869 
                 0.7760 
                 0.0192 
                 0.8589 
                 0.0321 
                 1.1955 
                 0.0583 
               
               
                 M_0943 
                 209.0555 
                 1.5244 
                 &lt;0.0001 
                 1.1174 
                 0.0615 
                 1.2614 
                 0.0207 
               
               
                 M_1034 
                 169.1075 
                 2.9547 
                 &lt;0.0001 
                 1.8887 
                 0.0110 
                 1.5405 
                 0.0315 
               
               
                 M_1131 
                 130.0863 
                 0.7631 
                 &lt;0.0001 
                 0.8427 
                 0.0188 
                 0.8580 
                 0.0252 
               
               
                 M_1133 
                 84.0809 
                 0.6868 
                 &lt;0.0001 
                 0.8004 
                 0.0099 
                 0.8035 
                 0.0117 
               
               
                 M_1174 
                 147.0443 
                 1.1629 
                 0.0179 
                 1.0552 
                 0.2445 
                 0.9313 
                 0.0835 
               
               
                 M_1185 
                 248.1491 
                 2.5233 
                 0.0007 
                 1.3870 
                 0.0788 
                 1.0831 
                 0.6350 
               
               
                 M_1253 
                 136.0153 
                 8.7313 
                 &lt;0.05 
                 1.1071 
                 0.1616 
                 0.9095 
                 0.0826 
               
               
                 M_1257 
                 263.1137 
                 8.1430 
                 &lt;0.05 
                 1.0430 
                 0.7413 
                 1.3713 
                 0.0497 
               
               
                 M_1326 
                 153.0659 
                 1.6787 
                 0.0406 
                 1.2339 
                 0.3834 
                 1.4802 
                 0.0963 
               
               
                 M_1463 
                 150.0588 
                 2.5757 
                 &lt;0.0001 
                 1.4064 
                 0.0040 
                 1.4420 
                 0.0017 
               
               
                 M_1465 
                 153.0659 
                 1.6336 
                 0.0114 
                 1.1852 
                 0.3109 
                 1.1744 
                 0.2405 
               
               
                 M_1611 
                 319.1363 
                 72.6607 
                 &lt;0.05 
                 1.7386 
                 0.3694 
                 0.8971 
                 0.8219 
               
               
                 M_1707 
                 146.0601 
                 1.3923 
                 0.0002 
                 0.9956 
                 0.9566 
                 0.9595 
                 0.5402 
               
               
                 M_1728 
                 120.0814 
                 1.2416 
                 &lt;0.0001 
                 1.1452 
                 0.0061 
                 1.0329 
                 0.3064 
               
               
                 M_1731 
                 93.0701 
                 1.2707 
                 &lt;0.0001 
                 1.1065 
                 0.0366 
                 1.0118 
                 0.7153 
               
               
                 M_1732 
                 77.039 
                 1.2538 
                 &lt;0.0001 
                 1.1568 
                 0.0036 
                 1.0339 
                 0.3102 
               
               
                 M_1733 
                 149.06 
                 1.2139 
                 &lt;0.0001 
                 1.1148 
                 0.0077 
                 1.0326 
                 0.2130 
               
               
                 M_1871 
                 126.0914 
                 2.2405 
                 &lt;0.0001 
                 1.3054 
                 0.0352 
                 1.2222 
                 0.1179 
               
               
                 M_2020 
                 574.9177 
                 5.2426 
                 0.001 
                 3.1454 
                 0.2943 
                 0.0000 
                 1.0000 
               
               
                 M_2217 
                 167.0335 
                 4.9645 
                 0.0166 
                 3.4615 
                 0.1669 
                 1.8851 
                 0.1975 
               
               
                 M_2253 
                 236.1834 
                 111.2498 
                 &lt;0.05 
                 316.5225 
                 0.0411 
                 689.2129 
                 0.0898 
               
               
                 M_2262 
                 130.0498 
                 1.6234 
                 0.0236 
                 1.2367 
                 0.2023 
                 1.1722 
                 0.3532 
               
               
                 M_2319 
                 373.122 
                 8176.337 
                 &lt;0.05 
                 6706.942 
                 0.3255 
                 6784.352 
                 0.3211 
               
               
                 M_2698 
                 357.1271 
                 18.5978 
                 &lt;0.05 
                 18.5726 
                 0.3249 
                 18.7389 
                 0.3052 
               
               
                 M_2833 
                 93.5765 
                 0.3091 
                 0.0030 
                 0.5402 
                 0.0883 
                 0.7212 
                 0.3630 
               
               
                 M_3029 
                 368.0871 
                 31.4366 
                 &lt;0.05 
                 2.2780 
                 0.2328 
                 1.6483 
                 0.1478 
               
               
                 M_3184 
                 336.0631 
                 10.1884 
                 &lt;0.05 
                 1.5120 
                 0.3697 
                 9.6124 
                 0.0097 
               
               
                 M_3293 
                 314.2325 
                 0.6385 
                 0.0033 
                 0.4211 
                 &lt;0.0001 
                 0.4280 
                 0.0000 
               
               
                 M_3342 
                 389.1235 
                 6.3609 
                 &lt;0.05 
                 1.4481 
                 0.3614 
                 5.0206 
                 0.2646 
               
               
                 M_3369 
                 316.2484 
                 0.4181 
                 &lt;0.0001 
                 0.3851 
                 &lt;0.0001 
                 0.3963 
                 0.0000 
               
               
                 M_3371 
                 328.2479 
                 0.6430 
                 0.0369 
                 0.5125 
                 0.0014 
                 0.5434 
                 0.0050 
               
               
                 M_3610 
                 288.2893 
                 29.0521 
                 0.0001 
                 9.4909 
                 0.0046 
                 0.8718 
                 0.3705 
               
               
                 M_3727 
                 91.0547 
                 5.5180 
                 &lt;0.05 
                 0.9161 
                 0.5246 
                 1.3875 
                 0.4406 
               
               
                 M_3728 
                 404.2072 
                 139.3227 
                 &lt;0.05 
                 1.2042 
                 0.6347 
                 1.1604 
                 0.5660 
               
               
                 M_3769 
                 356.2791 
                 0.5046 
                 0.0108 
                 0.3819 
                 0.0006 
                 0.5858 
                 0.0465 
               
               
                 M_3809 
                 358.2947 
                 0.6305 
                 &lt;0.05 
                 0.4494 
                 0.0154 
                 0.7409 
                 0.3529 
               
               
                 M_3871 
                 185.0959 
                 104.0675 
                 &lt;0.05 
                 1.1257 
                 0.2847 
                 1.1538 
                 0.2132 
               
               
                 M_3872 
                 231.1024 
                 16.9689 
                 &lt;0.05 
                 0.8612 
                 0.0904 
                 0.9964 
                 0.9743 
               
               
                 M_3911 
                 316.3205 
                 20.3052 
                 0.0031 
                 5.6069 
                 0.0393 
                 0.4379 
                 0.3228 
               
               
                 M_4074 
                 209.1171 
                 0.6845 
                 0.0499 
                 0.6871 
                 0.0200 
                 0.9313 
                 0.6958 
               
               
                 M_4133 
                 268.2633 
                 0.9246 
                 &lt;0.05 
                 0.9359 
                 0.2256 
                 0.9984 
                 0.9759 
               
               
                 M_4148 
                 376.2587 
                 0.0020 
                 &lt;0.0001 
                 0.2143 
                 &lt;0.0001 
                 0.6099 
                 0.0124 
               
               
                 M_4151 
                 421.3162 
                 0.0040 
                 &lt;0.0001 
                 0.2213 
                 &lt;0.0001 
                 0.6212 
                 0.0716 
               
               
                 M_4231 
                 119.0854 
                 1.8895 
                 0.0489 
                 1.4290 
                 0.0186 
                 1.0436 
                 0.6283 
               
               
                 M_4264 
                 512.3343 
                 1.3509 
                 0.0070 
                 1.1561 
                 0.2250 
                 1.1989 
                 0.1077 
               
               
                 M_4604 
                 249.1847 
                 0.9847 
                 &lt;0.05 
                 1.0110 
                 0.3092 
                 1.0066 
                 0.6168 
               
               
                 M_4780 
                 399.2507 
                 0.9782 
                 &lt;0.05 
                 0.9902 
                 0.5343 
                 1.0076 
                 0.6723 
               
               
                 M_4781 
                 299.1614 
                 0.9735 
                 &lt;0.05 
                 0.9836 
                 0.2881 
                 1.0066 
                 0.6868 
               
               
                 M_4889 
                 646.2779 
                 0.6875 
                 0.0194 
                 0.8420 
                 0.1843 
                 0.8011 
                 0.0389 
               
               
                 M_4900 
                 626.296 
                 0.6965 
                 0.0035 
                 0.8115 
                 0.0311 
                 0.7268 
                 0.0002 
               
               
                 M_5003 
                 380.2488 
                 1.5854 
                 0.0004 
                 1.0729 
                 0.6958 
                 1.7496 
                 &lt;0.0001 
               
               
                 M_0116 
                 331.8446 
                 — 
                 — 
                 1.0948 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_0396 
                 162.1129 
                 — 
                 — 
                 1.0443 
                 &lt;0.05 
                 — 
                 — 
               
               
                 M_0417 
                 122.027 
                 — 
                 — 
                 2.0851 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_0527 
                 242.9827 
                 — 
                 — 
                 1.2182 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_0528 
                 301.0738 
                 — 
                 — 
                 0.8475 
                 0.0008 
                 — 
                 — 
               
               
                 M_0667 
                 383.1133 
                 — 
                 — 
                 2.0964 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_0899 
                 452.9094 
                 — 
                 — 
                 0.8593 
                 0.0017 
                 — 
                 — 
               
               
                 M_1106 
                 146.0271 
                 — 
                 — 
                 1.4223 
                 0.0008 
                 — 
                 — 
               
               
                 M_1176 
                 91.0545 
                 — 
                 — 
                 0.9009 
                 0.0053 
                 — 
                 — 
               
               
                 M_1178 
                 205.0286 
                 — 
                 — 
                 0.7732 
                 0.0016 
                 — 
                 — 
               
               
                 M_1496 
                 86.0968 
                 — 
                 — 
                 1.0969 
                 0.0436 
                 — 
                 — 
               
               
                 M_1639 
                 367.1497 
                 — 
                 — 
                 3.4365 
                 0.0004 
                 — 
                 — 
               
               
                 M_1916 
                 130.0652 
                 — 
                 — 
                 0.8769 
                 0.0001 
                 — 
                 — 
               
               
                 M_2356 
                 340.031 
                 — 
                 — 
                 3.1049 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2402 
                 260.1854 
                 — 
                 — 
                 0.6278 
                 0.0047 
                 — 
                 — 
               
               
                 M_2435 
                 304.2116 
                 — 
                 — 
                 0.5018 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2498 
                 340.0362 
                 — 
                 — 
                 3.1534 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2563 
                 121.0281 
                 — 
                 — 
                 3.3815 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2803 
                 308.0509 
                 — 
                 — 
                 666.3764 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2804 
                 310.0481 
                 — 
                 — 
                 344.3028 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2805 
                 198.0316 
                 — 
                 — 
                 26.2854 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_2806 
                 262.0455 
                 — 
                 — 
                 11.1538 
                 0.0002 
                 — 
                 — 
               
               
                 M_3102 
                 332.2427 
                 — 
                 — 
                 0.3681 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3201 
                 235.9849 
                 — 
                 — 
                 5.2144 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3205 
                 237.9557 
                 — 
                 — 
                 18.6739 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3443 
                 340.2479 
                 — 
                 — 
                 0.3948 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3507 
                 342.2635 
                 — 
                 — 
                 0.3958 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3759 
                 368.2787 
                 — 
                 — 
                 0.3769 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3882 
                 370.0874 
                 — 
                 — 
                 924.6001 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_3883 
                 372.0844 
                 — 
                 — 
                 234.7603 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_4313 
                 321.1415 
                 — 
                 — 
                 4.9596 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_4556 
                 546.3553 
                 — 
                 — 
                 1.1106 
                 &lt;0.05 
                 — 
                 — 
               
               
                 M_4886 
                 240.0995 
                 — 
                 — 
                 0.8120 
                 0.0033 
                 — 
                 — 
               
               
                 M_4948 
                 502.3707 
                 — 
                 — 
                 1.4365 
                 &lt;0.0001 
                 — 
                 — 
               
               
                 M_5000 
                 337.1676 
                 — 
                 — 
                 1.7275 
                 &lt;0.0001 
                 — 
                 — 
               
               
                   
               
               
                 Essential AA: essential amino acids. 
               
               
                 P-value 1 , the difference between stage C and normal controls; 
               
               
                 P-value 2 , the difference between stage A and normal controls; 
               
               
                 P-value 3 , the difference between stage B and normal controls. 
               
             
          
         
       
     
       3. Identification of Abnormal Metabolic Pathways in HF 
       [0075]    Different categories of metabolites changed at different stages of HF (Table 2). These metabolites include purines, amino acids, biogenic amines, and phospholipids. Compared to the controls, arginine metabolism, urea cycle, purine metabolism, and nitric oxide synthesis pathways were markedly affected in the stage C patients. Levels of some metabolites related to the arginine metabolism, such as glutamine and citrulline, were lowered in the stage C patients. Levels of hypoxanthine, xanthine, uric acid, glutamate, proline, ornithine, spermine and spermidine were elevated in the stage C patients. Aromatic amino acids, such as tyrosine and phenylalanine, were higher in the stage C patients. In addition, levels of several phosphatidylcholines decreased whilst taurine increased. These findings of global metabolites changes in patients at stage C were mapped onto the biochemical pathways through KEGG and HMDB databases ( FIG. 2 ). These findings could demonstrate the abnormalities of metabolites changes behind the clinical presentation of HF, and provide more information related to the disease mechanisms. 
       4. Different Combinations of Global Metabolites to Discriminate Patients at Different Stages of HF, and the Normal Controls 
       [0076]    To discriminate patients at stage A from the normal subjects, a few good metabolite combinations were found as shown in the Table 3. The diagnosis values of these combinations are analyzed by receiver operating characteristic (ROC) curves, and are presented by the area under the curve (AUC). The diagnostic values of metabolomics-derived parameters are better than BNP. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Different combinations of global metabolites to discriminate 
               
               
                 patients at stage A from the normal subjects 
               
             
          
           
               
                   
                 Area under 
               
               
                   
                 the curve 
               
               
                 Metabolites and BNP 
                 (AUC) 
               
               
                   
               
               
                 BNP 
                 0.762 
               
               
                 Gln + PCaeC34:2 + C18:2 + Ile + Hypoxanthine 
                 0.973 
               
               
                 Gln + PCaeC34:2 + C18:2 + Hypoxanthine 
                 0.971 
               
               
                 Gln + PCaeC34:2 + Hypoxanthine 
                 0.969 
               
               
                 Gln + PCaeC34:2 + C3 + TyrPhe + ILe + Hypoxanthine 
                 0.974 
               
               
                 Gln + PCaeC34:2 + C3 + TyrPhe + Hypoxanthine 
                 0.972 
               
               
                 Gln + PCaeC34:2 + C3 + Hypoxanthine 
                 0.970 
               
               
                 Gln + Spermidine + PCaeC34:3 + Hypoxanthine 
                 0.973 
               
               
                 Gln + Spermidine + PCaeC34:3 + Xanthine 
                 0.974 
               
               
                 Gln + Spermidine + PCaeC34:2 + Hypoxanthine 
                 0.973 
               
               
                 Gln + Spermidine + PCaeC34:2 + Xanthine 
                 0.974 
               
               
                 Creatinine + Met + PCaeC34:3 + Essential 
                 0.972 
               
               
                 AA + C5:1 + C3OH + His + Pro 
                   
               
               
                 Creatinine + Met + PCaeC34:3 + C5:1 + Pro 
                 0.933 
               
               
                 Creatinine + Met + PCaeC34:3 + Pro 
                 0.914 
               
               
                 Met + PCaeC34:3 + Pro 
                 0.826 
               
               
                 Gln + PCaeC34:2 + C3 + Creatinine 
                 0.899 
               
               
                 Gln + PCaeC34:2 + Creatinine 
                 0.868 
               
               
                 Gln + PCaeC34:2 + C18:2 + Ile 
                 0.892 
               
               
                 Gln + PCaeC34:2 + C18:2 
                 0.862 
               
               
                 Gln + PCaeC34:2 
                 0.806 
               
               
                 Gln + PCaeC34:2 + C3 + TyrPhe + Ile 
                 0.801 
               
               
                 Gln + PCaeC34:2 + C3 + TyrPhe 
                 0.895 
               
               
                 Gln + PCaeC34:2 + C3 
                 0.869 
               
               
                   
               
               
                 BNP: B-type natriuretic peptide, Met: Methionine; Met: Methionine; Essential AA: Essential amino acid; PCaeC34:3: Phosphatidylcholine acyl-alkyl C34:3, C5:1: Tiglylcarnitine, C3OH: Hydroxypropionylcarnitine, His: Histidine, Pro: Proline, Gln: Glutamine, PCaeC34:2: Phosphatidylcholine acyl-alkyl C34:2, C3: Propionylcarnitine, Tyr: Tyrosine, Phe: Phenylalanine, Ile: Isoleucine, C18:2: Octadecadienylcarnitine. 
               
             
          
         
       
     
         [0077]    To discriminate patients at stage A from patients at stage C, a few good metabolite combinations were found as shown in the Table 4. The diagnosis values of these combinations are analyzed by ROC curves, and are presented by the area under the curve (AUC). The diagnostic values of metabolomics-derived parameters are better than BNP. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Different combinations of global metabolites to discriminate 
               
               
                 patients at stage A from patients at stage C 
               
             
          
           
               
                   
                 Area under 
                 P value 
               
               
                   
                 the curve 
                 (compared 
               
               
                 Metabolites and BNP 
                 (AUC) 
                 to BNP) 
               
               
                   
               
               
                 BNP 
                 0.942 
                 — 
               
               
                 Fisher Ratio + His + Phe + Spermidine 
                 0.966 
                 0.05 
               
               
                 His + Phe + Spermidine 
                 0.957 
                 0.12 
               
               
                 His + Phe + Spermidine + PCaeC34:3 
                 0.971 
                 0.04 
               
               
                 Fisher Ratio + His + Phe + Spermidine + 
                 0.973 
                 0.04 
               
               
                 Hypoxanthine 
                   
                   
               
               
                 His + Phe + Spermidine + Hypoxanthine 
                 0.970 
                 0.04 
               
               
                 His + Phe + Spermidine + PCaeC34:3 + 
                 0.972 
                 0.04 
               
               
                 Hypoxanthine 
               
               
                   
               
               
                 BNP: B-type natriuretic peptide, Fisher Ratio: ratio of branched chain amino acids to aromatic amino acids, PCaeC34:3: Phosphatidylcholine acyl-alkyl C34:3, His: Histidine, Phe: Phenylalanine. 
               
             
          
         
       
     
         [0078]    To discriminate patients at stage C from the normal subjects, a few good metabolite combinations were found as shown in the Table 5. The diagnosis values of these combinations are analyzed by ROC curves, and are presented by the area under the curve (AUC). The diagnostic values of metabolomics-derived parameters are similar to BNP. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Different combinations of global metabolites to discriminate 
               
               
                 patients at stage C from the normal subjects 
               
             
          
           
               
                   
                 Area under 
               
               
                   
                 the curve 
               
               
                 Metabolites and BNP 
                 (AUC) 
               
               
                   
               
             
          
           
               
                 1. 
                 BNP 
                 0.971 
               
               
                 2. 
                 His + Phe 
                 0.815 
               
               
                 3. 
                 His + Phe + Spermidine 
                 0.969 
               
               
                 4. 
                 His + Phe + Spermidine + PCaaC34:4 
                 0.971 
               
               
                 5. 
                 His + Phe + PCaaC34:4 + Hypoxanthine 
                 0.970 
               
               
                 6. 
                 His + Phe + Hypoxanthine 
                 0.958 
               
               
                 7. 
                 His + Phe + Spermidine + Hypoxanthine 
                 0.971 
               
               
                 8. 
                 His + Phe + Spermidine + PCaaC34:4 + Hypoxanthine 
                 0.973 
               
               
                   
               
               
                 BNP: B-type natriuretic peptide, PCaaC34:4: Phosphatidylcholine diacyl C34:4, His: Histidine, Phe: Phenylalanine. 
               
             
          
         
       
     
         [0079]    To discriminate patients at stage B from the normal subjects, a few good metabolite combinations were found as shown in the Table 6. The diagnosis values of these combinations are analyzed by ROC curves, and are presented by the area under the curve (AUC). The diagnostic values of metabolomics-derived parameters are better than BNP. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Different combinations of metabolites to discriminate 
               
               
                 patients at stage B from the normal subjects. 
               
             
          
           
               
                   
                 Area under 
                 P value 
               
               
                   
                 the curve 
                 (compared 
               
               
                 Metabolites 
                 (AUC) 
                 to BNP) 
               
               
                   
               
               
                 BNP 
                 0.943 
                 — 
               
               
                 PCaeC34:2 + C3 + His + Phe + Glu + 
                 0.982 
                 0.02 
               
               
                 Fisher ratio 
                   
                   
               
               
                 C3 + His + Phe + Glu + Fisher ratio + 
                 0.977 
                 0.03 
               
               
                 Total PCae 
                   
                   
               
               
                 PCaeC34:2 + C3 + His + Phe + Glu 
                 0.961 
                 0.045 
               
               
                 PCaeC34:2 + C3 + His + Phe + Glu + Tyr 
                 0.950 
                 0.05 
               
               
                 PCaeC34:2 + C3 + His + Phe + Glu + 
                 0.985 
                   
               
               
                 Hypoxanthine 
                   
                   
               
               
                 PCaeC34:2 + C3 + His + Phe + Glu + 
                 0.994 
                   
               
               
                 Tyr + Hypoxanthine 
                   
                   
               
               
                 Glu + Tyr/Phe + His + C0 + Total 
                 0.973 
                 0.03 
               
               
                 PCae + Essential AA 
                   
                   
               
               
                 Glu + Tyr/Phe + His + C0 + Total 
                 0.981 
                 0.02 
               
               
                 PCae + Total ACOH/Total AC 
                   
                   
               
               
                 Glu + Tyr/Phe + His + C0 + Total 
                 0.997 
                 0.001 
               
               
                 PCae + Essential AA + Total ACOH/Total AC 
               
               
                   
               
               
                 BNP: B-type natriuretic peptide, Essential AA: Essential amino acid; PCaeC34:2: Phosphatidylcholine acyl-alkyl C34:2, C3: Propionylcarnitine, His: Histidine, Pro: Proline, Glu: Glutamate, Tyr: Tyrosine, Phe: Phenylalanine, C0: Carnitine, Total ACOH: Total hydroxylacylcarnitine, Total PCae: Total phosphatidylcholine, Fisher Ratio: (Leucine + Isoleucine + Valine)/(Phenylalanine + Tryptophan + Tyrosine). 
               
               
                 Total AC: Total acylcarnitine. 
               
             
          
         
       
     
         [0080]    To discriminate patients at stage B from patients at stage A, a few good metabolite combinations were found as shown in the Table 7. The diagnosis values of these combinations are analyzed by ROC curves, and are presented by the area under the curve (AUC). The diagnostic values of metabolomics-derived parameters are better than BNP. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Different combinations of global metabolites to discriminate 
               
               
                 patients at stage B from patients at stage A 
               
             
          
           
               
                   
                 Area under 
                 P value 
               
               
                   
                 the curve 
                 (compared 
               
               
                 Metabolites 
                 (AUC) 
                 to BNP 
               
               
                   
               
               
                 BNP 
                 0.770 
                 — 
               
               
                 PCaaC34:4 + Ala + SDMA + C5:1 
                 0.883 
                 0.004 
               
               
                 PCaaC34:4 + Ala + SDMA + C3 
                 0.874 
                 0.01 
               
               
                 PCaaC34:4 + Ala + SDMA + C5:1 + 
                 0.915 
                   
               
               
                 Hypoxanthine 
                   
                   
               
               
                 PCaaC34:4 + Ala + SDMA + C3 + 
                 0.969 
                   
               
               
                 Hypoxanthine 
                   
                   
               
               
                 PCaaC34:4 + Ala + SDMA + Sarcosine 
                 0.937 
                 0.0001 
               
               
                 PCaaC34:4 + Ala + SDMA + Sarcosine 
                 0.982 
                 &lt;0.0001 
               
               
                   
               
               
                 BNP: B-type natriuretic peptide, PCaaC34:4: Phosphatidylcholine diacyl C34:4, Ala: Alanine, SDMA: Symmetric dimethylarginine, C5:1: Tiglylcarnitine, C3: Propionylcarnitine. 
               
             
          
         
       
     
         [0081]    To discriminate patients at stage B from patients at stage C, a few good metabolite combinations were found as shown in the Table 8. The diagnosis values of these combinations are analyzed by ROC curves, and are presented by the area under the curve (AUC). The diagnostic values of metabolomics-derived parameters are better than BNP. 
         [0000]                                  TABLE 8                   Different combinations of global metabolites to discriminate       patients at stage B from patients at stage C                Area under   P value           the curve   (compared       Metabolites   (AUC)   to BNP)               BNP   0.836   —       Spermidine + PCaaC34:4 + PCaeC34:2 +   0.892   0.01       His + Fisher ratio               Spermidine + PCaeC34:2 + His + Fisher   0.917   0.001       ratio               Spermidine + His + Fisher ratio   0.873   0.02       Spermidine + PCaeC34:2 + His + Fisher   0.939           ratio + Hypoxanthine               Spermidine + His + Fisher ratio +   0.891           Hypoxanthine               Spermidine + SM C16:0 + C14:2 + Fisher   0.924   0.001       ratio               BNP: B-type natriuretic peptide, PCaaC34:4: Phosphatidylcholine diacyl C34:4, PCaeC34:2: Phosphatidylcholine acyl-alkyl C34:2, His: Histidine, SM C16:0: Sphingomyelin C16:0, C14:2: Tetradecadienoylcarnitine, Fisher Ratio: a ratio of branched chain amino acids to aromatic amino acids.            
5. Use of Metabolomics in Serial Estimations for Patients from Acute HF Status to Stabilized Status
 
         [0082]    Based on the data described in table 5, it was tried to verify the diagnostic value of combining four metabolites (Histidine, Phenylalanine, Spermidine and Hypoxanthine). A parameter derived from the calculation of these 4 metabolites was produced, called tPS[1]. For this purpose, metabolomics analysis along with BNP measurement was further performed in 32 patients (22 males and 10 females, aged 54±11 years) at stage C. These patients were initially hospitalized due to acute cardiogenic pulmonary edema, got improved to NYHA functional classes I, and survived longer than one year. Plasma was analyzed before, and 6 and 12 months after discharged. The serial changes in tPS[ ] values were presented. As shown in  FIG. 3 , the tPS[1] values in the 32 patients at pre-discharge were significantly higher than the normal controls. Although the tPS[1] values remarkably decreased at 6 months, increases of tPS[1] were noted in part of the patients at 12 months. Compared to the levels at pre-discharge, BNP levels remarkably decreased at 6 months and remained steady at 12 months. These findings suggest that metabolomics analysis is a more sensitive tool than BNP in estimating HF status after acute HF 
       Example 2 
     Targeted Metabolomics Analyses for Diagnosing and Staging Heart Failure 
     1. Patients 
       [0083]    A total of 145 subjects were enrolled in this example. This included 62 normal subjects and 83 patients at stage C. 
       2. Targeted Metabolomics Analyses in HF and Normal Controls 
       [0084]    For the quantitation of metabolite concentrations, the Biocrates kit was applied in this example. Plasma was subjected to metabolomics analysis according to the targeted metabolomics workflow and datasets were bioinformatically analyzed using OPLS-DA model. To test whether these targeted metabolite profiles could discriminate stage C HF patients from normal controls, a total of 201 variables was used in the analysis. The metabolites responsible for the discrimination between these 2 groups (those metabolites with a VIP score&gt;1.0) are listed in Table 9. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Statistical analysis of targeted metabolites in the normal controls and patients at HF stage C 
               
             
          
           
               
                 Metabolite ID 
                 Normal control 
                 Stage C 
                 P-value 
               
               
                 (μM) 
                 (n = 62) 
                 (n = 83) 
                 (t-test) 
               
               
                   
               
             
          
           
               
                 1. 
                 Histidine 
                 99.303 ± 18.043 
                 77.736 ± 18.423 
                 2.15E−09 
               
               
                 2. 
                 Phenylalanine 
                 57.437 ± 8.956  
                 76.960 ± 22.607 
                 1.42E−09 
               
               
                 3. 
                 Ornithine/Arginine 
                 1.027 ± 0.229 
                 2.107 ± 1.320 
                 1.51E−09 
               
               
                 4. 
                 Phosphatidylcholine diacyl C34:4 
                 0.973 ± 0.362 
                 0.596 ± 0.355 
                 6.09E−08 
               
               
                 5. 
                 Ornithine 
                 56.921 ± 18.711 
                 83.954 ± 32.530 
                 4.50E−08 
               
               
                 6. 
                 Phosphatidylcholine diacyl C36:2 
                 226.400 ± 58.442  
                 172.928 ± 52.451  
                 4.60E−07 
               
               
                 7. 
                 Octadecadienylcarnitine 
                 0.053 ± 0.034 
                 0.105 ± 0.070 
                 2.42E−07 
               
               
                 8. 
                 Phosphatidylcholine diacyl C36:1 
                 44.837 ± 11.149 
                 35.375 ± 10.244 
                 3.24E−06 
               
               
                 9. 
                 Glutamate 
                 53.219 ± 43.497 
                 147.612 ± 135.093 
                 2.50E−07 
               
               
                 10. 
                 Phosphatidylcholine diacyl C36:0 
                 4.981 ± 1.166 
                 3.855 ± 1.435 
                 9.09E−06 
               
               
                 11. 
                 Spermidine 
                 0.258 ± 0.034 
                 0.372 ± 0.176 
                 6.93E−07 
               
               
                 12. 
                 Spermine 
                 0.051 ± 0.161 
                 0.301 ± 0.373 
                 1.70E−06 
               
               
                 13. 
                 Phosphatidylcholine diacyl C40:5 
                 9.322 ± 3.251 
                 6.971 ± 2.600 
                 1.79E−05 
               
               
                 14. 
                 Phosphatidylcholine diacyl C36:6 
                 0.908 ± 0.468 
                 0.562 ± 0.394 
                 1.95E−05 
               
               
                 15. 
                 Phosphatidylcholine diacyl C33:3 
                 0.231 ± 0.069 
                 0.172 ± 0.079 
                  0.00003 
               
               
                 16. 
                 Citrulline/Ornithine 
                 1.163 ± 0.737 
                 0.677 ± 0.510 
                 0.0001 
               
               
                 17. 
                 Phosphatidylcholine diacyl C38:5 
                 44.186 ± 12.629 
                 33.627 ± 14.051 
                 3.60E−05 
               
               
                 18. 
                 Creatinine 
                 103.998 ± 41.635  
                 185.324 ± 135.578 
                 5.98E−06 
               
               
                 19. 
                 Phosphatidylcholine diacyl C36:3 
                 94.046 ± 21.592 
                 78.162 ± 21.183 
                 8.19E−05 
               
               
                 20. 
                 Sphingomyeline C20:2 
                 1.119 ± 0.298 
                 0.873 ± 0.355 
                 9.29E−05 
               
               
                 21. 
                 Phosphatidylcholine diacyl C38:3 
                 31.133 ± 8.561  
                 24.971 ± 8.234  
                 9.59E−05 
               
               
                 22. 
                 Phosphatidylcholine acyl-alkyl C38:6 
                 7.104 ± 2.281 
                 5.519 ± 2.142 
                 0.0001 
               
               
                 23. 
                 Putrescine/Ornithine 
                 0.008 ± 0.005 
                 0.005 ± 0.003 
                 0.0005 
               
               
                 24. 
                 Phosphatidylcholine acyl-alkyl C34:2 
                 8.297 ± 2.463 
                 6.568 ± 2.375 
                 0.0001 
               
               
                 25. 
                 Spermine/Spermidine 
                 0.178 ± 0.564 
                 0.677 ± 0.778 
                 0.0001 
               
               
                 26. 
                 Aromatic amino acids 
                 163.971 ± 24.682  
                 193.288 ± 49.833  
                 3.34E−05 
               
               
                 27. 
                 Hydroxybutyrylcarnitine 
                 0.030 ± 0.015 
                 0.049 ± 0.032 
                 3.47E−05 
               
               
                 28. 
                 Phosphatidylcholine diacyl C40:4 
                 2.758 ± 0.839 
                 2.221 ± 0.711 
                 0.0002 
               
               
                 29. 
                 Phosphatidylcholine acyl-alkyl C36:2 
                 9.792 ± 2.232 
                 8.096 ± 2.632 
                 0.0003 
               
               
                 30. 
                 Hexose 
                 5284.765 ± 1986.055 
                 6993.400 ± 2813.938 
                 0.0001 
               
               
                 31. 
                 Phosphatidylcholine acyl-alkyl C36:3 
                 6.427 ± 1.800 
                 5.243 ± 1.712 
                 0.0003 
               
               
                 32. 
                 Octadecenoylcarnitine 
                 0.122 ± 0.074 
                 0.183 ± 0.102 
                 0.0002 
               
               
                 33. 
                 Butrylcarnitine 
                 0.233 ± 0.065 
                 0.352 ± 0.230 
                 5.95E−05 
               
               
                 34. 
                 Phosphatidylcholine diacyl C36:5 
                 18.999 ± 9.949  
                 12.797 ± 9.264  
                 0.0005 
               
               
                 35. 
                 Phosphatidylcholine acyl-alkyl C36:4 
                 12.616 ± 4.188  
                 10.299 ± 3.158  
                 0.0006 
               
               
                 36. 
                 Phosphatidylcholine acyl-alkyl C34:3 
                 5.202 ± 1.929 
                 4.079 ± 1.721 
                 0.0009 
               
               
                 37. 
                 Glutamine 
                 733.303 ± 134.001 
                 624.401 ± 199.760 
                 0.0009 
               
               
                 38. 
                 Fisher ratio 
                 2.878 ± 0.419 
                 2.502 ± 0.711 
                 0.0003 
               
               
                 39. 
                 Phosphatidylcholine acyl-alkyl C40:1 
                 1.319 ± 0.362 
                 1.065 ± 0.445 
                 0.0010 
               
               
                 40. 
                 Phosphatidylcholine diacyl C28:1 
                 1.402 ± 0.324 
                 1.169 ± 0.418 
                 0.0011 
               
               
                 41. 
                 Phosphatidylcholine acyl-alkyl C36:5 
                 9.951 ± 3.435 
                 8.114 ± 2.781 
                 0.0021 
               
               
                 42. 
                 Total Phosphatidylcholine acyl-alkyl 
                 129.041 ± 28.626  
                 111.007 ± 32.806  
                 0.0019 
               
               
                 43. 
                 Decanoylcarnitine 
                 0.278 ± 0.117 
                 0.218 ± 0.094 
                 0.0020 
               
               
                 44. 
                 Phosphatidylcholine diacyl C42:6 
                 0.545 ± 0.152 
                 0.465 ± 0.135 
                 0.0024 
               
               
                 45. 
                 Tyrosine 
                 67.599 ± 12.932 
                 79.768 ± 25.909 
                 0.0008 
               
               
                 46. 
                 Taurine 
                 44.966 ± 8.465  
                 58.678 ± 31.475 
                 0.0006 
               
               
                 47. 
                 Phosphatidylcholine diacyl C34:3 
                 10.689 ± 3.391  
                 8.680 ± 3.807 
                 0.0031 
               
               
                 48. 
                 Phosphatidylcholine diacyl C38:0 
                 3.821 ± 1.190 
                 3.131 ± 1.298 
                 0.0031 
               
               
                 49. 
                 Phosphatidylcholine diacyl C42:5 
                 0.282 ± 0.100 
                 0.228 ± 0.098 
                 0.0032 
               
               
                 50. 
                 Phosphatidylcholine diacyl C38:4 
                 81.812 ± 22.096 
                 68.326 ± 26.379 
                 0.0034 
               
               
                 51. 
                 Phosphatidylcholine acyl-alkyl C30:0 
                 0.272 ± 0.075 
                 0.230 ± 0.080 
                 0.0034 
               
               
                 52. 
                 Phosphatidylcholine acyl-alkyl C30:1 
                 0.308 ± 0.068 
                 0.266 ± 0.087 
                 0.0044 
               
               
                 53. 
                 Phosphatidylcholine diacyl C40:6 
                 28.904 ± 11.144 
                 23.258 ± 10.341 
                 0.0045 
               
               
                 54. 
                 Phosphatidylcholine acyl-alkyl C38:1 
                 3.324 ± 0.671 
                 2.856 ± 1.014 
                 0.0047 
               
               
                 55. 
                 Phosphatidylcholine acyl-alkyl C44:5 
                 0.922 ± 0.398 
                 1.119 ± 0.359 
                 0.0047 
               
               
                 56. 
                 Phosphatidylcholine acyl-alkyl C40:6 
                 3.805 ± 0.986 
                 3.223 ± 1.204 
                 0.0051 
               
               
                 57. 
                 Phosphatidylcholine diacyl C34:2 
                 398.289 ± 93.260  
                 347.433 ± 101.429 
                 0.0053 
               
               
                 58. 
                 Isoleucine 
                 72.689 ± 17.269 
                 84.387 ± 26.271 
                 0.0062 
               
               
                 59. 
                 DMA/Arginine 
                 0.004 ± 0.011 
                 0.013 ± 0.020 
                 0.0028 
               
               
                 60. 
                 Phosphatidylcholine diacyl C32:0 
                 9.482 ± 1.963 
                 10.622 ± 2.477  
                 0.0071 
               
               
                 61. 
                 Valerycarnitine 
                 0.101 ± 0.036 
                 0.149 ± 0.121 
                 0.0021 
               
               
                 62. 
                 Phosphatidylcholine acyl-alkyl C38:0 
                 2.071 ± 1.106 
                 1.508 ± 1.170 
                 0.0080 
               
               
                 63. 
                 Glycine 
                 225.479 ± 58.069  
                 261.766 ± 84.559  
                 0.0054 
               
               
                 64. 
                 Octanoylcarnitine 
                 0.197 ± 0.079 
                 0.165 ± 0.056 
                 0.0142 
               
               
                 65. 
                 Tetradecenoylcarnitine 
                 0.084 ± 0.057 
                 0.110 ± 0.050 
                 0.0097 
               
               
                 66. 
                 Phosphatidylcholine diacyl C38:6 
                 81.443 ± 25.515 
                 67.862 ± 31.996 
                 0.0130 
               
               
                 67. 
                 Phosphatidylcholine acyl-alkyl C32:2 
                 0.419 ± 0.140 
                 0.356 ± 0.140 
                 0.0151 
               
               
                 68. 
                 Phosphatidylcholine diacyl C30:0 
                 2.134 ± 0.537 
                 1.907 ± 0.496 
                 0.0170 
               
               
                 69. 
                 Phosphatidylcholine acyl-alkyl C38:4 
                 9.389 ± 2.402 
                 8.347 ± 2.350 
                 0.0176 
               
               
                 70. 
                 Phosphatidylcholine acyl-alkyl C38:2 
                 3.909 ± 0.808 
                 3.415 ± 1.326 
                 0.0195 
               
               
                 71. 
                 Phosphatidylcholine acyl-alkyl C38:5 
                 12.521 ± 3.380  
                 11.122 ± 3.174  
                 0.0203 
               
               
                 72. 
                 Spermidine/Putrescine 
                 0.731 ± 0.530 
                 1.081 ± 0.917 
                 0.0226 
               
               
                 73. 
                 Phosphatidylcholine diacyl C36:4 
                 135.325 ± 38.086  
                 117.295 ± 46.025  
                 0.0231 
               
               
                 74. 
                 Arginine 
                 56.363 ± 18.312 
                 48.226 ± 20.136 
                 0.0212 
               
               
                 75. 
                 Propionylcarnitine 
                 0.357 ± 0.108 
                 0.447 ± 0.268 
                 0.0116 
               
               
                 76. 
                 DMA 
                 0.199 ± 0.494 
                 0.452 ± 0.705 
                 0.0203 
               
               
                 77. 
                 Phosphatidylcholine acyl-alkyl C44:6 
                 0.549 ± 0.265 
                 0.641 ± 0.199 
                 0.0294 
               
               
                 78. 
                 Phosphatidylcholine diacyl C40:3 
                 0.622 ± 0.192 
                 0.534 ± 0.240 
                 0.0308 
               
               
                 79. 
                 Hexadecanoylcarnitine 
                 0.087 ± 0.043 
                 0.112 ± 0.071 
                 0.0199 
               
               
                 80. 
                 LysoPhosphatidylcholine acyl C14:0 
                 2.774 ± 0.565 
                 2.551 ± 0.578 
                 0.0354 
               
               
                 81. 
                 Alpha-Aminoadipic acid 
                 1.009 ± 0.664 
                 1.263 ± 0.654 
                 0.0365 
               
               
                 82. 
                 Hydroxysphingomyeline C14:1 
                 4.212 ± 0.847 
                 3.802 ± 1.205 
                 0.0282 
               
               
                 83. 
                 Phosphatidylcholine acyl-alkyl C38:3 
                 3.713 ± 1.112 
                 3.248 ± 1.363 
                 0.0462 
               
               
                 84. 
                 Phosphatidylcholine acyl-alkyl C36:1 
                 6.186 ± 1.336 
                 5.589 ± 1.817 
                 0.0483 
               
               
                 85. 
                 Asymmetric dimethylarginine/Arginine 
                 0.000 ± 0.001 
                 0.011 ± 0.037 
                 0.0207 
               
               
                 86. 
                 Asymmetric dimethylarginine 
                 0.028 ± 0.116 
                 0.369 ± 1.236 
                 0.0222 
               
               
                 87. 
                 Glucogenic amino acids 
                 717.168 ± 109.617 
                 784.870 ± 237.345 
                 0.0352 
               
               
                 88. 
                 Symmetric dimethylarginine/Arginine 
                 0.004 ± 0.011 
                 0.010 ± 0.019 
                 0.0433 
               
               
                 89. 
                 Symmetric dimethylarginine 
                 0.128 ± 0.327 
                 0.267 ± 0.461 
                 0.0524 
               
               
                 90. 
                 Proline 
                 166.166 ± 61.575  
                 189.780 ± 78.301  
                 0.0745 
               
               
                 91. 
                 L-Dopa 
                 0.173 ± 0.264 
                 0.099. ± 0.210  
                 0.0963 
               
               
                 92. 
                 Decadienylcarnitine 
                 0.399 ± 0.126 
                 0.342 ± 0.232 
                 0.0836 
               
               
                 93. 
                 Valine 
                 250.655 ± 56.145  
                 234.922 ± 70.229  
                 0.1861 
               
               
                 94. 
                 Tryptophan 
                 38.935 ± 9.790  
                 36.560 ± 11.643 
                 0.2359 
               
               
                 95. 
                 Tiglylcarnitine 
                 0.032 ± 0.019 
                 0.036 ± 0.023 
                 0.2839 
               
               
                 96. 
                 Citrulline 
                 58.040 ± 27.607 
                 53.524 ± 41.084 
                 0.4656 
               
               
                 97. 
                 Putrescine 
                 0.430 ± 0.256 
                 0.404 ± 0.228 
                 0.5499 
               
               
                 98. 
                 Leucine 
                 149.621 ± 44.640  
                 147.630 ± 47.565  
                 0.8145 
               
               
                 99. 
                 Threonine 
                 111.582 ± 20.515  
                 112.500 ± 33.305  
                 0.8500 
               
               
                 100. 
                 Lysine 
                 207.936 ± 47.620  
                 209.698 ± 66.436  
                 0.8712 
               
               
                   
               
               
                 Data are presented as mean ± SD. 
               
               
                 DMA: DMA represents total dimethylarginine 
               
             
          
         
       
     
       3. Discrimination of Patients at Stage C and Normal Controls 
       [0085]    To discriminate patients at stage C and normal controls (diagnostic value), the ROC curves were drawn for both BNP and t[2](by taking all targeted metabolites into account by using the principal component analysis) ( FIG. 4 ). The areas under the curves were 0.998 and 1.0, respectively. In the targeted metabolomics data set, it was found four important metabolites that significantly contributed to the diagnostic value of metabolomics for HF, including histidine, phenylalanine, spermidine, and phosphatidylcholine diacyl C34:4 (Table 10). A combination of these 4 metabolites reached an area under curve of 0.995, which was better than each metabolite alone ( FIG. 4 ). Based on the combination of these 4 metabolites, a parameter was produced, called tPS[2]. The diagnostic values of the BNP and tPS[2] levels for identifying stage C HF (vs. normal controls) were presented in Table 10. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Diagnostic values of BNP and targeted metabolites in patients 
               
               
                 with heart failure at stage C (versus normal controls) 
               
             
          
           
               
                   
                 Area under 
                 P value 
               
               
                   
                 the curve 
                 (compared to 
               
               
                   
                 (AUC) 
                 BNP) 
               
               
                   
               
             
          
           
               
                   
                 BNP 
                 0.998 
                 — 
               
               
                   
                 t[2](all targeted metabolites) 
                 1.000 
                 0.92 
               
               
                   
                 Histidine 
                 0.752 
                 &lt;0.001 
               
               
                   
                 Phenylalanine 
                 0.733 
                 &lt;0.001 
               
               
                   
                 PC aa C34:4 
                 0.715 
                 &lt;0.001 
               
               
                   
                 Spermidine 
                 0.712 
                 &lt;0.001 
               
               
                   
                 tPS[2] (Four metabolites: 
                 0.995 
                 0.97 
               
               
                   
                 Histidine, Phenylalanine, PC 
                   
                   
               
               
                   
                 aa C34:4, Spermidine) 
               
               
                   
               
               
                 BNP, B-type natriuretic peptide level; PC aa, phosphatidylcholine diacyl. 
               
               
                 “t[2]” is a parameter derived from the whole targeted metabolites dataset; “tPS[2]” is a parameter derived from the 4 metabolites: Histidine, Phenylalanine, PC aa C34:4 (Phosphatidylcholine diacyl C34:4) and Spermidine. 
               
             
          
         
       
     
       Example 3 
     Targeted Metabolomics Analyses for Evaluating a Prognosis for Heart Failure 
     1. Prognostic Values of Metabolomic Signature 
       [0086]    To estimate the prognostic values of metabolomics and BNP, the following analyses focused on patients at stages B and C. To look for potential metabolic predictors of a composite event of all-cause death and HF-related re-hospitalization, extensive analyses on the targeted metabolite dataset revealed that a combination of 4 classes of metabolites (Dimethylarginine/Arginine ratio, spermidine, butyrylcarnitine, and total amount of essential amino acids) gave rise to an optimal prognostic value remarkably better than BNP. By combining these 4 classes of metabolites, a parameter was produced, called tPS[3]. The AUC of ROC curves were 0.853, 0.792, and 0.744, respectively to tPS[3], tPS[2](derived from the whole targeted metabolomics dataset), and BNP levels ( FIG. 5A ). Table 11 showed the data of AUC (by ROC curves) and Log Rank values (by Kaplan-Meier analysis) for these parameters on prognosis. 
         [0087]    The mean of tPS[3](2.9, range 0.04-5.63) was set as the cutoff value for prognostic prediction. In  FIG. 5B , the Kaplan-Meier curves demonstrated that a tPS[3] of ≧2.9 at pre-discharge was associated with a higher composite event rate of HF-related re-hospitalization and all-cause death (Log rank=17.5, p&lt;0.0001). In comparison, the prognostic values of a BNP of ≧350 pg/ml is shown in  FIG. 5C  (Log rank=9.9, p=0.002). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Prognostic values of BNP and targeted metabolites 
               
               
                 in patients with heart failure 
               
             
          
           
               
                   
                   
                   
                 P value 
               
               
                   
                 Area under the 
                 Log Rank 
                 (Compared 
               
               
                   
                 curve (by ROC 
                 (by Kaplan- 
                 to BNP by 
               
               
                   
                 curve) 
                 Meire) 
                 chi-square) 
               
               
                   
               
             
          
           
               
                 BNP 
                 0.744 
                 9.95 
                 — 
               
               
                 t[2] 
                 0.781 
                 10.7 
                 0.04 
               
               
                 tPS[2] 
                 0.792 
                 12.1 
                 &lt;0.001 
               
               
                 DMA/Arginine ratio 
                 0.730 
                 8.8 
                 0.06 
               
               
                 Spermidine 
                 0.712 
                 8.2 
                 0.042 
               
               
                 Butyrylcarnitine 
                 0.698 
                 7.65 
                 0.04 
               
               
                 Total essential amino acids 
                 0.674 
                 7.33 
                 0.03 
               
               
                 tPS[3] (Four metabolites: 
                 0.853 
                 17.5 
                 &lt;0.0001 
               
               
                 DMA/Arginine ratio, 
                   
                   
                   
               
               
                 Spermidine, 
                   
                   
                   
               
               
                 butyrylcarnitine, Total 
                   
                   
                   
               
               
                 essential amino acids) 
               
               
                   
               
               
                 BNP represents B-type natriuretic peptide level; t[2] is a parameter derived from all targeted metabolomics analysis; tPS[2] is a parameter derived from 4 targeted metabolites (Histidine, Phenylalanine, PC aa C34:4 (phosphatidylcholine diacyl C34:4), Spermidine); DMA represents total dimethylarginine. 
               
               
                 The essential amino acids comprise phenylalanine, valine, threonine, tryptophan, isoleucine, leucine, methionine, lysine and histidine. 
               
             
          
         
       
     
       Example 4 
     The Prognostic Values of Global Metabolites Analyses for the Patients with Heart Failure 
       [0088]    Global metabolomics analysis was performed in this example. A total of 157 patients were enrolled, including patients at stages B (n=81), and C (n=76). A global metabolomics analysis was used to identify different combinations of metabolites with good values on predicting a composite event of death and heart failure-related re-hospitalization. 
         [0089]    For the prognostic values of metabolomics and BNP, the estimations were based on AUC (derived from ROC curves), and “Log Rank” values (derived from Kaplan-Meier analysis). The data were shown in Table 12. 
       1. Comparisons of BNP and Different Combinations of Global Metabolites on Prognostic Values: 
       [0090]    (1). By AUC (Derived from ROC Curves): 
         [0091]    Initially, it was found that the prognostic values of metabolomics is better than BNP while combining 4 classes of metabolites, including dimethylarginine/arginine, butyrylcarnitine, spermidine, and total essential amino acid. 
         [0092]    A combination of dimethylarginine/arginine and butyrylcarnitine is already better than BNP based on Table 12. A combination of dimethylarginine/arginine, butyrylcarnitine, and spermidine is already better than BNP. A combination of dimethylarginine/arginine, butyrylcarnitine, and xanthine is already better than BNP. A combination of dimethylarginine/arginine and xanthine is already better than BNP. A combination of dimethylarginine/arginine, xanthine, and tryptophan is already better than BNP. A combination of dimethylarginine/arginine, xanthine, and spermidine/spermine is already better than BNP. Xanthine alone is already better than BNP. A combination of SDMA (symmetric dimethylarginine)/arginine and xanthine is already better than BNP. A combination of SDMA/arginine, xanthine, and tryptophan is already better than BNP. A combination of SDMA/arginine, xanthine, and spermidine/spermine is already better than BNP. SDMA alone is already better than BNP. SDMA/arginine alone is already better than BNP. P-cresyl sulfate alone is already better than BNP. A combination of SDMA, and P-cresyl sulfate is already better than BNP. A combination of SDMA, P-cresyl sulfate, and Phosphatidylcholine diacyl C38:6 is already better than BNP. A combination of SDMA, P-cresyl sulfate, and butyrylcarnitine is already better than BNP. A combination of SDMA, P-cresyl sulfate, and spermidine is already better than BNP. A combination of DMA/arginine, and P-cresyl sulfate is already better than BNP. A combination of DMA/arginine, P-cresyl sulfate, and Phosphatidylcholine diacyl C38:6 is already better than BNP. A combination of DMA/arginine, P-cresyl sulfate, and butyrylcarnitine is already better than BNP. A combination of DMA/arginine, P-cresyl sulfate, and spermidine is already better than BNP. A combination of dimethylarginine/arginine and spermidine is already better than BNP. A combination of SDMA/arginine and spermidine is already better than BNP. A combination of SDMA/arginine and butyrylcarnitine is already better than BNP. A combination of tryptophan and xanthine is already better than BNP. A combination of tryptophan and spermidine is already better than BNP. A combination of tryptophan and butyrylcarnitine is already better than BNP. A combination of leucine and xanthine is already better than BNP. A combination of leucine and spermidine is already better than BNP. A combination of leucine and butyrylcarnitine is already better than BNP. A combination of threonine and xanthine is already better than BNP. A combination of threonine and spermidine is already better than BNP. A combination of threonine and butyrylcarnitine is already better than BNP. 
         [0093]    However, it was noted that the dimethylarginine/arginine only is worse than BNP. 
         [0000]    (2). By “Log Rank” Value (Derived from Kaplan-Meier Analysis): (the Cutoff is Set at the Mean Value of Each Parameter) 
         [0094]    Initially, it was found that the prognostic value of metabolomics is better than BNP while combining 4 classes of metabolites, including dimethylarginine/arginine, butyrylcarnitine, spermidine, and total essential amino acid. 
         [0095]    A combination of dimethylarginine/arginine and butyrylcarnitine is already better than BNP. A combination of dimethylarginine/arginine, butyrylcarnitine, and spermidine is already better than BNP. A combination of dimethylarginine/arginine, butyrylcarnitine, and xanthine is already better than BNP. The dimethylarginine/arginine only is still better than BNP. A combination of dimethylarginine/arginine and xanthine is already better than BNP. A combination of dimethylarginine/arginine, xanthine, and tryptophan is already better than BNP. A combination of dimethylarginine/arginine, xanthine, and spermidine/spermine is already better than BNP. Xanthine alone is already better than BNP. A combination of SDMA (symmetric dimethylarginine)/arginine, and xanthine is already better than BNP. A combination of SDMA/arginine, xanthine, and tryptophan is already better than BNP. A combination of SDMA/arginine, xanthine, and spermidine/spermine is already better than BNP. SDMA alone is already better than BNP. SDMA/arginine alone is already better than BNP. P-cresyl sulfate alone is already better than BNP. A combination of SDMA, and P-cresyl sulfate is already better than BNP. A combination of SDMA, P-cresyl sulfate, and Phosphatidylcholine diacyl C38:6 is already better than BNP. A combination of SDMA, P-cresyl sulfate, and butyrylcarnitine is already better than BNP. A combination of SDMA, P-cresyl sulfate, and spermidine is already better than BNP. A combination of DMA/arginine, and P-cresyl sulfate is already better than BNP. A combination of DMA/arginine, P-cresyl sulfate, and Phosphatidylcholine diacyl C38:6 is already better than BNP. A combination of DMA/arginine, P-cresyl sulfate, and butyrylcarnitine is already better than BNP. A combination of DMA/arginine, P-cresyl sulfate, and spermidine is already better than BNP. A combination of dimethylarginine/arginine and spermidine is already better than BNP. A combination of SDMA/arginine and spermidine is already better than BNP. A combination of SDMA/arginine and butyrylcarnitine is already better than BNP. A combination of tryptophan and xanthine is already better than BNP. A combination of tryptophan and spermidine is already better than BNP. A combination of tryptophan and butyrylcarnitine is already better than BNP. A combination of leucine and xanthine is already better than BNP. A combination of leucine and spermidine is already better than BNP. A combination of leucine and butyrylcarnitine is already better than BNP. A combination of threonine and xanthine is already better than BNP. A combination of threonine and spermidine is already better than BNP. A combination of threonine and butyrylcarnitine is already better than BNP. 
       (3). Replacement of the Total Essential Amino Acids by 2 or 3 Essential Amino Acids: 
       [0096]    For evaluating the prognosis of heart failure, when the total essential amino acid was used in the combinations of metabolites as described above, it was noted that the total essential amino acids (9 amino acids) could be replaced by using only three amino acids (leucine, threonine and tryptophan) with the similar prognostic values. Furthermore, it was noted that the total essential amino acids (9 amino acids) could also be replaced by using only two amino acids (leucine and threonine, or leucine and tryptophan) with the similar prognostic values. (see Table 12) 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Comparisons of different combinations of global metabolites to B-type natriuretic 
               
               
                 peptide on the prognostic values in patients with heart failure 
               
             
          
           
               
                   
                   
                   
                 P value 
               
               
                   
                 Area under the 
                 Log Rank 
                 (Compared 
               
               
                   
                 curve (by ROC 
                 (by Kaplan- 
                 to BNP by 
               
               
                 Biomarkers 
                 curve) 
                 Meire) 
                 chi-square) 
               
               
                   
               
             
          
           
               
                 B-type natriuretic peptide (BNP) 
                 0.744 
                 9.95 
                 — 
               
               
                 Metabolites from global 
                   
                   
                   
               
               
                 metabolomics analysis 
                   
                   
                   
               
               
                 DMA/Arginine, Butyrylcarnitine, and 
                 0.971 
                 23.7 
                 &lt;0.0001 
               
               
                 Xanthine 
                   
                   
                   
               
               
                 DMA/Arginine and Xanthine 
                 0.966 
                 22.5 
                 &lt;0.0001 
               
               
                 DMA/Arginine, Xanthine, and Tryptophan 
                 0.973 
                 23.1 
                 &lt;0.0001 
               
               
                 DMA/Arginine, Xanthine, and 
                 0.972 
                 23.9 
                 &lt;0.0001 
               
               
                 Spermidine/Spermine 
                   
                   
                   
               
               
                 Xanthine 
                 0.902 
                 19.7 
                 &lt;0.0001 
               
               
                 SDMA/Arginine, Xanthine, and Tryptophan 
                 0.974 
                 24.0 
                 &lt;0.0001 
               
               
                 SDMA, Xanthine, and Tryptophan 
                 0.972 
                 23.8 
                 &lt;0.0001 
               
               
                 SDMA/Arginine, and Xanthine 
                 0.969 
                 22.4 
                 &lt;0.0001 
               
               
                 SDMA/Arginine, Xanthine, and 
                 0.970 
                 22.9 
                 &lt;0.0001 
               
               
                 Spermidine/Spermine 
                   
                   
                   
               
               
                 SDMA 
                 0.861 
                 18.2 
                 &lt;0.01 
               
               
                 SDMA/Arginine 
                 0.863 
                 18.3 
                 &lt;0.01 
               
               
                 P-cresyl sulfate 
                 0.766 
                 10.0 
                 0.04 
               
               
                 SDMA, and P-cresyl sulfate 
                 0.975 
                 24.2 
                 &lt;0.0001 
               
               
                 SDMA, P-cresyl sulfate, and PCaaC38:6 
                 0.981 
                 25.5 
                 &lt;0.0001 
               
               
                 SDMA, P-cresyl sulfate, and Butyrylcarnitine 
                 0.977 
                 24.7 
                 &lt;0.0001 
               
               
                 SDMA, P-cresyl sulfate, and spermidine 
                 0.979 
                 24.9 
                 &lt;0.0001 
               
               
                 DMA/Arginine, and P-cresyl sulfate 
                 0.965 
                 22.1 
                 &lt;0.0001 
               
               
                 DMA/Arginine, P-cresyl sulfate, and 
                 0.972 
                 24.0 
                 &lt;0.0001 
               
               
                 PCaaC38:6 
                   
                   
                   
               
               
                 DMA/Arginine, P-cresyl sulfate, and 
                 0.966 
                 22.3 
                 &lt;0.0001 
               
               
                 Butyrylcarnitine 
                   
                   
                   
               
               
                 DMA/Arginine, P-cresyl sulfate, and 
                 0.961 
                 20.3 
                 &lt;0.0001 
               
               
                 spermidine 
                   
                   
                   
               
               
                 DMA/Arginine, Butyrylcarnitine, Spermidine, 
                 0.912 
                 20.3 
                 &lt;0.0001 
               
               
                 and Total essential amino acid 
                   
                   
                   
               
               
                 DMA/Arginine, Butyrylcarnitine, Spermidine, 
                 0.952 
                 18.3 
                 &lt;0.0001 
               
               
                 Leucine, Threonine, and Tryptophan 
                   
                   
                   
               
               
                 DMA/Arginine, Butyrylcarnitine, Spermidine, 
                 0.969 
                 19.7 
                 &lt;0.0001 
               
               
                 Leucine, and Threonine 
                   
                   
                   
               
               
                 DMA/Arginine, Butyrylcarnitine, Spermidine, 
                 0.963 
                 19.1 
                 &lt;0.0001 
               
               
                 Leucine, and Tryptophan 
                   
                   
                   
               
               
                 DMA/Arginine, Butyrylcarnitine, and 
                 0.951 
                 15.4 
                 &lt;0.0001 
               
               
                 Spermidine 
                   
                   
                   
               
               
                 DMA/Arginine and Butyrylcarnitine 
                 0.947 
                 15.1 
                 &lt;0.0001 
               
               
                 DMA/Arginine 
                 0.757 
                 9.7 
                 0.11 
               
               
                 DMA/Arginine, Butyrylcarnitine, and 
                 0.940 
                 13.2 
                 &lt;0.0001 
               
               
                 PCaaC38:6 
                   
                   
                   
               
               
                 DMA/Arginine + Spermidine 
                 0.861 
                 18.1 
                 0.0001 
               
               
                 SDMA/Arginine + Spermidine 
                 0.874 
                 18.9 
                 0.0001 
               
               
                 SDMA/Arginine + Butyrylcarnitine 
                 0.936 
                 21.5 
                 0.0001 
               
               
                 SDMA/Arginine + Xanthine 
                 0.970 
                 22.1 
                 0.0001 
               
               
                 Tryptophan + Xanthine 
                 0.825 
                 15.1 
                 0.006 
               
               
                 Tryptophan + Spermidine 
                 0.813 
                 14.4 
                 0.012 
               
               
                 Tryptophan + Butyrylcarnitine 
                 0.775 
                 10.1 
                 0.032 
               
               
                 Leucine + Xanthine 
                 0.782 
                 10.3 
                 0.003 
               
               
                 Leucine + Spermidine 
                 0.755 
                 9.98 
                 0.137 
               
               
                 Leucine + Butyrylcarnitine 
                 0.754 
                 9.97 
                 0.094 
               
               
                 Threonine + Xanthine 
                 0.820 
                 14.7 
                 0.006 
               
               
                 Threonine + Spermidine 
                 0.817 
                 14.2 
                 0.087 
               
               
                 Threonine + Butyrylcarnitine 
                 0.849 
                 16.5 
                 0.109 
               
               
                   
               
               
                 DMA: Total dimethylarginine, 
               
               
                 SDMA: Symmetric dimethylarginine, 
               
               
                 PCaaC38:6: Phosphatidylcholine diacyl C38:6 
               
             
          
         
       
     
       Example 5 
     Kits for Diagnosing Heart Failure 
     1. Sample Extraction 
     (1). Plasma Sample Preparation for Global Metabolites Analysis 
       [0097]    To 100 μl of plasma, 400 μl acetonitrile (ACN) is added. The mixture will be vortexed for 30 s, sonicated for 15 min. and centrifuged at 10,000×g for 25 min. The supernatant will be collected into a separate tube. The pellets will be re-extracted once. To the residual pellets, an equivalent volume of aqueous methanol (1:1 methanol/water, volume to volume) will be added. The suspension will be vortexed for 30 s, sonicated for 15 min and again centrifuged to remove the precipitates. The aqueous methanolic supernatant and acetonitrile supernatant will be pooled and dried in a nitrogen evaporator. The residues will be saved and stored at −80° C. Residues can be suspended in 100 μl of 95:5 water/acetonitrile and centrifuged at 14,000×g for 5 min, the clear supernatant will be collected a for LC-MS analysis. 
       (2). Plasma Sample Preparation for Lipid Analysis 
       [0098]    For extraction of lipids, a modification of Folch&#39;s method will be employed. 
         [0099]    Briefly stated, the 100 μl plasma will be transferred to a glass tube. Six milliliters of chloroform/methanol (2:1, v/v) solution and 1.5 ml of water are added. The sample will be vortexed 4 times for 30 s, and subsequently centrifuged at 700×g for 30 min at 4□. The upper phase is removed as completely as possible, and the lower phase is sonicated for 10 min. The sample will be centrifuged at 700×g for 10 min at 4□. The upper phase can be removed as completely as possible, and the lower phase was allowed to stand still at 4□:. Three milliliters of this sample will be dried under nitrogen gas, and stored at −80□. Prior to analysis, the sample will be dissolved in 200 μl of 40% methanol. 
       2. Metabolites Identification by Diagnostic Device 
     (1). MS/MS Analyses 
       [0100]    For structural identification of target metabolite, standards will be operated under identical chromatographic conditions with that of the profiling experiment. MS and MS/MS analyses are performed in the same conditions. MS/MS spectra are collected at 0.1 spectra per second, with a medium isolation window of ˜4 m/z. The collision energy will be set from 5 to 35 V. Several metabolites will be further confirmed by an ion mobility mass spectrometer under similar chromatographic conditions. 
       (2). Fluorescence Spectroscopy 
       [0101]    The concentration of histidine (or other metabolites, such as xanthine, spermidine, propionylcarnitine, butyrylcarnitine, P-cresyl sulfate and a combination thereof) in plasma will be determined with a method in which histidine (or other metabolites, such as xanthine, spermidine, propionylcarnitine, butyrylcarnitine, P-cresyl sulfate and a combination thereof) and o-phthaldialdehyde react in alkali to form a fluorescent product which is measured in a fluorescence spectrometer. The method is linear in the range used. 
         [0102]    A diagnostic device used herein is not limited to the above examples. Based on the nature of a metabolite, other diagnostic device, such as biochip, ELISA, LC-MS, etc. can also be employed for detecting the metabolites identified herein. 
         [0103]    Metabolomics analysis explores the global metabolic abnormalities in patients with heart failure. By using metabolomics analysis, this patent provides information associated with heart failure more than BNP and traditional markers provide. Analysis of the abundant metabolites in plasma explored the global sophisticated metabolic perturbation behind an abnormal BNP level, including up-regulation of glutamate-ornithine-proline, polyamine, purine and taurine synthesis pathways; down-regulation of nitric oxide, dopamine, and phosphatidylcholines synthesis pathways during progression of HF (see  FIG. 2 ) and changes in urea cycle, biopterin cycle, MTA cycle, methionine cycle, ornithine-proline-glutamate, polyamine synthesis, dopamine synthesis, methylation (creatinine and phosphatidylcholine), transsulfuration (taurine) and purine metabolism. The plasma concentrations of a few metabolites (for example, xanthine, histidine, phenylalanine, ornithine, arginine, spermine, spermidine, taurine, and phosphatidylcholines) changed at different stages of HF, and these metabolites changes are potential biomarkers. 
         [0104]    By using metabolomics analysis, this patent provides more sensitive and specific metabolic evaluation for HF staging than ACC/AHA classification, BNP and other traditional markers provide. The methods provided in this patent are able to discriminate patients at HF stage C from the healthy subjects, patients at HF stage A from the healthy subjects, and patients at HF stage C from patients at HF stage A. By the methods provided in this patent, the discrimination among patients at different HF stages is more scientific than the way by ACC/AHA classification. 
         [0105]    By using metabolomics analysis, this patent identifies novel biomarkers (for example, by combining xanthine, spermidine, butyrylcarnitine, some phosphatidylcholines, and other metabolites) to provide better diagnostic and prognostic values for patients with heart failure than BNP and traditional markers provide. 
         [0106]    While some of the embodiments of the present invention have been described in detail in the above, it is, however, possible for those of ordinary skill in the art to make various modifications and changes to the particular embodiments shown without substantially departing from the teaching and advantages of the present invention. Such modifications and changes are encompassed in the spirit and scope of the present invention as set forth in the appended claim.