# Patent

## Title
Biomarkers for insulin resistance and methods using the same

## Abstract

Biomarkers relating to insulin resistance are provided, as well as methods for using the same as biomarkers for insulin resistance. Also provided are suites of small molecule entities as biomarkers for insulin resistance.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/966,475, filed Aug. 14, 2013, which is a divisional of U.S. patent application Ser. No. 13/457,620, filed Apr. 27, 2012, which is a divisional of U.S. patent application Ser. No. 12/218,980, filed Jul. 17, 2008, which claims the benefit of U.S. Provisional Application No. 60/950,286, filed Jul. 17, 2007, and U.S. Provisional Application No. 61/037,628, filed Mar. 18, 2008, the entireties of which are hereby incorporated by reference herein.
FIELD
The invention generally relates to biomarkers, methods for identifying biomarkers correlated to insulin resistance, cardiovascular disease, and metabolic-syndrome-related disorders and methods based on the same biomarkers.


BACKGROUND
Diabetes is classified as either type 1 (early onset) or type 2 (adult onset), with type 2 comprising 90-95% of the cases of diabetes. Diabetes is the final stage in a disease process that begins to affect individuals long before the diagnosis of diabetes is made. Type 2 diabetes develops over 10 to 20 years and results from an impaired ability to utilize glucose (glucose utilization) due to impaired sensitivity to insulin (insulin resistance).
In pre-diabetes, insulin becomes less effective at helping tissues metabolize glucose. Pre-diabetics may be detectable as early as 20 years before diabetic symptoms become evident. Studies have shown that although patients show very few symptoms, long-term physiological damage is already occurring at this stage. Up to 60% of these individuals will progress to type 2 diabetes within 10 years.
The American Diabetes Association (ADA) has recommended routine screening to detect patients with pre-diabetes. Current screening methods for pre-diabetes include the fasting plasma glucose (FPG) test, the oral glucose tolerance test (OGTT), the fasting insulin test and the hyperinsulinemic euglycemic clamp (HI clamp). The first two tests are used clinically whereas the latter two tests are used extensively in research but rarely in the clinic. In addition, mathematical means (e.g., HOMA, QUICKI) that consider the fasting glucose and insulin levels together have been proposed. However, normal plasma insulin concentrations vary considerably between individuals as well as within an individual throughout the day. Further, these methods suffer from variability and methodological differences between laboratories and do not correlate rigorously with glucose clamp studies.
Worldwide, an estimated 194 million adults have type 2 diabetes and this number is expected to increase to 333 million by 2025, largely due to the epidemic of obesity in westernized societies. In the United States, it is estimated that over 54 million adults are pre-diabetic, depending on the level of insulin resistance. There are approximately 1.5 million new cases of type 2 diabetes a year in the United States. The annual US healthcare cost for diabetes is estimated at $174 billion. This figure has risen more than 32% since 2002. In industrialized countries such as the U.S., about 25% of medical expenditures treat glycemic control, 50% is associated with general medical care associated with diabetes, and the remaining 25% of the costs go to treat long-term complications, primarily cardiovascular disease. Considering the distribution of the healthcare costs and the fact that insulin resistance is a direct causal factor in cardiovascular disease and diabetes progression, it is no surprise that cardiovascular disease accounts for 70-80% of the mortality observed for diabetic patients. Detecting and preventing type 2 diabetes has become a major health care priority.
Diabetes may also lead to the development of other diseases or conditions, or is a risk factor in the development of conditions such as Metabolic Syndrome and cardiovascular diseases. Metabolic Syndrome is the clustering of a set of risk factors in an individual. According to the American Heart Association these risk factors include: abdominal obesity, decreased ability to properly process glucose (insulin resistance or glucose intolerance), dyslipidemia (high triglycerides, high LDL, low HDL cholesterol), hypertension, prothrombotic state (high fibrinogen or plasminogen activator inhibitor-1 in the blood) and proinflammatory state (elevated C-reactive protein in the blood). Metabolic Syndrome is also known as syndrome X, insulin resistance syndrome, obesity syndrome, dysmetabolic syndrome and Reaven's syndrome. Patients diagnosed with Metabolic Syndrome are at an increased risk of developing diabetes, cardiac and vascular disease. It is estimated that, in the United States, 20% of the adults (>50 million people) have metabolic syndrome. While it can affect anyone at any age, the incidence increases with increasing age and in individuals who are inactive, and significantly overweight, especially with excess abdominal fat.
Type 2 diabetes is the most common form of diabetes in the United States. According to the American Diabetes Foundation over 90% of the US diabetics suffer from Type 2 diabetes. Individuals with Type 2 diabetes have a combination of increased insulin resistance and decreased insulin secretion that combine to cause hyperglycemia. Most persons with Type 2 diabetes have Metabolic Syndrome.
The diagnosis for Metabolic Syndrome is based upon the clustering of three or more of the risk factors in an individual. There are no well-accepted criteria for diagnosing the metabolic syndrome. The criteria proposed by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III), with minor modifications, are currently recommended and widely used.
The American Heart Association and the National Heart, Lung, and Blood Institute recommend that the metabolic syndrome be identified as the presence of three or more of these components: increased waist circumference (Men—equal to or greater than 40 inches (102 cm), Women—equal to or greater than 35 inches (88 cm); elevated triglycerides (equal to or greater than 150 mg/dL); reduced HDL (“good”) cholesterol (Men—less than 40 mg/dL, Women—less than 50 mg/dL); elevated blood pressure (equal to or greater than 130/85 mm Hg); elevated fasting glucose (equal to or greater than 100 mg/dL).
Type 2 diabetes develops slowly and often people first learn they have type 2 diabetes through blood tests done for another condition or as part of a routine exam. In some cases, type 2 diabetes may not be detected before damage to eyes, kidneys or other organs has occurred. A need exists for an objective, biochemical evaluation (e.g. lab test) that can be administered by a primary care provider to identify individuals that are at risk of developing Metabolic Syndrome or Type 2 diabetes.
Newer, more innovative molecular diagnostics that reflect the mechanisms of the patho-physiological progression to pre-diabetes and diabetes are needed because the prevalence of pre-diabetes and diabetes is increasing in global epidemic proportions. Mirroring the obesity epidemic, pre-diabetes and diabetes are largely preventable but are frequently undiagnosed or diagnosed too late due to the asymptomatic nature of the progression to clinical disease.
Therefore there is an unmet need for diagnostic biomarkers and tests that can identify pre-diabetics at risk of developing type 2 diabetes and to determine the risk of disease progression in subjects with insulin resistance. Insulin resistance biomarkers and diagnostic tests can better identify and determine the risk of diabetes development in a pre-diabetic subject, can monitor disease development and progression and/or regression, can allow new therapeutic treatments to be developed and can be used to test therapeutic agents for efficacy on reversing pre-diabetes and/or preventing diabetes. Further, a need exists for diagnostic biomarkers to more effectively assess the efficacy and safety of pre-diabetic and diabetic therapeutic candidates.
SUMMARY OF THE INVENTION
In one embodiment, the present disclosure provides a method of diagnosing insulin resistance in a subject, the method comprising analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for insulin resistance in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, 29 and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to insulin resistance-positive and/or insulin resistance-negative reference levels of the one or more biomarkers in order to diagnose whether the subject is insulin resistant.
In another embodiment, the present disclosure provides a method of predicting the glucose disposal rate in a subject, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for insulin resistance in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to glucose disposal reference levels of the one or more biomarkers in order to predict the glucosal disposal rate in the subject.
The disclosure also provides a method of classifying a subject according to glucose tolerance from normal glucose tolerance (NGT), impaired fasting glucose tolerance (IFG), or impaired glucose tolerance (IGT), to type-2 diabetes, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for glucose tolerance in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to glucose tolerance reference levels of the one or more biomarkers in order to classify the subject as having NGT, IFG, IGT, or diabetic.
Further provided is a method of determining susceptibility of a subject to developing type-2 diabetes, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for pre-diabetes in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to diabetes-positive and/or diabetes-negative reference levels of the one or more biomarkers in order to diagnose whether the subject is susceptible to developing type-2 diabetes.
The present disclosure also provides a method of determining an insulin resistance score in a subject, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to insulin resistance reference levels of the one or more biomarkers in order to determine an insulin resistance score for the subject.
In another embodiment, the present disclosure provides a method of monitoring the progression or regression of pre-diabetes in a subject, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for pre-diabetes in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to pre-diabetes progression and/or pre-diabetes-regression reference levels of the one or more biomarkers in order to monitor the progression or regression of pre-diabetes in a subject.
In yet another embodiment, the present disclosure provides a method of monitoring the efficacy of insulin resistance treatment, the method comprising: analyzing a first biological sample from a subject to determine the level(s) of one or more biomarkers for pre-diabetes, the first sample obtained from the subject at a first time point wherein the one or more biomarkers are selected from one or more biomarkers listed in Tables 4, 5, 6, 7, and 8, and combinations thereof; treating the subject for insulin resistance; analyzing a second biological sample from the subject to determine the level(s) of the one or more biomarkers, the second sample obtained from the subject at a second time point after treatment; comparing the level(s) of one or more biomarkers in the first sample to the level(s) of the one or more biomarkers in the second sample to assess the efficacy of the treatment for treating insulin resistance.
The present disclosure further provides a method of diagnosing whether a subject has metabolic syndrome, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for metabolic syndrome in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 12 and 13, analyzing the biological sample to determine the level(s) of one or more biomarkers for glucose disposal, obesity, and/or cardiovascular disease, wherein the one or more biomarkers for glucose disposal, obesity, and/or cardiovascular disease are selected from one or more biomarkers identified in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to the respective disorder-positive and/or disorder-negative reference levels of the one or more biomarkers in order to diagnose whether the subject has the metabolic syndrome.
In another embodiment, the present disclosure provides a method of diagnosing a cardiovascular disease in a subject, the method comprising, analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for a cardiovascular disease in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Tables 14, 15, 16, 17, 21, 22, 23, 25, and combinations thereof; and comparing the level(s) of the one or more biomarkers in the sample to disease-positive and/or disease-negative reference levels of the one or more biomarkers in order to diagnose whether the subject has cardiomyopathy or atherosclerosis.
The disclosure further provides a method for determining whether a subject is predisposed to becoming obese, the method comprising: analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for obesity in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Table 26; and comparing the level(s) of the one or more biomarkers in the sample to obesity-positive and/or obesity-negative and/or lean-positive and/or lean-negative reference levels of the one or more biomarkers in order to determine whether the subject is susceptible to obesity.
In yet a further embodiment, the disclosure provides a method for determining whether a therapeutic agent is capable of inducing weight gain in a subject, the method comprising: analyzing a biological sample from a subject receiving a therapeutic agent to determine the level(s) of one or more biomarkers for obesity in the sample, where the one or more biomarkers are selected from one or more biomarkers listed in Table 26; and comparing the level(s) of the one or more biomarkers in the sample to obesity-positive and/or obesity-negative and/or lean-positive and/or lean-negative reference levels of the one or more biomarkers in order to determine whether the subject is susceptible to gaining weight.
The present disclosure also provides a method for predicting a subject's response to a course of treatment for pre-diabetes or diabetes, the method comprising: analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for pre-diabetes, where the one or more biomarkers are selected from one or more biomarkers listed in Table 27; comparing the level(s) of one or more biomarkers in the sample to treatment-positive and/or treatment-negative reference levels of the one or more biomarkers to predict whether the subject is likely to respond to a course of treatment.
The disclosure also provides a method for monitoring a subject's response to a treatment for pre-diabetes or diabetes, the method comprising: analyzing a first biological sample from a subject to determine the level(s) of one or more biomarkers for pre-diabetes, the first sample obtained from the subject at a first time point where the one or more biomarkers are selected from one or more biomarkers listed in Table 28; administering the composition to the subject; analyzing a second biological sample from the subject to determine the level(s) of the one or more biomarkers, the second sample obtained from the subject at a second time point after administration of the composition; comparing the level(s) of one or more biomarkers in the first sample to the level(s) of the one or more biomarkers in the second sample to assess the efficacy of the composition for treating pre-diabetes or diabetes.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a graph showing the mean R2 values of Rd correlation as a function of the number of metabolites. As the number of compounds increases (from right to left), the r-square value for Rd correlation (Y) increases until it reaches an optimal number (n<30), indicating that variable selection is more or less stable for the approximately first 30 variables.
FIG. 2 provides a graph showing the mean R2 values of Rd correlation as a function of the number of metabolites. As the number of compounds increases (from right to left), test error for Rd correlation (Y) decreases until it reach an optimal number (n<30), indicating that variable selection is more or less stable for the approximately first 30 variables.
FIG. 3 provides a graph showing the mean R-square values (Y-axis) of Rd correlation as a function of the number of metabolites (X-axis).
FIG. 4 provides a graph showing the mean test error values (Y-axis) of Rd correlation as a function of the number of metabolites (X-axis).
FIG. 5 provides a schematic example of a report describing prophetic results of an algorithm using insulin resistance biomarkers to determine a subject's level of insulin resistance that is reported as an “IR Score” and containing additional clinical information (e.g. BMI, demographic information).
FIG. 6 provides a schematic illustration comparing the use of biomarkers for patient stratification according to the level of insulin resistance and the use of biomarkers for patient risk stratification for the progression of insulin resistance.
FIG. 7 provides a Random Forest Analysis Importance plot of one embodiment of metabolites that are useful biomarkers for predicting glucose disposal.
FIG. 8 provides a Random Forest Analysis Importance Plot of one embodiment of serum metabolites that are useful biomarkers for predicting metabolic syndrome.
FIG. 9 provides a Random Forest Analysis Importance Plot of one embodiment of plasma metabolites that are useful biomarkers for predicting metabolic syndrome.
FIG. 10 provides a Random Forest Analysis Importance Plots of embodiments of metabolites from plasma that are useful biomarkers for predicting atherosclerosis at early (initiation) (FIG. 10A), mid (FIG. 10B), later (FIG. 10C), or all (FIG. 10D) stages of the disease.
FIG. 11 provides a Random Forest Analysis Importance Plots of embodiments of metabolites from aorta tissue that are useful biomarkers for predicting atherosclerosis at early (initiation) (FIG. 11A), mid (FIG. 11B), later (FIG. 11C), or all (FIG. 11D) stages of the disease.
FIG. 12 provides a Random Forest Analysis Importance Plots of embodiments of metabolites from liver tissue that are useful biomarkers for predicting atherosclerosis at early (initiation) (FIG. 12A), mid (FIG. 12B), later (FIG. 12C), or all (FIG. 12D) stages of the disease.
FIG. 13 provides an example of plasma levels of cholesterol in atherosclerosis subjects and control subjects at different ages.
FIG. 14 provides an example of plasma levels of docosahexaenoic acid in atherosclerosis subjects and control subjects at different ages.
FIG. 15 provides an example of plasma levels of Metabolite-7888 in atherosclerosis subjects and control subjects at different ages.
FIG. 16 provides an example of plasma levels of Metabolite-X8403 in atherosclerosis subjects and control subjects at different ages.
FIG. 17 provides an example of plasma levels of Metabolite-X1834 in atherosclerosis subjects and control subjects at different ages.
FIG. 18 provides an example of plasma levels of p-cresol-sulfate in atherosclerosis subjects and control subjects at different ages.
FIG. 19 provides an example of plasma levels of Metabolite-4887 in atherosclerosis subjects and control subjects at different ages.
FIG. 20 provides an example of recursive partitioning of DCM biomarker metabolites.
FIG. 21 provides an example of model validation using plasma from metabolic syndrome and healthy subjects.
FIG. 22 provides an example of a model validation using serum from metabolic syndrome and healthy subjects.
FIG. 23 provides an example of a regression analysis showing the predictive power of the ten models combined on the glucose disposal rate (Rd).
FIG. 24 provides and illustration of the inter-relationships of the various risk factors for metabolic syndrome.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to biomarkers of pre-diabetes (e.g. impaired glucose tolerance, impaired fasting glucose tolerance, insulin resistance) and type-2 diabetes; methods for diagnosis of pre-diabetes and type 2 diabetes; methods of determining predisposition to pre-diabetes and type 2 diabetes; methods of monitoring progression/regression of pre-diabetes and type 2 diabetes; methods of assessing efficacy of compositions for treating pre-diabetes and type 2 diabetes; methods of screening compositions for activity in modulating biomarkers of pre-diabetes and type 2 diabetes; methods of treating pre-diabetes and type 2 diabetes; as well as other methods based on biomarkers of pre-diabetes and type 2 diabetes.
Current blood tests for insulin resistance perform poorly for early detection of insulin resistance or involve significant medical procedures.
Using metabolomic analysis, panels of metabolites that can be used in a simple blood test to predict insulin resistance as measured by the “gold standard” of hyperinsulinemic euglycemic clamps in at least two independent cohorts of subjects were discovered.
Independent studies were carried out to identify a set of biomarkers that when used with a polynomic algorithm will enable the early detection of changes in insulin resistance in a subject. The instant invention provides the subject with a score indicating the level of insulin resistance (“IR Score”) of the subject. The score can be based upon clinically significant changed reference level for a biomarker and/or combination of biomarkers. The reference level can be derived from an algorithm or computed from indices for impaired glucose tolerance and can be presented in a report as shown in FIG. 5. The IR Score places the subject in the range of insulin resistance from normal to high. Disease progression or remission can be monitored by periodic determination and monitoring of the IR Score. Response to therapeutic intervention can be determined by monitoring the IR Score. The IR Score can also be used to evaluate drug efficacy.
The present invention also relates to biomarkers of metabolic syndrome and cardiovascular diseases, such as atherosclerosis and cardiomyopathy; methods for diagnosis of such diseases and syndromes; methods of determining predisposition to such diseases and syndromes; methods of monitoring progression/regression of such diseases and syndromes; methods of assessing efficacy of compositions for treating such diseases and syndromes; methods of screening compositions for activity in modulating biomarkers of such diseases and syndromes; methods of treating such diseases and syndromes; as well as other methods based on biomarkers of such diseases and syndromes.
Prior to describing this invention in further detail, however, the following terms will first be defined.
Definitions:
“Biomarker” means a compound, preferably a metabolite, that is differentially present (i.e., increased or decreased) in a biological sample from a subject or a group of subjects having a first phenotype (e.g., having a disease) as compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having the disease). A biomarker may be differentially present at any level, but is generally present at a level that is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more; or is generally present at a level that is decreased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent). A biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value less than 0.05 and/or a q-value of less than 0.10 as determined using either Welch's T-test or Wilcoxon's rank-sum Test). Alternatively, the biomarkers demonstrate a correlation with pre-diabetes, or particular levels of pre-diabetes. The range of possible correlations is between negative (−) 1 and positive (+) 1. A result of negative (−) 1 means a perfect negative correlation and a positive (+) 1 means a perfect positive correlation, and 0 means no correlation at all. A “substantial positive correlation” refers to a biomarker having a correlation from +0.25 to +1.0 with a disorder or with a clinical measurement (e.g., Rd), while a “substantial negative correlation” refers to a correlation from −0.25 to −1.0 with a given disorder or clinical measurement. A “significant positive correlation” refers to a biomarker having a correlation of from +0.5 to +1.0 with a given disorder or clinical measurement (e.g., Rd), while a “significant negative correlation” refers to a correlation to a disorder of from −0.5 to −1.0 with a given disorder or clinical measurement.
The “level” of one or more biomarkers means the absolute or relative amount or concentration of the biomarker in the sample.
“Sample” or “biological sample” or “specimen” means biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material from the subject. The sample can be isolated from any suitable biological tissue or fluid such as, for example, adipose tissue, aortic tissue, liver tissue, blood, blood plasma, serum, or urine.
“Subject” means any animal, but is preferably a mammal, such as, for example, a human, monkey, non-human primate, rat, mouse, cow, dog, cat, pig, horse, or rabbit.
A “reference level” of a biomarker means a level of the biomarker that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof. A “positive” reference level of a biomarker means a level that is indicative of a particular disease state or phenotype. A “negative” reference level of a biomarker means a level that is indicative of a lack of a particular disease state or phenotype. For example, a “pre-diabetes-positive reference level” of a biomarker means a level of a biomarker that is indicative of a positive diagnosis of pre-diabetes in a subject, and a “pre-diabetes-negative reference level” of a biomarker means a level of a biomarker that is indicative of a negative diagnosis of pre-diabetes in a subject. As another example, a “pre-diabetes-progression-positive reference level” of a biomarker means a level of a biomarker that is indicative of progression of the pre-diabetes in a subject, and a “pre-diabetes-regression-positive reference level” of a biomarker means a level of a biomarker that is indicative of regression of the pre-diabetes. A “reference level” of a biomarker may be an absolute or relative amount or concentration of the biomarker, a presence or absence of the biomarker, a range of amount or concentration of the biomarker, a minimum and/or maximum amount or concentration of the biomarker, a mean amount or concentration of the biomarker, and/or a median amount or concentration of the biomarker; and, in addition, “reference levels” of combinations of biomarkers may also be ratios of absolute or relative amounts or concentrations of two or more biomarkers with respect to each other. Appropriate positive and negative reference levels of biomarkers for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired biomarkers in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between biomarker levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of biomarkers in biological samples (e.g., LC-MS, GC-MS, etc.), where the levels of biomarkers may differ based on the specific technique that is used.
“Non-biomarker compound” means a compound that is not differentially present in a biological sample from a subject or a group of subjects having a first phenotype (e.g., having a first disease) as compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having the first disease). Such non-biomarker compounds may, however, be biomarkers in a biological sample from a subject or a group of subjects having a third phenotype (e.g., having a second disease) as compared to the first phenotype (e.g., having the first disease) or the second phenotype (e.g., not having the first disease).
“Metabolite”, or “small molecule”, means organic and inorganic molecules which are present in a cell. The term does not include large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). The small molecules of the cell are generally found free in solution in the cytoplasm or in other organelles, such as the mitochondria, where they form a pool of intermediates which can be metabolized further or used to generate large molecules, called macromolecules. The term “small molecules” includes signaling molecules and intermediates in the chemical reactions that transform energy derived from food into usable forms. Examples of small molecules include sugars, fatty acids, amino acids, nucleotides, intermediates formed during cellular processes, and other small molecules found within the cell.
“Metabolic profile”, or “small molecule profile”, means a complete or partial inventory of small molecules within a targeted cell, tissue, organ, organism, or fraction thereof (e.g., cellular compartment). The inventory may include the quantity and/or type of small molecules present. The “small molecule profile” may be determined using a single technique or multiple different techniques.
“Metabolome” means all of the small molecules present in a given organism.
“Metabolic disorder” refers to disorders or diseases that result in perturbation of the normal physiological state of homeostasis due to an alteration in metabolism (anabolism and/or catabolism). An alteration in metabolism can result from an inability to break down (catabolize) a substance that should be broken down (e.g. phenylalanine) and as a result the substance and/or an intermediate substance builds up to toxic levels, or from an inability to produce (anabolize) some essential substance (e.g. insulin).
“Metabolic syndrome” refers to the concept of a clustering of metabolic risk factors that come together in a single individual and lead to a high risk of developing diabetes and/or cardiovascular diseases. The main features of metabolic syndrome include insulin resistance, hypertension (high blood pressure), cholesterol abnormalities, dyslipidemia, triglyceride abnormalities, an increased risk for clotting and excess body weight, especially in the abdomen, or obesity. Metabolic syndrome is also known as syndrome X, insulin resistance syndrome, obesity syndrome, dysmetabolic syndrome and Reaven's syndrome. The inter-relationships of the various risk factors for metabolic syndrome are illustrated in FIG. 24. The presence of three or more of the risk factors in a single individual is indicative of metabolic syndrome. The American Heart Association suggests that metabolic syndrome be diagnosed by the presence of three or more of the following components: (1) an elevated waste circumference (men, equal to or greater than 40 inches (102 cm); women, equal to or greater than 35 inches (88 cm)); (2) elevated triglycerides (equal to or greater than 150 mg/dL); (3) reduced High Density Lipids or HDL (men, less than 40 mg/dL; women, less than 50 mg/dL); (4) elevated blood pressure (equal to or greater than 130/85 mm Hg); and (5) elevated fasting glucose (equal to or greater than 100 mg/dL).
“Metabolic syndrome-related metabolic disorder” as used herein refers to metabolic syndrome as well as obesity, insulin resistance, type-2 diabetes, atherosclerosis, and cardiomyopathy.
“Diabetes” refers to a group of metabolic diseases characterized by high blood sugar (glucose) levels which result from defects in insulin secretion or action, or both.
“Type 2 diabetes” refers to one of the two major types of diabetes, the type in which the beta cells of the pancreas produce insulin, at least in the early stages of the disease, but the body is unable to use it effectively because the cells of the body are resistant to the action of insulin. In later stages of the disease the beta cells may stop producing insulin. Type 2 diabetes is also known as insulin-resistant diabetes, non-insulin dependent diabetes and adult-onset diabetes.
“Pre-diabetes” refers to one or more early diabetic conditions including impaired glucose utilization, abnormal or impaired fasting glucose levels, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance.
“Insulin resistance” refers to the condition when cells become resistant to the effects of insulin—a hormone that regulates the uptake of glucose into cells—or when the amount of insulin produced is insufficient to maintain a normal glucose level. Cells are diminished in the ability to respond to the action of insulin in promoting the transport of the sugar glucose from blood into muscles and other tissues(i.e. sensitivity to insulin decreases). Eventually, the pancreas produces far more insulin than normal and the cells continue to be resistant. As long as enough insulin is produced to overcome this resistance, blood glucose levels remain normal. Once the pancreas is no longer able to keep up, blood glucose starts to rise, resulting in diabetes. Insulin resistance ranges from normal (insulin sensitive) to insulin resistant (IR).
“Insulin sensitivity” refers to the ability of cells to respond to the effects of insulin to regulate the uptake and utilization of glucose. Insulin sensitivity ranges from normal to Insulin Resistant (IR).
The “IR Score” is a measure of insulin resistance based upon the insulin resistance biomarkers and algorithms of the instant invention that will allow the physician to place the patient on the spectrum of glucose tolerance, from normal to high.
“Glucose utilization” refers to the absorption of glucose from the blood by muscle and fat cells and utilization of the sugar for cellular metabolism. The uptake of glucose into cells is stimulated by insulin.
“Rd” refers to glucose disposal rate, a metric for glucose utilization. The rate at which glucose disappears from the blood (disposal rate) is an indication of the ability of the body to respond to insulin (i.e. insulin sensitivity). There are several methods to determine Rd and the hyperinsulinemic euglycemic clamp is regarded as the “gold standard” method. In this technique, while a fixed amount of insulin is infused, the blood glucose is “clamped” at a predetermined level by the titration of a variable rate of glucose infusion. The underlying principle is that upon reaching steady state, by definition, glucose disposal is equivalent to glucose appearance. During hyperinsulinemia, glucose disposal (Rd) is primarily accounted for by glucose uptake into skeletal muscle, and glucose appearance is equal to the sum of the exogenous glucose infusion rate plus the rate of hepatic glucose output (HGO). The rate of glucose infusion during the last 30 minutes of the test determines insulin sensitivity. If high levels (Rd=7.5 mg/min or higher) are required, the patient is insulin-sensitive. Very low levels (Rd=4.0 mg/min or lower) indicate that the body is resistant to insulin action. Levels between 4.0 and 7.5 mg/min (Rd values between 4.0 mg/min and 7.5 mg/min) are not definitive and suggest “impaired glucose tolerance,” an early sign of insulin resistance.
“Impaired fasting glucose (IFG)” and “impaired glucose tolerance (IGT)” are the two clinical definitions of “pre-diabetes”. IFG is defined as a fasting blood glucose concentration of 100-125 mg/dL. IGT is defined as a postprandial (after eating) blood glucose concentration of 140-199 mg/dL. It is known that IFG and IGT do not always detect the same pre-diabetic populations. Between the two populations there is approximately a 60% overlap observed. Fasting plasma glucose levels are a more efficient means of inferring a patient's pancreatic function, or insulin secretion, whereas postprandial glucose levels are more frequently associated with inferring levels of insulin sensitivity or resistance. IGT is known to identify a greater percentage of the pre-diabetic population compared to IFG. The IFG condition is associated with lower insulin secretion, whereas the IGT condition is known to be strongly associated with insulin resistance. Numerous studies have been carried out that demonstrate that IGT individuals with normal FPG values are at increased risk for cardiovascular disease. Patients with normal FPG values may have abnormal postprandial glucose values and are often unaware of their risk for pre-diabetes, diabetes, and cardiovascular disease.
“Fasting plasma glucose (FPG) test” is a simple test measuring blood glucose levels after an 8 hour fast. According to the ADA, blood glucose concentration of 100-125 mg/dL is considered IFG and defines pre-diabetes whereas ≧126 mg/dL defines diabetes. As stated by the ADA, FPG is the preferred test to diagnose diabetes and pre-diabetes due to its ease of use, patient acceptability, lower cost, and relative reproducibility. The weakness in the FPG test is that patients are quite advanced toward Type 2 Diabetes before fasting glucose levels change.
“Oral glucose tolerance test (OGTT)”, a dynamic measurement of glucose, is a postprandial measurement of a patient's blood glucose levels after oral ingestion of a 75 g glucose drink. Traditional measurements include a fasting blood sample at the beginning of the test, a one hour time point blood sample, and a 2 hour time point blood sample. A patient's blood glucose concentration at the 2 hour time point defines the level of glucose tolerance: Normal glucose tolerance (NGT) ≦140 mg/dL blood glucose; Impaired glucose tolerance (IGT)=140-199 mg/dL blood glucose; Diabetes ≧200 mg/dL blood glucose. As stated by the ADA, even though the OGTT is known to be more sensitive and specific at diagnosing pre-diabetes and diabetes, it is not recommended for routine clinical use because of its poor reproducibility and difficulty to perform in practice.
“Fasting insulin test” measures the circulating mature form of insulin in plasma. The current definition of hyperinsulinemia is difficult due to lack of standardization of insulin immunoassays, cross-reactivity to proinsulin forms, and no consensus on analytical requirements for the assays. Within-assay CVs range from 3.7%-39% and among-assay CVs range from 12%-66%. Therefore, fasting insulin is not commonly measured in the clinical setting and is limited to the research setting.
The “hyperinsulinemic euglycemic clamp (HI clamp)” is considered worldwide as the “gold standard” for measuring insulin resistance in patients. It is performed in a research setting, requires insertion of two catheters into the patient and the patient must remain immobilized for up to six hours. The HI clamp involves creating steady-state hyperinsulinemia by insulin infusion, along with parallel glucose infusion in order to quantify the required amount of glucose to maintain euglycemia (normal concentration of glucose in the blood; also called normoglycemia). The result is a measure of the insulin-dependent glucose disposal rate (Rd), measuring the peripheral uptake of glucose by the muscle (primarily) and adipose tissues. This rate of glucose uptake is notated by M, whole body glucose metabolism by insulin action under steady state conditions. Therefore, a high M indicates high insulin sensitivity and a lower M value indicates reduced insulin sensitivity, i.e. insulin resistance. The HI clamp requires three trained professionals to carry out the procedure, including simultaneous infusions of insulin and glucose over 2-4 hours and frequent blood sampling every 5 minutes for analysis of insulin and glucose levels. Due to the high cost, complexity, and time required for the HI clamp, this procedure is strictly limited to the clinical research setting.
“Obesity” refers to a chronic condition defined by an excess amount body fat. The normal amount of body fat (expressed as percentage of body weight) is between 25-30% in women and 18-23% in men. Women with over 30% body fat and men with over 25% body fat are considered obese.
“Body Mass Index, (or BMI)” refers to a calculation that uses the height and weight of an individual to estimate the amount of the individual's body fat. Too much body fat (e.g. obesity) can lead to illnesses and other health problems. BMI is the measurement of choice for many physicians and researchers studying obesity. BMI is calculated using a mathematical formula that takes into account both height and weight of the individual. BMI equals a person's weight in kilograms divided by height in meters squared. (BMI=kg/m2). Subjects having a BMI less than 19 are considered to be underweight, while those with a BMI of between 19 and 25 are considered to be of normal weight, while a BMI of between 25 to 29 are generally considered overweight, while individuals with a BMI of 30 or more are typically considered obese. Morbid obesity refers to a subject having a BMI of 40 or greater.
“Cardiovascular disease” refers to any disease of the heart or blood vessels. Cardiovascular or heart disease includes but is not limited to, for example, angina, arrhythmia, coronary artery disease (CAD), coronary heart disease, cardiomyopathy (including dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and diabetic cardiomyopathy) heart attack (myocardial infarction), heart failure, hypertrophic cardiomyopathy, mitral regurgitation, mitral valve prolapse, pulmonary stenosis, etc. Blood vessel disease includes but is not limited to, for example, peripheral vascular disease, artery disease, carotid artery disease, deep vein thrombosis, venous diseases, atherosclerosis, etc.
I. Biomarkers
The biomarkers described herein were discovered using metabolomic profiling techniques. Such metabolomic profiling techniques are described in more detail in the Examples set forth below as well as in U.S. Pat. Nos. 7,005,255 and 7,329,489 and U.S. patent application Ser. No. 11/357,732 (Publication No. 2007/0026389), Ser. No. 11/301,077 (Publication No. 2006/0134676), Ser. No. 11/301,078 (Publication No. 2006/0134677), Ser. No. 11/301,079 (Publication No. 2006/0134678), and Ser. No. 11/405,033 (Publication No. US 2007/0072203), the entire contents of which are hereby incorporated herein by reference.
Generally, metabolic profiles may be determined for biological samples from human subjects diagnosed with a condition such as pre-diabetes as well as from one or more other groups of human subjects (e.g., healthy control subjects with normal glucose tolerance, subjects with impaired glucose tolerance, subjects with insulin resistance). The metabolic profile for a pre-diabetes disorder may then be compared to the metabolic profile for biological samples from the one or more other groups of subjects. The comparisons may be conducted using models or algorithms, such as those described herein. Those molecules differentially present, including those molecules differentially present at a level that is statistically significant, in the metabolic profile of samples from subjects with a pre-diabetes disorder as compared to another group (e.g., healthy control subjects not pre-diabetic) may be identified as biomarkers to distinguish those groups.
Biomarkers for use in the methods disclosed herein may be obtained from any source of biomarkers related to pre-diabetes and/or type-2 diabetes. Biomarkers for use in methods disclosed herein relating to pre-diabetes include those listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, 29, and combinations and subsets thereof. In one embodiment, the biomarkers include those listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, and combinations thereof. Additional biomarkers include those disclosed in U.S. Application No. 60/950,286, the entirety of which is hereby incorporated by reference in its entirety. In one aspect, the biomarkers correlate to insulin resistance.
Biomarkers for use in methods disclosed herein relating to metabolic syndrome-related metabolic disorders include those listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 12, 13, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, 29, and combinations thereof. For example, biomarkers for use in distinguishing, or aiding in distinguishing, between subjects having metabolic syndrome and subjects not having metabolic syndrome include those biomarkers identified in Tables 4, 5, 6, 7, 8, 9A, 9B, 12, 13, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, 29, and combinations thereof. In one aspect, biomarkers for use in methods relating to metabolic syndrome include one or more of those listed in Tables 12 and 13. In another aspect biomarkers for use in methods relating to metabolic syndrome using plasma samples obtained from a subject include one or more of those listed in Table 12. In a preferred aspect, the biomarkers for use in methods disclosed herein related to metabolic syndrome using plasma samples obtained from a subject include one or more of the biomarkers N-acetylglycine, metabolite-6346, metabolite-8792, gamma-glu-leu, metabolite-4806, metabolite-3165, metabolite-7762, metabolite-3030, metabolite-5978, metabolite-3218, metabolite-2000, metabolite-5848, metabolite-3370, malic acid, metabolite-3843, metabolite-4275, metabolite-3094, metabolite-4167, metabolite-3073, aldosterone, metabolite-1320, metabolite-2185, phenylalanine, metabolite-2139, glutamic acid, alpha-tocopherol, metabolite-5767, metabolite-5346, metabolite-9855, and 1-octadecanol, and combinations thereof. In yet another aspect, biomarkers for use in methods relating to metabolic syndrome using serum samples obtained from a subject include one or more of those listed in Table 13. In a preferred aspect, the biomarkers for use in metabolic syndrome methods disclosed herein using serum samples obtained from a subject include one or more of the biomarkers metabolite-8792, metabolite-5767, metabolite-2139, metabolite-8402, metabolite-3073, phenylalanine, metabolite-4929, metabolite-3370, nonanate, N-acetylglycine, metabolite-5848, metabolite-3077, monopalmitin, dioctyl-phthalate, octadecanoic acid, cholesterol, metabolite-2608, metabolite-6272, metabolite-3012, D-glucose, metabolite-2986, metabolite-4275, metabolite-6268, tyrosine, metabolite-10683, metabolite-2000, alpha-tocopherol, metabolite-2469, xanthine, and metabolite-2039, and combinations thereof.
In another aspect, biomarkers for use in methods disclosed herein relating to metabolic syndrome may include the use of one or more biomarkers listed in Tables 12 and/or 13 in combination with one or more biomarkers in one or more of Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, or combinations thereof. For example, biomarkers for use in methods relating to metabolic syndrome may include one or more biomarkers listed in Tables 12 and/or 13 in combination with one or more biomarkers associated with insulin resistance, such as those listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, or combinations thereof.
Biomarkers for use in methods disclosed herein relating to pre-diabetic or diabetic conditions, such as impaired insulin sensitivity, insulin resistance, or type-2 diabetes include one or more of those listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, and combinations thereof. Such biomarkers allow subjects to be classified as insulin resistant, insulin impaired, or insulin sensitive. In one aspect, biomarkers for use in distinguishing or aiding in distinguishing, between subjects having impaired insulin sensitivity from subjects not having impaired insulin sensitivity include one or more of those listed in Table 4, 5, 6, 7, 8, 9A, and/or 9B. In another aspect, biomarkers for use in diagnosing insulin resistance include one or more of those listed in Tables 4, 5, 6, 7, 8, 9A, and/or 9B. In another example, biomarkers for use in distinguishing subjects having insulin resistance from subject not having insulin resistance include one or more of those listed in Tables 4, 5, 6, 7, 8, 9A, and/or 9B. In another example, biomarkers for use in categorizing, or aiding in categorizing, a subject as having impaired fasting glucose levels or impaired glucose tolerance include one or more of those listed in Tables 4, 5, 6, 7, 8, 9A, and/or 9B.
Biomarkers for use in methods disclosed herein relating to atherosclerosis include one or more of those listed in Tables 14, 15, 16, and/or 17 and combinations thereof. For example, biomarkers for use in distinguishing, or aiding in distinguishing, atherosclerotic subjects from non-atherosclerotic subjects include one or more of those biomarkers listed in Tables 14, 15, 16, 17, 3-methylhistidine, p-cresol sulfate, mannose, glucose, and/or gluconate, and combinations thereof. In one aspect biomarkers for use in methods relating to atherosclerosis using plasma samples from a subject include one or more of 3-methylhistidine, p-cresol sulfate, mannose, glucose, gluconate, and those listed in Tables 14 and 17. In another aspect biomarkers for use in methods relating to atherosclerosis using aortic samples from a subject include one or more of those listed in Table 15. In yet another aspect, biomarkers for use in methods relating to atherosclerosis using liver samples from a subject include one or more of those listed in Table 16. In one aspect, preferred biomarkers for use in methods involving subjects in an early stage of atherosclerosis include the biomarkers identified in FIGS. 10A, 11A, and 12A. Preferred biomarkers for use in methods involving subjects in a mid-stage of atherosclerosis include the biomarkers identified in FIGS. 10B, 11B, and 12B. Preferred biomarkers for use in methods involving subjects in a later stage of atherosclerosis include the biomarkers identified in FIGS. 10C, 11C, and 12C. Preferred biomarkers for use in methods involving subjects in any stage of atherosclerosis include the biomarkers identified in FIGS. 10D, 11D, and 12D.
Biomarkers for use in methods disclosed herein relating to cardiomyopathy include one or more of those biomarkers listed in Tables 21, 22, 23, and/or 25. Such markers may be used, for example, to distinguish, or aiding in distinguishing, between subjects having cardiomyopathy from subjects not having cardiomyopathy. In one aspect, biomarkers for use in methods relating to cardiomyopathy using cardiac tissue samples from a subject include one or more of those listed in Table 21. In another aspect, biomarkers for use in methods relating to cardiomyopathy using plasma samples from a subject include one or more of those listed in Table 22 and/or 23.
Biomarkers for use in methods disclosed herein relating to obesity include one or more of those biomarkers listed in Table 26. Such markers may be used, for example, to distinguish obese subjects from lean subjects. Such markers may also be used in combination with biomarkers for pre-diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy. In another aspect, the markers may be used, for example, to determine susceptibility to obesity or weight gain. In another aspect, the markers may be used, for example, to determine if a therapeutic agent is likely to induce weight gain in a subject.
Any number of biomarkers may be used in the methods disclosed herein. That is, the disclosed methods may include the determination of the level(s) of one biomarker, two or more biomarkers, three or more biomarkers, four or more biomarkers, five or more biomarkers, six or more biomarkers, seven or more biomarkers, eight or more biomarkers, nine or more biomarkers, ten or more biomarkers, fifteen or more biomarkers, etc., including a combination of all of the biomarkers in each or all of Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, 29, or any fraction thereof. In another aspect, the number of biomarkers for use in the disclosed methods include the levels of about thirty or less biomarkers, twenty-five or less, twenty or less, fifteen or less, ten or less, nine or less, eight or less, seven or less, six or less, five or less biomarkers. In another aspect, the number of biomarkers for use in the disclosed methods include the levels of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, or thirty biomarkers.
Although the identities of some of the biomarkers compounds are not known at this time, such identities are not necessary for the identification of the biomarkers in biological samples from subjects, as the “unnamed” compounds have been sufficiently characterized by analytical techniques to allow such identification. The analytical characterization of all such “unnamed” compounds is listed in the Examples. Such “unnamed” biomarkers are designated herein using the nomenclature “Metabolite” followed by a specific metabolite number.
In addition, the methods disclosed herein using the biomarkers listed in the tables may be used in combination with clinical diagnostic measures of the respective conditions. Combinations with clinical diagnostics may facilitate the disclosed methods, or confirm results of the disclosed methods (for example, facilitating or confirming diagnosis, monitoring progression or regression, and/or determining predisposition to pre-diabetes).
Finally, where the potential identity of a compound is proposed for an “unnamed” metabolite and such identity has not been confirmed, the nomenclature of “possible” (along with the potential compound identity) follows the “Metabolite” number. Such proposed identity should not be considered as limiting the analytical characterization of the otherwise “unnamed” compounds.
II. Diagnostic Methods
The biomarkers described herein may be used to diagnose, or to aid in diagnosing, whether a subject has a disease or condition, such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy. For example, biomarkers for use in diagnosing, or aiding in diagnosing, whether a subject has a insulin resistance include one or more of those identified in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, 29, and combinations thereof. In one embodiment, the biomarkers include one or more of those identified in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, and combinations thereof. In another embodiment, combinations of biomarkers include those, such as 2-hydroxybutyrate in combination with one or more biomarkers indentified in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, and/or 29.
Methods for diagnosing, or aiding in diagnosing, whether a subject has a disease or condition, such as pre-diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, may performed using one or more of the biomarkers identified in the respective tables provided herein. A method of diagnosing (or aiding in diagnosing) whether a subject has a disease or condition, such as pre-diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of the disease or condition in the sample and (2) comparing the level(s) of one or more biomarkers in the sample to disease- or condition-positive and/or disease- or condition-negative reference levels of the one or more biomarkers to diagnose (or aid in the diagnosis of) whether the subject has the disease or condition. For example, a method of diagnosing (or aiding in diagnosing) whether a subject is pre-diabetic comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of pre-diabetes in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to pre-diabetes-positive and/or pre-diabetes-negative reference levels of the one or more biomarkers in order to diagnose (or aid in the diagnosis of) whether the subject has pre-diabetes. The one or more biomarkers that are used are selected from Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof. When such a method is used in aiding in the diagnosis of a disease or condition, such as insulin resistance, pre-diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, the results of the method may be used along with other methods (or the results thereof) useful in the clinical determination of whether a subject has a given disease or condition. Methods useful in the clinical determination of whether a subject has a disease or condition such as pre-diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy are known in the art. For example, methods useful in the clinical determination of whether a subject has pre-diabetes include, for example, glucose disposal rates (Rd), body weight measurements, waist circumference measurements, BMI determinations, Peptide YY measurements, Hemoglobin A1C measurements, adiponectin measurements, fasting plasma glucose measurements, free fatty acid measurements, fasting plasma insulin measurements, and the like. Methods useful for the clinical determination of atherosclerosis and/or cardiomyopathy in a subject include angiography, stress-testing, blood tests (e.g. to measure homocysteine, fibrinogen, lipoprotein (a), small LDL particles, and c-reactive protein levels), electrocardiography, echocardiography, computed tomography (CT) scans, ankle/brachial index, and intravascular ultrasounds.
In another example, the identification of biomarkers for diseases or conditions such as insulin resistance, pre-diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy, allows for the diagnosis of (or for aiding in the diagnosis of) such diseases or conditions in subjects presenting one or more symptoms of the disease or condition. For example, a method of diagnosing (or aiding in diagnosing) whether a subject has insulin resistance comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of insulin resistance in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to insulin resistance-positive and/or insulin resistance-negative reference levels of the one or more biomarkers in order to diagnose (or aid in the diagnosis of) whether the subject has insulin resistance. The one or more biomarkers that are used are selected from Tables 4, 5, 6, 7, 8, 9A, and/or 9B. The biomarkers for insulin resistance may also be used to classify subjects as being either insulin resistant, insulin sensitive, or having impaired insulin sensitivity. As described in Example 2, below, biomarkers are identified that may be used to classify subjects as being insulin resistant, insulin sensitive, or having impaired insulin sensitivity. The biomarkers in Tables 4, 5, 6, 7, 8, 9A, and/or 9B, may also be used to classify subjects as having impaired fasting glucose levels or impaired glucose tolerance or normal glucose tolerance. Thus, the biomarkers may indicate compounds that increase and decrease as the glucose disposal rate increases. By determining appropriate reference levels of the biomarkers for each group (insulin resistant, insulin impaired, insulin sensitive), subjects can be diagnosed appropriately. The results of this method may be combined with the results of clinical measurements to aid in the diagnosis of insulin resistance or for categorizing the subject as having NGT, IFG, or IGT.
Increased insulin resistance correlates with the glucose disposal rate (Rd) as measured by the HI clamp. As exemplified below, metabolomic analysis was carried out to identify biomarkers that correlate with the glucose disposal rate (Rd). These biomarkers can be used in a mathematical model to determine the glucose disposal rate of the subject. The insulin sensitivity of the individual can be determined using this model. Using metabolomic analysis, panels of metabolites that can be used in a simple blood test to predict insulin resistance as measured by the “gold standard” of hyperinsulinemic euglycemic clamps in at least two independent cohorts of subjects were discovered. In another example, biomarkers are identified that correlate with the results of oral glucose tolerance tests (OGTT) for use in categorizing subjects as having normal glucose tolerance (NGT), impaired fasting glucose levels (IFG), or impaired glucose tolerance (IGT).
Independent studies were carried out to identify a set of biomarkers that when used with a polynomic algorithm will enable the early detection of changes in insulin resistance in a subject. In one aspect, the instant invention provides the subject with a score indicating the level of insulin resistance (“IR Score”) of the subject. The score is based upon clinically significant changed reference level for a biomarker and/or combination of biomarkers. The reference level can be derived from an algorithm or computed from indices for impaired glucose tolerance and can be presented in a report as shown in FIG. 5. The IR Score places the subject in the range of insulin resistance from normal (i.e. insulin sensitive) to high. Disease progression or remission can be monitored by periodic determination and monitoring of the IR Score. Response to therapeutic intervention can be determined by monitoring the IR Score. The IR Score can also be used to evaluate drug efficacy.
Methods for determining a subject's insulin resistance score (IR score) may be performed using one or more of the biomarkers identified in the respective Tables provided herein. For example, a method for determining the IR score of a subject comprises the steps of: (1) analyzing a biological sample from a subject to determine the level(s) of one or more insulin resistance biomarkers in the sample, and (2) comparing the level(s) of the one or more insulin resistance biomarkers in the sample to insulin resistance reference levels of the one or more biomarkers in order to determine the subject's insulin resistance score. The one or more biomarkers that are used may be selected from Tables 4, 5, 6, 7, 8, 9A, 9B, and combinations thereof. The method may employ any number of markers selected from Tables 4, 5, 6, 7, 8, 9A, and/or 9B, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more markers. Multiple biomarkers may be correlated with a given condition, such as insulin resistance, by any method, including statistical methods such as regression analysis.
Also as exemplified below, metabolomic analysis was carried out to identify biomarkers that correlate with metabolic syndrome, atherosclerosis, cardiomyopathy, and other diseases or conditions. Such biomarkers may be used in the methods of the present invention to analyze biological samples to identify or measure the level of the biomarkers in the sample.
Any suitable method may be used to analyze the biological sample in order to determine the level(s) of the one or more biomarkers in the sample. Suitable methods include chromatography (e.g., HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g., MS, MS-MS), enzyme-linked immunosorbent assay (ELISA), antibody linkage, other immunochemical techniques, and combinations thereof. Further, the level(s) of the one or more biomarkers may be measured indirectly, for example, by using an assay that measures the level of a compound (or compounds) that correlates with the level of the biomarker(s) that are desired to be measured.
After the level(s) of the one or more biomarker(s) is determined, the level(s) may be compared to disease or condition reference level(s) of the one or more biomarker(s) to determine a rating for each of the one or more biomarker(s) in the sample. The rating(s) may be aggregated using any algorithm to create a score, for example, an insulin resistance (IR) score, for the subject. The algorithm may take into account any factors relating to the disease or condition, such as insulin resistance, including the number of biomarkers, the correlation of the biomarkers to the disease or condition, etc.
In one example, the subject's insulin resistance score may be correlated to any index indicative of a level insulin resistance, from normal glucose tolerance to insulin resistant. For example, a subject having an insulin resistance score of less than 25 may indicate that the subject has normal glucose tolerance; a score of between 26 and 50 may indicate that the subject has low impaired glucose tolerance; a score of between 51 and 75 may indicate that the subject has medium impaired glucose tolerance; a score of between 76 and 100 may indicate that the subject has high impaired glucose tolerance; and a score above 100 may indicate that the subject has type-2 diabetes.
III. Monitoring Disease or Condition Progression/Regression
The identification of biomarkers herein allows for monitoring progression/regression of the respective diseases or conditions (e.g. pre-diabetes, metabolic syndrome, atherosclerosis, cardiomyopathy, insulin resistance, etc.) in a subject. A method of monitoring the progression/regression of disease or condition, such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy, in a subject comprises (1) analyzing a first biological sample from a subject to determine the level(s) of one or more biomarkers for the respective disease or condition selected from Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and combinations thereof in the first sample obtained from the subject at a first time point, (2) analyzing a second biological sample from a subject to determine the level(s) of the one or more biomarkers, the second sample obtained from the subject at a second time point, and (3) comparing the level(s) of one or more biomarkers in the first sample to the level(s) of the one or more biomarkers in the second sample in order to monitor the progression/regression of the disease or condition in the subject. The results of the method are indicative of the course of the disease or condition (i.e., progression or regression, if any change) in the subject.
In one embodiment, the results of the method may be based on Insulin Resistance (IR) Score which is indicative of the insulin resistance in the subject and which can be monitored over time. By comparing the IR Score from a first time point sample to the IR Score from at least a second time point sample the progression or regression of IR can be determined. Such a method of monitoring the progression/regression of pre-diabetes and/or type-2 diabetes in a subject comprises (1) analyzing a first biological sample from a subject to determine an IR score for the first sample obtained from the subject at a first time point, (2) analyzing a second biological sample from a subject to determine a second IR score, the second sample obtained from the subject at a second time point, and (3) comparing the IR score in the first sample to the IR score in the second sample in order to monitor the progression/regression of pre-diabetes and/or type-2 diabetes in the subject.
Using the biomarkers and algorithm of the instant invention for progression monitoring may guide, or assist a physician's decision to implement preventative measures such as dietary restrictions, exercise, or early-stage drug treatment.
IV. Determining Predisposition to a Disease or Condition
The biomarkers identified herein may also be used in the determination of whether a subject not exhibiting any symptoms of a disease or condition, such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy. The biomarkers may be used, for example, to determine whether a subject is predisposed to developing, for example, insulin resistance. Such methods of determining whether a subject having no symptoms of a particular disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, is predisposed to developing a particular disease or condition comprise (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers listed in the respective tables (e.g. Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, and combinations thereof) in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to disease- or condition-positive and/or disease- or condition-negative reference levels of the one or more biomarkers in order to determine whether the subject is predisposed to developing the respective disease or condition. For example, the identification of biomarkers for insulin resistance allows for the determination of whether a subject having no symptoms of insulin resistance is predisposed to developing insulin resistance. A method of determining whether a subject having no symptoms of insulin resistance is predisposed to developing insulin resistance comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, and 9B, and combinations thereof in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to insulin resistance-positive and/or insulin resistance-negative reference levels of the one or more biomarkers in order to determine whether the subject is predisposed to developing insulin resistance. The results of the method may be used along with other methods (or the results thereof) useful in the clinical determination of whether a subject is predisposed to developing the disease or condition.
After the level(s) of the one or more biomarkers in the sample are determined, the level(s) are compared to disease- or condition-positive and/or disease- or condition-negative reference levels in order to predict whether the subject is predisposed to developing a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy. Levels of the one or more biomarkers in a sample corresponding to the disease- or condition-positive reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of the subject being predisposed to developing the disease or condition. Levels of the one or more biomarkers in a sample corresponding to disease- or condition-negative reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of the subject not being predisposed to developing the disease or condition. In addition, levels of the one or more biomarkers that are differentially present (especially at a level that is statistically significant) in the sample as compared to disease- or condition-negative reference levels may be indicative of the subject being predisposed to developing the disease or condition. Levels of the one or more biomarkers that are differentially present (especially at a level that is statistically significant) in the sample as compared to disease-condition-positive reference levels are indicative of the subject not being predisposed to developing the disease or condition.
By way of example, after the level(s) of the one or more biomarkers in the sample are determined, the level(s) are compared to insulin resistance-positive and/or insulin resistance-negative reference levels in order to predict whether the subject is predisposed to developing insulin resistance. Levels of the one or more biomarkers in a sample corresponding to the insulin resistance-positive reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of the subject being predisposed to developing insulin resistance. Levels of the one or more biomarkers in a sample corresponding to the insulin resistance-negative reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of the subject not being predisposed to developing insulin resistance. In addition, levels of the one or more biomarkers that are differentially present (especially at a level that is statistically significant) in the sample as compared to insulin resistance-negative reference levels are indicative of the subject being predisposed to developing insulin resistance. Levels of the one or more biomarkers that are differentially present (especially at a level that is statistically significant) in the sample as compared to insulin resistance-positive reference levels are indicative of the subject not being predisposed to developing insulin resistance. Although insulin resistance is discussed in this example, predisposition to the other diseases or conditions may also be determined in accordance with this method by using one or more of the respective biomarkers as set forth above.
Furthermore, it may also be possible to determine reference levels specific to assessing whether or not a subject that does not have a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, is predisposed to developing a disease or condition. For example, it may be possible to determine reference levels of the biomarkers for assessing different degrees of risk (e.g., low, medium, high) in a subject for developing a disease or condition. Such reference levels could be used for comparison to the levels of the one or more biomarkers in a biological sample from a subject.
V. Monitoring Therapeutic Efficacy:
The biomarkers provided also allow for the assessment of the efficacy of a composition for treating a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy. For example, the identification of biomarkers for insulin resistance also allows for assessment of the efficacy of a composition for treating insulin resistance as well as the assessment of the relative efficacy of two or more compositions for treating insulin resistance. Such assessments may be used, for example, in efficacy studies as well as in lead selection of compositions for treating the disease or condition.
Thus, also provided are methods of assessing the efficacy of a composition for treating a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy comprising (1) analyzing, from a subject (or group of subjects) having a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy and currently or previously being treated with a composition, a biological sample (or group of samples) to determine the level(s) of one or more biomarkers for the disorder selected from Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and combinations thereof, and (2) comparing the level(s) of the one or more biomarkers in the sample to (a) level(s) of the one or more biomarkers in a previously-taken biological sample from the subject, wherein the previously-taken biological sample was obtained from the subject before being treated with the composition, (b) disease- or condition-positive reference levels of the one or more biomarkers, (c) disease- or condition-negative reference levels of the one or more biomarkers, (d) disease- or condition-progression-positive reference levels of the one or more biomarkers, and/or (e) disease- or condition-regression-positive reference levels of the one or more biomarkers. The results of the comparison are indicative of the efficacy of the composition for treating the respective disease or condition.
The change (if any) in the level(s) of the one or more biomarkers over time may be indicative of progression or regression of the disease or condition in the subject. To characterize the course of a given disease or condition in the subject, the level(s) of the one or more biomarkers in the first sample, the level(s) of the one or more biomarkers in the second sample, and/or the results of the comparison of the levels of the biomarkers in the first and second samples may be compared to the respective disease- or condition-positive and/or disease- or condition-negative reference levels of the one or more biomarkers. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time (e.g., in the second sample as compared to the first sample) to become more similar to the disease- or condition-positive reference levels (or less similar to the disease- or condition-negative reference levels), then the results are indicative of the disease's or condition's progression. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time to become more similar to the disease- or condition-negative reference levels (or less similar to the disease- or condition-positive reference levels), then the results are indicative of the disease's or condition's regression.
For example, in order to characterize the course of insulin resistance in the subject, the level(s) of the one or more biomarkers in the first sample, the level(s) of the one or more biomarkers in the second sample, and/or the results of the comparison of the levels of the biomarkers in the first and second samples may be compared to insulin resistance-positive and/or insulin resistance-negative reference levels of the one or more biomarkers. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time (e.g., in the second sample as compared to the first sample) to become more similar to the insulin resistance-positive reference levels (or less similar to the insulin resistance-negative reference levels), then the results are indicative of insulin resistance progression. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time to become more similar to the insulin resistance-negative reference levels (or less similar to the insulin resistance-positive reference levels), then the results are indicative of insulin resistance regression.
The second sample may be obtained from the subject any period of time after the first sample is obtained. In one aspect, the second sample is obtained 1, 2, 3, 4, 5, 6, or more days after the first sample or after the initiation of the administration of a composition. In another aspect, the second sample is obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks after the first sample or after the initiation of the administration of a composition. In another aspect, the second sample may be obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months after the first sample or after the initiation of the administration of a composition.
The course of a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy in a subject may also be characterized by comparing the level(s) of the one or more biomarkers in the first sample, the level(s) of the one or more biomarkers in the second sample, and/or the results of the comparison of the levels of the biomarkers in the first and second samples to disease- or condition-progression-positive and/or disease- or condition-regression-positive reference levels. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time (e.g., in the second sample as compared to the first sample) to become more similar to the disease- or condition-progression-positive reference levels (or less similar to the disease- or condition-regression-positive reference levels), then the results are indicative of the disease or condition progression. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time to become more similar to the disease- or condition-regression-positive reference levels (or less similar to the disease- or condition-progression-positive reference levels), then the results are indicative of disease or condition regression.
As with the other methods described herein, the comparisons made in the methods of monitoring progression/regression of a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy in a subject may be carried out using various techniques, including simple comparisons, one or more statistical analyses, and combinations thereof.
The results of the method may be used along with other methods (or the results thereof) useful in the clinical monitoring of progression/regression of the disease or condition in a subject.
As described above in connection with methods of diagnosing (or aiding in the diagnosis of) a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, any suitable method may be used to analyze the biological samples in order to determine the level(s) of the one or more biomarkers in the samples. In addition, the level(s) one or more biomarkers, including a combination of all of the biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, and/or 28, or any fraction thereof, may be determined and used in methods of monitoring progression/regression of the respective disease or condition in a subject.
Such methods could be conducted to monitor the course of disease or condition development in subjects, for example the course of pre-diabetes to type-2 diabetes in a subject having pre-diabetes, or could be used in subjects not having a disease or condition (e.g., subjects suspected of being predisposed to developing the disease or condition) in order to monitor levels of predisposition to the disease or condition.
Clinical studies from around the world have been carried out to test whether anti-diabetic therapies, such as metformin or acarbose, can prevent diabetes progression in pre-diabetic patients. These studies have shown that such therapies can prevent diabetes onset. From the U.S. Diabetes Prevention Program (DPP), metformin reduced the rate of progression to diabetes by 38% and lifestyle and exercise intervention reduced the rate of progression to diabetes by 56%. Because of such successes, the ADA has revised its 2008 Standards of Medical Care in Diabetes to include the following statements in the section on Prevention/Delay of Type 2 Diabetes: “In addition to lifestyle counseling, metformin may be considered in those who are at very high risk (combined IFG and IGT plus other risk factors) and who are obese and under 60 years of age.”
Pharmaceutical companies have carried out studies to assess whether certain classes of drugs, such as the PPARγ class of insulin sensitizers, can prevent diabetes progression. Similar to the DPP trial, some of these studies have shown great promise and success for preventing diabetes, whereas others have exposed a certain amount of risk associated with certain anti-diabetic pharmacologic treatments when given to the general pre-diabetic population as defined by current IR diagnostics. Pharmaceutical companies are in need of diagnostics that can identify and stratify high risk pre-diabetics so they can assess the efficacy of their pre-diabetic therapeutic candidates more effectively and safely.
Considering the infrequency of the oral glucose tolerance test (OGTT) procedures in the clinical setting, a new diagnostic test that directly measures insulin resistance in a fasted sample would enable a physician to identify and stratify patients who are moving toward the etiology of pre-diabetes and cardiovascular disease much earlier.
VI. Identification of Responders and Non-responders to Therapeutic:
The biomarkers provided also allow for the identification of subjects in whom the composition for treating a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy is efficacious (i.e. patient responds to therapeutic). For example, the identification of biomarkers for insulin resistance also allows for assessment of the subject response to a composition for treating insulin resistance as well as the assessment of the relative patient response to two or more compositions for treating insulin resistance. Such assessments may be used, for example, in selection of compositions for treating the disease or condition for certain subjects.
Thus, also provided are methods of predicting the response of a patient to a composition for treating a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy comprising (1) analyzing, from a subject (or group of subjects) having a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy and currently or previously being treated with a composition, a biological sample (or group of samples) to determine the level(s) of one or more biomarkers for the disorder selected from Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, and 28, and combinations thereof, and (2) comparing the level(s) of the one or more biomarkers in the sample to (a) level(s) of the one or more biomarkers in a previously-taken biological sample from the subject, wherein the previously-taken biological sample was obtained from the subject before being treated with the composition, (b) disease- or condition-positive reference levels of the one or more biomarkers, (c) disease- or condition-negative reference levels of the one or more biomarkers, (d) disease- or condition-progression-positive reference levels of the one or more biomarkers, and/or (e) disease- or condition-regression-positive reference levels of the one or more biomarkers. The results of the comparison are indicative of the response of the patient to the composition for treating the respective disease or condition.
The change (if any) in the level(s) of the one or more biomarkers over time may be indicative of response of the subject to the therapeutic. To characterize the course of a given therapeutic in the subject, the level(s) of the one or more biomarkers in the first sample, the level(s) of the one or more biomarkers in the second sample, and/or the results of the comparison of the levels of the biomarkers in the first and second samples may be compared to the respective disease- or condition-positive and/or disease- or condition-negative reference levels of the one or more biomarkers. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time (e.g., in the second sample as compared to the first sample) to become more similar to the disease- or condition-positive reference levels (or less similar to the disease- or condition-negative reference levels), then the results are indicative of the patient not responding to the therapeutic. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time to become more similar to the disease- or condition-negative reference levels (or less similar to the disease- or condition-positive reference levels), then the results are indicative of the patient responding to the therapeutic.
For example, in order to characterize the patient response to a therapeutic for insulin resistance, the level(s) of the one or more biomarkers in the first sample, the level(s) of the one or more biomarkers in the second sample, and/or the results of the comparison of the levels of the biomarkers in the first and second samples may be compared to insulin resistance-positive and/or insulin resistance-negative reference levels of the one or more biomarkers. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time (e.g., in the second sample as compared to the first sample) to become more similar to the insulin resistance-positive reference levels (or less similar to the insulin resistance-negative reference levels), then the results are indicative of non-response to the therapeutic. If the comparisons indicate that the level(s) of the one or more biomarkers are increasing or decreasing over time to become more similar to the insulin resistance-negative reference levels (or less similar to the insulin resistance-positive reference levels), then the results are indicative of response to the therapeutic.
The second sample may be obtained from the subject any period of time after the first sample is obtained. In one aspect, the second sample is obtained 1, 2, 3, 4, 5, 6, or more days after the first sample. In another aspect, the second sample is obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks after the first sample or after the initiation of treatment with the composition. In another aspect, the second sample may be obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months after the first sample or after the initiation of treatment with the composition.
As with the other methods described herein, the comparisons made in the methods of determining a patient response to a therapeutic for a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy in a subject may be carried out using various techniques, including simple comparisons, one or more statistical analyses, and combinations thereof.
The results of the method may be used along with other methods (or the results thereof) useful in determining a patient response to a therapeutic for the disease or condition in a subject.
As described above in connection with methods of diagnosing (or aiding in the diagnosis of) a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, any suitable method may be used to analyze the biological samples in order to determine the level(s) of the one or more biomarkers in the samples. In addition, the level(s) one or more biomarkers, including a combination of all of the biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, and/or 28, or any fraction thereof, may be determined and used in methods of monitoring progression/regression of the respective disease or condition in a subject.
Such methods could be conducted to monitor the patient response to a therapeutic for a disease or condition development in subjects, for example the course of pre-diabetes to type-2 diabetes in a subject having pre-diabetes, or could be used in subjects not having a disease or condition (e.g., subjects suspected of being predisposed to developing the disease or condition) in order to monitor levels of predisposition to the disease or condition.
Pharmaceutical companies have carried out studies to assess whether certain classes of drugs, such as the PPARγ class of insulin sensitizers, can prevent diabetes progression. Some of these studies have shown great promise and success for preventing diabetes, whereas others have exposed a certain amount of risk associated with certain anti-diabetic pharmacologic treatments when given to the general pre-diabetic population as defined by current IR diagnostics. Pharmaceutical companies are in need of diagnostics that can identify responders and non-responders in order to stratify high risk pre-diabetics to assess the efficacy of their pre-diabetic therapeutic candidates more effectively and safely. A new diagnostic test that discriminates non-responding from responding patients to a therapeutic would enable pharmaceutical companies to identify and stratify patients that are likely to respond to the therapeutic agent and target specific therapeutics for certain cohorts that are likely to respond to the therapeutic.
VII. Methods of Screening a Composition for Activity in Modulating Biomarkers
The biomarkers provided herein also allow for the screening of compositions for activity in modulating biomarkers associated with a disease or condition, such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy, which may be useful in treating the disease or condition. Such methods comprise assaying test compounds for activity in modulating the levels of one or more biomarkers selected from the respective biomarkers listed in the respective tables. Such screening assays may be conducted in vitro and/or in vivo, and may be in any form known in the art useful for assaying modulation of such biomarkers in the presence of a test composition such as, for example, cell culture assays, organ culture assays, and in vivo assays (e.g., assays involving animal models). For example, the identification of biomarkers for insulin resistance also allows for the screening of compositions for activity in modulating biomarkers associated with insulin resistance, which may be useful in treating insulin resistance. Methods of screening compositions useful for treatment of insulin resistance comprise assaying test compositions for activity in modulating the levels of one or more biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, and/or 28. Although insulin resistance is discussed in this example, the other diseases and conditions such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy may also be diagnosed or aided to be diagnosed in accordance with this method by using one or more of the respective biomarkers as set forth above.
The methods for screening a composition for activity in modulating one or more biomarkers of a disease or condition such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy comprise (1) contacting one or more cells with a composition, (2) analyzing at least a portion of the one or more cells or a biological sample associated with the cells to determine the level(s) of one or more biomarkers of a disease or condition selected from the biomarkers provided in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, and/or 26; and (3) comparing the level(s) of the one or more biomarkers with predetermined standard levels for the one or more biomarkers to determine whether the composition modulated the level(s) of the one or more biomarkers. In one embodiment, a method for screening a composition for activity in modulating one or more biomarkers of insulin resistance comprises (1) contacting one or more cells with a composition, (2) analyzing at least a portion of the one or more cells or a biological sample associated with the cells to determine the level(s) of one or more biomarkers of insulin resistance selected from Tables 4, 5, 6, 7, 8, 9A, and/or 9B; and (3) comparing the level(s) of the one or more biomarkers with predetermined standard levels for the one or more biomarkers to determine whether the composition modulated the level(s) of the one or more biomarkers. As discussed above, the cells may be contacted with the composition in vitro and/or in vivo. The predetermined standard levels for the one or more biomarkers may be the levels of the one or more biomarkers in the one or more cells in the absence of the composition. The predetermined standard levels for the one or more biomarkers may also be the level(s) of the one or more biomarkers in control cells not contacted with the composition.
In addition, the methods may further comprise analyzing at least a portion of the one or more cells or a biological sample associated with the cells to determine the level(s) of one or more non-biomarker compounds of a disease or condition, such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy. The levels of the non-biomarker compounds may then be compared to predetermined standard levels of the one or more non-biomarker compounds.
Any suitable method may be used to analyze at least a portion of the one or more cells or a biological sample associated with the cells in order to determine the level(s) of the one or more biomarkers (or levels of non-biomarker compounds). Suitable methods include chromatography (e.g., HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g., MS, MS-MS), ELISA, antibody linkage, other immunochemical techniques, biochemical or enzymatic reactions or assays, and combinations thereof. Further, the level(s) of the one or more biomarkers (or levels of non-biomarker compounds) may be measured indirectly, for example, by using an assay that measures the level of a compound (or compounds) that correlates with the level of the biomarker(s) (or non-biomarker compounds) that are desired to be measured.
VIII. Method of Identifying Potential Drug Targets
The disclosure also provides methods of identifying potential drug targets for diseases or conditions such as insulin resistance, pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy, using the biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, and/or 28. A method for identifying a potential drug target for a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis and cardiomyopathy comprises (1) identifying one or more biochemical pathways associated with one or more biomarkers for a metabolic syndrome-related metabolic disorder selected from the respective tables (Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, and/or 28); and (2) identifying a protein (e.g., an enzyme) affecting at least one of the one or more identified biochemical pathways, the protein being a potential drug target for the disease or condition. For example, the identification of biomarkers for insulin resistance also allows for the identification of potential drug targets for insulin resistance. A method for identifying a potential drug target for insulin resistance comprises (1) identifying one or more biochemical pathways associated with one or more biomarkers for insulin resistance selected from Tables 4, 5, 6, 7, 8, 9A, 9B, 27, and/or 28, and (2) identifying a protein (e.g., an enzyme) affecting at least one of the one or more identified biochemical pathways, the protein being a potential drug target for insulin resistance. Although insulin resistance is discussed in this example, the other diseases or conditions such as type-2 diabetes, metabolic syndrome, atherosclerosis and cardiomyopathy, may also be diagnosed or aided to be diagnosed in accordance with this method by using one or more of the respective biomarkers as set forth above.
Another method for identifying a potential drug target for a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy comprises (1) identifying one or more biochemical pathways associated with one or more biomarkers for a metabolic syndrome-related metabolic disorder selected from the respective table(s) (Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, and/or 28) and one or more non-biomarker compounds of the disease or condition and (2) identifying a protein affecting at least one of the one or more identified biochemical pathways, the protein being a potential drug target for the disease or condition. For example, a method for identifying a potential drug target for insulin resistance comprises (1) identifying one or more biochemical pathways associated with one or more biomarkers for insulin resistance selected from Tables 4, 5, 6, 7, 8, 9A, 9B, 27, and/or 28, and one or more non-biomarker compounds of insulin resistance and (2) identifying a protein affecting at least one of the one or more identified biochemical pathways, the protein being a potential drug target for insulin resistance.
One or more biochemical pathways (e.g., biosynthetic and/or metabolic (catabolic) pathway) are identified that are associated with one or more biomarkers (or non-biomarker compounds). After the biochemical pathways are identified, one or more proteins affecting at least one of the pathways are identified. Preferably, those proteins affecting more than one of the pathways are identified.
A build-up of one metabolite (e.g., a pathway intermediate) may indicate the presence of a ‘block’ downstream of the metabolite and the block may result in a low/absent level of a downstream metabolite (e.g. product of a biosynthetic pathway). In a similar manner, the absence of a metabolite could indicate the presence of a ‘block’ in the pathway upstream of the metabolite resulting from inactive or non-functional enzyme(s) or from unavailability of biochemical intermediates that are required substrates to produce the product. Alternatively, an increase in the level of a metabolite could indicate a genetic mutation that produces an aberrant protein which results in the over-production and/or accumulation of a metabolite which then leads to an alteration of other related biochemical pathways and result in dysregulation of the normal flux through the pathway; further, the build-up of the biochemical intermediate metabolite may be toxic or may compromise the production of a necessary intermediate for a related pathway. It is possible that the relationship between pathways is currently unknown and this data could reveal such a relationship.
The proteins identified as potential drug targets may then be used to identify compositions that may be potential candidates for treating a particular disease or condition, such as insulin resistance, including compositions for gene therapy.
IX. Methods of Treatment
In another aspect, methods for treating a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy are provided. The methods generally involve treating a subject having a disease or condition such as pre-diabetes, type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy with an effective amount of one or more biomarker(s) that are lowered in a subject having the disease or condition as compared to a healthy subject not having the disease or condition. The biomarkers that may be administered may comprise one or more of the biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 that are decreased in a disease or condition state as compared to subjects not having that disease or condition. Such biomarkers could be isolated based on the identity of the biomarker compound (i.e. compound name). The biomarkers that are currently unnamed metabolites could be isolated based on the analytical characterizations for the biomarkers listed in the Examples below (e.g. Table 29). In some embodiments, the biomarkers that are administered are one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 that are decreased in a metabolic syndrome-related metabolic disorder and that have a p-value less than 0.05 or a q-value less than 0.10, or both a p-value less than 0.05 and a q-value less than 0.10, as determined by using a Welch's T-test or a Wilcoxon's rank sum Test. In other embodiments, the biomarkers that are administered are one or biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 that are decreased in a disease or condition and that have either a positive or negative correlation with a disease or condition. In one embodiment, the biomarkers have a positive or negative correlation either ≧+0.5 or ≦−0.5, respectively, with a disease or condition. In other embodiments, the biomarkers that are administered are one or more biomarkers listed in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 that are decreased in a disease or condition by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent). In one example, the identification of biomarkers for insulin resistance also allows for the treatment of insulin resistance. For example, in order to treat a subject having insulin resistance, an effective amount of one or more insulin resistance biomarkers that are lowered in subjects having insulin resistance as compared to a healthy subject not having insulin resistance may be administered to the subject. The biomarkers that may be administered may comprise one or more of the biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 27, 28, and/or 29 that are decreased in a subject having insulin resistance. Although insulin resistance is discussed in this example, the other diseases or conditions, such as type-2 diabetes, metabolic syndrome, atherosclerosis, and cardiomyopathy, may also be treated in accordance with this method by using one or more of the respective biomarkers as set forth above.
X. Methods of Using the Biomarkers for Other Diseases or Conditions
In another aspect, at least some of the biomarkers disclosed herein for a particular disease or condition may also be biomarkers for other diseases or conditions. For example, it is believed that at least some of the insulin resistance biomarkers may be used in the methods described herein for other diseases or conditions (e.g., metabolic syndrome). That is, the methods described herein with respect to insulin resistance may also be used for diagnosing (or aiding in the diagnosis of) a disease or condition such as type-2 diabetes, metabolic syndrome, atherosclerosis, or cardiomyopathy, methods of monitoring progression/regression of such a disease or condition, methods of assessing efficacy of compositions for treating such a disease or condition, methods of screening a composition for activity in modulating biomarkers associated with such a disease or condition, methods of identifying potential drug targets for such diseases and conditions, and methods of treating such diseases and conditions. Such methods could be conducted as described herein with respect to insulin resistance.
XI. Other methods
Other methods of using the biomarkers discussed herein are also contemplated. For example, the methods described in U.S. Pat. No. 7,005,255 and U.S. patent application Ser. Nos. 11/357,732, 10/695,265 (Publication No. 2005/0014132), Ser. No. 11/301,077 (Publication No. 2006/0134676), Ser. No. 11/301,078 (Publication No. 2006/0134677), Ser. No. 11/301,079 (Publication No. 2006/0134678), and Ser. No. 11/405,033 may be conducted using a small molecule profile comprising one or more of the biomarkers disclosed herein.
In any of the methods listed herein, the biomarkers that are used may be selected from those biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 having p-values of less than 0.05 and/or those biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 having q-values less than 0.10, and/or having a positive or negative correlation either ≧+0.5 or ≦−0.5, respectively, with a disorder. The biomarkers that are used in any of the methods described herein may also be selected from those biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 that are decreased in a metabolic syndrome-related metabolic disorder (as compared to the control or remission) or that are decreased in remission (as compared to control or a particular disease or condition) by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent); and/or those biomarkers in Tables 4, 5, 6, 7, 8, 9A, 9B, 14, 15, 16, 17, 21, 22, 23, 25, 26, 27, 28, and/or 29 that are increased in a given disease or condition (as compared to the control or remission) or that are increased in remission (as compared to the control or a given disease or condition) by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more.
EXAMPLES
I. General Methods
A. Identification of Metabolic Profiles
Each sample was analyzed to determine the concentration of several hundred metabolites. Analytical techniques such as GC-MS (gas chromatography-mass spectrometry) and LC-MS (liquid chromatography-mass spectrometry) were used to analyze the metabolites. Multiple aliquots were simultaneously, and in parallel, analyzed, and, after appropriate quality control (QC), the information derived from each analysis was recombined. Every sample was characterized according to several thousand characteristics, which ultimately amount to several hundred chemical species. The techniques used were able to identify novel and chemically unnamed compounds.
B. Statistical Analysis:
The data was analyzed using several statistical methods to identify molecules (either known, named metabolites or unnamed metabolites) present at differential levels in a definable population or subpopulation (e.g., biomarkers for metabolic syndrome biological samples compared to control biological samples or compared to patients in remission from insulin resistance) useful for distinguishing between the definable populations (e.g., insulin resistance and control, insulin resistance and remission, remission and control). Other molecules (either known, named metabolites or unnamed metabolites) in the definable population or subpopulation were also identified.
Random forest analyses were used for classification of samples into groups (e.g. disease or healthy, insulin resistant or normal insulin sensitivity, atherosclerosis or normal, metabolic syndrome or obese but not metabolic syndrome). Random forests give an estimate of how well we can classify individuals in a new data set into each group, in contrast to a t-test, which tests whether the unknown means for two populations are different or not. Random forests create a set of classification trees based on continual sampling of the experimental units and compounds. Then each observation is classified based on the majority votes from all the classification trees.
Regression analysis was performed using the Random Forest Regression method and the Univariate Correlation/Linear Regression method to build models that are useful to identify the biomarker compounds that are associated with disease or disease indicators (e.g. atherosclerosis, metabolic syndrome, Rd) and then to identify biomarker compounds useful to classify individuals according to for example, the level of glucose utilization as normal, insulin impaired, or insulin resistant. Biomarker compounds that are useful to predict disease or measures of disease (e.g. atherosclerosis, metabolic syndrome, Rd) and that are positively or negatively correlated with disease or measures of disease (e.g. atherosclerosis, metabolic syndrome, Rd) were identified in these analyses. All of the biomarker compounds identified in these analyses were statistically significant (p<0.05, q<0.1).
Recursive partitioning relates a ‘dependent’ variable (Y) to a collection of independent (‘predictor’) variables (X) in order to uncover—or simply understand—the elusive relationship, Y=f(X). The analysis was performed with the JMP program (SAS) to generate a decision tree. The statistical significance of the “split” of the data can be placed on a more quantitative footing by computing p-values, which discern the quality of a split relative to a random event. The significance level of each “split” of data into the nodes or branches of the tree was computed as p-values, which discern the quality of the split relative to a random event. It was given as LogWorth, which is the negative log 10 of a raw p-value.
Statistical analyses were performed with the program “R” available on the worldwide web at the website cran.r-project.org and in JMP 6.0.2 (SAS® Institute, Cary, N.C.).
Example 2
Biomarkers of Pre-Diabetes
2A: Identification of Biomarkers that Correlate with Glucose Disposal
A combination of biomarkers were discovered that, when used in an algorithm, correlate with the glucose disposal rate (i.e. Rd). Further, the initial panel of biomarkers can be narrowed for the development of targeted assays comprised of 15-30 candidate metabolites. An algorithm to predict insulin resistance was developed.
Several studies were conducted to identify biomarkers that correlate with glucose disposal. In a first study, plasma samples were collected from 113 lean, obese or diabetic subjects that had received treatment with one of three different thiazolidinedione drugs (T=troglitazone, R=rosiglitazone, or P=pioglitazone) (Table 1). Base line samples obtained from the subjects prior to treatment (S=baseline) served as controls.
One to three plasma samples were obtained from each subject, with samples collected at baseline (all subjects; A), and after 12 weeks (B) or 4 weeks (C) of drug treatment (Table 2). Glucose disposal rate was measured in every subject by the hyperinsulinemic euglycemic (HI) clamp following each blood draw. A total of 198 plasma samples were collected for analysis.







TABLE 1







Gender and treatments of the study 1 cohort.















Group
Gender
P
R
S
T
Total



















Lean
F
1
0
1
1
3




M
7
0
12
8
27



Obese
F
2
0
3
1
6




M
7
0
14
8
29



Diabetic
F
0
7
3
1
11




M
8
13
7
9
37














Total
25
20
40
28
113

















TABLE 2







Treatment and collection time of the study 1 cohort.













GROUP
TIME
P
R
S
T
Total
















L
A
8
0
13
9
30



B
8
0
0
8
16


O
A
9
0
17
9
35



B
9
0
0
9
18



C
9
0
0
0
9


D
A
8
19
10
9
46



B
8
20
0
10
38



C
6
0
0
0
6



Total
65
39
40
54
198









In a second study, plasma samples were collected from 402 subjects that were balanced for age and gender. The subjects underwent HI clamp to determine the glucose disposal rate (Rd) of each individual. Based upon an Oral Glucose Tolerance Test (OGTT) or a Fasting Plasma Glucose Test (FPGT) the glucose tolerance of the subjects was designated as Normal glucose tolerance (NGT), Impaired Fasting Glucose (IFG) or Impaired Glucose Tolerance (IGT). The cohort is described in Table 3.







TABLE 3







Cohort Description, Study 2










Age
Rd













Group 
Gender 
N
Mean
Std Dev
Mean
Std Dev
















NGT
female
155
44.64
8.02
8.5
3.09



male
148
44.03
8.62
8.38
2.77


IFG
female
5
46.8
6.53
6.13
3.32



male
12
45.25
9.63
4.67
2.57


IGT
female
45
45.56
7.81
4.19
1.81



male
37
45.73
7.8
4.73
2.27





Abbreviations


Rd: Glucose disposal rate


NGT: Normal Glucose Tolerant (OGTT, <140 mg/dL or <7.8 mmol/L)


IFG: Impaired Fasting Glucose (Fasting plasma glucose, 100-125 mg/dL or 5.6-6.9 mmol/L)


IGT: Impaired Glucose Tolerant (OGTT, 140-199 mg/dL or 7.8-11.0 mmol/L)






All samples from both studies were analyzed by GC-MS and LC-MS to identify and quantify the small molecules present in the samples. Over 400 compounds were detected in the samples.
Statistical analyses were performed to determine the compounds that are useful as biomarkers. Linear regression was used to correlate the baseline levels of individual compounds with the glucose disposal rate (Rd) as measured by the euglycemic hyperinsulinemic clamp for each individual. This analysis was followed by Random Forest analysis to identify variables most useful for Rd modeling. Then, LASSO regression analysis was performed on the cross-validated variables from the Random Forest analysis to pick the combination of variables useful to predict Rd.
2B: Biomarkers of Glucose Utilization, Molecules Positively and Negatively Correlated with Glucose Disposal Rate (Rd)
Biomarkers were discovered by (1) analyzing blood samples drawn from different groups of human subjects to determine the levels of metabolites in the samples and then (2) statistically analyzing the results to determine those metabolites that were differentially present in the groups of subjects and the metabolites that correlate with the glucose disposal rate, an indicator of insulin sensitivity.
The plasma samples used for the analysis were from the cohorts described in Tables 1, 2 and 3; the subjects had various rates of glucose disposal (Rd). Based on the Rd value subjects were classified as insulin resistant (Rd≦4), insulin impaired (4<Rd<7.5) or insulin sensitive (Rd>7.5). After the levels of metabolites were determined, the data was analyzed using Univariate Correlation/Linear Regression.
As listed below in Table 4, biomarkers were discovered that were correlated with the glucose disposal rate (Rd), an indicator of insulin sensitivity.
Table 4 includes, for each listed biomarker, the p-value determined in the statistical analysis of the data concerning the biomarkers, and the correlation (Con) with Rd. A positive correlation indicates that the level of the biomarker increases as the glucose disposal rate increases. A negative correlation indicates that the level of the biomarker decreases as the glucose disposal rate increases. The range of possible correlations is between negative (−)1.0 and positive (+)1.0. A result of negative (−)1.0 means a perfect negative correlation, a positive (+)1.0 means a perfect positive correlation, and 0 means no correlation at all. The term “Isobar” as used in the table indicates the compounds that could not be distinguished from each other on the analytical platform used in the analysis (i.e., the compounds in an isobar elute at nearly the same time and have similar (and sometimes exactly the same) quant ions, and thus cannot be distinguished).
The results of this analysis showed the individual compounds that are correlated with Rd in both study 1 and study 2; these biomarkers are listed in Table 4. For each biomarker the study, compound name, database identifier, median importance are given. The Library ID (LIB_ID) indicates the analytical platform that was used to measure the biomarker compound. GC-MS is indicated by Library ID (Lib_ID) 50 whereas LC-MS is indicated by Library ID 61, 200, and 201. The biomarker compounds are ordered in the table by the statistical significance of the correlation (P-value). “RF_Rank” refers to the importance score obtained for the biomarker from Random Forest analysis. “Comp_ID” refers to the internal database identifier for that compound in our internal compound library.







TABLE 4







Biomarker correlation with Rd














Study
Comp_ID
Compound Name
Lib_ID
RF_Rank
Correlation
R-square
P-value

















2
21044
2-hydroxybutyrate (AHB)
50
1
−4.47E−01
0.200
1.21E−14


1
20488
glucose
50
1
3.69E−01
0.136
1.21E−14


1
587
gluconate
50
2
−6.11E−01
0.373
8.73E−13


1
1336
palmitate (16:0)
201
5
−5.95E−01
0.355
4.37E−12


1
33416
Metabolite-12064
201
9
−5.94E−01
0.353
5.09E−12


1
20675
1,5-anhydroglucitol (1,5-AG)
201
13
5.93E−01
0.352
5.62E−12


1
12751
glutamate-2
50
3
−5.82E−01
0.339
1.66E−11


1
1121
margarate (17:0)
50
4
−5.52E−01
0.304
2.88E−10


1
584
mannose
50
6
−5.50E−01
0.303
3.28E−10


1
31535
Bradykinin, hydroxyproline 
61
6
1.22E−08
0.295
7.09E−10




form







1
21044
2-hydroxybutyrate (AHB)
50
14
−5.40E−01
0.292
7.75E−10


1
27719
galactonic acid
50
1
−5.40E−01
0.291
8.28E−10


1
16235
Isobar.19 (1,5-AG etc)
61
31
1.96E−08
0.287
1.43E−09


1
33388
Metabolite-12037
201
19
−5.32E−01
0.283
1.58E−09


2
1359
oleate (18:1(n-9))
201
3
−3.54E−01
0.125
2.15E−09


1
1358
stearate (18:0)
201
15
−5.22E−01
0.273
3.57E−09


1
27392
dipalmitin
50
8
−5.18E−01
0.268
4.97E−09


1
32569
Metabolite-11252
200
56
−5.10E−01
0.260
9.09E−09


1
33232
Metabolite-11887
201
41
−5.03E−01
0.253
1.60E−08


1
21047
3-methyl-2-oxobutyrate
201
42
−5.03E−01
0.253
1.64E−08


1
10737
Isobar.1 (mannose, glucose 
61
36
1.72E−07
0.254
1.75E−08




etc)







1
22570
Metabolite-9033
50
62
−4.98E−01
0.248
2.24E−08


1
32566
Metabolite-11249
200
70
−4.95E−01
0.245
2.82E−08


1
18369
gamma-glutamylleucine
200
12
−4.95E−01
0.245
2.95E−08


1
25602
Metabolite-10432
50
39
−4.91E−01
0.241
3.79E−08


1
27722
erythrose
50
43
−4.91E−01
0.241
3.84E−08


1
32630
oleate (18:1(n-9))
201
54
−4.86E−01
0.236
5.47E−08


1
27888
Metabolite-10609
50
10
−4.85E−01
0.235
5.87E−08


1
24077
Metabolite-9727
50
22
−4.85E−01
0.235
6.15E−08


1
32696
Metabolite-11379
201
37
−4.84E−01
0.234
6.50E−08


1
12666
Threonine
50
9
4.82E−07
0.235
7.02E−08


1
32551
Metabolite-11234
201
34
−4.83E−01
0.233
7.11E−08


1
30288
Metabolite-10750
50
25
−4.82E−01
0.232
7.39E−08


1
33413
Metabolite-12061
201
16
−4.80E−01
0.231
8.27E−08


1
60
leucine
200
58
−4.80E−01
0.230
8.52E−08


1
21127
palmitoylglycerol
50
60
−4.75E−01
0.225
1.23E−07




(monopalmitin)







1
32393
glutamylvaline
200
11
−4.74E−01
0.225
1.26E−07


1
19462
Metabolite-6446
50
30
−4.74E−01
0.225
1.28E−07


1
16120
Metabolite-4055
50
38
−4.73E−01
0.223
1.44E−07


1
32515
valine
200
71
−4.72E−01
0.223
1.49E−07


1
5628
Metabolite-1086
61
5
1.12E−06
0.221
1.96E−07


1
32704
Metabolite-11387
200
115
−4.68E−01
0.219
1.99E−07


1
32571
Metabolite-11254
200
97
−4.66E−01
0.217
2.25E−07


1
32501
dihomo-alpha-linolenate
201
26
−4.66E−01
0.217
2.31E−07




(20:3(n-3))







1
17627
Metabolite-4986
50
24
−4.61E−01
0.213
3.05E−07


1
32402
gondoate (20:1(n-9))
201
101
−4.48E−01
0.201
7.10E−07


1
32575
Metabolite-11258
200
241
−4.47E−01
0.200
7.75E−07


1
32970
Metabolite-11653
201
51
−4.45E−01
0.198
8.81E−07


1
16512
Metabolite-4275
50
54
3.95E−06
0.2
8.91E−07


1
12757
Metabolite-3078
50
52
4.44E−01
0.197
9.64E−07


1
27718
creatine
200
27
−4.41E−01
0.194
1.16E−06


1
30832
Metabolite-10814
50
47
−4.40E−01
0.193
1.23E−06


1
30290
Metabolite-10752
50
121
−4.38E−01
0.192
1.36E−06


1
32703
Metabolite-11386
200
94
−4.37E−01
0.191
1.47E−06


1
577
fructose
50
93
−4.36E−01
0.190
1.53E−06


1
22116
4-methyl-2-oxopentanoate
201
74
−4.35E−01
0.189
1.65E−06


1
33172
Metabolite-11827
201
61
4.35E−01
0.189
1.67E−06


1
22600
Metabolite-9043
50
90
−4.31E−01
0.186
2.05E−06


1
1125
isoleucine
200
137
−4.31E−01
0.185
2.14E−06


1
19490
Metabolite-6488
50
32
−4.28E−01
0.183
2.46E−06


1
16518
Metabolite-4276
50
18
−4.26E−01
0.182
2.78E−06


1
15122
glycerol
50
45
−4.25E−01
0.181
2.93E−06


1
12782
Metabolite-3100
50
17
−4.23E−01
0.179
3.39E−06


1
33242
Metabolite-11897
201
77
−4.21E−01
0.177
3.75E−06


1
17330
Metabolite-4769
50
35
4.21E−01
0.177
3.86E−06


1
32672
Metabolite-02546_200
200
67
4.14E−01
0.171
5.69E−06


1
33237
Metabolite-11892
201
124
−4.12E−01
0.170
6.48E−06


1
32673
linoleate (18:2(n-6))
201
28
−4.07E−01
0.166
8.49E−06


1
32545
Metabolite-11228
201
64
−4.03E−01
0.163
1.05E−05


1
32751
Metabolite-11434
201
63
−4.03E−01
0.162
1.06E−05


1
22895
Metabolite-9299
50
72
−3.99E−01
0.159
1.34E−05


2
33488
5-alpha-Cholest-7-en-3-
50
11
−2.61E−01
0.068
1.40E−05




beta-ol







1
32517
Metabolite-11203
200
82
3.98E−01
0.158
1.42E−05


1
33415
Metabolite-12063
201
44
−3.97E−01
0.158
1.46E−05


1
32682
Metabolite-11365
201
29
−3.94E−01
0.155
1.72E−05


2
32761
Metabolite-11444
201
12
−2.58E−01
0.067
1.77E−05


2
32338
glycine
50
13
2.58E−01
0.066
1.83E−05


1
32749
Metabolite-11432
201
107
−3.91E−01
0.153
2.04E−05


1
1110
arachidonate (20:4(n-6))
50
49
−3.90E−01
0.152
2.12E−05


1
33138
Metabolite-11793
200
96
−3.88E−01
0.150
2.39E−05


2
33447
palmitoleate (16:1(n-7))
201
15
−2.53E−01
0.064
2.59E−05


1
32504
n-3 DPA (22:5(n-3))
201
31
−3.83E−01
0.147
3.05E−05


1
18868
Metabolite-5847
50
80
−3.80E−01
0.144
3.56E−05


1
27738
threonate
50
53
3.75E−01
0.141
4.62E−05


1
32552
Metabolite-11235
201
73
−3.75E−01
0.140
4.68E−05


1
27279
Metabolite-10511
50
7
−3.69E−01
0.136
6.11E−05


1
32547
Metabolite-11230
201
163
−3.68E−01
0.135
6.55E−05


1
19377
Metabolite-6272
50
194
−3.66E−01
0.134
7.40E−05


1
32416
alpha-linolenate (18:3(n-3)) 
201
65
-3.66E−01
0.134
7.41E−05


1
33080
Metabolite-11735
200
50
3.63E−01
0.132
8.27E−05


1
22320
Metabolite-8889
50
177
3.62E−01
0.131
8.90E−05


1
32945
Metabolite-11628
201
197
−3.60E−01
0.130
9.48E−05


2
599
pyruvate
50
18
−2.31E−01
0.053
1.00E−04


2
33453
alpha-ketoglutarate
50
17
−2.35E−01
0.055
1.00E−04


2
1105
linoleate (18:2(n-6))
201
16
−2.44E−01
0.059
1.00E−04


1
527
lactate
50
76
−3.58E−01
0.128
1.06E−04


1
15676
3-methyl-2-oxovalerate
201
216
−3.53E−01
0.125
1.35E−04


1
32836
peptide-HWESASXX
200
33
−3.47E−01
0.121
1.76E−04


1
32637
Metabolite-11320
201
100
3.45E−01
0.119
1.91E−04


2
15749
hydrocinnamic acid
201
21
2.26E−01
0.051
2.00E−04


2
1648
serine
50
19
2.27E−01
0.052
2.00E−04


1
15500
carnitine
200
310
−3.38E−01
0.114
2.64E−04


1
16496
pyruvate
50
119
−3.35E−01
0.113
3.00E−04


1
32559
Metabolite-11242
201
87
−3.35E−01
0.112
3.11E−04


1
32632
Metabolite-11315
200
110
3.31E−01
0.110
3.61E−04


2
33587
Isobar-cis-9-cis-11-trans-11- 
201
23
-2.16E−01
0.046
4.00E−04




eicosenoate







2
32401
trigonelline (N-
200
22
2.16E−01
0.047
4.00E−04




methylnicotinate)







1
21630
Metabolite-8402
50
36
−3.27E−01
0.107
4.40E−04


1
16666
Metabolite-4365
50
272
−3.25E−01
0.105
4.79E−04


1
16665
Metabolite-4364
50
105
3.24E−01
0.105
4.98E−04


1
19983
Metabolite-6955
50
122
−3.24E−01
0.105
4.99E−04


2
32405
3-indolepropionate
50
24
2.10E−01
0.044
5.00E−04


1
31509
Metabolite-10931
50
220
−3.21E−01
0.103
5.66E−04


1
27889
Metabolite-10610
50
95
−3.19E−01
0.102
6.12E−04


1
16650
Metabolite-4360
50
125
−3.18E−01
0.101
6.37E−04


1
32644
Metabolite-11327
200
111
−3.18E−01
0.101
6.39E−04


2
32445
3-methylxanthine
201
26
2.06E−01
0.042
7.00E−04


1
19370
Metabolite-6268
50
179
3.14E−01
0.099
7.47E−04


1
32702
Metabolite-11385
200
221
−3.13E−01
0.098
7.73E−04


1
19576
Metabolite-6627
50
135
−3.12E−01
0.098
7.97E−04


2
32757
Metabolite-11440
201
28
−2.03E−01
0.041
8.00E−04


1
12663
serine-2
50
20
3.11E−01
0.097
8.44E−04


1
19494
Metabolite-6506
50
236
−3.10E−01
0.096
8.82E−04


1
32628
palmitoleate (16:1(n-7))
201
83
−3.10E−01
0.096
8.90E−04


1
59
histidine
201
355
−3.09E−01
0.096
9.00E−04


1
32516
Metabolite-11202
200
134
3.08E−01
0.095
9.69E−04


1
33087
peptide-RPPGFSPF
200
127
−3.04E−01
0.093
1.11E−03


2
31453
cysteine
50
29
−1.95E−01
0.038
1.30E−03


1
16138
Metabolite-4080
50
371
−3.00E−01
0.090
1.30E−03


2
24074
Metabolite-9706
50
30
1.94E−01
0.038
1.40E−03


1
32595
Metabolite-08993_200
200
48
−2.97E−01
0.088
1.46E−03


1
16509
Metabolite-4273
50
369
2.96E−01
0.088
1.50E−03


1
32735
Metabolite-01911_200
200
146
−2.96E−01
0.088
1.53E−03


1
30281
glycine-2
50
154
2.95E−01
0.087
1.58E−03


1
32519
Metabolite-11205
200
120
2.95E−01
0.087
1.59E−03


2
33531
Metabolite-12116
200
31
1.91E−01
0.036
1.60E−03


1
64
phenylalanine
200
21
−2.90E−01
0.084
1.89E−03


1
32548
Metabolite-11231
201
131
−2.90E−01
0.084
1.89E−03


1
22154
bradykinin
200
55
−2.89E−01
0.084
2.00E−03


1
32348
2-aminobutyrate
200
297
−2.86E−01
0.082
2.25E−03


1
31537
peptide-HWESASXXR
200
99
−2.84E−01
0.081
2.41E−03


1
32747
Metabolite-01142_201
201
148
−2.83E−01
0.080
2.48E−03


1
32550
Metabolite-02272_201
201
176
2.82E−01
0.080
2.59E−03


2
15753
hippurate
200
35
1.82E−01
0.033
2.70E−03


2
32198
acetylcarnitine
200
34
−1.82E−01
0.033
2.70E−03


1
21188
stearoylglycerol
50
196
−2.78E−01
0.077
2.97E−03




(monostearin)







1
12626
Metabolite-3003
50
198
2.77E−01
0.077
3.10E−03


1
32654
Metabolite-11337
200
84
−2.73E−01
0.075
3.53E−03


1
21421
Metabolite-8214
50
98
−2.73E−01
0.075
3.58E−03


2
32807
Metabolite-11490
201
38
−1.74E−01
0.030
4.10E−03


1
606
uridine
201
230
−2.69E−01
0.072
4.16E−03


1
19487
Metabolite-6486
50
78
−2.69E−01
0.072
4.18E−03


1
32412
butyrylcarnitine
200
389
−2.69E−01
0.072
4.20E−03


2
32616
Metabolite-11299
201
41
1.72E−01
0.030
4.60E−03


1
25459
Metabolite-10395
50
285
−2.62E−01
0.068
5.35E−03


1
33210
Metabolite-11865
201
171
2.61E−01
0.068
5.47E−03


1
27264
Metabolite-10503
50
181
2.59E−01
0.067
5.85E−03


1
32578
Metabolite-11261
200
223
−2.58E−01
0.067
5.93E−03


1
32609
Metabolite-01345_201
201
57
2.58E−01
0.067
5.94E−03


1
25609
Metabolite-10439
50
150
−2.57E−01
0.066
6.19E−03


1
12768
Metabolite-3088
50
91
2.56E−01
0.065
6.49E−03


1
18120
Metabolite-5348
50
108
−2.56E−01
0.065
6.53E−03


2
3147
xanthine
50
44
−1.65E−01
0.027
6.60E−03


2
15990
glycerophosphorylcholine
200
45
1.64E−01
0.027
6.90E−03




(GPC)







1
2730
gamma-glutamylglutamine
200
109
2.51E−01
0.063
7.60E−03


1
32701
urate
200
85
−2.50E−01
0.063
7.80E−03


1
33227
Metabolite-11882
201
23
−2.49E−01
0.062
8.01E−03


1
19934
inositol
50
227
−2.44E−01
0.059
9.62E−03


1
25402
Metabolite-10360
50
129
−2.44E−01
0.059
9.63E−03


1
32520
Metabolite-11206
200
218
−2.43E−01
0.059
9.91E−03


2
32877
Metabolite-11560
201
49
1.56E−01
0.024
1.02E−02


1
32753
Metabolite-09789_201
201
193
2.42E−01
0.058
1.03E−02


2
1494
5-oxoproline
200
50
1.56E−01
0.024
1.04E−02


1
12774
Metabolite-3094
50
199
−2.41E−01
0.058
1.05E−02


1
32635
Metabolite-11318
201
175
2.41E−01
0.058
1.05E−02


1
20950
Metabolite-7846
50
162
−2.40E−01
0.058
1.07E−02


1
32606
bilirubin
201
289
2.40E−01
0.058
1.08E−02


1
32752
Metabolite-11435
201
184
−2.38E−01
0.057
1.16E−02


1
32754
Metabolite-11437
201
186
2.34E−01
0.055
1.29E−02


1
12129
beta-hydroxyisovalerate
50
140
−2.34E−01
0.055
1.30E−02


1
17028
Metabolite-4611
50
130
−2.34E−01
0.055
1.31E−02


1
33132
Metabolite-11787
200
251
−2.34E−01
0.055
1.31E−02


1
12067
undecanoate
201
183
−2.33E−01
0.054
1.34E−02


1
542
3-hydroxybutyrate (BHBA)
50
206
−2.33E−01
0.054
1.35E−02


2
33323
Metabolite-11977
200
51
−1.49E−01
0.022
1.41E−02


1
512
asparagine
50
75
2.31E−01
0.053
1.44E−02


2
54
tryptophan
200
52
1.47E−01
0.022
1.53E−02


1
22032
Metabolite-8766
50
208
−2.28E−01
0.052
1.57E−02


2
32792
Metabolite-11475
201
53
−1.46E−01
0.021
1.64E−02


1
32625
Metabolite-11308
201
301
−2.26E−01
0.051
1.65E−02


1
32813
Metabolite-11496
201
116
−2.25E−01
0.051
1.69E−02


1
18477
glycodeoxycholate
201
192
−2.24E−01
0.050
1.76E−02


2
12795
Metabolite-3113
50
54
1.44E−01
0.021
1.78E−02


1
32553
Metabolite-03832_201
201
256
−2.23E−01
0.050
1.82E−02


2
33364
gamma-glutamylthreonine-
200
56
1.43E−01
0.020
1.88E−02


2
2342
serotonin (5HT)
200
57
1.40E−01
0.020
2.12E−02


1
32855
Metabolite-11538
201
46
2.17E−01
0.047
2.13E−02


1
32197
3-(4-hydroxyphenyl) lactate
201
190
−2.14E−01
0.046
2.38E−02


2
2132
citrulline
200
58
1.37E−01
0.019
2.41E−02


1
21049
1,6-anhydroglucose
50
136
2.13E−01
0.045
2.43E−02


1
33362
gamma-
200
173
−2.12E−01
0.045
2.49E−02




glutamylphenylalanine







1
32452
propionylcarnitine
200
252
−2.12E−01
0.045
2.51E−02


1
32656
Metabolite-11339
201
142
2.11E−01
0.045
2.55E−02


2
1365
myristate (14:0)
201
59
−1.36E−01
0.018
2.59E−02


1
25532
Metabolite-10413
50
226
−2.10E−01
0.044
2.63E−02


2
3141
betaine
200
60
1.35E−01
0.018
2.64E−02


1
32648 
Metabolite-11331
201
132
2.10E−01
0.044
2.66E−02


1
25548 
Metabolite-10419
50
128
2.08E−01
0.043
2.76E−02


1
32748 
Metabolite-11431
201
178
−2.08E−01
0.043
2.79E−02


1
33135 
Metabolite-11790
200
138
−2.07E−01
0.043
2.84E−02


1
31518 
Metabolite-10933
50
152
−2.07E−01
0.043
2.85E−02


1
19478 
Metabolite-6467
50
118
−2.06E−01
0.042
2.95E−02


2
33226 
Metabolite-11881
201
65
1.32E−01
0.018
2.97E−02


1
32561 
Metabolite-11244
201
243
−2.05E−01
0.042
2.98E−02


1
11438
phosphate
50
273
−2.05E−01
0.042
3.02E−02


2
1572
glycerate
50
66
1.31E−01
0.017
3.10E−02


2
33477 
erythronate-
50
67
1.31E−01
0.017
3.13E−02


1
12781 
Metabolite-3099
50
141
−2.03E−01
0.041
3.18E−02


1
32732 
Metabolite-11415
201
155
2.03E−01
0.041
3.22E−02


1
1299
tyrosine
200
147
−2.02E−01
0.041
3.31E−02


2
27256 
Metabolite-10500
50
69
−1.29E−01
0.017
3.37E−02


1
32346 
glycochenodeoxycholate
201
202
−2.00E−01
0.040
3.45E−02


1
27710
N-acetylglycine
50
126
2.00E−01
0.040
3.47E−02


1
22842 
cholate
201
165
−1.99E−01
0.040
3.56E−02


1
31373 
Metabolite-10878
50
274
1.98E−01
0.039
3.59E−02


2
16511 
Metabolite-4274
50
72
1.27E−01
0.016
3.71E−02


2
15996 
aspartate
50
73
1.27E−01
0.016
3.72E−02


1
33228 
Metabolite-11883
200
332
1.97E−01
0.039
3.72E−02


1
18929 
Metabolite-5907
50
172
−1.96E−01
0.039
3.80E−02


2
569
caffeine
200
74
−1.26E−01
0.016
3.87E−02


2
32971 
Metabolite-11654
200
76
−1.25E−01
0.016
3.98E−02


1
32795
Metabolite-11478
201
189
−1.94E−01
0.038
4.00E−02


1
32868 
glycocholate
201
225
−1.93E−01
0.037
4.12E−02


2
18335 
quinate
50
78
1.24E−01
0.015
4.24E−02


1
32587 
Metabolite-02249_201
201
89
−1.91E−01
0.036
4.38E−02


1
22548 
Metabolite-9026
50
337
−1.91E−01
0.036
4.39E−02


1
32829
Metabolite-03653_200
200
102
1.90E−01
0.036
4.48E−02


1
33185 
Metabolite-11840
201
104
−1.90E−01
0.036
4.49E−02


1
20699 
erythritol
50
133
−1.90E−01
0.036
4.50E−02


1
32588 
Metabolite-01327_201
201
233
−1.90E−01
0.036
4.52E−02


1
32848 
Metabolite-11531
201
245
−1.89E−01
0.036
4.54E−02


1
17389 
Metabolite-4796
50
79
1.89E−01
0.036
4.57E−02


2
1493
ornithine
50
82
1.22E−01
0.015
4.60E−02


2
32418 
myristoleate*14-1-n-5-
201
84
−1.21E−01
0.015
4.68E−02


2
15140 
kynurenine
200
83
1.21E−01
0.015
4.68E−02


1
32596 
Metabolite-02250_200
200
151
−1.88E−01
0.035
4.69E−02


1
33380 
Metabolite-12029
201
68
1.87E−01
0.035
4.78E−02


1
27272 
Metabolite-10505
50
393
1.86E−01
0.035
4.96E−02


1
63
cholesterol
50
212
−1.86E−01
0.035
4.96E−02


1
33198 
Metabolite-11853
201
314
−1.84E−01
0.034
5.21E−02


2
15630 
N-acetylornithine
200
87
1.17E−01
0.014
5.52E−02


2
19323 
docosahexaenoate
201
90
−1.16E−01
0.013
5.66E−02




(DHA) 22-6-n-3-







2
32760 
Metabolite-11443
201
91
−1.16E−01
0.013
5.68E−02


2
18392 
theobromine
200
92
1.16E−01
0.013
5.73E−02


1
32514 
Metabolite-11200
200
201
1.79E−01
0.032
5.88E−02


2
16634
Metabolite-4357
50
96
−1.13E−01
0.013
6.37E−02


1
32518
Metabolite-11204
200
396
−1.75E−01
0.031
6.46E−02


1
33081
Metabolite-11736
200
276
−1.75E−01
0.031
6.47E−02


1
32850
Metabolite-11533
201
217
−1.73E−01
0.030
6.82E−02


1
32671
Metabolite-11354
200
139
1.73E−01
0.030
6.86E−02


2
32786
Metabolite-11469
200
98
1.11E−01
0.012
6.92E−02


2
25522
Metabolite-10407
50
99
−1.10E−01
0.012
7.11E−02


2
1649
valine
200
100
−1.10E−01
0.012
7.21E−02


1
32684
Metabolite-11367
201
224
−1.70E−01
0.029
7.23E−02


1
553
cotinine
200
266
1.70E−01
0.029
7.24E−02


1
33389
Metabolite-12038
201
214
−1.70E−01
0.029
7.24E−02


1
1563
chenodeoxycholate
201
370
−1.70E−01
0.029
7.39E−02


2
22481
Metabolite-8988
50
102
1.09E−01
0.012
7.48E−02


1
12593
Metabolite-2973
50
149
1.67E−01
0.028
7.76E−02


1
32793
Metabolite-11476
200
383
−1.67E−01
0.028
7.80E−02


1
32564
Metabolite-11247
201
249
1.67E−01
0.028
7.84E−02


2
1508
pantothenate
200
104
−1.07E−01
0.012
7.84E−02


2
16829
Metabolite-4503
50
105
1.07E−01
0.011
7.99E−02


1
32652
Metabolite-11335
200
388
1.66E−01
0.028
8.03E−02


1
15365
glycerol 3-phosphate (G3P)
50
348
−1.65E−01
0.027
8.15E−02


2
32875
Metabolite-11558
200
107
−1.06E−01
0.011
8.29E−02


2
15506
choline
200
108
1.06E−01
0.011
8.32E−02


2
32492
caprylate-8-0-
201
109
1.05E−01
0.011
8.51E−02


1
16287
Metabolite-2800
50
209
−1.63E−01
0.027
8.51E−02


1
1114
deoxycholate
201
106
−1.63E−01
0.027
8.63E−02


2
32619
Metabolite-11302
201
110
1.05E−01
0.011
8.64E−02


1
27275
Metabolite-10507
50
182
1.62E−01
0.026
8.74E−02


1
32718
Metabolite-01342_200
200
167
−1.61E−01
0.026
8.94E−02


2
33403
Metabolite-12051
200
112
−1.03E−01
0.011
8.96E−02


1
32769
Metabolite-11452
201
278
−1.59E−01
0.025
9.31E−02


2
32756
Metabolite-02276_201
201
116
1.01E−01
0.010
9.72E−02


2
33225
Metabolite-11880
201
118
−1.01E−01
0.010
9.88E−02


1
32839
Metabolite-11522
201
144
1.57E−01
0.025
9.89E−02









2C: Variable Selection with Random Forest for RD Modeling

50 iterations of a random forest analysis with complete 5-fold cross-validation regressions (for Study 1 this analysis included only baseline data, n=111; while for Study 2 all samples were included, n=402) was carried out as follows:
80% of the data was used as the training set to run 1000 regression random forests, record the importance scores and rank the variables according to their importance scores;
Next, four variables at a time were deleted starting from the lowest ranked variables, then the random forest was run with the remaining variables on the training set to predict the remaining 20% of the data (i.e., test set). The error and R-square for each was recorded.
For each variable, the mean/median importance score and rank across all run was calculated.
Variable selection is more or less stable for the approximately first 30-60 variables.
2D: Estimate of the Number of Metabolites Considered Significant for Rd Correlation
The mean R-square values remain constantly high and the corresponding errors remain consistently low as the number of metabolites reaches approximately 30 or more (FIGS. 1 and 2), suggesting that a total of approximately 30 metabolites may be sufficient for construction of an algorithm to correlate with Rd, although it may also be possible to construct an algorithm to correlate with Rd based on a combination of less than seven metabolites. As a result, only the top 30 to 50 cross-validated compounds were selected for regression analyses.
Based on random forest variable selection procedures, the biomarker compounds that are considered reliably significant for construction of an algorithm for Rd correlation were identified. The RF score for each of the biomarker compounds is listed in the column headed “RF_Rank” in Table 4.
2E: Modeling of Rd Correlation with Top Compounds
Based on the modeling experiments, the mean R-square values remain constantly high and the corresponding errors remain consistently low as the number of metabolites reaches seven and above (FIGS. 3 and 4), suggesting that a combination of seven metabolites will be sufficient for construction of an algorithm to correlate with Rd, although it may also be possible to construct an algorithm to correlate with Rd based on a combination of less than seven metabolites.
2F: LASSO Regression
Only cross-validated variables from the random forest analyses above were used for LASSO regression to pick the best combination of variables to predict Rd. The most appropriate transformation of the cross-validated variables was also considered for the LASSO regression.
LASSO regression analysis based upon the cohort in study 1 provided one of the best models of Rd regression with three to nine variables and cross-validated r-square values for the correlation. The best r-square value approaches 0.68 with seven to eight metabolites using the non-transformed data (Table 5) and approaches 0.69-0.70 with the same number of metabolites with appropriate transformation of each variable (Table 6).







TABLE 5







LASSO regression with non-transformed data.









Number of Variables















3
4
5
6
7
8
9





LASSO 
0.355
0.422
0.600
0.643
0.670
0.720
0.779


Maximum R-square









Cross-Validated R-square
0.586
0.600
0.653
0.651
0.681
0.687
0.665


1,5-Anhydro-D-glucitol
√
√
√
√
√
√
√


Bradykinin-
√
√
√
√
√
√
√


hydroxyproline









Palmitate

√
√
√
√
√
√


Metabolite-9727


√
√
√
√
√


Glu-Val


√
√
√
√
√


Threonine

√


√
√
√


Dihydroimidazole-




√
√
√


4-acetate









Mannose





√



2-Hydroxybutyrate



√





Isobar-56**






√


Serine






√





Note:


**Isobar 56 includes DL-pipecolic acid and 1-amino-1-cyclopentanecarboxylic acid that can be separated













TABLE 6







LASSO regression with transformed data.









Number of Variables















3
4
5
6
7
8
9

















LASSO Maximum R-square
0.355
0.422
0.600
0.643
0.670
0.720
0.779


Cross-Validated R-square
0.592
0.603
0.662
0.657
0.692
0.702
0.684


1,5-Anhydro-D-glucitol
√
√
√
√
√
√
√


Bradykinin-hydroxyproline
√
√
√
√
√
√
√


form









Log (Palmitate)
√
√
√
√
√
√
√


Log (Metabolite-9727)


√
√
√
√
√


Glu-Val


√
√
√
√
√


Threonine

√


√
√
√


Log(Dihydroimidazole-4-




√
√
√


acetate)









Log (Mannose)





√



Log (2-Hydroxybutyrate)



√





Isobar-56**






√


Serine






√





Note:


**Isobar 56 includes DL-pipecolic acid and 1-amino-l-cyclopentanecarboxylic






The R-square for the correlation of Rd with 7-8 metabolites approaches 0.70 with cross-validation in an independent cohort.
LASSO analysis based on the cohort in study 1 provided the best models of Rd regression with 3-9 variables with cross-validated r-square values for the correlation of another set approaching 0.68 with 7-8 metabolites using the non-transformed data as shown in Table 7.







TABLE 7







LASSO regression with non-transformed data.









Number of Variables















3
4
5
6
7
8
9





Cross-Validated
0.617
0.636
0.650
0.656
0.678
0.681
0.685


R-square









1,5-Anhydro-
✓
✓
✓
✓
✓
✓
✓


D-glucitol









Palmitate
✓
✓
✓
✓
✓
✓
✓


Glu-Val
✓
✓
✓
✓
✓
✓
✓


Serine

✓
✓
✓
✓
✓
✓


Margarate


✓
✓
✓
✓
✓


X-9727



✓
✓
✓
✓


X-10511




✓
✓



Etio cholanolone





✓
✓


sulfate (X-1345)









Gamma tocopherol






✓


(X-4276)









Creatine






✓









2G: Models Predictive of Insulin Resistance.

In study 2, compounds identified as important in building models to predict Rd by Random Forest and Lasso Regression are listed in Table 4. The cross-validated compounds were then selected for regression analysis along with clinical measurements (e.g. fasting insulin, fasting pro-insulin, fasting free fatty acids (FFA), fasting C-peptide, HDL cholesterol, LDL cholesterol, fasting plasma glucose, adiponectin, BMI, PYY, etc.) Each regression method and the Univariate Correlation/Linear Regression method model was then used to predict Rd for each individual, which was in turn used to classify individuals according to the level of glucose utilization as normal, insulin impaired, or insulin resistant. Samples from ninety percent of the subjects were used to build the model and samples from the remaining ten percent of the subjects were used to test the predictive power of the model. Biomarker compounds that are useful to predict Rd and that are positively or negatively correlated with Rd were identified in these analyses. These markers are useful to predict insulin resistance. All of the biomarker compounds are statistically significant (p<0.05) in each of the regression models.
The models generated using this analytical approach are summarized in Table 8. The sensitivity, specificity and predictive power (positive, PPV and negative, NPV) of the models are shown in Table 8. The sensitivity of the models ranges from about 54% to about 63% and the specificity ranges from about 63% to greater than 95%. The PPV range is from about 78% to about 94% and the NPV from greater than about 79% to greater than about 83%.







TABLE 8





Metabolite Biomarkers and models that are predictive of Insulin Resistance as determined by glucose disposal rate (Rd).
























Model
No.










No.
Variables
R-square
Sensitivity
Specificity
PPV
NPV
Variable 1
Variable 2
Variable 3





1
8
0.5486
59.84
95.51
87.36
82.11
BMI
Fasting Insulin
Fasting_Proinsulin


2
9
0.4937
55.12
95.51
86.42
80.41
Fasting Insulin
Oleate
BMI


3
7
0.5398
59.06
95.10
86.21
81.75
BMI
Fasting Insulin
Fasting Proinsulin


4
9
0.5137
56.69
95.10
85.71
80.90
BMI
Fasting Insulin
2Hydroxybutyrate


5
9
0.5308
60.63
95.10
86.52
82.33
Fasting Insulin
Fasting Proinsulin
Glul.Val


6
7
0.5122
56.69
94.69
84.71
80.84
BMI
Fasting Insulin
Pyruvate


7
8
0.5179
56.69
94.69
84.71
80.84
BMI
Fasting Insulin
Pyruvate


8
9
0.5179
56.69
94.69
84.71
80.84
BMI
Fasting Insulin
Lactate


9
7
0.5380
62.20
94.69
85.87
82.86
BMI
Fasting Insulin
Fasting Proinsulin


10
7
0.5458
61.42
94.69
85.71
82.56
BMI
Fasting Insulin
Fasting Proinsulin


11
8
0.5531
60.63
94.69
85.56
82.27
BMI
Fasting Insulin
Fasting Proinsulin


12
8
0.5534
59.84
94.69
85.39
81.98
BMI
Fasting Insulin
Fasting Proinsulin


13
9
0.5596
62.99
94.69
86.02
83.15
BMI
Fasting Insulin
Fasting Proinsulin


14
9
0.5584
59.84
94.69
85.39
81.98
BMI
Fasting Insulin
Fasting Proinsulin


15
9
0.5580
59.84
94.69
85.39
81.98
BMI
Fasting Insulin
Fasting Proinsulin


16
9
0.5223
56.69
94.69
84.71
80.84
BMI
Fasting Insulin
2Hydroxybutyrate


17
9
0.5317
60.63
94.69
85.56
82.27
Fasting Insulin
Glul.Val
BMI


18
9
0.5106
57.48
94.29
93.91
81.05
BMI
Fasting Insulin
2Hydroxybutyrate


19
9
0.5167
55.91
94.29
83.53
80.49
BMI
Fasting Insulin
2Hydroxybutyrate


20
9
0.5368
61.42
94.29
84.78
82.50
Fasting Insulin
Fasting Proinsulin
BMI


21
9
0.5253
59.84
94.29
84.44
81.91
Creatine
Glycine
Fasting Insulin


22
9
0.5260
58.27
94.29
84.09
81.34
Fasting Insulin
Fasting Proinsulin
Pyruvate


23
9
0.4943
56.69
94.29
83.72
80.77
Fasting Insulin
BMI
FPG


24
9
0.4864
55.12
94.29
83.33
80.21
Fasting Insulin
BMI
FPG


25
9
0.4958
54.33
94.29
83.13
79.93
Triglycerides
Linolenate
Fasting Insulin


26
7
0.5401
57.48
94.29
83.91
81.05
BMI
Fasting Insulin
Fasting FFA


27
8
0.5499
59.06
94.29
84.27
81.63
BMI
Fasting Insulin
Fasting Proinsulin


28
8
0.5492
59.06
94.29
84.27
81.63
BMI
Fasting Insulin
Fasting Proinsulin


29
9
0.5578
62.99
94.29
85.11
83.09
BMI
Fasting Insulin
Fasting Proinsulin


30
9
0.5576
62.99
94.29
85.11
83.09
BMI
Fasting Insulin
Fasting Proinsulin


31
9
0.5615
59.84
94.29
84.44
81.91
BMI
Fasting Insulin
Fasting Proinsulin


32
9
0.4972
60.63
93.88
83.70
82.14
BMI
Fasting Insulin
2Hydroxybutyrate


33
9
0.5060
55.91
93.88
82.56
80.42
BMI
Fasting Insulin
2Hydroxybutyrate


34
9
0.5635
62.99
93.88
84.21
83.03
Fasting Insulin
Fasting Proinsulin
Gamma.Glu.Leu


35
9
0.4996
55.12
93.88
82.35
80.14
Fasting Insulin
Fasting_C_Peptide
Lactate


36
9
0.4983
54.33
93.88
82.14
79.86
Fasting Insulin
Lactate
BMI


37
7
0.5136
55.12
93.88
82.35
80.14
BMI
Fasting Insulin
.Betaine


38
8
0.5177
55.91
93.88
82.56
80.42
BMI
Fasting Insulin
Pyruvate


39
8
0.5183
54.33
93.88
82.14
79.86
BMI
Fasting Insulin
.Betaine


40
9
0.5177
55.91
93.88
82.56
80.42
BMI
Fasting Insulin
.Galactonate


41
9
0.5183
54.33
93.88
82.14
79.86
BMI
Fasting Insulin
Pyruvate


42
7
0.5390
62.99
93.88
84.21
83.03
BMI
Fasting Insulin
Fasting Proinsulin


43
7
0.5382
62.20
93.88
84.04
82.73
BMI
Fasting Insulin
Fasting Proinsulin


44
8
0.5479
61.42
93.88
83.87
82.44
BMI
Fasting Insulin
Fasting Proinsulin


45
8
0.5513
59.84
93.88
83.52
81.85
BMI
Fasting Insulin
Fasting Proinsulin


46
9
0.5595
61.42
93.88
83.87
82.44
BMI
Fasting Insulin
Fasting Proinsulin


47
9
0.5575
59.06
93.88
83.33
81.56
BMI
Fasting Insulin
Fasting Proinsulin


48
9
0.4936
56.69
93.47
81.82
80.63
BMI
Fasting Insulin
2Hydroxybutyrate


49
9
0.5198
55.91
93.47
81.61
80.35
BMI
Fasting Insulin
2Hydroxybutyrate


50
9
0.5211
58.27
93.47
82.22
81.21
Fasting Insulin
Glutamate
BMI


51
9
0.5167
58.27
93.47
82.22
81.21
Fasting Insulin
Oleoyl.LPC
Pyruvate


52
7
0.5148
57.48
93.47
82.02
80.92
BMI
Fasting Insulin
.Gamma.Glu.Leu


53
7
0.5128
55.91
93.47
81.61
80.35
BMI
Fasting Insulin
.Glul.Val


54
7
0.5139
55.12
93.47
81.40
80.07
BMI
Fasting Insulin
.Gamma.Glu.Leu


55
8
0.5188
57.48
93.47
82.02
80.92
BMI
Fasting Insulin
.Betaine


56
8
0.5194
55.91
93.47
81.61
80.35
BMI
Fasting Insulin
.Gamma.Glu.Leu


57
8
0.5178
54.33
93.47
81.18
79.79
BMI
Fasting Insulin
.Betaine


58
9
0.5188
57.48
93.47
82.02
80.92
BMI
Fasting Insulin
.Betaine


59
9
0.5194
55.91
93.47
81.61
80.35
BMI
Fasting Insulin
.Betaine


60
9
0.5178
54.33
93.47
81.18
79.79
BMI
Fasting Insulin
Pyruvate


61
7
0.5396
62.99
93.47
83.33
82.97
BMI
Fasting Insulin
Fasting Proinsulin


62
8
0.5547
62.99
93.47
83.33
82.97
BMI
Fasting Insulin
Fasting Proinsulin


63
8
0.5493
62.20
93.47
83.16
82.67
BMI
Fasting Insulin
Fasting Proinsulin


64
8
0.5525
60.63
93.47
82.80
82.08
BMI
Fasting Insulin
Fasting Proinsulin


65
9
0.5615
62.20
93.47
83.16
82.67
BMI
Fasting Insulin
Fasting Proinsulin


66
9
0.5589
61.42
93.47
82.98
82.37
BMI
Fasting Insulin
Fasting Proinsulin


67
7
0.5127
55.12
93.06
80.46
80.00
BMI
Fasting Insulin
.Gamma.Glu.Leu


68
8
0.5179
55.91
93.06
80.68
80.28
BMI
Fasting Insulin
.Betaine


69
9
0.5179
55.91
93.06
80.68
80.28
BMI
Fasting Insulin
.Gamma.Glu.Leu


70
7
0.5388
57.48
93.06
81.11
80.85
BMI
Fasting Insulin
Fasting FFA


71
9
0.5081
59.06
93.06
81.52
81.43
Fasting Insulin
Fasting_C_Peptide
BMI


72
7
0.5157
56.69
92.65
80.00
80.50
BMI
Fasting Insulin
.Betaine


73
7
0.5124
56.69
92.65
80.00
80.50
BMI
Fasting Insulin
.Betaine


74
7
0.5131
55.91
92.65
79.78
80.21
BMI
Fasting Insulin
.Betaine


75
7
0.5121
55.91
92.65
79.78
80.21
BMI
Fasting Insulin
Pyruvate


76
8
0.5187
58.27
92.65
80.43
81.07
BMI
Fasting Insulin
.Betaine


77
8
0.5183
55.12
92.65
79.55
79.93
BMI
Fasting Insulin
Pyruvate


78
8
0.5176
55.12
92.65
79.55
79.93
BMI
Fasting Insulin
.Betaine


79
9
0.5187
58.27
92.65
80.43
81.07
BMI
Fasting Insulin
Pyruvate


80
9
0.5183
55.12
92.65
79.55
79.93
BMI
Fasting Insulin
.Gamma.Glu.Leu


81
9
0.5176
55.12
92.65
79.55
79.93
BMI
Fasting Insulin
.Betaine


82
7
0.5383
57.48
92.65
80.22
80.78
BMI
Fasting Insulin
Fasting FFA


83
9
0.5071
55.91
92.65
79.78
80.21
BMI
Fasting Insulin
2Hydroxybutyrate


84
9
0.5137
58.27
92.65
80.43
81.07
Fasting Insulin
Oleoyl.LPC
BMI


85
9
0.5153
53.54
92.65
79.07
79.37
Fasting Insulin
BMI
Galactonate


86
9
0.4879
56.69
92.24
79.12
80.43
BMI
Fasting Insulin
2Hydroxybutyrate


87
9
0.5032
55.91
91.84
78.02
80.07
Fasting Insulin
Fasting_C_Peptide
Lactate


88
9
0.4877
55.91
91.84
78.02
80.07
Fasting Insulin
Lactate
Oleate


89
7
0.5390
57.48
91.84
78.49
80.65
Fasting Insulin
Fasting Insulin
Fasting FFA


90
9
0.5122
52.87
62.63
86.05
81.47
Fasting Insulin
Fasting Insulin
2Hydroxybutyrate



















Model








No.
Variable 4
Variable 5
Variable 6
Variable 7
Variable 8
Variable 9





1
Fasting FFA
Adiponectin
GlulVal
Betaine
2Hydroxybutyrate



2
LDL_Cholesterol
FPG
Linoleate
2Hydroxybutyrate
Linolenate
HDL








Cholesterol


3
Fasting FFA
Adiponectin
GlulVal
2Hydroxybutyrate




4
Glul.Val.
Creatine
Pyruvate
Gluconate
Glycine
Linolenate


5
BMI
Betaine
2Hydroxybutyrate
Oleate
Adiponectin
Gluconate


6
Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate
Gluconate




7
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Creatine
Gluconate



8
Pyruvate_
Betaine
.Galactonate
2Hydroxybutyrate
Linoleoyl LPC
Gluconate


9
Fasting FFA
HDL Cholesterol
.Gamma.Glu.Leu
2Hydroxybutyrate




10
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate




11
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Creatine



12
Fasting FFA
Adiponectin
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate



13
Fasting FFA
Adiponectin
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate
Gluconate


14
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Creatine
Gluconate


15
.LDL_Cholesterol
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate


16
Gamma.Glu.Leu
lin_Linoleoyl LPC
Betaine
Pyruvate
Gluconate
X9033


17
Betaine
Fasting Proinsulin
2Hydroxybutyrate
Linoleoyl LPC
Adiponectin
Gluconate


18
Pyruvate
Oleoyl.LPC
Gluconate
Oleate
Galactonate
Linoleate


19
Betaine
Glul.Val
Creatine
Gluconate
lin_Glycine
X9033


20
Betaine
Gamma.Glu.Leu
2Hydroxybutyrate
Oleate
Adiponectin
Gluconate


21
Fasting Proinsulin
Glul.Val
BMI
2Hydroxybutyrate
Adiponectin
Gluconate


22
BMI
FPG
2Hydroxybutyrate
Lactate
Adiponectin
Gluconate


23
Linoleate
2Hydroxybutyrate
Linolenate
HDL Cholesterol
Hepadecenate
Oleate


24
Galactonate
1.5.Anhydroglucitol
2Hydroxybutyrate
Lactate
Oleate
HDL Cholesterol


25
Lactate
BMI
LDL_Cholesterol
FPG
1.5.Anhydroglucitol
2Hydroxybutyrate


26
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Creatine




27
.FPG
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate



28
.LDL_Cholesterol
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate



29
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate
.Linolenate


30
.FPG
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate


31
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate
Creatine


32
Gluconate
Glycine
Lactate
Galactonate
Threonine
X1.5.Anhydroglucitol


33
Glutamate
Pyruvate
Oleoyl.LPC
Gluconate
Glycine
Galactonate


34
Linoleate
BMI
Fasting FFA
2Hydroxybutyrate
Adiponectin
Gluconate


35
BMI
LDL_Cholesterol
FPG
Glycine
Triglycerides
2Hydroxybutyrate


36
LDL_Cholesterol
FPG
Glycine
Triglycerides
1.5.Anhydroglucitol
2Hydroxybutyrate


37
.Gamma.Glu.Leu
2Hydroxybutyrate
Creatine
Gluconate




38
.Betaine
.Galactonate
2Hydroxybutyrate
Linoleoyl LPC
Gluconate



39
.Gamma.Glu.Leu
2Hydroxybutyrate
.Glycine
Creatine
Gluconate



40
.Gamma.Glu.Leu
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Creatine
Gluconate


41
.Gamma.Glu.Leu
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Creatine
Gluconate


42
Fasting FFA
.Gamma.Glu.Leu
2Hydroxybutyrate
Gluconate




43
Fasting FFA
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate




44
Fasting FFA
Adiponectin
.Glul.Val
.Linoleate
2Hydroxybutyrate



45
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC



46
Fasting FFA
Adiponectin
.Betaine
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate


47
Fasting FFA
Adiponectin
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate
Hepadecenate


48
Linolenate
Oleate
Hepadecenate
Linoleate
Serine
Threonine


49
Linoleoyl LPC
Betaine
Glul.Val
Pyruvate
Gluconate
Galactonate


50
2Hydroxybutyrate
Glycine
Oleate
Adiponectin
Creatine
Gluconate


51
BMI
Galactonate
2Hydroxybutyrate
Oleate
Adiponectin
Gluconate


52
2Hydroxybutyrate
Linoleoyl LPC
Creatine
Gluconate




53
.Betaine
2Hydroxybutyrate
Linoleoyl LPC
Gluconate




54
2Hydroxybutyrate
.Glycine
Creatine
Gluconate




55
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Creatine
Gluconate



56
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Creatine
Gluconate



57
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Gluconate
Threonine



58
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
.Oleate
Gluconate
Hepadecenate


59
.Gamma.Glu.Leu
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Creatine
Gluconate


60
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Creatine
Gluconate


61
Fasting FFA
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate




62
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate



63
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Hepadecenate



64
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Gluconate.



65
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate
Gluconate


66
Fasting FFA
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate
Linoleoyl LPC


67
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Creatine




68
.Gamma.Glu.Leu
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Gluconate



69
2Hydroxybutyrate
.Glycine
.Linolenate
Linoleoyl LPC
Creatine
Gluconate


70
Adiponectin
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate




71
LDL Cholesterol
Galactonate
2Hydroxybutyrate
Oleate
Adiponectin
Gluconate


72
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Gluconate




73
.Galactonate
.Gamma.Glu.Leu
2Hydroxybutyrate
Gluconate




74
.Gamma.Glu.Leu
2Hydroxybutyrate
.Glycine
Gluconate




75
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Gluconate




76
.Galactonate
.Gamma.Glu.Leu
2Hydroxybutrate
Linoleoyl LPC
Gluconate



77
.Betaine
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Gluconate



78
.Gamma.Glu.Leu
2Hydroxybutyrate
.Linolenate
Linoleoyl LPC
Gluconate



79
.Betaine
.Galactonate
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Gluconate


80
2Hydroxybutyrate
.Glycine
Linoleoyl LPC
Creatine
Gluconate
Threonine


81
.Galactonate
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC
Creatine
Gluconate


82
Adiponectin
.Gamma.Glu.Leu
.Linoleate
2Hydroxybutyrate




83
Oleoyl.LPC
Gluconate
Oleate
Galactonate
Hepadecenate
Linoleate


84
Galactonate
Linoleate
2Hydroxybutyrate
Oleate
Adiponectin
Gluconate


85
Linoleate
2Hydroxybutyrate
Glutamate
Oleate
Adiponectin
Gluconate


86
Lactate
Oleate
Arginine
Hepadecenate
Serine
Threonine


87
BMI
LDL_Cholesterol
Galactonate
Triglycerides
2Hydroxybutyrate
Gluconate


88
Serine
BMI
Arginine
2Hydroxybutyrate
HDL Cholesterol
Threonine


89
Adiponectin
.Gamma.Glu.Leu
2Hydroxybutyrate
Linoleoyl LPC




90
Creatine
Glutamate
Pyruvate
Gluconate
Glycine
Galactonate





Abbreviations: BMI, Body Mass Index; FFA, Free Fatty Acids; FPG, Fasting Plasma Glucose






2H: Correlated Biomarker Compounds:

Many biomarker compounds were correlated as shown in Tables 9A and 9B. Table 9A contains the pair-wise correlation analysis of biomarkers identified in Study 1 and Table 9B contains the pair-wise correlation analysis of biomarkers identified in Study 2. Correlated compounds are often mutually exclusive in regression models and thus can be used (i.e. substituted for a correlated compound) in different models that had similar prediction powers as those shown in Table 8 above. This aspect is useful when developing biochemical assays that are targeted to specific biomarkers since certain biomarkers may be more amenable to assay development than other biomarkers.







TABLE 9A







Correlated Biomarkers in Study 1.











Pairwise Correlation
N
Correlation
P-value
R-square














1,5-anhydroglucitol-1,5 (AG) *Metabolite-11234
112
−0.5242
2.99E−09
0.2748


1,5-anhydroglucitol-1,5 (AG) *Metabolite-11249
112
−0.5041
1.46E−08
0.2541


1,5-anhydroglucitol-1,5 (AG) *Metabolite-11252
112
−0.5114
8.32E−09
0.2615


1,5-anhydroglucitol-1,5 (AG) *Metabolite-12061
112
−0.5521
2.78E−10
0.3049


1,5-anhydroglucitol-1,5 (AG) *Metabolite-12064
112
−0.5046
1.40E−08
0.2546


2-hydroxybutyrate (AHB)*1,5-anhydroglucitol-1,5 (AG)
112
−0.5413
7.19E−10
0.2930


2-hydroxybutyrate (AHB)*2-aminobutyrate
112
0.7651
<0.000
0.5854


2-hydroxybutyrate (AHB)*3-hydroxybutyrate (BHBA)
112
0.6517
7.11E−15
0.4247


2-hydroxybutyrate (AHB)*3-methyl-2-oxobutyrate
112
0.6750
2.22E−16
0.4557


2-hydroxybutyrate (AHB)*3-methyl-2-oxovalerate
112
0.5970
3.71E−12
0.3565


2-hydroxybutyrate (AHB)*4-methyl-2-oxopentanoate
112
0.6599
2.44E−15
0.4355


2-hydroxybutyrate (AHB)*creatine
112
0.5123
7.75E−09
0.2624


2-hydroxybutyrate (AHB)*erythrose
112
0.5156
5.96E−09
0.2659


2-hydroxybutyrate (AHB)*galactonic acid
112
0.7137
<0.000
0.5094


2-hydroxybutyrate (AHB)*gluconate
112
0.5427
6.35E−10
0.2945


2-hydroxybutyrate (AHB)*gondoate-20-1-n-9-
112
0.5765
2.91E−11
0.3323


2-hydroxybutyrate (AHB)*isoleucine
112
0.6025
2.09E−12
0.3630


2-hydroxybutyrate (AHB)*leucine
112
0.6472
1.27E−14
0.4188


2-hydroxybutyrate (AHB)*mannose
112
0.7043
<0.000
0.4960


2-hydroxybutyrate (AHB)*margarate (17:0)
112
0.5270
2.38E−09
0.2777


2-hydroxybutyrate (AHB)*palmitate (16:0)
112
0.5191
4.54E−09
0.2694


2-hydroxybutyrate (AHB)*stearate (18:0)
112
0.5888
8.57E−12
0.3467


2-hydroxybutyrate (AHB)*uridine
112
0.5282
2.15E−09
0.2790


2-hydroxybutyrate (AHB)*valine*
112
0.6705
6.66E−16
0.4496


2-hydroxybutyrate (AHB)*Metabolite-10432
112
0.6826
2.22E−16
0.4660


2-hydroxybutyrate (AHB)*Metabolite-10752
112
0.5221
3.55E−09
0.2726


2-hydroxybutyrate (AHB)*Metabolite-11228
112
0.5810
1.87E−11
0.3376


2-hydroxybutyrate (AHB)*Metabolite-11887
112
0.5314
1.66E−09
0.2824


2-hydroxybutyrate (AHB)*Metabolite-11897
112
0.5037
1.51E−08
0.2537


2-hydroxybutyrate (AHB)*Metabolite-12037
112
0.5768
2.82E−11
0.3327


2-hydroxybutyrate (AHB)*Metabolite-12061
112
0.5053
1.33E−08
0.2553


2-hydroxybutyrate (AHB)*Metabolite-12064
112
0.8857
<0.000
0.7844


2-hydroxybutyrate (AHB)*glutamate
112
0.7745
<0.000
0.5998


2-hydroxybutyrate (AHB)*Metabolite-3100
112
0.5619
1.14E−10
0.3158


2-hydroxybutyrate (AHB)*Metabolite-4055
112
0.6836
<0.000
0.4672


2-hydroxybutyrate (AHB)*Metabolite-6488
112
0.5779
2.54E−11
0.3339


2-hydroxybutyrate (AHB)*Metabolite-6627
112
0.5193
4.44E−09
0.2697


2-hydroxybutyrate (AHB)*Metabolite-9033
112
0.5608
1.27E−10
0.3145


2-hydroxybutyrate (AHB)*Metabolite-9043
112
0.5879
9.46E−12
0.3456


2-hydroxybutyrate (AHB)*Metabolite-9727
112
0.7077
<0.000
0.5009


3-hydroxybutyrate (BHBA)*glutamate
112
0.5506
3.18E−10
0.3032


3-methyl-2-oxobutyrate*palmitate (16:0)
112
0.5683
6.31E−11
0.3230


4-methyl-2-oxopentanoate*palmitate (16:0)
112
0.5424
6.53E−10
0.2942


alpha linolenate (18:3(n-3))*dihomo-alpha-linolenate-20-3-n-3-
112
0.5295
1.94E−09
0.2804


alpha linolenate (18:3(n-3))*gonodoate-20-1-n-9-
112
0.7264
<0.000
0.5277


alpha linolenate (18:3(n-3))*linoleate (18:2(n-6))
112
0.7877
<0.000
0.6204


alpha linolenate (18:3(n-3))*n-3-DPA-22-5-n-3-
112
0.5722
4.37E−11
0.3274


alpha linolenate (18:3(n-3))*oleate (18:1(n-9))
112
0.7490
<0.000
0.5610


alpha linolenate (18:3(n-3))*palmitate (16:0)
112
0.7354
<0.000
0.5409


alpha linolenate (18:3(n-3))*palmitoleate (16:1(n-7))
112
0.6224
2.36E−13
0.3874


alpha linolenate (18:3(n-3))*stearate (18:0)
112
0.6939
<0.000
0.4815


alpha linolenate (18:3(n-3))*Metabolite-11365
112
0.5054
1.32E−08
0.2555


alpha linolenate (18:3(n-3))*Metabolite-11379
112
0.7245
0
0.5249


alpha linolenate (18:3(n-3))*Metabolite-11653
112
0.5578
1.66E−10
0.3112


alpha linolenate (18:3(n-3))*Metabolite-11887
112
0.7730
0
0.5975


alpha linolenate (18:3(n-3))*Metabolite-12037
112
0.6665
1.11E−15
0.4443


BMI*gamma-glutamylleucine
112
0.5215
3.74E−09
0.2719


BMI*glutamylvaline
112
0.5425
6.47E−10
0.2943


bradykinin*bradykinin, hydroxyproline form-
112
0.5212
3.83E−09
0.2716


creatine*Metabolite-02546_200
112
−0.5849
1.27E−11
0.3421


dipalmitin*palmitate (16:0)
112
0.5630
1.04E−10
0.3170


erythrose*1,5-anhydroglucitol-1,5 (AG)
112
−0.5099
9.35E−09
0.2600


erythrose*galactonic acid
112
0.6691
6.66E−16
0.4476


erythrose*gluconate
112
0.6461
1.44E−14
0.4174


erythrose*glutamate
112
0.7334
0
0.5378


fructose*galactonic acid
112
0.6151
5.35E−13
0.3784


fructose*gluconate
112
0.7100
0
0.5042


fructose*glutamate
112
0.6623
1.78E−15
0.4387


galactonic acid*1,5-anhydroglucitol-1,5 (AG)
112
−0.6211
2.77E−13
0.3857


galactonic acid*2-aminobutyrate
112
0.5246
2.91E−09
0.2752


galactonic acid*3-methyl-2-oxobutyrate
112
0.5517
2.89E−10
0.3044


galactonic acid*4-methyl-2-oxopentanoate
112
0.5288
2.05E−09
0.2797


galactonic acid*gluconate
112
0.7653
0
0.5857


galactonic acid*gondoate-20-1-n-9-
112
0.5981
3.33E−12
0.3577


galactonic acid*isoleucine
112
0.5461
4.74E−10
0.2982


galactonic acid*mannose
112
0.8354
0
0.6978


galactonic acid*margarate (17:0)
112
0.5473
4.26E−10
0.2995


galactonic acid*palmitate (16:0)
112
0.5279
2.21E−09
0.2787


galactonic acid*stearate (18:0)
112
0.5986
3.16E−12
0.3583


galactonic acid*Metabolite-10360
112
0.5113
8.39E−09
0.2614


galactonic acid*Metabolite-10432
112
0.6635
1.55E−15
0.4403


galactonic acid*Metabolite-10609
112
0.5803
2.01E−11
0.3367


galactonic acid*Metabolite-10750
112
0.6247
1.83E−13
0.3903


galactonic acid*Metabolite-10752
112
0.6833
0
0.4668


galactonic acid*Metabolite-11228
112
0.6299
9.99E−14
0.3968


galactonic acid*Metabolite-11230
112
0.5353
1.19E−09
0.2866


galactonic acid*Metabolite-11234
112
0.7072
0
0.5002


galactonic acid*Metabolite-11235
112
0.5168
5.43E−09
0.2671


galactonic acid*Metabolite-11242
112
0.5837
1.43E−11
0.3407


galactonic acid*Metabolite-11249
112
0.5771
2.72E−11
0.3331


galactonic acid*Metabolite-11252
112
0.6003
2.65E−12
0.3603


galactonic acid*Metabolite-11258
112
0.5045
1.42E−08
0.2545


galactonic acid*Metabolite-11387
112
0.5520
2.82E−10
0.3047


galactonic acid*Metabolite-11432
112
0.6147
5.64E−13
0.3778


galactonic acid*Metabolite-11434
112
0.6344
5.93E−14
0.4025


galactonic acid*Metabolite-11628
112
0.5803
2.01E−11
0.3367


galactonic acid*Metabolite-11887
112
0.5451
5.16E−10
0.2971


galactonic acid*Metabolite-11897
112
0.6229
2.25E−13
0.3880


galactonic acid*Metabolite-12037
112
0.5794
2.18E−11
0.3357


galactonic acid*Metabolite-12061
112
0.7387
0
0.5456


galactonic acid*Metabolite-12064
112
0.6051
1.59E−12
0.3662


galactonic acid*glutamate
112
0.9200
0
0.8464


galactonic acid*Metabolite-3100
112
0.5343
1.30E−09
0.2855


galactonic acid*Metabolite-4055
112
0.6292
1.09E−13
0.3958


galactonic acid*Metabolite-5847
112
0.6075
1.23E−12
0.3690


galactonic acid*Metabolite-6446
112
0.6367
4.53E−14
0.4053


galactonic acid*Metabolite-6488
112
0.5753
3.26E−11
0.3309


galactonic acid*Metabolite-6506
112
0.5094
9.72E−09
0.2595


galactonic acid*Metabolite-9033
112
0.7427
0
0.5517


galactonic acid*Metabolite-9043
112
0.5751
3.32E−11
0.3307


galactonic acid*Metabolite-9727
112
0.5195
4.37E−09
0.2699


gamma-glutamylleucine*glutamylvaline
112
0.9404
0
0.8844


gamma-glutamylleucine*peptide-HWESASXX
112
0.7379
0
0.5445


gamma-glutamylleucine*peptide-HWESASXXR
112
0.5766
2.87E−11
0.3325


gluconate*1,5-anhydroglucitol-1,5 (AG)
112
−0.6774
2.22E−16
0.4588


gluconate*mannose
112
0.7419
0
0.5504


gluconate*Metabolite-10432
112
0.6367
4.51E−14
0.4054


gluconate*Metabolite-10609
112
0.5759
3.08E−11
0.3316


gluconate*Metabolite-10610
112
0.5098
9.39E−09
0.2599


gluconate*Metabolite-10750
112
0.7024
0
0.4934


gluconate*Metabolite-10752
112
0.6996
0
0.4894


gluconate*Metabolite-11228
112
0.6652
1.33E−15
0.4425


gluconate*Metabolite-11230
112
0.5881
9.21E−12
0.3459


gluconate*Metabolite-11231
112
0.5374
1.00E−09
0.2888


gluconate*Metabolite-11234
112
0.7041
0
0.4958


gluconate*Metabolite-11235
112
0.5807
1.92E−11
0.3372


gluconate*Metabolite-11242
112
0.6321
7.75E−14
0.3996


gluconate*Metabolite-11249
112
0.5309
1.72E−09
0.2819


gluconate*Metabolite-11252
112
0.5145
6.51E−09
0.2647


gluconate*Metabolite-11387
112
0.5015
1.78E−08
0.2515


gluconate*Metabolite-11432
112
0.5697
5.56E−11
0.3245


gluconate*Metabolite-11434
112
0.6693
6.66E−16
0.4480


gluconate*Metabolite-11435
112
0.5294
1.96E−09
0.2802


gluconate*Metabolite-11628
112
0.5278
2.24E−09
0.2785


gluconate*Metabolite-11897
112
0.6460
1.47E−14
0.4173


gluconate*Metabolite-12061
112
0.7448
0
0.5548


gluconate*Metabolite-12064
112
0.5160
5.78E−09
0.2663


gluconate*glutamate
112
0.7895
0
0.6234


gluconate*Metabolite-3100
112
0.5649
8.70E−11
0.3191


gluconate*Metabolite-4055
112
0.5887
8.67E−12
0.3466


gluconate*Metabolite-4986
112
0.5742
3.63E−11
0.3297


gluconate*Metabolite-5847
112
0.5735
3.88E−11
0.3289


gluconate*Metabolite-6446
112
0.6913
0
0.4779


gluconate*Metabolite-6488
112
0.5778
2.55E−11
0.3339


gluconate*Metabolite-9033
112
0.7458
0
0.5562


gluconate*Metabolite-9043
112
0.5257
2.64E−09
0.2764


glutamylvaline*peptide-HWESASXX
112
0.7816
0
0.6109


glutamylvaline*peptide-HWESASXXR
112
0.5946
4.75E−12
0.3536


glycerate*threonate
112
0.6260
1.57E−13
0.3919


glycerol*alpha linolenate (18:3(n-3))
112
0.6345
5.86E−14
0.4026


glycerol*linoleate (18:2(n-6))
112
0.7008
0
0.4911


glycerol*oleate (18:1(n-9))
112
0.7338
0
0.5385


glycerol*palmitate (16:0)
112
0.6943
0
0.4821


gondoate-20-1-n-9-*linolenate (18:2(n-6))
112
0.7862
0
0.6181


gondoate-20-1-n-9-*oleate (18:1(n-9))
112
0.8756
0
0.7666


gondoate-20-1-n-9-*palmitate (16:0)
112
0.8128
0
0.6607


lactate*pyruvate
112
0.7722
0
0.5963


lactate*Metabolite-4357
112
0.6960
0
0.4844


lactate*Metabolite-4360
112
0.8576
0
0.7355


lactate*Metabolite-4986
112
0.6386
3.60E−14
0.4078


lactate*Metabolite-5348
112
0.5750
3.35E−11
0.3306


linoleate (18:2(n-6))*n-3-DPA-22-5-n-3-
112
0.5966
3.86E−12
0.3560


linoleate (18:2(n-6))*oleate (18:1(n-9))
112
0.8621
0
0.7433


linoleate (18:2(n-6))*palmitate (16:0)
112
0.8248
0
0.6803


linoleate (18:2(n-6))*palmitoleate (16:1(n-7))
112
0.6826
2.22E−16
0.4659


linoleate (18:2(n-6))*stearate (18:0)
112
0.7065
0
0.4991


linoleate (18:2(n-6))*Metabolite-11379
112
0.7858
0
0.6175


linoleate (18:2(n-6))*Metabolite-11653
112
0.5638
9.63E−11
0.3179


linoleate (18:2(n-6))*Metabolite-11887
112
0.8301
0
0.6891


linoleate (18:2(n-6))*Metabolite-12037
112
0.7066
0
0.4992


mannose*1,5-anhydroglucitol-1,5 (AG)
112
−0.6115
8.00E−13
0.3739


mannose*margarate (17:0)
112
0.5273
2.33E−09
0.2780


mannose*glutamate
112
0.8909
0
0.7937


margarate (17:0)*creatine
112
0.5567
1.85E−10
0.3099


margarate (17:0)*stearate (18:0)
112
0.5258
2.63E−09
0.2765


margarate (17:0)*Metabolite-10750
112
0.5003
1.94E−08
0.2503


margarate (17:0)*Metabolite-12037
112
0.5123
7.75E−09
0.2624


margarate (17:0)*Metabolite-12064
112
0.5374
1.00E−09
0.2888


margarate (17:0)*glutamate
112
0.5499
3.38E−10
0.3024


margarate (17:0)*Metabolite-6446
112
0.5082
1.07E−08
0.2582


margarate (17:0)*Metabolite-9033
112
0.5051
1.35E−08
0.2551


myristate (14:0)*oleate (18:1(n-9))
112
0.5118
8.05E−09
0.2619


myristate (14:0)*palmitate (16:0)
112
0.6230
2.22E−13
0.3881


n-3-DPA-22-5-n-3-*oleate (18:1(n-9))
112
0.6538
5.55E−15
0.4275


n-3-DPA-22-5-n-3-*palmitate (16:0)
112
0.6285
1.18E−13
0.3950


oleate (18:1(n-9))*palmitate (16:0)
112
0.9032
0
0.8158


oleate (18:1(n-9))*palmitoleate (16:1(n-7))
112
0.7218
0
0.5209


oleate (18:1(n-9))*stearate (18:0)
112
0.7798
0
0.6081


oleate (18:1(n-9))*Metabolite-11252
112
0.5052
1.34E−08
0.2552


oleate (18:1(n-9))*Metabolite-11379
112
0.8566
0
0.7337


oleate (18:1(n-9))*Metabolite-11653
112
0.6108
8.61E−13
0.3731


oleate (18:1(n-9))*Metabolite-11887
112
0.8989
0
0.8081


oleate (18:1(n-9))*Metabolite-12037
112
0.8335
0
0.6947


ornithine*EDTA*
112
0.6101
9.31E−13
0.3722


ornithine*Metabolite-10812
112
0.5121
7.86E−09
0.2623


ornithine*Metabolite-3091
112
0.5529
2.59E−10
0.3057


ornithine*Metabolite-3103
112
0.5739
3.73E−11
0.3293


ornithine*Metabolite-3108
112
0.5599
1.37E−10
0.3135


ornithine*Metabolite-4274
112
0.5198
4.29E−09
0.2702


palmitate (16:0)*palmitoleate (16:1(n-7))
112
0.7277
0
0.5296


palmitate (16:0)*stearate (18:0)
112
0.8313
0
0.6911


palmitate (16:0)*Metabolite-11252
112
0.5102
9.13E−09
0.2603


palmitate (16:0)*Metabolite-11379
112
0.8594
0
0.7386


palmitate (16:0)*Metabolite-11653
112
0.6739
4.44E−16
0.4542


palmitate (16:0)*Metabolite-11887
112
0.8789
0
0.7724


palmitate (16:0)*Metabolite-12037
112
0.7972
0
0.6356


palmitate (16:0)*Metabolite-12064
112
0.5431
6.13E−10
0.2950


palmitoleate (16:1(n-7))*Metabolite-11379
112
0.8744
0
0.7646


stearate (18:0)*Metabolite-11249
112
0.5247
2.88E−09
0.2753


stearate (18:0)*Metabolite-11252
112
0.5606
1.30E−10
0.3142


stearate (18:0)*Metabolite-11258
112
0.5185
4.74E−09
0.2689


stearate (18:0)*Metabolite-11379
112
0.6025
2.10E−12
0.3630


stearate (18:0)*Metabolite-11653
112
0.5560
1.97E−10
0.3091


stearate (18:0)*Metabolite-11887
112
0.7628
0
0.5818


stearate (18:0)*Metabolite-12037
112
0.7230
0
0.5228


stearate (18:0)*Metabolite-12064
112
0.5859
1.15E−11
0.3433


Metabolite-10432*1,5-anhydroglucitol-1,5 (AG)
112
−0.5674
6.88E−11
0.3220


Metabolite-10432*glutamate
112
0.7402
0
0.5479


Metabolite-10609*glutamate
112
0.5732
3.99E−11
0.3285


glycine*Metabolite-3003
112
0.9861
0
0.9725


Metabolite-10750*1,5-anhydroglucitol-1,5 (AG)
112
−0.5347
1.26E−09
0.2859


Metabolite-10750*glutamate
112
0.6864
0
0.4711


Metabolite-10752*1,5-anhydroglucitol-1,5 (AG)
112
−0.5387
8.96E−10
0.2902


Metabolite-10752*glutamate
112
0.7503
0
0.5629


Metabolite-10814*glutamate
112
0.5121
7.85E−09
0.2623


serine*Metabolite-3078
112
0.6279
1.26E−13
0.3943


serine*Metabolite-3088
112
0.5175
5.15E−09
0.2678


serine*Metabolite-4364
112
0.5899
7.72E−12
0.3480


serine*Metabolite-4769
112
0.6668
1.11E−15
0.4447


glutamate*1,5-anhydroglucitol-1,5 (AG)
112
−0.6945
0
0.4823


glutamate*2-aminobutyrate
112
0.5296
1.92E−09
0.2805


glutamate*3-methyl-2-oxobutyrate
112
0.5857
1.18E−11
0.3430


glutamate*4-methyl-2-oxopentanoate
112
0.5366
1.07E−09
0.2879


glutamate*gondoate-20-1-n-9-
112
0.5823
1.65E−11
0.3390


glutamate*isoleucine
112
0.5442
5.59E−10
0.2961


glutamate*leucine
112
0.5311
1.70E−09
0.2820


glutamate*palmitate (16:0)
112
0.5134
7.08E−09
0.2636


glutamate*stearate (18:0)
112
0.5742
3.63E−11
0.3297


glutamate*valine*
112
0.5511
3.04E−10
0.3037


glutamate*Metabolite-11228
112
0.6659
1.11E−15
0.4435


glutamate*Metabolite-11230
112
0.5964
3.98E−12
0.3557


glutamate*Metabolite-11231
112
0.5182
4.85E−09
0.2686


glutamate*Metabolite-11234
112
0.7245
0
0.5249


glutamate*Metabolite-11235
112
0.5706
5.09E−11
0.3256


glutamate*Metabolite-11242
112
0.6197
3.21E−13
0.3841


glutamate*Metabolite-11249
112
0.6040
1.80E−12
0.3648


glutamate*Metabolite-11252
112
0.6137
6.24E−13
0.3767


glutamate*Metabolite-11258
112
0.5232
3.24E−09
0.2738


glutamate*Metabolite-11387
112
0.5860
1.14E−11
0.3434


glutamate*Metabolite-11432
112
0.6245
1.87E−13
0.3900


glutamate*Metabolite-11434
112
0.6707
6.66E−16
0.4498


glutamate*Metabolite-11435
112
0.5301
1.84E−09
0.2810


glutamate*Metabolite-11628
112
0.5606
1.30E−10
0.3142


glutamate*Metabolite-11887
112
0.5450
5.21E−10
0.2970


glutamate*Metabolite-11897
112
0.6695
6.66E−16
0.4482


glutamate*Metabolite-12037
112
0.5761
3.01E−11
0.3319


glutamate*Metabolite-12061
112
0.7919
0
0.6271


glutamate*Metabolite-12064
112
0.6742
4.44E−16
0.4545


glutamate*Metabolite-3078
112
−0.5143
6.62E−09
0.2645


glutamate*Metabolite-3100
112
0.6438
1.91E−14
0.4145


glutamate*Metabolite-4055
112
0.7284
0
0.5305


glutamate*Metabolite-4986
112
0.5229
3.34E−09
0.2734


glutamate*Metabolite-5847
112
0.6457
1.51E−14
0.4170


glutamate*Metabolite-6446
112
0.7149
0
0.5111


glutamate*Metabolite-6488
112
0.6599
2.44E−15
0.4355


glutamate*Metabolite-6627
112
0.5376
9.88E−10
0.2890


glutamate*Metabolite-9033
112
0.8164
0
0.6665


glutamate*Metabolite-9043
112
0.6741
4.44E−16
0.4544


glutamate*Metabolite-9299
112
0.5571
1.78E−10
0.3103


glutamate*Metabolite-9727
112
0.5597
1.41E−10
0.3132


Metabolite-3078*1,5-anhydroglucitol-1,5 (AG)
112
0.5166
5.54E−09
0.2668


Metabolite-3078*gamma-glutamylglutamine
112
0.5975
3.54E−12
0.3570


Metabolite-4055*1,5-anhydroglucitol-1,5 (AG)
112
−0.5505
3.20E−10
0.3031


pyruvate*Metabolite-4357
112
0.5054
1.32E−08
0.2554


pyruvate*Metabolite-4360
112
0.7299
0
0.5328


Metabolite-4769*gamma-glutamylglutamine
112
0.6458
1.49E−14
0.4171


Metabolite-5847*1,5-anhydroglucitol-1,5 (AG)
112
−0.5083
1.05E−08
0.2584


Metabolite-6446*1,5-anhydroglucitol-1,5 (AG)
112
−0.5887
8.71E−12
0.3465


Metabolite-9033*1,5-anhydroglucitol-1,5 (AG)
112
−0.5674
6.87E−11
0.3220


Metabolite-9033*Metabolite-11228
112
0.6119
7.64E−13
0.3744


Metabolite-9033*Metabolite-11230
112
0.6313
8.50E−14
0.3986


Metabolite-9033*Metabolite-11231
112
0.5342
1.31E−09
0.2854


Metabolite-9033*Metabolite-11234
112
0.6833
0
0.4669


Metabolite-9033*Metabolite-11235
112
0.5267
2.44E−09
0.2774


Metabolite-9033*Metabolite-11242
112
0.5712
4.84E−11
0.3262


Metabolite-9033*Metabolite-11249
112
0.5127
7.51E−09
0.2629


Metabolite-9033*Metabolite-11252
112
0.5285
2.10E−09
0.2794


Metabolite-9033*Metabolite-11387
112
0.5259
2.60E−09
0.2766


Metabolite-9033*Metabolite-11432
112
0.6192
3.42E−13
0.3834


Metabolite-9033*Metabolite-11434
112
0.6631
1.55E−15
0.4396


Metabolite-9033*Metabolite-11435
112
0.5165
5.56E−09
0.2668


Metabolite-9033*Metabolite-11628
112
0.5228
3.36E−09
0.2733


Metabolite-9033*Metabolite-11897
112
0.6761
2.22E−16
0.4572


Metabolite-9033*Metabolite-12061
112
0.7544
0
0.5691


Metabolite-9033*Metabolite-12064
112
0.5456
4.93E−10
0.2977


Metabolite-9033*Metabolite-9043
112
0.5697
5.53E−11
0.3246


Metabolite-9033*Metabolite-9045
112
0.5596
1.42E−10
0.3131


Metabolite-9033*Metabolite-9299
112
0.6908
0
0.4772
















TABLE 9B







Correlated Biomarkers in Study 2.















p-


Compounds
N
Correlation
R-square
values














HDL_Cholesterol*Adiponectin
397
0.511148
0.261272
<0.001


Fat_Mass*BMI
402
0.843078
0.710781
<0.001


Weight*BMI
402
0.804681
0.647512
<0.001


Waist*BMI
398
0.800452
0.640724
<0.001


Hip*BMI
398
0.705318
0.497473
<0.001


Fat_Mass_pcnt*BMI
402
0.602829
0.363403
<0.001


BMI*HOMA
388
0.590842
0.349094
<0.001


BMI*Fasting_Insulin
388
0.589749
0.347804
<0.001


BMI*QUICKI
388
−0.580267
0.336710
<0.001


RD*BMI
402
−0.551166
0.303784
<0.001


BMI*Fasting_C_Peptide
401
0.542661
0.294480
<0.001


Fasting_C_Peptide*HOMA
388
0.829625
0.688277
<0.001


Fasting_Insulin*Fasting_C_Peptide
388
0.828392
0.686233
<0.001


Fasting_C_Peptide*QUICKI
388
−0.768811
0.591070
<0.001


Fasting_Proinsulin*Fasting_C_Peptide
398
0.570761
0.325768
<0.001


Fat_Mass*Fasting_C_Peptide
401
0.519632
0.270017
<0.001


RD*Fasting_C_Peptide
401
−0.506727
0.256773
<0.001


Waist*Fasting_C_Peptide
397
0.501492
0.251495
<0.001


Fasting_Insulin*HOMA
388
0.979376
0.959178
<0.001


Fasting_Insulin*QUICKI
388
−0.880137
0.774641
<0.001


Fasting_Insulin*Fasting_Proinsulin
386
0.509757
0.259853
<0.001


Fat_Mass*Fasting_Insulin
388
0.576818
0.332719
<0.001


Waist*Fasting_Insulin
384
0.502325
0.252330
<0.001


Fasting_Proinsulin*HOMA
386
0.525130
0.275761
<0.001


Fasting_FFA*palmitate (16:0)
393
0.552703
0.305480
<0.001


Fasting_FFA*oleate (18:1(n-9))
393
0.519978
0.270377
<0.001


Fasting_FFA*linoleate (18:2(n-6))
393
0.504094
0.254111
<0.001


Fasting_FFA*Heptadecenate
393
0.503364
0.253375
<0.001


2-aminobutyrate*2-hydroxybutyrate (AHB)
270
0.526705
0.277419
<0.001


alpha linolenate (18:3(n-3))*Isobar-cis-9-cis-11-trans-11-
270
0.634441
0.402516
<0.001


eicosenoate






alpha linolenate (18:3(n-3))*linoleate (18:1(n-9)) (18:2(n-6))
270
0.561647
0.315447
<0.001


alpha linolenate (18:3(n-3))*myristate (14:0)
270
0.656699
0.431254
<0.001


alpha linolenate (18:3(n-3))*myristoleate (18:1(n-9))*14-1-n-5-
270
0.580375
0.336836
<0.001


alpha linolenate (18:3(n-3))*n-3-DPA-22-5-n-3-
270
0.730453
0.533562
<0.001


alpha linolenate (18:3(n-3))*oleate (18:1(n-9))
270
0.576371
0.332204
<0.001


alpha linolenate (18:3(n-3))*palmitate (16:0)
270
0.656120
0.430494
<0.001


alpha linolenate (18:3(n-3))*palmitoleate (16:1(n-7))
270
0.631278
0.398512
<0.001


alpha linolenate (18:3(n-3))*stearate (18:0)
270
0.592125
0.350612
<0.001


alpha linolenate (18:3(n-3))*Metabolite-11261
270
0.545276
0.297326
<0.001


alpha linolenate (18:3(n-3))*Heptadecenate
270
0.645969
0.417276
<0.001


alpha linolenate (18:3(n-3))*Metabolite-11521
270
0.536472
0.287803
<0.001


5-oxoproline*gamma-glutamylleucine
270
0.634304
0.402341
<0.001


aspartate*gamma-glutamylleucine
270
0.673200
0.453199
<0.001


erythronate-*gamma-glutamylleucine
270
0.645586
0.416781
<0.001


gamma-glutamylleucine*gamma-glutamylmethionine-
270
0.624245
0.389682
<0.001


gamma-glutamylleucine*gammaglutamylphenylalanine
270
0.797356
0.635776
<0.001


gamma-glutamylleucine*gamma-glutamylthreonine-
270
0.590454
0.348635
<0.001


gamma-glutamylleucine*gamma-glutamyltyrosine
270
0.709135
0.502873
<0.001


gamma-glutamylleucine*glutamine
270
−0.589607
0.347636
<0.001


gamma-glutamylleucine*glycerate
270
0.515968
0.266223
<0.001


gamma-glutamylleucine*Metabolite-10814
270
0.561643
0.315442
<0.001


gamma-glutamylleucine*Metabolite-11505
270
0.580041
0.336448
<0.001


gamma-glutamylleucine*Metabolite-11560
270
0.544984
0.297008
<0.001


gamma-glutamylleucine*Metabolite-12055
270
0.818261
0.669551
<0.001


gamma-glutamylleucine*Metabolite-3078
270
−0.544729
0.296730
<0.001


gamma-glutamylleucine*Metabolite-3114
270
0.610266
0.372424
<0.001


gamma-glutamylleucine*Glutamate
402
0.813405
0.661627
<0.001


gamma-glutamylleucine*glutamylvaline
270
0.980569
0.961516
<0.001


glucose*mannose
270
0.569026
0.323791
<0.001


glucose*galactonic acid
270
0.612109
0.374677
<0.001


5-oxoproline*gluconate
270
0.519968
0.270367
<0.001


5-oxoproline*Glutamate
270
0.598936
0.358724
<0.001


aspartate*Glutamate
270
0.647610
0.419399
<0.001


erythronate-*Glutamate
270
0.577551
0.333565
<0.001


gamma-glutamylleucine*Glutamate
270
0.702612
0.493664
<0.001


gamma-glutamylphenylalanine*Glutamate
270
0.683581
0.467283
<0.001


gamma-glutamylthreonine-*Glutamate
270
0.547683
0.299956
<0.001


gamma-glutamyltyrosine*Glutamate
270
0.656418
0.430885
<0.001


glutamine*Glutamate
270
−0.693796
0.481352
<0.001


glycerate*Glutamate
270
0.514091
0.264289
<0.001


Metabolite-10814*Glutamate
270
0.683648
0.467375
<0.001


Metabolite-11505*Glutamate
270
0.565409
0.319687
<0.001


Metabolite-11560*Glutamate
270
0.596566
0.355891
<0.001


Metabolite-12055*Glutamate
270
0.710157
0.504322
<0.001


Metabolite-3078*Glutamate
270
−0.573092
0.328434
<0.001


Metabolite-3114*Glutamate
270
0.717209
0.514389
<0.001


Glutamate*glutamylvaline
402
0.815543
0.665110
<0.001


5-oxoproline*glutamylvaline
270
0.567314
0.321845
<0.001


aspartate*glutamylvaline
270
0.650190
0.422746
<0.001


erythronate-*glutamylvaline
270
0.648632
0.420723
<0.001


gamma-glutamylmethionine-*glutamylvaline
270
0.682431
0.465712
<0.001


gammaglutamylphenylalanine*glutamylvaline
270
0.748588
0.560384
<0.001


gamma-glutamylthreonine-*glutamylvaline
270
0.613004
0.375774
<0.001


gamma-glutamyltyrosine*glutamylvaline
270
0.669454
0.448169
<0.001


glutamine*glutamylvaline
270
−0.586263
0.343704
<0.001


glycerate*glutamylvaline
270
0.500861
0.250862
<0.001


Metabolite-10814*glutamylvaline
270
0.544487
0.296466
<0.001


Metabolite-11505*glutamylvaline
270
0.571094
0.326149
<0.001


Metabolite-11560*glutamylvaline
270
0.520971
0.271411
<0.001


Metabolite-12055*glutamylvaline
270
0.818203
0.669456
<0.001


Metabolite-3078*glutamylvaline
270
−0.536866
0.288225
<0.001


Metabolite-3114*glutamylvaline
270
0.588849
0.346744
<0.001


Docosatetraenate*Heptadecenate
402
0.731992
0.535812
<0.001


Fasting_FFA*Heptadecenate
393
0.503364
0.253375
<0.001


Heptadecenate*palmitate (16:0)
402
0.902155
0.813884
<0.001


Heptadecenate*margarate (17:0)
402
0.827249
0.684341
<0.001


Heptadecenate*stearate (18:0)
402
0.719541
0.517740
<0.001


Heptadecenate*alpha linolenate (18:3(n-3))
402
0.605486
0.366614
<0.001


Isobar-cis-9-cis-11-trans-11-eicosenoate*Heptadecenate
270
0.717041
0.514147
<0.001


linoleate (18:2(n-6))*Heptadecenate
270
0.696473
0.485075
<0.001


myristate (14:0)*Heptadecenate
270
0.815585
0.665178
<0.001


myristoleate (18:1(n-9))*14-1-n-5-*Heptadecenate
270
0.764373
0.584266
<0.001


n-3-DPA-22-5-n-3-*Heptadecenate
270
0.600981
0.361178
<0.001


oleate (18:1(n-9))*Heptadecenate
270
0.826866
0.683707
<0.001


palmitoleate (16:1(n-7))*Heptadecenate
270
0.891137
0.794126
<0.001


Heptadecenate*Metabolite-11909
270
0.500849
0.250850
<0.001


Linoleoyl LPC*Oleoyl.LPC
270
0.503307
0.253318
<0.001


hypoxanthine*lactate
270
0.521393
0.271850
<0.001


dihomo-alpha-alpha linolenate (18:3(n-3))-20-3-n-3-*linoleate
270
0.513066
0.263237
<0.001


(18:2(n-6))






Isobar-cis-9-cis-11-trans-11-eicosenoate*linoleate (18:2(n-6))
270
0.614356
0.377433
<0.001


linoleate (18:2(n-6))*myristate (14:0)
270
0.777196
0.604033
<0.001


linoleate (18:2(n-6))*oleate (18:1(n-9))
270
0.764875
0.585034
<0.001


linoleate (18:2(n-6))*palmitate (16:0)
270
0.591405
0.349760
<0.001


linoleate (18:2(n-6))*palmitoleate (16:1(n-7))
270
0.667721
0.445851
<0.001


linoleate (18:2(n-6))*stearate (18:0)
402
0.688839
0.474500
<0.001


Docosatetraenate*linoleate (18:2(n-6))
402
0.718624
0.516421
<0.001


linoleate (18:2(n-6))*margarate (17:0)
402
0.658122
0.433124
<0.001


Docosatetraenate*oleate (18:1(n-9))
402
0.764928
0.585115
<0.001


margarate (17:0)*oleate (18:1(n-9))
270
0.510486
0.260596
<0.001


3-hydroxybutyrate (BHBA)*oleate (18:1(n-9))
270
0.576371
0.332204
<0.001


alpha linolenate (18:3(n-3))*oleate (18:1(n-9))
270
0.736518
0.542459
<0.001


Isobar-cis-9-cis-11-trans-11-eicosenoate*oleate (18:1(n-9))
270
0.777196
0.604033
<0.001


linoleate (18:1(n-9)) (18:2(n-6))*oleate (18:1(n-9))
270
0.709041
0.502739
<0.001


margarate (17:0)*oleate (18:1(n-9))
270
0.668674
0.447124
<0.001


myristate (14:0)*oleate (18:1(n-9))
270
0.587740
0.345438
<0.001


myristoleate (18:1(n-9))*14-1-n-5-*oleate (18:1(n-9))
270
0.907290
0.823175
<0.001


oleate (18:1(n-9))*palmitate (16:0)
270
0.766301
0.587217
<0.001


oleate (18:1(n-9))*palmitoleate (16:1(n-7))
270
0.765960
0.586695
<0.001


oleate (18:1(n-9))*stearate (18:0)
402
0.748928
0.560893
<0.001


pyruvate*Metabolite-4357
270
0.586698
0.344214
<0.001


asparagine*serine
270
0.638729
0.407974
<0.001


ornithine*serine
270
0.656649
0.431187
<0.001


serine*Metabolite-4274
270
0.578680
0.334870
<0.001


dihomo-alpha-alpha linolenate (18:3(n-3))-20-3-n-3-*palmitate
270
0.516782
0.267063
<0.001


(16:0)






Isobar-cis-9-cis-11-trans-11-eicosenoate*palmitate (16:0)
270
0.703264
0.494580
<0.001


margarate (17:0)*palmitate (16:0)
270
0.752390
0.566091
<0.001


myristate (14:0)*palmitate (16:0)
270
0.807589
0.652199
<0.001


myristoleate (18:1(n-9))*14-1-n-5-*palmitate (16:0)
270
0.658236
0.433274
<0.001


n-3-DPA-22-5-n-3-*palmitate (16:0)
270
0.553025
0.305836
<0.001


palmitate (16:0)*palmitoleate (16:1(n-7))
270
0.784704
0.615761
<0.001


palmitate (16:0)*stearate (18:0)
270
0.843751
0.711916
<0.001


palmitate (16:0)*Heptadecenate
270
0.851782
0.725532
<0.001


palmitate (16:0)*Docosatetraenate
270
0.533851
0.284997
<0.001


Metabolite-9033*Metabolite-10750
270
0.550669
0.303236
<0.001


bradykinin, hydroxyproline form-*peptide-HWESASXXR
270
0.587635
0.345314
<0.001









2I: Predicting and Monitoring Insulin Resistance:

The biomarker panel and algorithm will measure insulin resistance (IR) which is a root cause of type 2 diabetes. The results will be presented as an “IR ScoreTM” which represents the level of insulin resistance of the subject. IR Scores will range from Normal Glucose Tolerance (NGT) through increasing levels (Low, Medium, High) of Impaired Glucose Tolerance (IGT). The IR ScoreTM will allow the physician to place the patient on the spectrum of glucose tolerance, from normal to high. For example, an IR ScoreTM of 25 will put the patient in the Low IGT category while an IR ScoreTM of 80 will put the patient in the High IGT category.
By determining the IR Score on an annual or semi-annual basis, physicians can monitor a patient's progression toward diabetes. For example, an IR score of 25 was obtained at a first time point, an IR Score of 34 was obtained at a second time point, an
IR Score of 40 was obtained at a third time point, an IR Score of 40 was obtained at a third time point, an IR Score of 55 was obtained at a fourth time point, and an IR Score of 80 was obtained at a fourth time point indicating an increase in IR and progression of disease from normal to highly impaired glucose tolerance. Using the biomarkers and algorithm of the instant invention for progression monitoring will guide the physician's decision to implement preventative measures such as dietary restrictions, exercise, or early-stage drug treatment. An example of a report demonstrating the use of the IR Score to monitor IR status over time is shown in FIG. 5.







TABLE 10





IR Score


















IR Score 1 to 100
≦25 NGT




26 to 50 Low IGT




51 to 75 Medium IGT




76 to 100 High IGT




>100 Type 2 Diabetes









2J: Biomarkers that Correlate with Glucose Tolerance Tests

Another study will be carried out to test the biomarkers discovered in the instant invention with a new cohort and to discover additional biochemical biomarkers that correlate with insulin sensitivity (IS) and insulin resistance (IR) as measured by the hyperinsulinemic euglycemic (HI) clamp (Table 11). Using the following study design, baseline fasting EDTA-plasma samples collected from NGT, IGT, IFG IGT/IFG and diabetic subjects (total=250) will be analyzed.







TABLE 11







Summary of Study Subjects










Condition
Number of Subjects






NGT
50



IGT
50



IFG
50



IGT/IFG
50



T2D
50





Abbreviations


NGT: Normal Glucose Tolerant (OGTT, <140 mg/dL or <7.8 mmol/L)


IGT: Impaired Glucose Tolerant (OGTT, 140-199 mg/dL or 7.8-11.0 mmol/L)


IFG: Impaired Fasting Glucose (Fasting plasma glucose, 100-125 mg/dL or 5.6-6.9 mmol/L)


IGT/IFG: IGT and/or IFG


T2D: Type II Diabetes (OGTT, ≧200 mg/dL or ≧11.1 mmol/L)






Example 3
Biomarkers for Metabolic Syndrome Related Disorders
3A: Biomarkers of Metabolic Syndrome
Biomarkers were discovered by (1) analyzing plasma and serum samples drawn from different groups of subjects to determine the levels of metabolites in the samples and then (2) statistically analyzing the results to determine those metabolites that were differentially present in the two groups.
The samples used for the analysis were obtained from 19 Caucasian males aged 18-39, average age of 25.6, that had been diagnosed with metabolic syndrome and 19 healthy, age-matched, Caucasian males.
T-tests were used to determine differences in the mean levels of metabolites between the two populations (i.e., Metabolic syndrome vs. Healthy controls).
Biomarkers:
As listed below in Tables 12 and 13, biomarkers were discovered that were differentially present between samples from subjects with Metabolic Syndrome and Control (healthy) subjects.
Tables 12 and 13 include, for each listed biomarker, the p-value and q-value determined in the statistical analysis of the data concerning the biomarkers and an indication of the mean level in metabolic syndrome, the mean level in the control, and the percentage difference in the metabolic syndrome mean level as compared to the healthy mean level in plasma (Table 12) and serum (Table 13). The term “Isobar” as used in the tables indicates the compounds that could not be distinguished from each other on the analytical platform used in the analysis (i.e., the compounds in an isobar elute at nearly the same time and have similar (and sometimes exactly the same) quant ions, and thus cannot be distinguished). Comp_ID refers to the compound identification number used as a primary key for that compound in the in-house chemical database. Library indicates the chemical library that was used to identify the compounds. The number 50 refer to the GC library and the number 61 refers to the LC library.







TABLE 12







Metabolite biomarkers of Metabolic Syndrome in plasma





















% Change









Met.







Mean_Metabolic

Syn. vs


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
Syndrome
Mean_Ctrl
Control

















22290
2-propylpentanoic acid
50
0.2189
0.329
14.75
0.89
1557% 


10715
Metabolite - 2395
61
0.3183
0.3868
6.94
0.83
736% 


10327
Metabolite - 2281
61
0.0788
0.2083
1.97
0.72
174% 


10092
Metabolite - 2250
61
0.3313
0.3919
1.84
0.74
149% 


569
caffeine
61
0.0806
0.2083
1.92
0.78
146% 


22054
Metabolite - 8792
50
<0.0001
4.00E−04
1.71
0.76
125% 


12796
Metabolite - 3114
50
0.0559
0.1731
1.89
0.89
112% 


10286
Metabolite - 2272
61
0.0922
0.2131
2.09
1.01
107% 


12751
Metabolite - 3073
50
0.0055
0.0575
3.09
1.6
93%


10672
Metabolite - 2390
61
0.0045
0.0575
1.85
0.97
91%


18369
gamma-glu-leu
61
0.2496
0.3473
2.82
1.49
89%


14715
Metabolite - 3653
61
0.5859
0.5302
2.39
1.28
87%


11056
Metabolite - 2568
61
0.3525
0.3951
3.38
1.84
84%


57
glutamic acid
50
0.0403
0.1575
2.6
1.46
78%


9130
Metabolite - 2139
61
0.0027
0.0473
1.62
0.94
72%


1638
arginine
61
0.0795
0.2083
1.53
0.9
70%


24233
Metabolite - 9855
61
0.327
0.3896
1.59
0.95
67%


22130
DL-3-phenyllactic acid
61
0.1899
0.3052
1.82
1.1
65%


17492
Metabolite - 4906
61
0.1714
0.2843
1.62
0.98
65%


21630
Metabolite - 8402
50
0.0044
0.0575
1.48
0.9
64%


17557
Metabolite - 4929
61
0.0132
0.0946
1.25
0.77
62%


15253
Metabolite - 3832
61
0.4313
0.4494
2.22
1.38
61%


20842
Metabolite - 7765
61
0.2648
0.3613
2.17
1.35
61%


14837
Metabolite - 3707
61
0.8263
0.605
3.14
1.97
59%


3147
xanthine
61
0.0204
0.1174
1.54
0.98
57%


21127
monopalmitin
50
0.0025
0.0473
1.5
0.96
56%


2734
gamma-L-glutamyl-L-tyrosine
61
0.1081
0.2168
1.93
1.24
56%


6413
Metabolite - 1342-possible-
61
0.2081
0.3221
1.82
1.17
56%



phenylacetylglutamine-








2132
citrulline
50
0.4298
0.4494
0.34
0.22
55%


20830
Metabolite - 7762
61
0.0195
0.1168
1.43
0.93
54%


15996
aspartate
50
0.2785
0.3701
2.81
1.83
54%


18118
Metabolite - 5346
50
0.016
0.1008
1.52
0.99
54%


15113
Metabolite - 3783
61
0.0978
0.2144
0.85
0.56
52%


7171
Metabolite - 1643
61
0.3158
0.3868
2.06
1.36
51%


19377
Metabolite - 6272
50
0.0031
0.0497
1.16
0.77
51%


16337
Metabolite - 4167
61
0.0333
0.1436
1.41
0.94
50%


12756
Metabolite - 3077
50
4.00E−04
0.045
1.93
1.3
48%


17390
Metabolite - 4806
50
0.0276
0.1362
1.23
0.83
48%


21418
Isobar-56-includes-DL-
61
0.1632
0.2797
1.78
1.21
47%



pipecolic acid-1-amino-1-









cyclopentanecarboxylic acid








1125
isoleucine
50
0.0994
0.2148
1.13
0.77
47%


6847
Metabolite - 1496
61
0.0238
0.1264
1.45
0.99
46%


12658
Metabolite - 3026
50
0.0062
0.0575
1.66
1.15
44%


18392
theobromine
61
0.4972
0.4818
1.41
0.98
44%


13775
Metabolite - 3370
61
0.001
0.0473
1.52
1.06
43%


7933
Metabolite - 1911
61
0.5673
0.5208
1.44
1.01
43%


22320
Metabolite - 8889
50
0.0244
0.1264
0.72
0.51
41%


27278
Metabolite - 10510
50
0.0213
0.1195
1.48
1.05
41%


11178
Metabolite - 2608
61
0.0065
0.0575
1.24
0.88
41%


12656
Metabolite - 3025
50
0.0025
0.0473
1.59
1.13
41%


18882
taurodeoxycholic acid
61
0.2208
0.3294
1.87
1.33
41%


27513
indole-3-acetic acid
61
0.0439
0.1617
1.36
0.97
40%


13214
Metabolite - 3183-possible-
61
0.2025
0.3182
2.06
1.48
39%



gamma-L-glutamyl-L-









phenylalanine








1481
inositol-1-phosphate
50
0.0444
0.1617
1.72
1.24
39%


60
leucine
50
0.0726
0.2034
1.12
0.81
38%


12780
Metabolite - 3098
50
0.0023
0.0473
1.67
1.21
38%


12774
Metabolite - 3094
50
0.0057
0.0575
1.19
0.87
37%


1561
alpha-tocopherol
50
0.0599
0.1774
1.31
0.96
36%


12647
Metabolite - 3019
50
0.0024
0.0473
1.5
1.1
36%


17068
Metabolite - 4627
61
0.8349
0.6052
1.66
1.22
36%


12960
Metabolite - 3134
61
0.0558
0.1731
1.25
0.92
36%


9491
Metabolite - 2185
61
0.2076
0.3221
1.18
0.87
36%


9172
Metabolite - 2000
61
0.0197
0.1168
1.15
0.85
35%


1898
proline
61
0.0309
0.1425
1.36
1.01
35%


1299
tyrosine
61
0.0027
0.0473
1.3
0.97
34%


18829
phenylalanine
61
0.0014
0.0473
1.51
1.13
34%


12767
Metabolite - 3087
50
0.3192
0.3868
1.24
0.93
33%


9905
Metabolite - 2231
61
0.0482
0.1629
1.45
1.09
33%


19372
Metabolite - 6269
50
0.0255
0.1288
1.01
0.76
33%


19397
Metabolite - 6326
50
0.016
0.1008
1.38
1.04
33%


1649
valine
50
0.1994
0.3156
1.1
0.83
33%


12222
Metabolite - 2374
50
0.0068
0.0575
1.37
1.04
32%


15140
L-kynurenine
61
0.0123
0.0912
1.33
1.01
32%


5628
Metabolite - 1086
61
0.8915
0.6155
1.95
1.49
31%


5687
Metabolite - 1110
61
0.6883
0.573
1.54
1.18
31%


20699
meso-erythritol
50
0.0466
0.1629
1.29
0.99
30%


15990
L-alpha-
61
0.2682
0.3634
1.94
1.49
30%



glycerophosphorylcholine








27718
creatine
61
0.0922
0.2131
1.47
1.13
30%


12609
Metabolite - 2986
50
0.0331
0.1436
1.82
1.4
30%


18476
glycocholic acid
61
0.1687
0.283
1.91
1.47
30%


18010
Metabolite - 5231
61
0.2164
0.329
1.52
1.17
30%


12876
Metabolite - 3125
61
0.0652
0.1905
1.22
0.94
30%


19364
Metabolite - 6246
50
0.0105
0.0809
1.32
1.02
29%


10245
Metabolite - 2269-
61
0.84
0.6052
1.5
1.16
29%


6266
Metabolite - 1286
61
0.092
0.2131
1.59
1.23
29%


15506
choline
61
0.1324
0.2451
1.71
1.33
29%


12639
Metabolite - 3012
50
0.0024
0.0473
1.59
1.24
28%


16518
Metabolite - 4276
50
0.1013
0.2164
1.14
0.89
28%


17512
Metabolite - 4912
61
0.5453
0.5139
2.99
2.34
28%


29817
Metabolite - 10683
50
0.0151
0.1008
1.57
1.23
28%


24076
Metabolite - 9726
50
0.0364
0.1465
1.34
1.06
26%


584
mannose
50
0.1042
0.2168
1.39
1.1
26%


18524
6-hydroxydopamine
50
0.3352
0.3923
1.06
0.84
26%


1126
alanine
50
0.1098
0.2168
0.97
0.77
26%


10629
Metabolite - 2386
61
0.5698
0.5208
1.2
0.96
25%


1301
lysine
50
0.3451
0.3929
1.01
0.81
25%


27256
Metabolite - 10500
50
0.0367
0.1465
1.07
0.86
24%


9024
Metabolite - 2111
61
0.0925
0.2131
0.98
0.79
24%


10746
Isobar-6-includes-valine-
61
0.1592
0.2751
1.47
1.19
24%



betaine








12768
Metabolite - 3088
50
0.1478
0.2642
1.85
1.5
23%


1572
glyceric acid
50
0.2335
0.3381
1.6
1.3
23%


12650
Metabolite - 3022
50
0.0772
0.2083
1.45
1.18
23%


22337
Metabolite - 8893
61
0.1269
0.2414
1.08
0.88
23%


10087
Metabolite - 2249
61
0.3326
0.3919
1.36
1.11
23%


1670
urea
50
0.0445
0.1617
1.31
1.07
22%


527
lactate
50
0.2905
0.3718
1.54
1.26
22%


16496
Metabolite - 4251
50
0.5501
0.5161
0.88
0.72
22%


8336
Metabolite - 2005
61
0.2177
0.329
1.29
1.06
22%


1303
malic acid
50
0.754
0.5875
1.07
0.88
22%


15737
hydroxyacetic acid
50
0.0844
0.2083
1.08
0.89
21%


16819
Metabolite - 4496
50
0.0589
0.177
1.2
0.99
21%


1358
octadecanoic acid
50
0.0073
0.0584
1.15
0.95
21%


17665
p-hydroxybenzaldehyde
61
0.0329
0.1436
1.84
1.52
21%


7081
Metabolite - 1609
61
0.3512
0.3951
1.05
0.87
21%


10737
Isobar-1-includes-mannose-
61
0.0589
0.177
1.3
1.08
20%



fructose-glucose-galactose-









alpha-L-sorbopyranose-









Inositol-D-allose-D--altrose-









D-psicone-L--gulose-allo-









inositol








13557
Metabolite - 3323
61
0.5667
0.5208
1.26
1.05
20%


15122
glycerol
50
0.0822
0.2083
1.21
1.01
20%


16511
Metabolite - 4274
50
0.4771
0.4733
1.15
0.96
20%


1121
heptadecanoic acid
50
0.0531
0.1724
1.23
1.03
19%


11053
Metabolite - 2567
61
0.5088
0.4907
3.1
2.6
19%


22026
1-methylguanidine
50
0.0457
0.1629
1.19
1
19%


25609
Metabolite - 10439
50
0.3468
0.3929
1.63
1.37
19%


12035
nonanate
50
0.0892
0.2131
1.47
1.24
19%


1110
arachidonic acid
50
0.1038
0.2168
1.11
0.94
18%


54
tryptophan
61
0.0487
0.1629
1.28
1.09
17%


15278
Metabolite - 3843
61
0.4117
0.4378
1.15
0.98
17%


27570
Metabolite - 10569
61
0.0063
0.0575
1.09
0.93
17%


30178
Metabolite - 10705
61
0.7541
0.5875
1.64
1.4
17%


63
cholesterol
50
0.0343
0.1446
1.17
1
17%


10551
Metabolite - 2347
61
0.6957
0.573
1.96
1.68
17%


21188
1-stearoyl-rac-glycerol
50
0.4701
0.4687
1.14
0.98
16%


1365
tetradecanoic acid
50
0.071
0.2016
1.15
0.99
16%


5426
Metabolite - 1004
61
0.3901
0.4235
1.08
0.93
16%


19368
Metabolite - 6267
50
0.2381
0.3381
1.59
1.37
16%


27273
Metabolite - 10506
50
0.1692
0.283
1.38
1.19
16%


7029
Metabolite - 1597
61
0.0558
0.1731
1.77
1.53
16%


10156
Metabolite - 2259
61
0.7281
0.5856
1.04
0.9
16%


10700
Metabolite - 2393
61
0.6334
0.5495
2.23
1.93
16%


13142
Metabolite - 3165
61
0.0829
0.2083
1.34
1.16
16%


25602
Metabolite - 10432
50
0.9349
0.6253
2.17
1.88
15%


1431
p-hydroxyphenyllactic acid
50
0.2345
0.3381
1.23
1.07
15%


27271
Metabolite - 10504
50
0.2474
0.3466
1.08
0.94
15%


6398
Metabolite - 1335
61
0.8947
0.6155
1.89
1.66
14%


1336
n-hexadecanoic acid
50
0.1174
0.2297
1.09
0.96
14%


27672
3-indoxyl-sulfate
61
0.4812
0.4752
1.3
1.15
13%


22895
Metabolite - 9299
50
0.7239
0.5856
1.05
0.93
13%


12129
beta-hydroxyisovaleric acid
50
0.3035
0.3814
1.4
1.24
13%


19282
Metabolite - 6126
61
0.8029
0.6047
1.14
1.01
13%


21069
dioctyl-phthalate
50
0.0953
0.2144
1.07
0.95
13%


17064
Metabolite - 4624
50
0.0925
0.2131
1.25
1.11
13%


21128
1-octadecanol
50
0.0486
0.1629
1.08
0.96
13%


18232
Metabolite - 5403
50
0.1348
0.2473
1.19
1.06
12%


15529
Metabolite - 3951
61
0.1081
0.2168
1.29
1.15
12%


27675
4-nitrophenol
61
0.3162
0.3868
1.27
1.14
11%


9216
Metabolite - 2168
61
0.0835
0.2083
1.2
1.08
11%


10750
Isobar-8-includes-anthranilic
61
0.2166
0.329
1.2
1.08
11%



acid-salicylamide








7601
Metabolite - 1819
61
0.5864
0.5302
1.2
1.08
11%


1604
uric acid
61
0.2964
0.3747
1.04
0.94
11%


513
creatinine
61
0.1885
0.3052
1.06
0.96
10%


1361
pentadecanoic acid
50
0.2362
0.3381
1.17
1.06
10%


1642
decanoic acid
50
0.3114
0.3866
1.39
1.26
10%


18147
Metabolite - 5367
50
0.0753
0.2083
1.11
1.01
10%


22803
Isobar-66-includes-
61
0.41
0.4378
1.69
1.54
10%



glycochenodeoxycholic acid-









glycodeoxycholic acid








20267
Metabolite - 7187
61
0.9304
0.6243
2.16
1.97
10%


5531
Metabolite - 1095
61
0.5242
0.5009
0.69
0.63
10%


19363
Metabolite - 6227
50
0.3417
0.3923
1.3
1.19
 9%


1105
Linoleic acid
50
0.3425
0.3923
1.07
0.98
 9%


17228
Metabolite - 4727
61
0.7215
0.5856
1.56
1.43
 9%


1643
fumaric acid
50
0.9687
0.6344
1.46
1.34
 9%


16782
Metabolite - 4470
61
0.9699
0.6344
1.12
1.03
 9%


1302
methionine
61
0.2524
0.3489
1.27
1.17
 9%


13545
Metabolite - 3322
61
0.9964
0.6461
1.93
1.78
 8%


12083
D-ribose
50
0.6963
0.573
1.31
1.21
 8%


20950
Metabolite - 7846
50
0.4339
0.4499
1.32
1.22
 8%


5765
Metabolite - 1142
61
0.6088
0.5395
1.21
1.12
 8%


27719
galactonic acid
50
0.6632
0.5631
1.08
1
 8%


27409
oleamide
50
0.5882
0.5302
0.95
0.88
 8%


1507
palmitoleic acid
50
0.7754
0.5955
1.25
1.16
 8%


24077
Metabolite - 9727
50
0.5913
0.5308
1.28
1.19
 8%


20489
D-glucose
50
0.0349
0.1446
1.14
1.06
 8%


6422
Metabolite - 1320
61
0.1095
0.2168
1.02
0.95
 7%


19787
Metabolite - 6746
61
0.2617
0.3594
1.17
1.09
 7%


5632
Metabolite - 1138
61
0.156
0.2741
1.03
0.96
 7%


8098
Metabolite - 1867
61
0.6653
0.5631
1.03
0.96
 7%


30273
Metabolite - 10736
50
0.6061
0.5394
1.18
1.1
 7%


19934
inositol
50
0.6428
0.553
1.35
1.26
 7%


15676
3-methyl-2-oxovaleric acid
61
0.5389
0.5102
1.21
1.13
 7%


18349
DL-indole-3-lactic acid
61
0.8534
0.6083
1.22
1.14
 7%


15765
ethylmalonic acid
61
0.7431
0.5875
0.99
0.93
 6%


30282
Metabolite - 10744
50
0.4613
0.4621
1.17
1.1
 6%


16138
Metabolite - 4080
50
0.8136
0.6047
2.4
2.27
 6%


10544
Metabolite - 2329
61
0.9173
0.6207
1.11
1.05
 6%


15500
carnitine
61
0.6276
0.5468
0.94
0.89
 6%


12645
Metabolite - 3017
50
0.6906
0.573
1.32
1.25
 6%


16665
Metabolite - 4364
50
0.7407
0.5875
1.16
1.1
 5%


17648
Metabolite - 5007
61
0.886
0.6154
2.2
2.09
 5%


15365
sn-Glycerol-3-phosphate
50
0.8826
0.6154
1.8
1.71
 5%


10499
Metabolite - 2073
61
0.5624
0.5208
1.02
0.97
 5%


12638
Metabolite - 3011
50
0.9472
0.6287
1.31
1.25
 5%


12663
Metabolite - 3030
50
0.7315
0.5856
1.55
1.48
 5%


10065
Metabolite - 2244
61
0.6638
0.5631
0.95
0.91
 4%


1645
n-dodecanoate
50
0.451
0.4584
1.24
1.19
 4%


6305
Metabolite - 1254
61
0.7656
0.5901
0.82
0.79
 4%


18665
Metabolite - 5728
61
0.6202
0.5451
1.14
1.1
 4%


13065
Metabolite - 3146
61
0.6037
0.5394
1.21
1.17
 3%


7127
Metabolite - 1616
61
0.9018
0.6171
0.92
0.89
 3%


12673
Metabolite - 3040
50
0.638
0.5512
1.29
1.25
 3%


59
histidine
50
0.8614
0.6083
1
0.97
 3%


27275
Metabolite - 10507
50
0.8966
0.6155
1.36
1.32
 3%


12626
Metabolite - 3003
50
0.7594
0.5875
1.1
1.07
 3%


17627
Metabolite - 4986
50
0.8711
0.6123
1.23
1.2
 2%


13589
Metabolite - 3327
61
0.919
0.6207
1.51
1.48
 2%


12894
Metabolite - 2456
61
0.9618
0.6331
1.05
1.03
 2%


1648
serine
50
0.8095
0.6047
1.14
1.12
 2%


20248
Metabolite - 7177
61
0.7567
0.5875
1.23
1.21
 2%


25607
Metabolite - 10437
50
0.9076
0.6182
1.34
1.32
 2%


1564
citric acid
50
0.4876
0.4779
0.04
0.04
 0%


12726
Metabolite - 3058
50
0.693
0.573
1.15
1.15
 0%


12593
Metabolite - 2973
50
0.7576
0.5875
0.43
0.43
 0%


14988
Metabolite - 3756
61
0.9471
0.6287
1.1
1.1
 0%


10147
Metabolite - 2036
61
0.9971
0.6461
1.26
1.27
−1%


16829
Metabolite - 4503
50
0.8459
0.6069
1.19
1.2
−1%


27411
Metabolite - 10547
61
0.9605
0.6331
1.04
1.05
−1%


1410
1-Hexadecanol
50
0.8407
0.6052
0.97
0.98
−1%


10655
Metabolite - 2388
61
0.7806
0.5973
1.1
1.12
−2%


17327
Metabolite - 4767
50
0.8114
0.6047
1.09
1.11
−2%


12666
Metabolite - 3033-possible-
50
0.6852
0.573
1.23
1.26
−2%



threonine-deriv-








1366
trans-4-hydroxyproline
50
0.9284
0.6243
1.09
1.12
−3%


21047
3-methyl-2-oxobutyric-
61
0.6204
0.5451
0.99
1.02
−3%


10825
Metabolite - 2546
61
0.7995
0.6047
0.98
1.01
−3%


16070
Metabolite - 4019
50
0.7173
0.5856
1.19
1.23
−3%


22132
DL-alpha-hydroxyisocaproic
61
0.8586
0.6083
1.17
1.21
−3%



acid








17786
aldosterone
61
0.8368
0.6052
1.1
1.14
−4%


30265
Metabolite - 10732
50
0.6895
0.573
2.46
2.55
−4%


19097
Metabolite - 5969
61
0.9094
0.6182
0.83
0.87
−5%


22145
acetyl-L-carnitine
61
0.7304
0.5856
1.14
1.2
−5%


1494
5-oxoproline
50
0.8287
0.605
1.33
1.4
−5%


22309
Metabolite - 8887
61
0.8136
0.6047
1.89
1.99
−5%


6571
Metabolite - 1397
61
0.7119
0.5834
0.93
0.98
−5%


16509
Metabolite - 4273
50
0.4072
0.4378
1.3
1.37
−5%


19623
Metabolite - 6671
50
0.563
0.5208
0.33
0.35
−6%


6517
Metabolite - 1338
61
0.4487
0.4583
0.98
1.04
−6%


12162
Metabolite - 2339
50
0.427
0.4494
0.62
0.66
−6%


5733
Metabolite - 1127
61
0.4107
0.4378
1.2
1.28
−6%


27272
Metabolite - 10505
50
0.2265
0.3355
1.38
1.48
−7%


58
glycine
50
0.4963
0.4818
0.96
1.03
−7%


12777
Metabolite - 3097
50
0.5284
0.5025
3.1
3.33
−7%


17568
Metabolite - 4931
61
0.7564
0.5875
1.05
1.13
−7%


13038
Metabolite - 3143
61
0.2742
0.3667
1.29
1.39
−7%


20299
Metabolite - 7266
50
0.341
0.3923
0.86
0.93
−8%


12720
Metabolite - 3056
61
0.5179
0.4971
1.1
1.19
−8%


12782
Metabolite - 3100
50
0.7853
0.5986
1.94
2.1
−8%


22609
Metabolite - 9047
50
0.8612
0.6083
1.45
1.57
−8%


2761
thyroxine
61
0.3227
0.3868
1.2
1.3
−8%


1284
threonine
50
0.4887
0.4779
0.99
1.08
−8%


22548
Metabolite - 9026
50
0.8228
0.605
1.01
1.11
−9%


6851
Metabolite - 1497
61
0.7431
0.5875
0.69
0.76
−9%


7644
Metabolite - 1831-
61
0.1522
0.2697
1.05
1.16
−9%


22880
Metabolite - 9286
50
0.3811
0.4159
0.38
0.42
−10% 


12533
Metabolite - 2915
50
0.0688
0.1983
0.9
1
−10% 


25402
Metabolite - 10360
50
0.4214
0.4458
0.7
0.78
−10% 


20676
maleic acid
61
0.3809
0.4159
0.62
0.7
−11% 


6362
Metabolite - 1323-possible-p-
61
0.8874
0.6154
1.22
1.38
−12% 



cresol-sulfate








22133
DL-hexanoyl-carnitine
61
0.289
0.3718
0.53
0.6
−12% 


17304
Metabolite - 4759
61
0.2874
0.3718
0.98
1.11
−12% 


16468
Metabolite - 4236
61
0.1187
0.23
0.87
0.99
−12% 


5618
Metabolite - 1085
61
0.3652
0.4028
0.97
1.11
−13% 


30555
Metabolite - 10781
61
0.8625
0.6083
1.28
1.47
−13% 


6373
Metabolite - 1304
61
0.2945
0.3747
1.18
1.36
−13% 


22175
l-aspartyl-l-phenylalanine
61
0.8236
0.605
1.03
1.19
−13% 


16071
Metabolite - 4020
50
0.1303
0.2435
0.93
1.08
−14% 


19402
Metabolite - 6346
50
0.0066
0.0575
0.97
1.13
−14% 


22600
Metabolite - 9043
50
0.3407
0.3923
1.09
1.27
−14% 


17330
Metabolite - 4769
50
0.2308
0.3381
0.92
1.08
−15% 


2342
serotonin
61
0.4557
0.4609
0.89
1.05
−15% 


3127
hypoxanthine
61
0.2372
0.3381
1.54
1.82
−15% 


15128
DL-homocysteine
61
0.0794
0.2083
0.81
0.96
−16% 


16512
Metabolite - 4275
50
0.2404
0.339
1.04
1.27
−18% 


17494
Metabolite - 4907
61
0.9491
0.6287
1.53
1.87
−18% 


19370
Metabolite - 6268
50
0.2874
0.3718
0.91
1.12
−19% 


14672
Metabolite - 3615
61
0.984
0.6416
0.88
1.09
−19% 


577
fructose
50
0.2716
0.3657
0.96
1.19
−19% 


11499
Metabolite - 2753
61
0.459
0.4619
0.69
0.86
−20% 


6374
Metabolite - 1327
61
0.3602
0.4016
0.94
1.18
−20% 


22053
3-hydroxydecanoic acid
61
0.3226
0.3868
1.04
1.31
−21% 


27738
threonic acid
50
0.1963
0.3131
1.02
1.29
−21% 


542
3-hydroxybutanoic acid
50
0.3099
0.3866
1.24
1.57
−21% 


12757
Metabolite - 3078
50
0.1683
0.283
1
1.27
−21% 


12781
Metabolite - 3099
50
0.2872
0.3718
2.88
3.67
−22% 


24074
Metabolite - 9706
50
0.2808
0.3708
0.85
1.1
−23% 


53
glutamine
50
0.0982
0.2144
0.71
0.92
−23% 


14239
Metabolite - 3474
61
0.1845
0.3012
1.02
1.33
−23% 


12625
Metabolite - 3002
50
0.0973
0.2144
0.68
0.9
−24% 


19110
Metabolite - 5978
50
0.1078
0.2168
0.64
0.85
−25% 


17540
Metabolite - 4926
61
0.4366
0.4504
2.34
3.13
−25% 


22570
Metabolite - 9033
50
0.1374
0.2477
0.25
0.34
−26% 


10961
Metabolite - 2561
61
0.5701
0.5208
1.31
1.81
−28% 


10604
Metabolite - 2370
61
0.1251
0.2402
0.68
0.95
−28% 


5657
Metabolite - 1092
61
0.6524
0.5589
3.89
5.46
−29% 


18705
Metabolite - 5768
61
0.158
0.2751
1.03
1.48
−30% 


16044
Metabolite - 4005
50
0.0144
0.0992
0.55
0.81
−32% 


17091
Metabolite - 4641
61
0.1742
0.2866
0.76
1.12
−32% 


10066
Metabolite - 2029
61
0.4461
0.4579
1.19
1.77
−33% 


22159
dehydroisoandrosterone-3-
61
0.0303
0.1425
0.93
1.4
−34% 



sulfate








22649
Metabolite - 9108
50
0.0532
0.1724
0.66
1
−34% 


17306
Metabolite - 4760
61
0.098
0.2144
0.82
1.25
−34% 


9165
Metabolite - 2150
61
0.1062
0.2168
0.76
1.16
−34% 


6239
Metabolite - 1264
61
0.1367
0.2477
2.92
4.84
−40% 


10781
Metabolite - 2469
61
0.0231
0.126
0.89
1.51
−41% 


10304
Metabolite - 2276
61
0.8813
0.6154
1.28
2.19
−42% 


5280
biliverdin
61
0.0289
0.1395
1.19
2.14
−44% 


18871
Metabolite - 5848
61
0.0069
0.0575
1.03
1.88
−45% 


18702
Metabolite - 5767
61
0.0017
0.0473
0.76
1.45
−48% 


12478
Metabolite - 2898
61
0.1285
0.2421
0.86
1.71
−50% 


27710
N-acetylglycine
50
0.0049
0.0575
0.78
1.64
−52% 


17495
Metabolite - 4908
61
0.044
0.1617
0.94
1.99
−53% 


10177
Metabolite - 2039
61
0.0067
0.0575
0.83
1.86
−55% 


12306
Metabolite - 2869
61
0.627
0.5468
1.18
2.72
−57% 
















TABLE 13







Metabolite biomarkers of Metabolic Syndrome in serum.





















% Change









Met.







Mean_Metabolic

Syn. vs


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
Syndrome
Mean_Control
Control

















19402
Metabolite - 6346
50
0
9.00E−04
0.88
1.07
−18% 


22054
Metabolite - 8792
50
0
9.00E−04
1.29
0.56
130% 


12663
Metabolite - 3030
50
4.00E−04
0.0304
0.61
0.83
−27% 


27710
N-acetylglycine
50
6.00E−04
0.0335
0.56
1.28
−56% 


18829
phenylalanine
61
0.0015
0.0629
0.95
0.71
34%


13257
Metabolite - 3218
61
0.0019
0.0675
0.94
0.57
65%


9172
Metabolite - 2000
61
0.0023
0.0675
1.24
0.86
44%


17390
Metabolite - 4806
50
0.0032
0.0827
1.2
0.69
74%


20830
Metabolite - 7762
61
0.005
0.1061
1.21
0.69
75%


10672
Metabolite - 2390
61
0.0051
0.1061
1.05
0.63
67%


13142
Metabolite - 3165
61
0.0066
0.1177
0.99
0.78
27%


18147
Metabolite - 5367
50
0.0068
0.1177
1.05
0.9
17%


19110
Metabolite - 5978
50
0.0075
0.1206
1.08
1.66
−35% 


16337
Metabolite - 4167
61
0.0087
0.1269
1.33
0.8
66%


27570
Metabolite - 10569
61
0.0095
0.1269
1.15
0.94
22%


6422
Metabolite - 1320
61
0.0098
0.1269
1.07
0.97
10%


21630
Metabolite - 8402
50
0.0104
0.1269
1.17
0.76
54%


1299
tyrosine
61
0.0113
0.1304
1.26
0.87
45%


9491
Metabolite - 2185
61
0.0119
0.1304
1.14
0.77
48%


18702
Metabolite - 5767
61
0.013
0.132
0.75
1.31
−43% 


13775
Metabolite - 3370
61
0.0138
0.132
0.93
0.68
37%


10177
Metabolite - 2039
61
0.014
0.132
0.82
1.59
−48% 


7081
Metabolite - 1609
61
0.0171
0.1442
1.02
0.84
21%


18871
Metabolite - 5848
61
0.0171
0.1442
0.86
1.32
−35% 


12658
Metabolite - 3026
50
0.0178
0.1442
0.98
0.79
24%


12647
Metabolite - 3019
50
0.0191
0.1442
0.92
0.79
16%


12656
Metabolite - 3025
50
0.0193
0.1442
0.88
0.75
17%


18118
Metabolite - 5346
50
0.0194
0.1442
1.16
0.86
35%


17786
aldosterone
61
0.0209
0.1498
1.14
0.91
25%


27273
Metabolite - 10506
50
0.0239
0.1655
0.77
0.91
−15% 


17665
p-
61
0.0265
0.1775
0.7
0.53
32%



hydroxybenzaldehyde








6374
Metabolite - 1327
61
0.0277
0.1781
1.23
1.92
−36% 


7029
Metabolite - 1597
61
0.0305
0.1781
0.67
0.5
34%


21188
1-stearoyl-rac-
50
0.0308
0.1781
1.16
0.76
53%



glycerol








16044
Metabolite - 4005
50
0.0317
0.1781
1.01
1.55
−35% 


5727
Metabolite - 1126
61
0.0341
0.1781
1.19
0.96
24%


10737
Isobar-1-includes-
61
0.0343
0.1781
0.93
0.75
24%



mannose-fructose-









glucose-galactose-









alpha-L-









sorbopyranose-









Inositol-D-allose-D--









altrose-D-psicone-L--









gulose-allo-inositol








1303
malic acid
50
0.0353
0.1781
1.05
1.23
−15% 


57
glutamic acid
50
0.0361
0.1781
1.86
1
86%


14491
Metabolite - 3530
61
0.0362
0.1781
0.66
1.02
−35% 


12478
Metabolite - 2898
61
0.0364
0.1781
0.64
1.64
−61% 


6266
Metabolite - 1286
61
0.0369
0.1781
1.01
0.79
28%


1638
arginine
61
0.0374
0.1781
1.49
0.81
84%


63
cholesterol
50
0.0378
0.1781
1.11
0.93
19%


15529
Metabolite - 3951
61
0.0386
0.1781
0.98
0.84
17%


15278
Metabolite - 3843
61
0.0403
0.181
1.17
0.8
46%


27275
Metabolite - 10507
50
0.0417
0.181
0.6
0.78
−23% 


22159
dehydroisoandrosterone-
61
0.0422
0.181
0.8
1.11
−28% 



3-sulfate








19397
Metabolite - 6326
50
0.0427
0.181
1.12
0.9
24%


12609
Metabolite - 2986
50
0.044
0.1812
0.88
0.7
26%


10087
Metabolite - 2249
61
0.0468
0.1812
1.21
0.84
44%


19377
Metabolite - 6272
50
0.0472
0.1812
1.24
0.95
31%


1604
uric acid
61
0.0485
0.1812
1.12
0.96
17%


54
tryptophan
61
0.0501
0.1812
0.98
0.82
20%


15140
L-kynurenine
61
0.0507
0.1812
1.11
0.85
31%


12666
Metabolite - 3033-
50
0.051
0.1812
0.82
0.95
−14% 



possible-threonine-









deriv-








21127
monopalmitin
50
0.0517
0.1812
1.31
0.82
60%


10629
Metabolite - 2386
61
0.0529
0.1812
1.08
0.82
32%


1125
isoleucine
50
0.0533
0.1812
1.57
1.05
50%


12035
nonanate
50
0.0542
0.1812
0.75
0.58
29%


60
leucine
50
0.0543
0.1812
1.36
1
36%


12751
Metabolite - 3073
50
0.0549
0.1812
0.74
0.64
16%


12781
Metabolite - 3099
50
0.0549
0.1812
0.52
0.75
−31% 


12774
Metabolite - 3094
50
0.0566
0.1828
1.11
0.89
25%


24076
Metabolite - 9726
50
0.059
0.1828
1.09
0.9
21%


6571
Metabolite - 1397
61
0.0593
0.1828
1.18
0.94
26%


10499
Metabolite - 2073
61
0.0594
0.1828
1.22
0.96
27%


27278
Metabolite - 10510
50
0.0598
0.1828
1.05
0.82
28%


513
creatinine
61
0.0623
0.1875
1.15
0.93
24%


18665
Metabolite - 5728
61
0.0632
0.1878
0.97
0.85
14%


18369
gamma-glu-leu
61
0.0679
0.1978
1.96
1.02
92%


19370
Metabolite - 6268
50
0.0685
0.1978
0.79
0.97
−19% 


20699
meso-erythritol
50
0.0697
0.1986
1.15
0.89
29%


9130
Metabolite - 2139
61
0.0709
0.1991
1.31
0.92
42%


18392
theobromine
61
0.0727
0.2015
1.24
0.69
80%


1649
valine
50
0.074
0.2015
1.5
1.08
39%


18882
taurodeoxycholic acid
61
0.0747
0.2015
2.13
1.13
88%


27718
creatine
61
0.0798
0.2098
1.28
0.94
36%


27513
indole-3-acetic acid
61
0.0815
0.2098
1.17
0.84
39%


9216
Metabolite - 2168
61
0.0832
0.2098
1.02
0.84
21%


9905
Metabolite - 2231
61
0.0833
0.2098
1.08
0.86
26%


19414
Metabolite - 6350
50
0.0838
0.2098
1.33
1.08
23%


20092
Metabolite - 7050
61
0.0844
0.2098
0.59
0.84
−30% 


6435
Metabolite - 1348
61
0.0848
0.2098
2.07
0.99
109% 


1648
serine
50
0.09
0.22
0.91
1.09
−17% 


13214
Metabolite - 3183-
61
0.0918
0.2214
1.4
0.97
44%



possible-gamma-L-









glutamyl-L-









phenylalanine








22649
Metabolite - 9108
50
0.0927
0.2214
0.93
1.17
−21% 


30689
Metabolite - 10790
61
0.0939
0.2217
0.97
1.07
−9%


22548
Metabolite - 9026
50
0.0989
0.2309
2.37
0.98
142% 


1358
octadecanoic acid
50
0.1003
0.2317
1.04
0.89
17%


16512
Metabolite - 4275
50
0.1067
0.2438
0.71
0.93
−24% 


21069
dioctyl-phthalate
50
0.1079
0.2438
1.34
0.88
52%


12960
Metabolite - 3134
61
0.1094
0.2446
1.27
1.05
21%


22609
Metabolite - 9047
50
0.1248
0.272
0.89
0.42
112% 


1898
proline
61
0.1257
0.272
1.12
0.88
27%


1642
decanoic acid
50
0.1266
0.272
0.85
0.74
15%


12726
Metabolite - 3058
50
0.1269
0.272
0.81
0.97
−16% 


11974
Metabolite - 2827
61
0.1311
0.2779
1.03
1.24
−17% 


20842
Metabolite - 7765
61
0.1349
0.2833
1.61
0.97
66%


17304
Metabolite - 4759
61
0.1386
0.2872
1.47
0.92
60%


15737
hydroxyacetic acid
50
0.1396
0.2872
2.37
0.81
193% 


18010
Metabolite - 5231
61
0.1433
0.2919
1.16
0.83
40%


59
histidine
50
0.1489
0.2956
0.87
1.03
−16% 


16665
Metabolite - 4364
50
0.1496
0.2956
0.78
0.93
−16% 


12757
Metabolite - 3078
50
0.15
0.2956
0.67
0.89
−25% 


1640
ascorbic acid
50
0.1509
0.2956
1.02
1.65
−38% 


21418
Isobar-56-includes-
61
0.1522
0.2956
1.17
0.74
58%



DL-pipecolic acid-1-









amino-1-









cyclopentanecarboxylic









acid








10327
Metabolite - 2281
61
0.1573
0.3028
1.44
0.85
69%


10700
Metabolite - 2393
61
0.1613
0.3032
1
0.8
25%


10961
Metabolite - 2561
61
0.1674
0.3032
0.91
0.72
26%


2734
gamma-L-glutamyl-
61
0.1684
0.3032
1.26
0.85
48%



L-tyrosine








27409
oleamide
50
0.169
0.3032
2.13
1.09
95%


6847
Metabolite - 1496
61
0.169
0.3032
1.27
1
27%


18705
Metabolite - 5768
61
0.1693
0.3032
0.68
0.97
−30% 


27742
aconitate
61
0.1696
0.3032
1.92
1.06
81%


19368
Metabolite - 6267
50
0.1697
0.3032
0.71
0.8
−11% 


30265
Metabolite - 10732
50
0.1716
0.3032
0.65
0.85
−24% 


542
3-hydroxybutanoic
50
0.1728
0.3032
0.91
1.24
−27% 



acid








21128
1-octadecanol
50
0.174
0.3032
1.93
0.91
112% 


1126
alanine
50
0.175
0.3032
1.28
1.11
15%


9165
Metabolite - 2150
61
0.1811
0.3111
0.7
0.95
−26% 


8098
Metabolite - 1867
61
0.1854
0.3159
1.15
0.93
24%


20299
Metabolite - 7266
50
0.1953
0.3285
0.92
1.04
−12% 


12129
beta-
50
0.196
0.3285
0.75
0.67
12%



hydroxyisovaleric









acid








12626
Metabolite - 3003
50
0.1999
0.3315
0.94
1.04
−10% 


18476
glycocholic acid
61
0.201
0.3315
1.75
1.57
11%


12222
Metabolite - 2374
50
0.2083
0.3378
1.04
0.91
14%


1361
pentadecanoic acid
50
0.2083
0.3378
2.3
0.96
140% 


53
glutamine
50
0.2096
0.3378
1.02
1.23
−17% 


22026
1-methylguanidine
50
0.2146
0.3408
1.01
0.92
10%


7127
Metabolite - 1616
61
0.2149
0.3408
1.02
1.28
−20% 


10746
Isobar-6-includes-
61
0.217
0.3408
0.88
0.49
80%



valine-betaine








5426
Metabolite - 1004
61
0.2181
0.3408
1.97
1.02
93%


1302
methionine
61
0.2235
0.3428
0.91
0.83
10%


1121
heptadecanoic acid
50
0.2248
0.3428
1.5
0.94
60%


6373
Metabolite - 1304
61
0.2252
0.3428
0.77
1.32
−42% 


25602
Metabolite - 10432
50
0.226
0.3428
0.64
0.8
−20% 


22145
acetyl-L-carnitine
61
0.2282
0.343
1.07
1.24
−14% 


18232
Metabolite - 5403
50
0.2307
0.343
1.02
0.93
10%


1365
tetradecanoic acid
50
0.231
0.343
1.31
0.86
52%


10750
Isobar-8-includes-
61
0.2353
0.346
0.93
0.81
15%



anthranilic acid-









salicylamide








24233
Metabolite - 9855
61
0.238
0.346
1.27
0.75
69%


6305
Metabolite - 1254
61
0.2381
0.346
2.42
1.05
130% 


30555
Metabolite - 10781
61
0.2409
0.3476
0.45
0.67
−33% 


569
caffeine
61
0.2479
0.3553
1.57
0.79
99%


12162
Metabolite - 2339
50
0.2496
0.3554
1.41
1.48
−5%


16829
Metabolite - 4503
50
0.2514
0.3554
0.83
0.96
−14% 


15113
Metabolite - 3783
61
0.2569
0.3592
1.97
1.34
47%


13211
Metabolite - 3182
61
0.2592
0.3592
1.63
1.05
55%


8336
Metabolite - 2005
61
0.261
0.3592
1.07
0.89
20%


17330
Metabolite - 4769
50
0.261
0.3592
0.79
0.91
−13% 


12645
Metabolite - 3017
50
0.2635
0.3603
0.81
0.91
−11% 


21047
3-methyl-2-
61
0.2676
0.3635
1
0.86
16%



oxobutyric-








5733
Metabolite - 1127
61
0.2766
0.3705
0.84
0.92
−9%


1670
urea
50
0.2793
0.3705
0.99
0.89
11%


1572
glyceric acid
50
0.2799
0.3705
0.9
0.69
30%


1507
palmitoleic acid
50
0.2804
0.3705
0.86
1.07
−20% 


1110
arachidonic acid
50
0.2843
0.3716
1.03
0.91
13%


1561
alpha-tocopherol
50
0.2874
0.3733
1.07
0.8
34%


19364
Metabolite - 6246
50
0.2912
0.3759
0.91
0.79
15%


5765
Metabolite - 1142
61
0.2932
0.3761
0.99
0.86
15%


6405
Metabolite - 1338
61
0.3015
0.3844
2.63
0.96
174% 


16071
Metabolite - 4020
50
0.3073
0.3884
0.92
1
−8%


16496
Metabolite - 4251
50
0.3085
0.3884
1.54
1.24
24%


12533
Metabolite - 2915
50
0.3102
0.3884
1.1
1.01
 9%


10286
Metabolite - 2272
61
0.316
0.3933
1.45
1
45%


8469
Metabolite - 2036-
61
0.3184
0.3939
0.88
0.55
60%



possible-Heme








10065
Metabolite - 2244
61
0.325
0.3991
1.02
0.95
 7%


19623
Metabolite - 6671
50
0.3265
0.3991
2.43
2.77
−12% 


27411
Metabolite - 10547
61
0.3324
0.4023
0.52
0.68
−24% 


12777
Metabolite - 3097
50
0.3329
0.4023
0.53
0.7
−24% 


18394
theophylline
61
0.3355
0.4031
0.97
0.71
37%


2761
thyroxine
61
0.3418
0.4082
0.86
0.93
−8%


18254
paraxanthine
61
0.3437
0.4082
0.97
0.58
67%


7933
Metabolite - 1911
61
0.3458
0.4083
1.13
0.77
47%


15996
aspartate
50
0.3567
0.417
0.63
0.49
29%


7601
Metabolite - 1819
61
0.3571
0.417
0.77
0.72
 7%


1336
n-hexadecanoic acid
50
0.3594
0.4174
1.07
0.92
16%


10147
Metabolite - 2036
61
0.3626
0.4186
0.9
0.99
−9%


5618
Metabolite - 1085
61
0.3702
0.4251
1.35
1.06
27%


15500
carnitine
61
0.377
0.429
1.07
0.97
10%


6413
Metabolite - 1342-
61
0.3778
0.429
1.1
0.94
17%



possible-









phenylacetylglutamine-








27738
threonic acid
50
0.3822
0.4317
0.89
0.96
−7%


22132
DL-alpha-
61
0.3947
0.4428
1
0.88
14%



hydroxyisocaproic









acid








17306
Metabolite - 4760
61
0.3968
0.4428
0.74
1.09
−32% 


20267
Metabolite - 7187
61
0.399
0.4428
2.07
1.37
51%


14368
Metabolite - 3489
61
0.4005
0.4428
0.84
0.94
−11% 


10825
Metabolite - 2546
61
0.405
0.4454
1.13
1.02
11%


6546
Metabolite - 1391
61
0.4074
0.4456
1.12
1.02
10%


27675
4-nitrophenol
61
0.4103
0.4465
0.96
0.88
 9%


17064
Metabolite - 4624
50
0.4189
0.4534
0.88
0.92
−4%


20489
D-glucose
50
0.4214
0.4538
0.93
0.95
−2%


20676
maleic acid
61
0.4265
0.4553
1.18
1.33
−11% 


13557
Metabolite - 3323
61
0.4306
0.4553
1.3
1.02
27%


10551
Metabolite - 2347
61
0.4307
0.4553
1.97
1.41
40%


15990
L-alpha-
61
0.4316
0.4553
0.71
0.58
22%



glycerophosphorylcholine








6517
Metabolite - 1338
61
0.4377
0.4591
0.86
0.8
 8%


19097
Metabolite - 5969
61
0.4396
0.4591
0.98
1.08
−9%


22803
Isobar-66-includes-
61
0.442
0.4593
1.63
1.38
18%



glycochenodeoxycholic









acid-









glycodeoxycholic









acid








1410
1-Hexadecanol
50
0.4484
0.4635
2.68
1.01
165% 


15765
ethylmalonic acid
61
0.4505
0.4635
0.95
0.81
17%


5280
biliverdin
61
0.4559
0.4663
0.86
1.01
−15% 


1284
threonine
50
0.4577
0.4663
1.14
1.24
−8%


30273
Metabolite - 10736
50
0.4611
0.4675
0.89
1
−11% 


16518
Metabolite - 4276
50
0.4687
0.4717
0.86
0.72
19%


12756
Metabolite - 3077
50
0.4698
0.4717
0.73
0.68
 7%


12639
Metabolite - 3012
50
0.4752
0.4748
0.79
0.73
 8%


12650
Metabolite - 3022
50
0.4937
0.4894
0.84
0.77
 9%


10544
Metabolite - 2329
61
0.4945
0.4894
1.05
1.4
−25% 


22337
Metabolite - 8893
61
0.5005
0.493
1.07
1.14
−6%


18524
6-hydroxydopamine
50
0.5075
0.4953
1.13
1.2
−6%


13065
Metabolite - 3146
61
0.5076
0.4953
1.01
0.97
 4%


15506
choline
61
0.5148
0.5
1.02
0.75
36%


14988
Metabolite - 3756
61
0.5198
0.5007
0.68
0.74
−8%


12894
Metabolite - 2456
61
0.5228
0.5007
0.9
0.94
−4%


22880
Metabolite - 9286
50
0.5255
0.5007
1.33
1.25
 6%


7171
Metabolite - 1643
61
0.5279
0.5007
2.54
2.05
24%


14715
Metabolite - 3653
61
0.5332
0.5007
1.92
1.07
79%


15000
Metabolite - 3758
61
0.5332
0.5007
0.96
0.91
 5%


29817
Metabolite - 10683
50
0.5335
0.5007
0.84
0.79
 6%


19372
Metabolite - 6269
50
0.535
0.5007
1.09
1.13
−4%


18091
Metabolite - 5306
61
0.5372
0.5007
1.06
0.96
10%


15676
3-methyl-2-
61
0.554
0.5065
0.93
0.89
 4%



oxovaleric acid








16468
Metabolite - 4236
61
0.5544
0.5065
1.1
0.98
12%


22053
3-hydroxydecanoic
61
0.5575
0.5065
0.92
1.07
−14% 



acid








10781
Metabolite - 2469
61
0.5584
0.5065
0.8
1
−20% 


17568
Metabolite - 4931
61
0.5585
0.5065
0.89
1.05
−15% 


12625
Metabolite - 3002
50
0.5605
0.5065
1.74
1.78
−2%


1643
fumaric acid
50
0.5626
0.5065
0.88
0.96
−8%


27272
Metabolite - 10505
50
0.5629
0.5065
0.72
0.74
−3%


19363
Metabolite - 6227
50
0.5725
0.5111
0.83
0.79
 5%


10655
Metabolite - 2388
61
0.5733
0.5111
0.93
1
−7%


58
glycine
50
0.5779
0.5111
0.9
0.98
−8%


15128
DL-homocysteine
61
0.5816
0.5111
1.27
1.36
−7%


17068
Metabolite - 4627
61
0.5821
0.5111
1.35
0.76
78%


1494
5-oxoproline
50
0.5828
0.5111
0.99
0.86
15%


6531
Metabolite - 1385
61
0.5892
0.5145
1.09
1.14
−4%


19282
Metabolite - 6126
61
0.605
0.5238
1.38
1.03
34%


22130
DL-3-phenyllactic
61
0.6056
0.5238
2.23
1.36
64%



acid








1301
lysine
50
0.6074
0.5238
1.53
1.32
16%


6851
Metabolite - 1497
61
0.616
0.5277
1.55
1.46
 6%


6398
Metabolite - 1335
61
0.6169
0.5277
1.34
1.56
−14% 


27256
Metabolite - 10500
50
0.6227
0.5304
1.1
1.05
 5%


21841
Metabolite - 8577
61
0.6366
0.54
1.08
0.99
 9%


5687
Metabolite - 1110
61
0.6394
0.5402
0.96
0.94
 2%


12306
Metabolite - 2869
61
0.6482
0.5455
2.58
1.89
37%


12767
Metabolite - 3087
50
0.6549
0.5488
1.44
1.18
22%


12638
Metabolite - 3011
50
0.6612
0.5519
0.91
0.99
−8%


5489
Metabolite - 1057
61
0.6673
0.5547
1.03
1.05
−2%


19462
Metabolite - 6446
50
0.6727
0.5556
1.08
1.04
 4%


10604
Metabolite - 2370
61
0.676
0.5556
0.86
0.88
−2%


15121
Metabolite - 3786
61
0.6764
0.5556
0.86
0.89
−3%


10092
Metabolite - 2250
61
0.6803
0.5561
1.63
0.76
114% 


22320
Metabolite - 8889
50
0.6822
0.5561
1.69
1.77
−5%


1645
n-dodecanoate
50
0.6858
0.5567
0.82
0.79
 4%


527
lactate
50
0.6911
0.5589
0.99
0.91
 9%


3127
hypoxanthine
61
0.7003
0.5629
0.54
0.51
 6%


24077
Metabolite - 9727
50
0.7015
0.5629
0.92
0.97
−5%


1366
trans-4-
50
0.7106
0.568
1.14
1.08
 6%



hydroxyproline








11499
Metabolite - 2753
61
0.7138
0.5684
1.31
0.8
64%


18349
DL-indole-3-lactic
61
0.7229
0.5722
0.91
0.86
 6%



acid








7644
Metabolite - 1831-
61
0.7346
0.5751
0.88
0.89
−1%


5531
Metabolite - 1095
61
0.7348
0.5751
1.34
1.34
 0%


12768
Metabolite - 3088
50
0.736
0.5751
0.65
0.68
−4%


1431
p-
50
0.7405
0.5764
1.19
0.91
31%



hydroxyphenyllactic









acid








12673
Metabolite - 3040
50
0.7654
0.5878
0.75
0.82
−9%


2132
citrulline
50
0.7659
0.5878
3.41
3.29
 4%


22309
Metabolite - 8887
61
0.7694
0.5878
0.58
0.54
 7%


30282
Metabolite - 10744
50
0.7699
0.5878
0.89
0.87
 2%


14672
Metabolite - 3615
61
0.7721
0.5878
0.82
0.89
−8%


25607
Metabolite - 10437
50
0.7765
0.589
0.76
0.87
−13% 


6820
Metabolite - 1554
61
0.7865
0.5944
1.06
1
 6%


14239
Metabolite - 3474
61
0.7901
0.5949
1
0.9
11%


20248
Metabolite - 7177
61
0.7984
0.5973
1.04
1.02
 2%


27326
Metabolite - 10527
50
0.799
0.5973
1.16
1.12
 4%


25402
Metabolite - 10360
50
0.8093
0.6029
1.29
1.3
−1%


19934
inositol
50
0.8135
0.6038
0.68
0.68
 0%


12593
Metabolite - 2973
50
0.8339
0.6152
1.66
1.66
 0%


23462
Metabolite - 9693
61
0.8348
0.6152
1.15
1.15
 0%


16819
Metabolite - 4496
50
0.8463
0.6215
0.88
0.85
 4%


16138
Metabolite - 4080
50
0.8667
0.6297
0.53
0.51
 4%


25609
Metabolite - 10439
50
0.8668
0.6297
0.72
0.66
 9%


584
mannose
50
0.87
0.6297
0.93
0.84
11%


15122
glycerol
50
0.8737
0.6297
0.93
0.89
 4%


22570
Metabolite - 9033
50
0.875
0.6297
1.52
1.51
 1%


20888
Metabolite - 7806
61
0.8756
0.6297
0.97
0.99
−2%


13038
Metabolite - 3143
61
0.8833
0.6316
1.32
1.14
16%


16509
Metabolite - 4273
50
0.8851
0.6316
0.69
0.7
−1%


16511
Metabolite - 4274
50
0.8873
0.6316
1.1
1.08
 2%


13589
Metabolite - 3327
61
0.8935
0.6338
1.19
1.06
12%


5628
Metabolite - 1086
61
0.9002
0.6364
1.73
1.6
 8%


10245
Metabolite - 2269-
61
0.9081
0.6398
1.08
0.87
24%


6362
Metabolite - 1323-
61
0.9178
0.6429
1.08
1.02
 6%



possible-p-cresol-









sulfate








5632
Metabolite - 1138
61
0.9201
0.6429
1.03
1.05
−2%


15365
sn-Glycerol-3-
50
0.9265
0.6429
0.82
0.82
 0%



phosphate








10304
Metabolite - 2276
61
0.9274
0.6429
1.36
1.77
−23% 


10156
Metabolite - 2259
61
0.9281
0.6429
1.06
1.03
 3%


27719
galactonic acid
50
0.9386
0.6449
1.13
1.1
 3%


22895
Metabolite - 9299
50
0.9394
0.6449
1.05
1.05
 0%


15253
Metabolite - 3832
61
0.9468
0.6449
1.48
1.41
 5%


1105
Linoleic acid
50
0.9483
0.6449
0.95
0.94
 1%


12780
Metabolite - 3098
50
0.9494
0.6449
0.87
0.83
 5%


10441
Metabolite - 2308
61
0.9506
0.6449
0.92
0.88
 5%


27672
3-indoxyl-sulfate
61
0.9527
0.6449
0.98
0.95
 3%


22133
DL-hexanoyl-
61
0.9585
0.646
1.36
1.35
 1%



carnitine








16070
Metabolite - 4019
50
0.9631
0.646
0.85
0.85
 0%


12720
Metabolite - 3056
61
0.9635
0.646
0.9
0.92
−2%


24074
Metabolite - 9706
50
0.9711
0.6479
1.12
1.11
 1%


13545
Metabolite - 3322
61
0.9736
0.6479
1.49
1.34
11%


1564
citric acid
50
0.9794
0.6479
3.72
3.73
 0%


17327
Metabolite - 4767
50
0.9803
0.6479
1.32
1.03
28%


20950
Metabolite - 7846
50
0.982
0.6479
0.93
0.97
−4%


14837
Metabolite - 3707
61
0.99
0.6511
2.12
1.66
28%









Random forests were generated for plasma and serum biomarkers. The models for the serum biomarkers correctly classified 81.5% of the subjects as either being healthy or having metabolic syndrome; 83% of the healthy subjects were classified correctly and 77% of the subjects having metabolic syndrome were correctly classified. For the models based on the biomarkers from plasma, the 89% of the subjects were correctly classified as either being healthy or having metabolic syndrome; 100% of the healthy subjects were correctly classified and 77% of the metabolic syndrome subjects were correctly classified. The most important biomarkers are shown in the importance plot in FIG. 8 (Serum) and FIG. 9 (Plasma).
3B: Biomarkers of Atherosclerosis
Biomarkers were discovered by (1) analyzing plasma, aorta and liver samples drawn from subjects with atherosclerosis and healthy subjects to determine the levels of metabolites in the samples and then (2) statistically analyzing the results to determine those metabolites that were differentially present in the two groups.
The samples used for the analysis were from wild-type and transgenic mice, C57BL/6 and LDb, respectively. The transgenic LDb mice provide a model for atherosclerosis in human subjects. Previous studies have shown that LDb transgenic mice in a C57BL/6 background have about 5-fold higher plasma cholesterol and triglyceride levels than C57BL/6 wild-type mice and start to develop atherosclerotic lesions at about 3 months of age. Plasma, ascending and descending aorta tissues and liver tissue from each group of mice at 2, 5 or 8 months were subjected to metabolomic analysis. These collection time points represent early (initiation), mid and late stage for atherosclerosis in the transgenic model.
T-tests were used to determine differences in the mean levels of metabolites between the two populations (i.e., LDb vs. C57BL/6). Classification analysis was carried out using recursive partitioning and random forest analyses to uncover the biomarkers that can best differentiate the 2 groups of mice. Recursive partitioning relates a ‘dependent’ variable (Y) to a collection of independent (‘predictor’) variables (X) in order to uncover—or simply understand—the elusive relationship, Y=f(X). It was performed with the JMP program (SAS) to generate a decision tree. The statistical significance of the “split” of the data can be placed on a more quantitative footing by computing p-values, which discern the quality of a split relative to a random event. The significance level of each “split” of data into the nodes or branches of the tree was computed as p-values, which discern the quality of the split relative to a random event. It was given as LogWorth, which is the negative log 10 of a raw p-value. Statistical analyses were performed with the program “R” available on the worldwide web at the website cran.r-project.org.
Random forests give an estimate of how well individuals can be classified in a new data set into each group, in contrast to a t-test, which tests whether the unknown means for two populations are different or not. Random forests create a set of classification trees based on continual sampling of the experimental units and compounds. Then each observation is classified based on the majority votes from all the classification trees. Statistical analyses were performed with the program “R” available on the worldwide web at the website cran.r-project.org.
Biomarkers:
As listed below in Tables 14, 15 and 16, biomarkers were discovered that were differentially present between samples from LDb (atherosclerotic) subjects and C57BL/6 (healthy) subjects.
Tables 14, 15 and 16 include, for each listed biomarker, the p-value and q-value determined in the statistical analysis of the data concerning the biomarkers and an indication of the percentage difference in the atherosclerotic mean level as compared to the healthy mean level in plasma (Table 14), aorta (Table 15) and liver (Table 16). The term “Isobar” as used in the tables indicates the compounds that could not be distinguished from each other on the analytical platform used in the analysis (i.e., the compounds in an isobar elute at nearly the same time and have similar (and sometimes exactly the same) quant ions, and thus cannot be distinguished). Comp_ID refers to the compound identification number used as a primary key for that compound in the in-house chemical database. Library indicates the chemical library that was used to identify the compounds. The number 50 refer to the GC library and the number 61 refers to the LC library.







TABLE 14







Metabolite biomarkers of Atherosclerosis in plasma.

















% Change


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
in LDb















21415
Metabolite - 8209
50
4.25E−07
2.40E−06
497%


21012
Metabolite - 7889
50
1.12E−22
6.95E−21
494%


21011
Metabolite - 7888
50
8.87E−24
8.25E−22
482%


25649
Metabolite - 10450
50
4.17E−15
7.76E−14
442%


18619
Metabolite - 5669
61
5.95E−06
2.84E−05
423%


27279
Metabolite - 10511
50
1.81E−19
6.74E−18
404%


63
cholesterol
50
1.56E−24
2.90E−22
400%


8469
Metabolite - 2036-possible-
61
1.00E−04
4.00E−04
371%



Heme






27278
Metabolite - 10510
50
5.00E−19
1.33E−17
351%


22993
Metabolite - 9448
50
4.73E−15
8.00E−14
350%


25366
Metabolite - 10286
61
1.65E−05
1.00E−04
339%


21631
Metabolite - 8403
50
9.85E−21
4.58E−19
306%


27414
beta-sitosterol
50
1.11E−12
1.29E−11
302%


21013
Metabolite - 7890
50
6.00E−14
7.73E−13
237%


12785
Metabolite - 3103
50
0.0011
0.0026
236%


16831
Metabolite - 4504
50
2.00E−04
5.00E−04
234%


18155
Metabolite - 5386
61
2.55E−05
1.00E−04
223%


8159
Metabolite - 1971
61
0.0026
0.0054
213%


27256
Metabolite - 10500
50
2.49E−19
7.72E−18
209%


21127
monopalmitin
50
6.23E−14
7.73E−13
204%


21184
1-oleoyl-rac-glycerol
50
6.14E−08
4.17E−07
196%


6380
Metabolite - 1330
61
0.0963
0.0914
177%


21188
1-stearoyl-rac-glycerol
50
5.43E−12
5.94E−11
177%


22032
Metabolite - 8766
50
1.79E−14
2.78E−13
163%


1561
alpha-tocopherol
50
4.00E−04
0.001
161%


6266
Metabolite - 1286
61
6.00E−04
0.0015
130%


19323
Docosahexaenoic-Acid
50
2.43E−17
5.65E−16
126%


27890
Metabolite - 10611
50
2.81E−09
2.26E−08
126%


6130
Metabolite - 1208
61
1.04E−05
4.62E−05
124%


6362
p-cresol-sulfate
61
2.77E−05
1.00E−04
123%


9172
Metabolite - 2000
61
1.43E−15
2.96E−14
121%


15991
L-alpha-
61
0.0014
0.0032
117%



glycerophosphorylcholine






9905
Metabolite - 2231
61
1.00E−04
2.00E−04
116%


15611
Metabolite - 3971
61
3.71E−06
1.82E−05
115%


24205
Metabolite - 9841
61
1.98E−06
1.02E−05
111%


12604
Metabolite - 2981
50
6.77E−09
5.04E−08
111%


27888
Metabolite - 10609
50
0.0116
0.0181
110%


27728
glycerol-2-phosphate
50
8.04E−06
3.65E−05
107%


12035
nonanate
50
3.95E−05
2.00E−04
104%


17251
Metabolite - 4732
61
0.0089
0.0147
103%


23079
Metabolite - 9647
61
1.18E−05
1.00E−04
101%


1110
arachidonic acid
50
0.0015
0.0032
100%


15753
hippuric acid
61
1.00E−04
2.00E−04
97%


17800
cortodoxone
61
1.00E−04
4.00E−04
92%


27773
Isobar-71[1]
61
0.0301
0.0379
92%


1359
oleic acid
50
0.0669
0.0711
90%


1358
octadecanoic acid
50
4.62E−14
6.61E−13
88%


12774
Metabolite - 3094
50
2.92E−09
2.26E−08
86%


12767
Metabolite - 3087
50
0.007
0.0124
85%


24330
Metabolite - 10126
61
1.00E−09
8.46E−09
84%


21150
sinapic acid
61
1.10E−10
9.75E−10
84%


20136
Metabolite - 7062
61
3.65E−05
1.00E−04
80%


10782
Metabolite - 2486
61
0.0854
0.0837
80%


6171
Metabolite - 1244
61
0.003
0.0059
78%


16138
Metabolite - 4080
50
3.02E−07
1.76E−06
77%


21418
Isobar-56[2]
61
6.28E−08
4.17E−07
77%


1105
linoleic acid
50
3.36E−11
3.29E−10
75%


1336
n-hexadecanoic acid
50
3.99E−11
3.71E−10
73%


12112
Metabolite - 2314
61
1.47E−06
7.82E−06
72%


57
glutamic acid
50
0.0083
0.0141
71%


22586
Metabolite - 9039
61
1.00E−04
3.00E−04
70%


21828
Metabolite - 8574
61
0.0027
0.0055
68%


26444
Metabolite - 10465
61
2.89E−11
2.99E−10
66%


5475
Metabolite - 1033
61
1.01E−07
6.06E−07
63%


10700
Metabolite - 2393
61
0.0101
0.0162
61%


22259
Isobar-59[3]
61
0.0013
0.003
58%


16992
Metabolite - 4603
61
0.0237
0.031
57%


18691
Metabolite - 5749
61
0.0854
0.0837
57%


19934
inositol
50
0.0067
0.012
55%


12609
Metabolite - 2986
50
0.0013
0.0029
55%


15365
sn-Glycerol-3-phosphate
50
8.00E−04
0.0018
53%


11499
Metabolite - 2753
61
0.0071
0.0125
51%


16074
Metabolite - 2758
50
6.00E−04
0.0016
49%


15670
2-methylhippuric acid
61
6.00E−04
0.0016
49%


8210
Metabolite - 1981
61
0.017
0.0238
47%


1121
heptadecanoic acid
50
8.42E−09
6.03E−08
46%


17488
Metabolite - 4887
61
2.00E−04
6.00E−04
45%


1493
ornithine
50
0.0482
0.0557
42%


6851
Metabolite - 1497
61
0.0676
0.0711
42%


22895
Metabolite - 9299
50
0.0133
0.0195
39%


13038
Metabolite - 3143
61
0.0117
0.0181
39%


5699
Metabolite - 1157
61
0.0036
0.0068
37%


18015
Metabolite - 3113
61
1.00E−04
4.00E−04
36%


24076
Metabolite - 9726
50
3.00E−04
0.001
36%


20031
Metabolite - 7007
61
0.0011
0.0026
36%


20488
D-glucose
50
2.00E−04
7.00E−04
34%


10401
Metabolite - 2058
61
0.0015
0.0032
34%


1507
palmitoleic acid
50
0.0168
0.0236
34%


1670
urea
50
4.00E−04
0.001
34%


15948
S-adenosyl-l-homocysteine
61
0.023
0.0303
33%


5465
Metabolite - 1029
61
8.00E−04
0.002
33%


2849
guanosine-5-
61
0.0418
0.0499
33%



monophosphate






1494
5-oxoproline
50
0.0039
0.0073
32%


11923
Metabolite - 2821
61
0.0089
0.0147
32%


18969
Metabolite - 5920
61
0.0033
0.0063
32%


20830
Metabolite - 7762
61
0.056
0.0621
31%


2832
adenosine-5-
61
0.0137
0.0199
30%



monophosphate






17083
Metabolite - 4634
50
0.0718
0.0742
28%


15996
aspartate
50
0.1053
0.0995
27%


14639
Metabolite - 3603
61
0.0225
0.0302
26%


25514
Metabolite - 10404
61
0.0747
0.0768
26%


17747
D-sphingosine
50
0.0239
0.0311
26%


27137
Metabolite - 10498
50
0.0511
0.0576
26%


30128
Metabolite - 10687
61
1.00E−04
4.00E−04
25%


16244
Isobar-21[4]
61
0.0038
0.0072
25%


15053
sorbitol
50
0.0718
0.0742
25%


15122
glycerol
50
0.0019
0.004
25%


14387
Metabolite - 3490
61
0.0754
0.0771
23%


1642
decanoic acid
50
0.0455
0.0536
23%


20675
1,5-anhydro-D-glucitol
50
0.1081
0.1016
22%


11438
phosphate
50
0.0022
0.0045
21%


584
mannose
50
0.0128
0.0191
19%


2129
L-5-Hydroxytryptophan
61
0.0809
0.0806
19%


9491
Metabolite - 2185
61
0.0162
0.023
18%


605
uracil
50
0.0468
0.0544
18%


10655
Metabolite - 2388
61
0.0112
0.0177
18%


17665
p-hydroxybenzaldehyde
61
0.0121
0.0186
18%


10746
Isobar-6[5]
61
0.0564
0.0621
16%


19372
Metabolite - 6269
50
0.0117
0.0181
16%


9216
Metabolite - 2168
61
0.0464
0.0543
15%


15113
Metabolite - 3783
61
0.0126
0.019
14%


27718
creatine
61
0.0245
0.0312
14%


1113
isocitrate
61
0.0132
0.0195
14%


18232
Metabolite - 5403
50
0.0493
0.0562
14%


10737
Isobar-1[6]
61
0.0229
0.0303
13%


7175
Metabolite - 1655
61
0.0099
0.016
13%


19402
Metabolite - 6346
50
0.0411
0.0496
10%


15677
3-methyl-L-histidine
61
0.0795
0.0799
−8%


9324
Metabolite - 2173
61
0.0842
0.0834
−9%


54
tryptophan
61
0.0808
0.0806
−11%


17007
Metabolite - 4609
61
0.0957
0.0914
−11%


1574
histamine
61
0.0574
0.0629
−12%


5765
Metabolite - 1142
61
0.0951
0.0913
−14%


27672
3-indoxyl-sulfate
61
0.0514
0.0576
−15%


10667
Metabolite - 2389
61
0.0939
0.0906
−15%


15765
ethylmalonic acid
61
0.0241
0.0311
−17%


16705
Metabolite - 4428
61
0.0153
0.0219
−17%


15529
Metabolite - 3951
61
0.0125
0.019
−18%


11053
Metabolite - 2567
61
0.0053
0.0098
−18%


24213
Metabolite - 9845
61
0.0189
0.0261
−19%


20950
Metabolite - 7846
50
0.0915
0.0887
−19%


13065
Metabolite - 3146
61
0.0021
0.0045
−20%


1643
fumaric acid
50
0.0485
0.0558
−20%


1512
picolinic acid
50
0.0322
0.0396
−21%


24197
Metabolite - 9838
61
0.0675
0.0711
−22%


12724
Metabolite - 3057
61
0.0174
0.0241
−22%


27130
Metabolite - 10493
61
0.0087
0.0146
−22%


15676
3-methyl-2-oxovaleric acid
61
0.0716
0.0742
−23%


18871
Metabolite - 5848
61
0.0385
0.0468
−24%


13328
Metabolite - 3238
61
0.0194
0.0266
−24%


6253
Metabolite - 1283
61
0.0595
0.0644
−25%


10347
Metabolite - 2285
61
0.0015
0.0032
−25%


16712
Metabolite - 4432
61
0.0081
0.0139
−26%


14786
Metabolite - 3697
61
0.0795
0.0799
−27%


15683
4-methyl-2-oxopentanoate
61
0.0128
0.0191
−28%


8644
Metabolite - 2051
61
0.0413
0.0496
−28%


7650
Metabolite - 1834
61
7.29E−08
4.52E−07
−28%


6398
Metabolite - 1335
61
0.0617
0.0664
−29%


1412
2′-deoxyuridine
61
4.00E−04
0.001
−30%


19405
Metabolite - 6347
50
0.0877
0.0855
−31%


22130
DL-3-phenyllactic acid
61
0.0441
0.0522
−34%


26449
Metabolite - 10467
61
0.0777
0.079
−34%


25584
Metabolite - 10425
50
0.0078
0.0136
−34%


18929
Metabolite - 5907
50
0.0253
0.032
−35%


28059
Metabolite - 10650
50
1.00E−04
5.00E−04
−36%


5466
Metabolite - 1030
61
0.0098
0.016
−37%


20169
Metabolite - 7092
61
0.0582
0.0633
−37%


25459
Metabolite - 10395
50
0.0542
0.0604
−38%


17091
Metabolite - 4641
61
8.22E−07
4.50E−06
−38%


19968
Metabolite - 6930
50
0.0318
0.0395
−39%


11299
Metabolite - 2706
61
6.83E−08
4.38E−07
−39%


25505
Metabolite - 10402
61
4.00E−04
0.0012
−39%


16653
Metabolite - 4361
50
0.0032
0.0063
−41%


18968
Metabolite - 5919
61
4.00E−04
0.0011
−41%


11292
Metabolite - 2703
61
0.0059
0.0107
−41%


13534
Metabolite - 3320
61
0.0055
0.01
−43%


6297
Metabolite - 1304
61
0.0334
0.0409
−43%


19367
Metabolite - 6266
50
0.0035
0.0068
−44%


16071
Metabolite - 4020
50
1.00E−04
4.00E−04
−45%


24206
Metabolite - 9842
61
0.0142
0.0204
−47%


25602
Metabolite - 10432
50
8.00E−04
0.0018
−52%


25598
Metabolite - 10428
50
0.0665
0.0711
−58%


25597
Metabolite - 10427
50
0.0028
0.0056
−58%


25599
Metabolite - 10429
50
0.0319
0.0395
−59%


22548
Metabolite - 9026
50
0.0202
0.0274
−61%


19362
Metabolite - 6226
50
0.0243
0.0312
−65%


16650
Metabolite - 4360
50
0.008
0.0138
−66%


25538
Metabolite - 10415
61
0.0495
0.0562
−66%


25546
Metabolite - 10418
61
0.0018
0.0038
−73%


25527
Metabolite - 10410
61
2.00E−04
5.00E−04
−78%


22566
Metabolite - 9029
61
2.00E−04
6.00E−04
−80%


25529
Metabolite - 10411
61
3.00E−04
0.001
−81%


25517
Metabolite - 10406
61
6.00E−04
0.0016
−83%


10148
Metabolite - 2257
61
1.00E−04
5.00E−04
−84%


21651
Metabolite - 8410
61
6.66E−06
3.10E−05
−87%


25541
Metabolite - 10417
61
2.84E−05
1.00E−04
−91%


25539
Metabolite - 10416
61
3.59E−06
1.81E−05
−93%





[1]Isobar-71 includes conduritol-beta-epoxide-3-deoxyglucosone


[2]Isobar-56 includes DL-pipecolic acid-1-amino-1-cyclopentanecarboxylic acid


[3]Isobar-59 includes N-6-trimethyl-L-lysine-H-homoarg-OH


[4]Isobar-21-includes gamma-aminobutyryl-L-histidine-L-anserine


[5]Isobar-6 includes valine-betaine


[6]Isobar-1 includes mannose, fructose, glucose, galactose, alpha-L-sorbopyranose, Inositol, D-allose, D-altrose, D-psicone, L-gulose, allo-inositol













TABLE 15







Metabolite biomarkers of Atherosclerosis in aorta.

















%







Change


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
in LDb















63
cholesterol
50
7.16E−07
8.78E−06
569%


22993
Metabolite-9448
50
1.06E−05
1.00E−04
565%


10655
Metabolite-2388
61
2.34E−07
4.83E−06
287%


25548
Metabolite-10419
50
1.00E−04
3.00E−04
277%


22320
Metabolite-8889
50
0.0015
0.0018
244%


15991
L-alpha-
61
0.0174
0.0135
227%



glycerophosphorylcholine






27256
Metabolite-10500
50
6.36E−07
8.78E−06
190%


19383
Metabolite-6286
50
6.00E−04
0.001
125%


9137
Metabolite-2141
61
0.0026
0.0028
121%


16028
Metabolite-3998
50
0.0012
0.0015
110%


10739
Metabolite-2407
61
0.0057
0.0053
94%


17987
Metabolite-5228
50
0.0087
0.0078
92%


22675
Metabolite-9126
61
9.00E−04
0.0013
89%


1481
inositol-1-phosphate
50
0.0149
0.0117
78%


30173
Metabolite-10701
61
0.002
0.0022
70%


12774
Metabolite-3094
50
0.0252
0.0186
66%


21421
Metabolite-8214
50
0.0482
0.0321
63%


22032
Metabolite-8766
50
0.0017
0.0019
62%


12604
Metabolite-2981
50
0.0027
0.0028
54%


16002
Metabolite-3992[1]
61
1.71E−05
1.00E−04
37%


20360
Metabolite-7326
61
0.0352
0.0243
−14%


7601
Metabolite-1819
61
0.0351
0.0243
−14%


18829
phenylalanine
61
0.0303
0.0216
−16%


1604
uric acid
50
0.0331
0.0234
−17%


13505
Metabolite-3313
61
0.0337
0.0237
−17%


19787
Metabolite-6746
61
0.0143
0.0115
−18%


16235
Isobar-19[2]
61
0.0433
0.0292
−18%


10743
Isobar-4[3]
61
0.0428
0.0292
−18%


54
tryptophan
61
0.0217
0.0164
−19%


3147
xanthine
61
0.0234
0.0175
−19%


27570
Metabolite-10569
61
0.0177
0.0135
−20%


15529
Metabolite-3951
61
0.0468
0.0313
−20%


1125
isoleucine
50
0.0109
0.0092
−22%


1648
serine
50
0.0071
0.0065
−23%


605
uracil
50
0.0416
0.0286
−23%


7029
Metabolite-1597
61
0.0045
0.0043
−24%


1299
tyrosine
61
0.008
0.0073
−24%


1592
N-acetylneuraminic acid
61
0.0087
0.0078
−24%


1508
pantothenic acid
61
0.0105
0.0089
−24%


13810
Metabolite-3379
61
0.0146
0.0116
−24%


24076
Metabolite-9726
50
1.00E−04
3.00E−04
−26%


8336
Metabolite-2005
61
0.0019
0.0021
−26%


22258
Isobar-58[4]
61
0.0292
0.0211
−26%


60
leucine
50
0.0045
0.0043
−28%


30128
Metabolite-10687
61
0.0114
0.0095
−28%


17048
Metabolite-4617
61
0.0014
0.0017
−29%


17960
Metabolite-5207
50
0.0015
0.0018
−29%


15677
3-methyl-L-histidine
61
5.00E−04
0.001
−30%


1431
(p-Hydroxyphenyl)lactic acid
50
0.001
0.0013
−30%


1649
valine
50
0.0039
0.004
−30%


1898
proline
50
0.0042
0.0042
−30%


1284
threonine
50
0.0047
0.0045
−31%


15948
S-adenosyl-l-homocysteine
61
0.0101
0.0088
−31%


26456
Metabolite-10470
61
0.0134
0.011
−31%


1302
methionine
61
0.0161
0.0125
−31%


22185
n-acetyl-l-aspartic acid
61
9.00E−04
0.0013
−32%


1414
3-phospho-d-glycerate
61
0.002
0.0022
−32%


1643
fumaric acid
50
0.0042
0.0042
−33%


10890
Metabolite-2554
61
0.0029
0.003
−34%


10746
Isobar-6[5]
61
0.0283
0.0208
−35%


15253
Metabolite-3832-possible-
61
0.0292
0.0211
−35%



phenol-sulfate






5821
3-phospho-l-serine
61
7.00E−04
0.0012
−36%


12102
o-phosphoethanolamine
50
0.0014
0.0018
−36%


8404
Metabolite-2027
61
0.0019
0.0021
−36%


1123
inosine
61
2.00E−04
5.00E−04
−37%


3127
hypoxanthine
61
8.00E−04
0.0012
−37%


6771
Metabolite-1460
61
0.0015
0.0018
−37%


14311
Metabolite-3481
61
0.0198
0.015
−37%


16233
Isobar-13[6]
61
6.00E−04
0.001
−38%


11222
Metabolite-2688
61
5.00E−04
0.001
−38%


1638
arginine
61
8.00E−04
0.0012
−38%


15497
arginino-succinate
61
0.0012
0.0015
−38%


15506
choline
61
2.00E−04
5.00E−04
−39%


16705
Metabolite-4428
61
0.0012
0.0016
−39%


8991
Metabolite-2105
61
0.0096
0.0085
−39%


514
cytidine
61
5.00E−04
9.00E−04
−40%


13018
Metabolite-3138
61
5.00E−04
0.001
−40%


527
lactate
50
7.00E−04
0.0012
−40%


23051
Metabolite-9566
61
0.001
0.0014
−40%


28131
Metabolite-10670
61
4.00E−04
8.00E−04
−41%


1494
5-oxoproline
50
9.00E−04
0.0013
−41%


606
uridine
61
5.00E−04
0.001
−42%


18374
methionine-sulfoxide
61
1.00E−04
4.00E−04
−43%


57
glutamic acid
50
6.00E−04
0.001
−44%


15996
aspartate
50
8.00E−04
0.0012
−44%


20489
D-glucose
50
0.0051
0.0048
−44%


18348
3-hydroxy-3methylglutaryl-
61
0.01
0.0087
−44%



coenzyme-A






27718
creatine
61
2.00E−04
5.00E−04
−45%


1303
malic acid
61
7.00E−04
0.0011
−45%


1412
2′-deoxyuridine
61
1.02E−06
1.09E−05
−46%


21430
Metabolite-8266
61
2.00E−04
6.00E−04
−47%


11544
Metabolite-2766
61
0.0013
0.0016
−47%


19372
Metabolite-6269
50
0.0027
0.0028
−47%


22145
acetyl-L-carnitine
61
2.00E−04
5.00E−04
−48%


10737
Isobar-1[7]
61
3.00E−04
6.00E−04
−48%


14247
Metabolite-3475
61
0.0146
0.0116
−48%


19110
Metabolite-5978
50
3.00E−04
7.00E−04
−49%


22730
Metabolite-9186
61
8.00E−04
0.0012
−49%


11777
glycine
50
5.00E−04
0.001
−50%


16228
Isobar-22[8]
61
1.00E−04
3.00E−04
−51%


16843
Metabolite-4510
50
1.00E−04
3.00E−04
−51%


53
glutamine
50
2.00E−04
6.00E−04
−51%


19708
Metabolite-6711
61
0.0124
0.0103
−51%


1416
GABA
50
1.38E−08
1.18E−06
−52%


15500
carnitine
61
5.00E−04
9.00E−04
−52%


2125
taurine
61
0.0017
0.002
−52%


22475
Metabolite-8986
61
0.0044
0.0043
−52%


28059
Metabolite-10650
50
1.87E−05
1.00E−04
−54%


20361
Metabolite-7327
61
3.32E−05
2.00E−04
−54%


17971
Metabolite-5210
50
1.00E−04
3.00E−04
−54%


22494
Metabolite-8994
50
4.00E−04
9.00E−04
−54%


7650
Metabolite-1834
61
2.06E−07
4.83E−06
−55%


27738
threonic acid
50
1.00E−04
3.00E−04
−55%


27678
Metabolite-10584
50
3.00E−04
7.00E−04
−55%


12459
Isobar-10[9]
61
3.43E−05
2.00E−04
−56%


15125
(2-Aminoethyl)phosphonate
61
1.00E−04
3.00E−04
−56%


19934
inositol
50
3.00E−04
7.00E−04
−56%


16860
Metabolite-4517
50
1.00E−04
3.00E−04
−57%


17064
Metabolite-4624
50
1.00E−04
4.00E−04
−57%


1107
allantoin
50
5.00E−04
0.001
−57%


1573
guanosine
61
4.00E−06
3.69E−05
−58%


1670
urea
50
1.00E−04
3.00E−04
−58%


27727
glutathione-oxidized
61
1.00E−04
4.00E−04
−61%


1519
sucrose
50
0.0104
0.0089
−62%


22702
Metabolite-9127
61
4.30E−06
3.69E−05
−63%


15336
tartaric acid
61
6.92E−08
2.97E−06
−66%


6172
Metabolite-1245
61
2.82E−07
4.83E−06
−70%


17975
Metabolite-5211
50
1.04E−05
1.00E−04
−70%


27773
Isobar-71[10]
61
3.00E−04
7.00E−04
−71%





[1] Possibly Cl-adduct of Formate dimmer


[2]Isobar-19 includes 1,5-anhydro-D-glucitol, 2′-deoxy-D-galactose, 2′-deoxy-D-glucose, L-fucose, L-rhamnose


[3]Isobar-4 includes Gluconic acid, DL-arabinose, D-ribose, L-xylose, DL-lyxose, D-xylulose, galactonic acid


[4]Isobar-58 includes bicine, 2-methylaminomethyl-tartronic acid


[5]Isobar-6 includes valine-betaine


[6]Isobar-13 includes 5-keto-D-gluconic acid, 2-keto-L-gulonic acid, D-glucuronic acid, D-galacturonic acid


[7]Isobar-1 includes mannose, fructose, glucose, galactose, alpha-L-sorbopyranose, Inositol, D-allose, D-altrose, D-psicone, L-gulose, allo-inositol


[8]Isobar-22-includes-glutamic acid-O-acetyl-L-serine


[9]Isobar-10-includes-glutamine-H-beta-ala-gly-OH-1-methylguanine-H-Gly-Sar-OH-lysine


[10]Isobar-71includes conduritol-beta-epoxide-3-deoxyglucosone













TABLE 16







Metabolite biomarkers of Atherosclerosis in liver.

















% 


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
Change in LDb















19788
Metabolite-6747
61
6.49E−06
0.001
895%


1564
citric acid
50
0.0069
0.0454
399%


9117
Metabolite-2135
61
0.0173
0.0851
206%


25626
Metabolite-10443
61
0.021
0.0964
180%


19597
Metabolite-6648
50
0.0055
0.0452
162%


15685
5-hydroxylysine
61
0.0049
0.0452
159%


16655
Metabolite-4362
50
0.0064
0.0454
131%


21418
Isobar-56[1]
61
1.36E−05
0.0011
129%


22475
Metabolite-8986
61
0.0012
0.0191
129%


15803
maltose
50
0.0224
0.0992
128%


18344
D-xyulose
50
1.00E−04
0.0071
126%


27678
Metabolite-10584
50
0.0083
0.049
126%


22020
Metabolite-8749
50
6.00E−04
0.0168
124%


30204
Metabolite-10713
61
0.0138
0.0703
122%


30203
Metabolite-10712
61
9.00E−04
0.0186
113%


15053
sorbitol
50
0.0048
0.0452
105%


1640
ascorbic acid
50
0.0032
0.0375
104%


19753
Metabolite-6718
61
0.0073
0.046
97%


21650
Metabolite-8409
61
0.0185
0.0866
91%


1516
sarcosine
50
0.0124
0.0659
85%


22309
Metabolite-8887
61
0.001
0.0191
85%


27299
Metabolite-10520
61
4.00E−04
0.0136
83%


1118
eicosanoic acid
50
0.0015
0.0191
78%


25429
Metabolite-10369
50
0.0045
0.0452
72%


8669
Metabolite-2055
61
0.0087
0.0498
69%


8210
Metabolite-1981
61
0.023
0.0992
65%


20488
D-glucose
50
0.0073
0.046
55%


15606
Metabolite-3968
61
0.0069
0.0454
51%


11379
Metabolite-2725
61
0.0224
0.0992
50%


11484
Metabolite-2752
61
7.00E−04
0.0168
50%


11292
Metabolite-2703
61
0.0133
0.0694
49%


8457
Metabolite-2035[2]
61
0.0066
0.0454
47%


16859
Metabolite-4516
50
0.0066
0.0454
44%


554
adenine
50
0.0076
0.0463
44%


7081
Metabolite-1609
61
0.0039
0.0412
41%


20795
Metabolite-7747
61
0.0033
0.0375
40%


16229
Isobar-24[3]
61
0.0055
0.0452
39%


18388
Metabolite-5491
50
0.0057
0.0452
39%


24360
Metabolite-10206
50
0.0178
0.086
38%


12080
D-ribose
50
0.0104
0.0575
37%


22993
Metabolite-9448
50
0.0156
0.0782
36%


10737
Isobar-1[4]
61
0.0084
0.049
36%


16060
Metabolite-4014
50
0.0181
0.0862
36%


24285
Metabolite-10026
61
0.0229
0.0992
23%


63
cholesterol
50
0.0104
0.0575
15%


22414
Metabolite-8933
61
0.0037
0.0406
−29%


1827
riboflavine
61
0.0052
0.0452
−29%


22320
Metabolite-8889
50
0.0056
0.0452
−29%


9002
Metabolite-2107
61
0.0012
0.0191
−31%


3138
pyridoxamine-phosphate
61
0.0054
0.0452
−34%


15964
D-arabitol
50
2.00E−04
0.0098
−34%


7432
Metabolite-1735
61
0.0014
0.0191
−35%


22185
n-acetyl-l-aspartic acid
61
0.0068
0.0454
−36%


23024
Metabolite-9458
61
0.0011
0.0191
−37%


21296
glucosamine-6-sulfate
61
0.0014
0.0191
−39%


7650
Metabolite-1834
61
7.40E−06
0.001
−41%


9468
Metabolite-2183
61
8.00E−04
0.0182
−46%


6530
Metabolite-1384
61
0.0115
0.0621
−57%


25561
Metabolite-10421
61
0.0069
0.0454
−58%


597
phosphoenolpyruvate
61
0.0014
0.0191
−59%


1414
3-phospho-d-glycerate
50
1.57E−05
0.0011
−60%


10148
Metabolite-2257
61
0.003
0.037
−68%


27794
Metabolite-10587
61
4.00E−04
0.0127
−69%


6146
alpha-amino-adipate
50
2.00E−04
0.0098
−73%





[1]Isobar-56 includes DL-pipecolic acid-1-amino-1-cyclopentanecarboxylic acid


[2] Possible 5-methyl-deoxycytidine-monophosphate


[3]Isobar-24 includes L-arabitol, adonitol, xylitol


[4]Isobar-1 includes mannose, fructose, glucose, galactose, alpha-L-sorbopyranose, Inositol, D-allose, D-altrose, D-psicone, L-gulose, allo-inositol






Identification of plasma biomarkers indicative of initiation and/or progression of atherosclerosis would help diagnosis and treatment of human patients with this disease. Recursive partitioning of plasma metabolites identified cholesterol as a biomarker that could differentiate the LDb and C57BL/6 mice perfectly, as expected. Three other metabolites were also identified by recursive partitioning to differentiate the LDb and C57BL/6 mice (Table 17). Plasma levels of these metabolites, like plasma cholesterol, were higher in LDb mice even at 2 months of age and remained consistently higher during the following 6 months, suggesting that earlier buildup of these metabolites is likely involved in the development of atherosclerosis and provide biomarkers for progression (FIGS. 13, 14, 15, and 16).







TABLE 17







Atherosclerosis biomarkers in plasma that differentiate Atherosclerosis


subjects (LDb) and healthy control subjects (C57BL/6 mice) without error.














Atherosclerosis
Control
R-



Cmpd_ID
Compound
level
level
Square
LogWorth















19323
Docosahexaenoic-
≧528666
<528666
1.000
21.11



Acid






21011
Metabolite-7888
≧2523794
<2523794
1.000
21.11


21631
Metabolite-8403
≧238349
<238349
1.000
21.11









Metabolite-1834 did not segregate LDb and C57BL/6 groups in 2-month-old mice, but started to segregate the 5-month-olds and segregated 8-month-old mice perfectly (FIG. 17). This metabolite is one of the biomarkers for atherosclerosis progression.
Several metabolites (p-cresol-sulfate, Metabolite-4887, Metabolite-5386) classified subjects as LDb or C57BL/6 very well in 2-month-old mice, but the power of differentiation diminished as the mice aged, with no segregation in 8-month-old mice (FIGS. 18 and 19). These metabolites are biomarkers for atherosclerosis initiation.
Random forest results show that the samples can be classified correctly with varying degrees of accuracy using the biomarkers. The confusion matrices demonstrate that LDb subjects can be distinguished from C57BL/6 subjects using plasma (Table 18), aorta (Table 19) and liver (Table 20) samples. The “Out-of-Bag” (OOB) Error rate gives an estimate of how accurately new observations can be predicted using the random forest model (e.g., whether a sample is from a subject having atherosclerosis or a control subject).







TABLE 18







Random Forest Confusion Matrices for Atherosclerosis in Plasma


Plasma











Control
Atherosclerosis
Error










Age Collected: 2 months












Control
10
0
0



Atherosclerosis
0
10 
0



OOB Error
0
 0%








Age Collected: 5 months












Control
10
0
0



Atherosclerosis
2
9
0



OOB Error
0
 0%








Age Collected: 8 months












Control
8
0
0



Atherosclerosis
0
8
0



OOB Error
0
 0%








ALL












Control
28
0
0



Atherosclerosis
0
27 
0



OOB Error
0
 0%
















TABLE 19







Random Forest Confusion Matrices for Atherosclerosis


in Aorta Tissues


Aorta Tissue











Control
Atherosclerosis
Error










Age Collected: 2 months












Control
6
1
0.14



Atherosclerosis
2
2
0.5



OOB Error
3/11 = 0.27
27%








Age Collected: 5 months












Control
7
0
0



Atherosclerosis
2
4
0.33



OOB Error
2/13 = 0.15
15%








Age Collected: 8 months












Control
6
0
0



Atherosclerosis
0
4
0



OOB Error
0/10 = 0.00
 0%








ALL












Control
18 
2
0.1



Atherosclerosis
3
11 
0.21



OOB Error
5/34 = 0.15
15%
















TABLE 20







Random Forest Confusion Matrices for Atherosclerosis in Liver


Liver











Control
Atherosclerosis
Error










Age Collected: 2 months












Control
7
1
0.13



Atherosclerosis
1
3
0.25



OOB Error
2/12 = 0.17
17%








Age Collected: 5 months












Control
3
1
0.25



Atherosclerosis
3
3
0.5



OOB Error
4/10 = 0.4
40%








Age Collected: 8 months












Control
5
0
0



Atherosclerosis
1
5
0.17



OOB Error
1/11 = 0.09
 9%








ALL












Control
15 
2
0.12



Atherosclerosis
3
13 
0.19



OOB Error
5/33 = 0.15
15%









In addition, a study was carried out on human subjects suffering from atherosclerosis (n=15) or healthy subjects (n=14). The biomarkers 3-methylhistidine, p-cresol sulfate, mannose, glucose, and gluconate showed the same alterations in human plasma from disease vs. healthy subjects as seen in the mouse model. Thus, these compounds were identified as important biomarkers useful to distinguish individuals with atherosclerosis from healthy subjects.
3C: Biomarkers of Cardiomyopathy
Biomarkers were discovered by (1) analyzing cardiac tissue samples (Table 21) or plasma samples (Table 22) from different groups of mouse subjects to determine the levels of metabolites in the samples and then (2) statistically analyzing the results to determine those metabolites that were differentially present in the two groups. These subjects provide an animal (mouse) model for human DCM.
Two groups of subjects were used. One group consisted of eight subjects exhibiting cardiac dilatation and depressed left ventricular systolic function (ejection fraction of less than 0.40), as determined by echocardiography (cTnT-W141 transgenic mice). Thirteen age- and gender-matched subjects (non-transgenic (wild-type background strain) mice) served as controls. All mice were 7-19 months old and weighed 23-40 gm.
T-tests were used to determine differences in the mean levels of metabolites between the two populations (i.e., Dilated Cardiomyopathy, DCM vs. Healthy control). Classification analysis was carried out using recursive partitioning and random forest analyses to uncover the biomarkers that can best differentiate the two groups. Recursive partitioning relates a ‘dependent’ variable (Y) to a collection of independent (‘predictor’) variables (X) in order to uncover—or simply understand—the elusive relationship, Y=f(X). It was performed with the JMP program (SAS) to generate a decision tree. The statistical significance of the “split” of the data can be placed on a more quantitative footing by computing p-values, which discern the quality of a split relative to a random event. The significance level of each “split” of data into the nodes or branches of the tree was computed as p-values, which discern the quality of the split relative to a random event. It was given as LogWorth, which is the negative log 10 of a raw p-value.
Biomarkers:
As listed below in Tables 21 and 22, biomarkers were discovered that were differentially present between cardiac tissue and plasma samples, respectively, collected from dilated cardiomyopathy subjects and healthy subjects.
Tables 21 and 22 include, for each listed biomarker, the p-value and q-value determined in the statistical analysis of the data concerning the biomarkers and an indication of the percentage difference in the dilated cardiomyopathy mean level as compared to the healthy mean level in cardiac tissue (Table 21) or plasma (Table 22). The term “Isobar” as used in the tables indicates the compounds that could not be distinguished from each other on the analytical platform used in the analysis (i.e., the compounds in an isobar elute at nearly the same time and have similar (and sometimes exactly the same) quant ions, and thus cannot be distinguished). Comp_ID refers to the compound identification number used as a primary key for that compound in the in-house chemical database. Library indicates the chemical library that was used to identify the compounds. The number 50 refer to the GC library and the number 61 refers to the LC library.







TABLE 21







Metabolite biomarkers of dilated cardiomyopathy (DCM) in cardiac


tissues.

















%







Change


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
in DCM















22185
n-acetyl-l-aspartic acid
61
0.0044
0.0144
100%


15996
aspartate
50
0.0002
0.0022
64%


1414
3-phospho-d-glycerate
50
0.0742
0.0888
52%


1898
proline
61
3.64E−05
0.0007
52%


1648
serine
50
0.0009
0.0057
49%


1299
tyrosine
61
3.36E−05
0.0007
39%


1284
threonine
50
0.0054
0.0173
28%


54
tryptophan
61
0.0383
0.0571
20%


1649
valine
50
0.0081
0.0227
19%


1125
isoleucine
50
0.0041
0.014
16%


11777
glycine
50
0.0109
0.0255
15%


13179
creatine
61
0.0011
0.0061
−17%


590
hypotaurine
61
0.0728
0.0879
−21%


5278
beta-nicotinamide adenine
61
0.0092
0.0239
−27%



dinucleotide






15500
carnitine
61
0.0331
0.0509
−32%


2127
glutathione, reduced
61
0.0033
0.0125
−62%


12080
D-ribose
50
0.0033
0.0125
39%


15122
glycerol
50
0.0663
0.0819
−16%


19934
inositol
50
0.0225
0.0417
−22%


18882
taurodeoxycholic acid
61
0.0584
0.0754
72%


15365
sn-Glycerol 3-phosphate
50
0.0109
0.0255
41%


63
cholesterol
50
0.011
0.0255
14%


1121
heptadecanoic acid
50
0.0594
0.0761
−15%


21127
monopalmitin
50
0.0149
0.0324
−23%


1336
n-hexadecanoic acid
50
0.0014
0.0072
−24%


19323
docosahexaenoic acid
50
0.0009
0.0057
−24%


1600
o-phosphoethanolamine
50
0.0253
0.0435
−30%


1365
tetradecanoic acid
50
0.0516
0.0691
−31%


1570
oleic acid
50
0.0269
0.0446
−31%


1105
linoleic acid
50
0.0014
0.0072
−32%


1518
squalene
50
0.0013
0.0072
−43%


15504
phosphopantheine
61
0.0186
0.0367
−49%


1827
riboflavine
61
0.0037
0.0129
85%


594
niacinamide
50
0.0091
0.0239
−17%


3138
pyridoxamine phosphate
61
0.0047
0.0153
−19%


1508
pantothenic acid
61
0.0093
0.0239
−32%


3127
hypoxanthine
50
0.0112
0.0255
92%


606
uridine
61
0.0262
0.0445
56%


1107
allantoin
50
0.0207
0.0398
39%


514
cytidine
61
0.0034
0.0126
33%


1573
guanosine
61
0.0326
0.0507
32%


605
uracil
50
0.0291
0.0457
27%


21031
hydroxyurea
61
0.0069
0.0204
−47%


2856
uridine 5′-monophosphate
61
0.0005
0.004
−68%


18360
adenylosuccinic acid
61
9.02E−06
0.0004
−85%


555
adenosine
61
0.0007
0.0051
−87%


2832
adenosine 5′-monophosphate
61
4.53E−06
0.0004
−90%


2849
guanosine 5′-monophosphate
61
2.06E−05
0.0006
−94%


20701
malitol
50
0.0167
0.0343
−32%


8469
Metabolite-2036
61
0.0144
0.0319
525%


10781
Metabolite-2469
61
0.007
0.0204
355%


10604
Metabolite-2370
61
0.0089
0.0239
213%


10401
Metabolite-2058
61
0.0006
0.0048
203%


5597
Metabolite-1073
61
0.0178
0.0357
203%


14639
Metabolite-3603
61
5.89E−05
0.001
194%


16019
Metabolite-3995
61
0.0001
0.0017
156%


22480
Metabolite-8987
50
1.78E−05
0.0006
150%


6130
Metabolite-1208
61
0.0277
0.0454
144%


9137
Metabolite-2141
61
0.0174
0.0353
127%


21418
Isobar-56[1]
61
0.0015
0.0072
122%


18015
Metabolite-3113
61
0.0015
0.0072
122%


22414
Metabolite-8933
61
0.0903
0.0028
108%


6122
Metabolite-1206[2]
61
0.0414
0.058
104%


9024
Metabolite-2111
61
0.0241
0.0432
104%


18073
Metabolite-5270
61
0.0159
0.0337
92%


12711
Metabolite-3053
61
0.0574
0.0752
89%


13512
Metabolite-3315
61
0.028
0.0454
89%


16471
Metabolite-4238
61
0.046
0.0627
89%


5618
Metabolite-1085[3]
61
0.0371
0.0558
85%


7654
Metabolite-1836
61
0.002
0.0085
82%


16860
Metabolite-4517
50
0.0027
0.011
79%


14715
Metabolite-3653[4]
61
0.0037
0.0129
72%


21410
Isobar-52[5]
61
0.0018
0.008
69%


17885
Metabolite-5147
61
0.0155
0.0332
56%


22494
Metabolite-8994
50
0.0003
0.003
56%


10850
Metabolite-2548[6]
61
0.0668
0.0819
52%


15213
Metabolite-3808
61
5.92E−06
0.0004
52%


21415
Metabolite-8209
50
0.011
0.0255
52%


6266
Metabolite-1286
61
9.64E−05
0.0013
49%


7127
Metabolite-1616
61
0.0019
0.0084
49%


7272
Metabolite-1679
61
0.0562
0.0746
45%


8509
Metabolite-2041
61
0.0104
0.0255
45%


9313
Metabolite-2172
61
0.0246
0.0432
45%


9905
Metabolite-2231
61
0.0167
0.0343
43%


16071
Metabolite-4020
50
0.002
0.0085
41%


22441
Metabolite-8950
61
0.0798
0.0925
39%


19273
Metabolite-6108
61
0.0405
0.058
37%


16233
Isobar-13[7]
61
0.0126
0.0282
33%


9324
Metabolite-2173
61
0.0577
0.0752
33%


19787
Metabolite-6746
61
0.0244
0.0432
30%


20299
Metabolite-7266
50
0.0212
0.0402
30%


9122
Metabolite-2137
61
0.0836
0.0961
28%


21404
Isobar-48[8]
61
0.0487
0.0658
27%


21011
Metabolite-7888
50
0.0031
0.012
27%


13142
Metabolite-3165
61
0.0412
0.058
18%


19372
Metabolite-6269
50
0.0192
0.0374
16%


16285
Metabolite-2798
50
0.045
0.062
15%


13505
Metabolite-3313
61
0.0338
0.0514
11%


17064
Metabolite-4624
50
0.0619
0.0779
−15%


19599
Metabolite-6649
50
0.0287
0.0457
−18%


16074
Metabolite-2758
50
0.0417
0.058
−19%


20361
Metabolite-7327
61
0.0008
0.0056
−20%


7081
Metabolite-1609
61
0.0604
0.0767
−21%


17919
Metabolite-5187
61
0.0072
0.0208
−22%


17978
Metabolite-5213
50
0.0689
0.0838
−23%


18273
Metabolite-5420
50
0.0784
0.0916
−23%


16984
Metabolite-4599
50
0.0624
0.0779
−24%


11545
Metabolite-2767
61
0.0412
0.058
−25%


22509
Metabolite-9011
61
0.0063
0.0191
−29%


12856
Metabolite-3123
61
0.0768
0.0904
−32%


16060
Metabolite-4014
50
0.0253
0.0435
−32%


16843
Metabolite-4510
50
0.0289
0.0457
−34%


17960
Metabolite-5207
50
0.0003
0.003
−35%


16116
Metabolite-4051
50
0.0226
0.0417
−36%


14595
Metabolite-3576
61
0.0106
0.0255
−36%


8176
Metabolite-1974
61
0.0008
0.0056
−37%


15085
Metabolite-3776
61
0.0093
0.0239
−39%


16705
Metabolite-4428
61
0.0059
0.0182
−40%


19505
Metabolite-6547
61
0.0267
0.0446
−41%


11056
Metabolite-2568
61
0.075
0.0891
−43%


22507
Metabolite-9010
50
0.0396
0.0578
−46%


9130
Metabolite-2139
61
0.001
0.006
−48%


22381
Metabolite-8908
61
0.0389
0.0574
−50%


18702
Metabolite-5767
61
0.0234
0.0428
−56%


11379
Metabolite-2725
61
6.71E−05
0.001
−69%


22501
Metabolite-9007
61
0.0007
0.0052
−79%


22534
Metabolite-9016
61
2.36E−05
0.0006
−86%





[1]Isobar-56 includes DL-pipecolic acid, 1-amino-1-cyclopentanecarboxylic acid.


[2]Possible methyltestosterone and others.


[3]Possible isolobinine or 4-aminoestra-1,3,5(10)-triene-3,17beta-diol.


[4]Possible stachydrine.


[5]Isobar-52 includes iminodiacetic acid, L-aspartic acid.


[6]Possible Cl adduct of uric acid.


[7]Isobar 13 includes 5-keto-D-gluconic acid, 2-keto-L-gulonic acid, D-glucuronic acid, D(+)-galacturonic acid.


[8]Isobar 48 includes Serine-2,2-amino-2-methyl-1,3-propanediol,diethanolamine.













TABLE 22







Metabolite biomarkers of dilated cardiomyopathy (DCM) in plasma.


















%








Change


COMP_ID
COMPOUND
LIB_ID
p-value
q-value
in DCM
TG/NTG
















17007
Metabolite - 4609
61
0.0003
0.0895
83%
1.83


1299
tyrosine
61
0.0005
0.0895
83%
1.83


20161
Metabolite - 7088
61
0.0008
0.0895
239% 
3.39


19787
Metabolite - 6746
61
0.0010
0.0895
62%
1.62


20699
meso-erythritol
50
0.0013
0.0895
46%
1.46


18968
Metabolite - 5919
61
0.0014
0.0895
100% 
2.00


1107
allantoin
50
0.0017
0.0901
54%
1.54


1431
(p-Hydroxyphenyl)lactic acid
61
0.0028
0.1283
114% 
2.14


584
mannose
50
0.0034
0.1411
−41% 
0.59


15632
Metabolite - 3980
61
0.0054
0.1997
64%
1.64


396
glutarate
61
0.0069
0.2328
181% 
2.81


11292
Metabolite - 2703
61
0.0080
0.2473
124% 
2.24


18829
phenylalanine
61
0.0098
0.2547
39%
1.39


15286
Metabolite - 3848
61
0.0103
0.2547
92%
1.92


13575
Metabolite - 3324
61
0.0112
0.2547
250% 
3.50


14786
Metabolite - 3697
61
0.0121
0.2547
52%
1.52


22597
Metabolite - 9041
61
0.0130
0.2547
−70% 
0.30


15611
Metabolite - 3971
61
0.0132
0.2547
66%
1.66


11813
Metabolite - 2809
61
0.0137
0.2547
−28% 
0.72


6571
Metabolite - 1397
61
0.0139
0.2547
75%
1.75


21418
Isobar 56 includes DL-
61
0.0151
0.2547
38%
1.38



pipecolic acid, 1-amino-1-



cyclopentanecarboxylic acid


11299
Metabolite - 2706
61
0.0159
0.2547
65%
1.65


6305
Metabolite - A-1254
61
0.0163
0.2547
64%
1.64


19857
Metabolite - 6783
61
0.0165
0.2547
135% 
2.35


21044
(s)-2-hydroxybutyric acid
50
0.0186
0.2722
105% 
2.05


16016
Metabolite - 3994
61
0.0209
0.2722
92%
1.92


5440
Metabolite - A-1014
61
0.0221
0.2722
41%
1.41


22555
Metabolite - 9027
50
0.0223
0.2722
−53% 
0.47


10737
Isobar 1 includes mannose,
61
0.0227
0.2722
−33% 
0.67



fructose, glucose, galactose,



alpha-L-sorbopyranose,



Inositol, D-allose, D-(+)-



altrose, D-psicone, L-(+)-



gulose, allo-inositol


20830
Metabolite - 7762
61
0.0231
0.2722
48%
1.48


14961
Metabolite - 3752
61
0.0234
0.2722
−46% 
0.54


15670
2-methylhippuric acid
61
0.0234
0.2722
53%
1.53


13142
Metabolite - 3165
61
0.0285
0.3038
37%
1.37


605
uracil
50
0.0286
0.3038
217% 
3.17


21011
Metabolite - 7888
50
0.0298
0.3038
37%
1.37


8959
Metabolite - 2100
61
0.0305
0.3038
−29% 
0.71


20169
Metabolite - 7092
61
0.0320
0.3038
88%
1.88


5776
Metabolite - A-1194
61
0.0322
0.3038
89%
1.89


6126
Metabolite - 1207
61
0.0326
0.3038
52%
1.52


12459
Isobar 10 includes glutamine,
61
0.0327
0.3038
−31% 
0.69



H-beta-ala-gly-OH, 1-



methylguanine, H-Gly-Sar-



OH lysine


3155
3-ureidopropionic acid
61
0.0351
0.3093
60%
1.60


15541
Metabolite - 3957
61
0.0360
0.3093
43%
1.43


1303
malic acid
50
0.0388
0.3093
108% 
2.08


15737
hydroxyacetic acid
50
0.0392
0.3093
38%
1.38


527
lactate
50
0.0399
0.3093
65%
1.65


1670
urea
50
0.0410
0.3093
23%
1.23


15949
2′-deoxycytidine
61
0.0413
0.3093
24%
1.24


7272
Metabolite - 1679
61
0.0415
0.3093
251% 
3.51


12011
Metabolite - 2848
61
0.0425
0.3093
53%
1.53


1643
fumaric acid
50
0.0437
0.3093
70%
1.70


1574
histamine
61
0.0452
0.3093
74%
1.74


22566
Metabolite - 9029
61
0.0458
0.3093
−63% 
0.37


15140
L-kynurenine
61
0.0458
0.3093
49%
1.49


22026
1-methylguanidine
50
0.0462
0.3093
19%
1.19



hydrochloride


7127
Metabolite - 1616
61
0.0473
0.3093
−38% 
0.62


1604
uric acid
50
0.0478
0.3093
146% 
2.46


7429
Metabolite - 1733
61
0.0492
0.3093
37%
1.37


12626
Metabolite - 3003
50
0.0500
0.3093
51%
1.51


2849
guanosine 5′-monophosphate
61
0.0509
0.3093
−58% 
0.42


16327
Metabolite - 4161
61
0.0514
0.3093
35%
1.35


14715
Metabolite - 3653 - Possible
61
0.0521
0.3093
29%
1.29



stachydrine


1507
palmitoleic acid
50
0.0522
0.3093
−54% 
0.46


9491
Metabolite - 2185
61
0.0525
0.3093
97%
1.97


2734
gamma-L-glutamyl-L-
61
0.0541
0.3112
34%
1.34



tyrosine


11235
Metabolite - 2690
61
0.0548
0.3112
−34% 
0.66


13775
Metabolite - 3370
61
0.0561
0.3112
38%
1.38


17960
Metabolite - 5207
50
0.0569
0.3112
−32% 
0.68


1587
N-acetyl-L-leucine
61
0.0577
0.3112
75%
1.75


20798
Metabolite - 7748
61
0.0581
0.3112
−41% 
0.59


2832
adenosine 5′-monophosphate
61
0.0589
0.3112
−80% 
0.20


19294
Metabolite - 6134
61
0.0596
0.3112
31%
1.31


15278
Metabolite - 3843
61
0.0603
0.3112
−31% 
0.69


15255
Metabolite - 3833
61
0.0614
0.3127
74%
1.74


16468
Metabolite - 4236
61
0.0644
0.3224
−31% 
0.69


10309
Metabolite - 2277
61
0.0651
0.3224
35%
1.35


1302
methionine
61
0.0705
0.3425
21%
1.21


17885
Metabolite - 5147
61
0.0710
0.3425
76%
1.76


13038
Metabolite - 3143
61
0.0726
0.3440
53%
1.53


11411
Metabolite - 2746
61
0.0732
0.3440
79%
1.79


6373
Metabolite - A-1304
61
0.0749
0.3454
−25% 
0.75


22259
Isobar 59 includes N(′6)-
61
0.0756
0.3454
−29% 
0.71



trimethyl-L-lysine, H-



homoarg-OH


7081
Metabolite - 1609
61
0.0762
0.3454
67%
1.67


2856
uridine 5′-monophosphate
61
0.0807
0.3484
−54% 
0.46


16983
Metabolite - 4598
50
0.0813
0.3484
34%
1.34


20092
Metabolite - 7050
61
0.0818
0.3484
−22% 
0.78


14439
Metabolite - 3498
61
0.0833
0.3484
26%
1.26


12682
Metabolite - 3044
61
0.0841
0.3484
77%
1.77


18281
2-hydroxyhippuric acid
61
0.0843
0.3484
21%
1.21


12129
beta-hydroxyisovaleric acid
50
0.0864
0.3484
17%
1.17


14117
Metabolite - 3441
61
0.0867
0.3484
−20% 
0.80


20488
D-glucose
50
0.0874
0.3484
−25% 
0.75


9216
Metabolite - 2168
61
0.0884
0.3484
−21% 
0.79


19596
Metabolite - 6647
50
0.0885
0.3484
28%
1.28


16819
Metabolite - 4496
50
0.0909
0.3484
19%
1.19


22584
Metabolite - 9038
61
0.0910
0.3484
−55% 
0.45


21650
Metabolite - 8409
61
0.0910
0.3484
−38% 
0.62


22598
Metabolite - 9042
61
0.0920
0.3484
−65% 
0.35


54
tryptophan
61
0.0927
0.3484
19%
1.19


16655
Metabolite - 4362
50
0.0946
0.3484
−37% 
0.63


19402
Metabolite - 6346
50
0.0969
0.3484
−21% 
0.79


11661
indole-3-pyruvic acid
61
0.0972
0.3484
60%
1.60


1561
alpha-tocopherol
50
0.0974
0.3484
38%
1.38


8180
Metabolite - 1975
61
0.0982
0.3484
35%
1.35


16666
Metabolite - 4365
50
0.0983
0.3484
−28% 
0.72


2132
citrulline
50
0.0985
0.3484
51%
1.51


1508
pantothenic acid
61
0.1014
0.3489
37%
1.37


1572
glyceric acid
50
0.1017
0.3489
14%
1.14


21732
Metabolite - 8475
61
0.1020
0.3489
−38% 
0.62


9130
Metabolite - 2139
61
0.1024
0.3489
105% 
2.05


21654
Metabolite - 8413
61
0.1043
0.3522
−25% 
0.75


10461
Metabolite - 2313
61
0.1057
0.3534
−27% 
0.73


16511
Metabolite - 4274
50
0.1072
0.3534
69%
1.69


1638
arginine
50
0.1075
0.3534
128% 
2.28


13345
Metabolite - 3244
61
0.1087
0.3543
24%
1.24


1493
ornithine
50
0.1183
0.3823
105% 
2.05


5809
3-indoxyl sulfate
61
0.1251
0.3991
80%
1.80


1648
serine
50
0.1261
0.3991
73%
1.73


1826
folic acid
61
0.1268
0.3991
−66% 
0.34


512
asparagine
50
0.1357
0.4181
64%
1.64


514
cytidine
61
0.1358
0.4181
69%
1.69


16244
Isobar 21 includes gamma-
61
0.1365
0.4181
−31% 
0.69



aminobutyryl-L-histidine, L-



anserine


1598
N-tigloylglycine
61
0.1373
0.4181
16%
1.16


17091
Metabolite - 4641
61
0.1396
0.4217
40%
1.40


17665
p-hydroxybenzaldehyde
61
0.1424
0.4242
18%
1.18


1494
5-oxoproline
50
0.1430
0.4242
24%
1.24


8336
Metabolite - 2005
61
0.1439
0.4242
82%
1.82


13214
Metabolite - 3183 - possible
61
0.1516
0.4351
28%
1.28



gamma-L-glutamyl-L-



phenylalanine


1336
n-hexadecanoic acid
50
0.1518
0.4351
−14% 
0.86


21701
Metabolite - 8454
61
0.1537
0.4351
−34% 
0.66


10141
Metabolite - A-2035
61
0.1540
0.4351
−15% 
0.85


17028
Metabolite - 4611
50
0.1542
0.4351
19%
1.19


1432
alphahydroxybenzeneacetic
61
0.1546
0.4351
−27% 
0.73



acid


6771
Metabolite - 1460
61
0.1581
0.4415
−25% 
0.75


12774
Metabolite - 3094
50
0.1632
0.4513
−13% 
0.87


20084
Metabolite - 7047
61
0.1656
0.4513
17%
1.17


1651
pyridoxal
61
0.1692
0.4513
24%
1.24


12924
Metabolite - 3131
61
0.1702
0.4513
20%
1.20


8072
Metabolite - 1958
61
0.1703
0.4513
17%
1.17


1126
alanine
50
0.1714
0.4513
61%
1.61


22567
Metabolite - 9030
61
0.1721
0.4513
−51% 
0.49


6413
Metabolite - 1342 - possible
61
0.1724
0.4513
29%
1.29



phenylacetylglutamine or



formyl-N-acetyl-5-



methoxykynurenamine


9137
Metabolite - 2141
61
0.1725
0.4513
23%
1.23


18232
Metabolite - 5403
50
0.1746
0.4537
16%
1.16


19372
Metabolite - 6269
50
0.1776
0.4565
19%
1.19


542
3-hydroxybutanoic acid
50
0.1785
0.4565
50%
1.50


11323
Metabolite - 2711
61
0.1806
0.4565
72%
1.72


606
uridine
61
0.1806
0.4565
93%
1.93


21631
Metabolite - 8403
50
0.1955
0.4908
24%
1.24


15118
Metabolite - 3784
61
0.2010
0.4996
22%
1.22


22572
Metabolite - 9034
50
0.2035
0.4996
28%
1.28


15121
Metabolite - 3786
61
0.2050
0.4996
−53% 
0.47


20950
Metabolite - 7846
50
0.2054
0.4996
−19% 
0.81


1649
valine
50
0.2076
0.4996
55%
1.55


1284
threonine
50
0.2098
0.4996
61%
1.61


16992
Metabolite - 4603
61
0.2133
0.4996
38%
1.38


7650
Metabolite - 1834
61
0.2138
0.4996
−19% 
0.81


14753
Metabolite - 3663
61
0.2148
0.4996
19%
1.19


17627
Metabolite - 4986
50
0.2153
0.4996
41%
1.41


19919
Metabolite - 6832
61
0.2160
0.4996
−34% 
0.66


18969
Metabolite - 5920
61
0.2161
0.4996
46%
1.46


22320
Metabolite - 8889
50
0.2165
0.4996
38%
1.38


12907
cGMP
61
0.2214
0.5078
27%
1.27


14759
Metabolite - 3667
61
0.2265
0.5163
10%
1.10


6379
Metabolite - 1329
61
0.2343
0.5308
28%
1.28


15872
malonic acid
61
0.2395
0.5345
46%
1.46


10825
Metabolite - 2546
61
0.2406
0.5345
−7%
0.93


14988
Metabolite - 3756
61
0.2407
0.5345
−10% 
0.90


15990
L-alpha-
61
0.2436
0.5345
−35% 
0.65



glycerophosphorylcholine


12780
Metabolite - 3098
50
0.2443
0.5345
72%
1.72


9002
Metabolite - 2107
61
0.2446
0.5345
−33% 
0.67


16044
Metabolite - 4005
50
0.2553
0.5546
58%
1.58


60
leucine
50
0.2579
0.5570
47%
1.47


1827
riboflavine
61
0.2609
0.5573
44%
1.44


22032
Metabolite - 8766
50
0.2610
0.5573
−13% 
0.87


16070
Metabolite - 4019
50
0.2653
0.5613
−15% 
0.85


1301
lysine
50
0.2661
0.5613
72%
1.72


6253
Metabolite - 1283
61
0.2689
0.5613
58%
1.58


14043
Metabolite - 3428
61
0.2689
0.5613
−26% 
0.74


19513
Metabolite - 6552
61
0.2776
0.5731
−31% 
0.69


1708
7,8-dihydrofolic acid
61
0.2791
0.5731
−32% 
0.68


8649
Metabolite - 2053
61
0.2796
0.5731
−15% 
0.85


15753
hippuric acid
61
0.2839
0.5731
29%
1.29


16232
Isobar 17 includes arginine,
61
0.2839
0.5731
21%
1.21



N-alpha-acetyl-ornithine


1591
N-acetyl-L-valine
61
0.2857
0.5731
42%
1.42


19374
Metabolite - 6270
50
0.2867
0.5731
−20% 
0.80


15122
glycerol
50
0.2870
0.5731
26%
1.26


15412
Metabolite - 3910
61
0.2889
0.5739
−34% 
0.66


2342
serotonin
61
0.2920
0.5770
−54% 
0.46


13512
Metabolite - 3315
61
0.2938
0.5776
22%
1.22


22590
Metabolite - 9040
61
0.2957
0.5782
−55% 
0.45


12789
Metabolite - 3107
50
0.3012
0.5859
39%
1.39


16138
Metabolite - 4080
50
0.3045
0.5893
−18% 
0.82


15681
4-Guanidinobutanoic acid
61
0.3070
0.5908
36%
1.36


1125
isoleucine
50
0.3094
0.5908
43%
1.43


14502
Metabolite - 3539
61
0.3109
0.5908
46%
1.46


14406
Metabolite - 3493
61
0.3119
0.5908
−16% 
0.84


1898
proline
61
0.3133
0.5908
10%
1.10


2129
oxitryptan
61
0.3172
0.5952
−17% 
0.83


15125
(2-Aminoethyl)phosphonate
61
0.3212
0.5961
−23% 
0.77


16226
Isobar 28 includes L-
61
0.3213
0.5961
−15% 
0.85



threonine, L-allothreonine, L-



homoserine, (S)-(−)-4-amino



2-hydroxybutyric acid


6104
tryptamine
50
0.3225
0.5961
26%
1.26


12719
Metabolite - 3055 - possible
61
0.3251
0.5980
32%
1.32



NH3 adduct of hippuric acid


15113
Metabolite - 3783
61
0.3303
0.6045
−17% 
0.83


16821
Metabolite - 4498
50
0.3358
0.6098
11%
1.11


15743
N,N-dimethylarginine
61
0.3377
0.6098
72%
1.72


12782
Metabolite - 3100
50
0.3397
0.6098
57%
1.57


22570
Metabolite - 9033
50
0.3430
0.6098
−24% 
0.76


10147
Metabolite - A-2036
61
0.3459
0.6098
−12% 
0.88


21046
digalacturonic acid
61
0.3472
0.6098
−25% 
0.75


12773
Metabolite - 3093
50
0.3483
0.6098
24%
1.24


18330
5-methyltetrahydrofolic acid
61
0.3499
0.6098
−21% 
0.79


20035
Metabolite - 7008
61
0.3521
0.6098
−13% 
0.87


22154
bradykinin
61
0.3530
0.6098
−45% 
0.55


22133
DL-hexanoyl-carnitine
61
0.3547
0.6098
50%
1.50


5628
Metabolite - 1086
61
0.3567
0.6098
−33% 
0.67


10743
Isobar 4 includes Gluconic
61
0.3597
0.6098
13%
1.13



acid, DL-arabinose, D-ribose,



L-xylose, DL-lyxose, D-



xylulose, galactonic acid


16985
Metabolite - 4600
61
0.3606
0.6098
32%
1.32


1476
glucarate
50
0.3609
0.6098
18%
1.18


17860
Metabolite - 5127
61
0.3632
0.6098
28%
1.28


5466
Metabolite - A-1030
61
0.3635
0.6098
23%
1.23


17845
Metabolite - 5108
61
0.3684
0.6098
17%
1.17


20308
Metabolite - 7270
61
0.3690
0.6098
19%
1.19


18761
Metabolite - 5793
61
0.3694
0.6098
57%
1.57


10476
Metabolite - 2316
61
0.3710
0.6098
−15% 
0.85


15382
Metabolite - 3898
61
0.3722
0.6098
17%
1.17


18467
cis-5,8,11,14,17-
61
0.3728
0.6098
32%
1.32



eicosapentaenoic acid


15529
Metabolite - 3951
61
0.3733
0.6098
14%
1.14


15336
tartaric acid
61
0.3743
0.6098
16%
1.16


21012
Metabolite - 7889
50
0.3786
0.6098
16%
1.16


12533
Metabolite - 2915
50
0.3809
0.6098
−14% 
0.86


10700
Metabolite - 2393
61
0.3831
0.6098
26%
1.26


12753
Metabolite - 3074
50
0.3842
0.6098
15%
1.15


10357
Metabolite - A-2055
61
0.3856
0.6098
−24% 
0.76


21763
Metabolite - 8507
61
0.3862
0.6098
−10% 
0.90


15074
Metabolite - 3774
61
0.3893
0.6098
18%
1.18


10286
Metabolite - 2272
61
0.3909
0.6098
47%
1.47


14480
Metabolite - 3521
61
0.3909
0.6098
−42% 
0.58


13872
Metabolite - 3393
61
0.3933
0.6098
52%
1.52


8300
Metabolite - 1988
61
0.3934
0.6098
−13% 
0.87


22586
Metabolite - 9039
61
0.3939
0.6098
−48% 
0.52


13920
Metabolite - 3404
61
0.3967
0.6107
16%
1.16


12645
Metabolite - 3017
50
0.3978
0.6107
24%
1.24


18369
gamma-glu-leu
61
0.3997
0.6111
11%
1.11


19368
Metabolite - 6267
50
0.4016
0.6115
29%
1.29


19613
Metabolite - 6670
61
0.4043
0.6131
−20% 
0.80


12756
Metabolite - 3077
50
0.4130
0.6237
25%
1.25


7644
Metabolite - 1831
61
0.4185
0.6289
18%
1.18


12639
Metabolite - 3012
50
0.4198
0.6289
46%
1.46


6130
Metabolite - 1208
61
0.4239
0.6324
−41% 
0.59


11053
Metabolite - 2567
61
0.4256
0.6324
10%
1.10


17390
Metabolite - 4806
50
0.4286
0.6335
−7%
0.93


14247
Metabolite - 3475
61
0.4319
0.6335
14%
1.14


20234
Metabolite - 7170
61
0.4326
0.6335
25%
1.25


17327
Metabolite - 4767
50
0.4364
0.6335
13%
1.13


17359
Metabolite - 4791
50
0.4383
0.6335
−21% 
0.79


22163
EDTA
50
0.4387
0.6335
47%
1.47


8176
Metabolite - 1974
61
0.4407
0.6335
−16% 
0.84


1647
glutamine
50
0.4446
0.6335
44%
1.44


17614
Metabolite - 4966
50
0.4467
0.6335
−29% 
0.71


5430
Metabolite - A-1008
61
0.4497
0.6335
−5%
0.95


19462
Metabolite - 6446
50
0.4509
0.6335
−9%
0.91


6398
Metabolite - 1335
61
0.4510
0.6335
−19% 
0.81


15639
Metabolite - 3984
61
0.4520
0.6335
54%
1.54


12777
Metabolite - 3097
50
0.4558
0.6335
25%
1.25


15805
maltose
50
0.4590
0.6335
25%
1.25


1366
trans-4-hydroxyproline
50
0.4603
0.6335
31%
1.31


4966
xylitol
50
0.4612
0.6335
10%
1.10


1419
5′-s-methyl-5′-thioadenosine
61
0.4617
0.6335
18%
1.18


11777
glycine
50
0.4623
0.6335
36%
1.36


22020
Metabolite - 8749
50
0.4627
0.6335
 9%
1.09


21047
3-methyl-2-oxobutyric
61
0.4640
0.6335
−12% 
0.88


16290
Metabolite - 4133
50
0.4655
0.6335
−7%
0.93


12803
Metabolite - A-2441
50
0.4671
0.6335
−12% 
0.88


8527
Metabolite - A-1931
61
0.4700
0.6335
−3%
0.97


19013
Metabolite - 5931
61
0.4701
0.6335
 7%
1.07


22568
Metabolite - 9031
61
0.4758
0.6335
−43% 
0.57


12783
Metabolite - 3101
50
0.4760
0.6335
27%
1.27


5533
Metabolite - A-1096
61
0.4762
0.6335
29%
1.29


16805
Metabolite - 4488
61
0.4772
0.6335
21%
1.21


22132
DL-alpha-hydroxyisocaproic
61
0.4774
0.6335
15%
1.15



acid


16071
Metabolite - 4020
50
0.4818
0.6360
 7%
1.07


22439
Metabolite - 8949
61
0.4839
0.6360
17%
1.17


5765
Metabolite - 1142 - possible
61
0.4877
0.6360
−13% 
0.87



5-hydroxypentanoate or beta-



hydroxyisovaleric acid


1481
inositol 1-phosphate
50
0.4879
0.6360
−13% 
0.87


13217
Metabolite - 3184
61
0.4913
0.6360
15%
1.15


5689
Metabolite - 1111 - possible
61
0.4925
0.6360
−19% 
0.81



methylnitronitrosoguanidine



or ethyl thiocarbamoylacetate


22166
glu-glu
61
0.4985
0.6360
29%
1.29


21127
monopalmitin
50
0.4987
0.6360
−10% 
0.90


22601
Metabolite - 9044
50
0.4990
0.6360
25%
1.25


18384
Metabolite - 5487
50
0.5030
0.6360
−12% 
0.88


18943
Metabolite - 5912
61
0.5031
0.6360
−13% 
0.87


22145
acetyl-L-carnitine
61
0.5078
0.6360
20%
1.20


17486
Metabolite - 4886
61
0.5102
0.6360
−14% 
0.86


14639
Metabolite - 3603
61
0.5123
0.6360
16%
1.16


12785
Metabolite - 3103
50
0.5144
0.6360
38%
1.38


9016
Metabolite - 2109
61
0.5147
0.6360
−6%
0.94


10136
Metabolite - A-2034
61
0.5160
0.6360
 4%
1.04


1417
kynurenic acid
61
0.5187
0.6360
12%
1.12


16893
Metabolite - 4530
61
0.5188
0.6360
107% 
2.07


1564
citric acid
50
0.5189
0.6360
12%
1.12


22001
3-hydroxyoctanoic acid
61
0.5214
0.6360
19%
1.19


18349
DL-indole-3-lactic acid
61
0.5214
0.6360
13%
1.13


16080
Metabolite - 4026
61
0.5228
0.6360
−26% 
0.74


15197
1-methylnicotinamide-1
61
0.5243
0.6360
24%
1.24


18172
Metabolite - 5391
61
0.5247
0.6360
−13% 
0.87


2240
homogentisate
61
0.5248
0.6360
−6%
0.94


15063
Metabolite - 3772
61
0.5263
0.6360
17%
1.17


5618
Metabolite - 1085 - possible
61
0.5287
0.6360
−8%
0.92



isolobinine or 4-aminoestra-



1,3,5(10)-triene-3,17beta-diol


59
histidine
61
0.5290
0.6360
−8%
0.92


5478
Metabolite - A-1036
61
0.5339
0.6394
−7%
0.93


21013
Metabolite - 7890
50
0.5363
0.6394
−15% 
0.85


22262
Isobar 61 includes 4-methyl-
61
0.5381
0.6394
−10% 
0.90



2-oxovaleric acid, 3-methyl-



2-oxovaleric acid


10850
Metabolite - 2548 - possible
61
0.5398
0.6394
21%
1.21



Cl adduct of uric acid


22360
Metabolite - 8901
61
0.5414
0.6394
16%
1.16


5475
Metabolite - A-1033
61
0.5423
0.6394
12%
1.12


8509
Metabolite - 2041
61
0.5445
0.6394
 7%
1.07


20694
oxalic acid
61
0.5456
0.6394
−7%
0.93


18010
Metabolite - 5231
61
0.5487
0.6411
33%
1.33


2183
thymidine
61
0.5516
0.6423
10%
1.10


2150
pyridoxamine
61
0.5544
0.6432
−4%
0.96


19708
Metabolite - 6711
61
0.5558
0.6432
−9%
0.91


22130
DL-3-phenyllactic acid
61
0.5598
0.6457
−16% 
0.84


16711
Metabolite - 4431
61
0.5614
0.6457
−15% 
0.85


20391
Metabolite - 7334
61
0.5785
0.6615
−13% 
0.87


16189
Metabolite - 4097
61
0.5787
0.6615
 9%
1.09


7177
Metabolite - 1656
61
0.5817
0.6623
36%
1.36


10655
Metabolite - 2388
61
0.5830
0.6623
11%
1.11


16506
Metabolite - 4271
50
0.5872
0.6651
26%
1.26


18146
Metabolite - 5366
50
0.5890
0.6651
−14% 
0.86


19494
Metabolite - 6506
50
0.6032
0.6752
−11% 
0.89


18109
isocitrate
61
0.6041
0.6752
20%
1.20


1110
arachidonic acid
50
0.6062
0.6752
 8%
1.08


17283
Metabolite - 4750
61
0.6064
0.6752
−15% 
0.85


13211
Metabolite - 3182
61
0.6084
0.6752
−13% 
0.87


12726
Metabolite - 3058
50
0.6104
0.6752
25%
1.25


15996
aspartate
50
0.6134
0.6752
27%
1.27


16705
Metabolite - 4428
61
0.6135
0.6752
13%
1.13


16865
Metabolite - 4522
50
0.6143
0.6752
 6%
1.06


12648
Metabolite - 3020
50
0.6166
0.6755
−6%
0.94


6266
Metabolite - 1286
61
0.6183
0.6755
 6%
1.06


1105
Linoleic acid
50
0.6208
0.6755
−6%
0.94


15730
suberic acid
61
0.6219
0.6755
19%
1.19


12650
Metabolite - 3022
50
0.6242
0.6761
24%
1.24


20031
Metabolite - 7007
61
0.6307
0.6811
−10% 
0.90


16860
Metabolite - 4517
50
0.6346
0.6815
−9%
0.91


17064
Metabolite - 4624
50
0.6349
0.6815
19%
1.19


19934
inositol
50
0.6365
0.6815
11%
1.11


12663
Metabolite - 3030
50
0.6414
0.6836
29%
1.29


20866
Metabolite - 7786
61
0.6422
0.6836
 4%
1.04


22602
Metabolite - 9045
50
0.6447
0.6843
−13% 
0.87


12593
Metabolite - 2973
50
0.6526
0.6907
 3%
1.03


15500
carnitine
61
0.6619
0.6957
−5%
0.95


7595
Metabolite - 1817
61
0.6633
0.6957
 5%
1.05


19364
Metabolite - 6246
50
0.6655
0.6957
13%
1.13


20248
Metabolite - 7177
61
0.6667
0.6957
10%
1.10


14054
Metabolite - 3430 - possible
61
0.6670
0.6957
 4%
1.04



gly-leu, acetyl-lys, ala-val


11438
phosphate
50
0.6686
0.6957
 5%
1.05


19511
Metabolite - 6551
61
0.6777
0.7032
 6%
1.06


20192
Metabolite - 7146
61
0.6833
0.7061
−9%
0.91


16949
Metabolite - 4592
61
0.6842
0.7061
−7%
0.93


2137
biliverdin
61
0.6893
0.7093
−18% 
0.82


19961
Metabolite - 6913
50
0.6913
0.7093
 2%
1.02


7107
Metabolite - A-1664
61
0.6945
0.7093
−22% 
0.78


18756
Metabolite - 5791
61
0.6950
0.7093
−8%
0.92


12768
Metabolite - 3088
50
0.7007
0.7125
14%
1.14


19362
Metabolite - 6226
50
0.7030
0.7125
−10% 
0.90


12791
Metabolite - 3109
50
0.7038
0.7125
18%
1.18


528
alpha-keto-glutarate
61
0.7155
0.7217
17%
1.17


10746
Isobar 6 includes valine,
61
0.7178
0.7217
10%
1.10



betaine


10304
Metabolite - 2276
61
0.7191
0.7217
24%
1.24


19377
Metabolite - 6272
50
0.7207
0.7217
 9%
1.09


18929
Metabolite - 5907
50
0.7249
0.7239
−9%
0.91


9060
Metabolite - A-1994
61
0.7284
0.7248
16%
1.16


22577
Metabolite - 9035
50
0.7323
0.7248
17%
1.17


1121
heptadecanoic acid
50
0.7328
0.7248
 4%
1.04


14840
Metabolite - 3708
61
0.7359
0.7248
 5%
1.05


16712
Metabolite - 4432
61
0.7373
0.7248
−3%
0.97


16285
Metabolite - A-2798
50
0.7393
0.7248
−11% 
0.89


1584
methyl indole-3-acetate
61
0.7401
0.7248
−13% 
0.87


18868
Metabolite - 5847
50
0.7421
0.7248
13%
1.13


16843
Metabolite - 4510
50
0.7452
0.7248
15%
1.15


19800
Metabolite - 6750
61
0.7544
0.7248
24%
1.24


12457
Metabolite - 2892
61
0.7565
0.7248
−9%
0.91


15677
3-methyl-L-histidine
61
0.7568
0.7248
−4%
0.96


57
glutamic acid
50
0.7577
0.7248
18%
1.18


21762
Metabolite - 8506
61
0.7591
0.7248
 7%
1.07


12770
Metabolite - 3090
50
0.7605
0.7248
 7%
1.07


19367
Metabolite - 6266
50
0.7620
0.7248
−6%
0.94


11770
Metabolite - 2806
61
0.7646
0.7248
 5%
1.05


10148
Metabolite - 2257
61
0.7649
0.7248
13%
1.13


10570
Metabolite - 2366
61
0.7663
0.7248
−6%
0.94


20267
Metabolite - 7187
61
0.7675
0.7248
14%
1.14


577
fructose
50
0.7684
0.7248
−7%
0.93


16308
Metabolite - 4147
50
0.7687
0.7248
 7%
1.07


6362
Metabolite - 1323 - possible
61
0.7707
0.7249
22%
1.22



p-cresol sulfate


12112
Metabolite - A-2314
61
0.7740
0.7261
 6%
1.06


12647
Metabolite - 3019
50
0.7767
0.7268
18%
1.18


13104
Metabolite - 3160
61
0.7839
0.7295
−5%
0.95


17060
Metabolite - 4622
61
0.7853
0.7295
 7%
1.07


1365
tetradecanoic acid
50
0.7859
0.7295
−5%
0.95


1512
picolinic acid
50
0.7875
0.7295
−5%
0.95


12673
Metabolite - 3040
50
0.7915
0.7315
 7%
1.07


12876
Metabolite - 3125
61
0.7960
0.7338
−8%
0.92


18892
Metabolite - 5866
61
0.8003
0.7360
−5%
0.95


12035
nonanate
50
0.8032
0.7368
 5%
1.05


18678
Metabolite - 5730
61
0.8065
0.7371
−7%
0.93


19955
Metabolite - 6907
50
0.8075
0.7371
 7%
1.07


19323
Docosahexaenoic Acid
50
0.8175
0.7428
 3%
1.03


12656
Metabolite - 3025
50
0.8225
0.7428
13%
1.13


12751
Metabolite - 3073
50
0.8227
0.7428
−10% 
0.90


18706
Metabolite - 5769
61
0.8236
0.7428
−6%
0.94


15064
Metabolite - 3773
61
0.8252
0.7428
10%
1.10


12666
Metabolite - 3033 - possible
50
0.8278
0.7428
10%
1.10



threonine deriv-


16829
Metabolite - 4503
50
0.8286
0.7428
−3%
0.97


12771
Metabolite - 3091
50
0.8298
0.7428
 7%
1.07


21188
1-stearoyl-rac-glycerol
50
0.8452
0.7498
 3%
1.03


16509
Metabolite - 4273
50
0.8458
0.7498
−8%
0.92


10544
Metabolite - 2329
61
0.8460
0.7498
−9%
0.91


15625
Metabolite - 3976
61
0.8476
0.7498
 3%
1.03


6461
Metabolite - A-1329
61
0.8499
0.7498
 7%
1.07


17207
Metabolite - 4707
61
0.8507
0.7498
 4%
1.04


12008
Metabolite - 2847
61
0.8532
0.7498
 6%
1.06


17389
Metabolite - 4796
50
0.8537
0.7498
−14% 
0.86


597
phosphoenolpyruvate
61
0.8558
0.7499
−4%
0.96


10111
Metabolite - A-2033
61
0.8587
0.7506
−5%
0.95


8796
Metabolite - 2074
61
0.8687
0.7575
 5%
1.05


7029
Metabolite - 1597
61
0.8755
0.7601
 2%
1.02


15253
Metabolite - 3832 - possible
61
0.8757
0.7601
 6%
1.06



phenol sulfate


20194
Metabolite - 7147
61
0.8787
0.7607
−2%
0.98


15535
Metabolite - 3955
61
0.8805
0.7607
 6%
1.06


15129
D-alanyl-D-alanine
50
0.8826
0.7608
 3%
1.03


12658
Metabolite - 3026
50
0.8933
0.7650
 8%
1.08


10604
Metabolite - 2370
61
0.8961
0.7650
−4%
0.96


13179
creatine
61
0.8973
0.7650
−4%
0.96


10781
Metabolite - 2469
61
0.8992
0.7650
 5%
1.05


1358
octadecanoic acid
50
0.9002
0.7650
 1%
1.01


15365
sn-Glycerol 3-phosphate
50
0.9018
0.7650
 3%
1.03


9172
Metabolite - A-2000
61
0.9060
0.7650
−1%
0.99


15227
trans-aconitic acid-1
61
0.9063
0.7650
 3%
1.03


5702
choline
61
0.9074
0.7650
−4%
0.96


12625
Metabolite - 3002
50
0.9081
0.7650
 2%
1.02


19860
Metabolite - 6784
61
0.9194
0.7716
 1%
1.01


12781
Metabolite - 3099
50
0.9201
0.7716
 4%
1.04


13273
Metabolite - 3224
61
0.9344
0.7759
 3%
1.03


21025
iminodiacetic acid
50
0.9345
0.7759
 2%
1.02


12912
Metabolite - 3129
61
0.9367
0.7759
−1%
0.99


6131
Metabolite - 1209
61
0.9387
0.7759
−3%
0.97


6380
Metabolite - 1330
61
0.9399
0.7759
−2%
0.98


2125
taurine
61
0.9400
0.7759
−3%
0.97


18118
Metabolite - 5346
50
0.9423
0.7759
−3%
0.97


19291
Metabolite - 6132
61
0.9430
0.7759
−2%
0.98


12667
Metabolite - 3034
50
0.9440
0.7759
 1%
1.01


20927
Metabolite - 7815
61
0.9519
0.7796
−1%
0.99


16665
Metabolite - 4364
50
0.9529
0.7796
−2%
0.98


20228
Metabolite - 7169
61
0.9548
0.7796
−2%
0.98


15600
Metabolite - 3964
61
0.9639
0.7799
−1%
0.99


9905
Metabolite - 2231
61
0.9664
0.7799
 0%
1.00


16496
Metabolite - 4251
50
0.9670
0.7799
 1%
1.01


513
creatinine
61
0.9687
0.7799
−1%
0.99


22581
Metabolite - 9037
61
0.9712
0.7799
 2%
1.02


18882
taurodeoxycholic acid
61
0.9715
0.7799
 2%
1.02


12754
Metabolite - 3075
50
0.9801
0.7799
 1%
1.01


13018
Metabolite - 3138
61
0.9829
0.7799
 1%
1.01


8196
Metabolite - 1979 - Cl adduct
61
0.9833
0.7799
 0%
1.00



of isobar 19


19397
Metabolite - 6326
50
0.9846
0.7799
−1%
0.99


13288
Metabolite - 3228
61
0.9860
0.7799
 0%
1.00


19490
Metabolite - 6488
50
0.9863
0.7799
 1%
1.01


12757
Metabolite - 3078
50
0.9867
0.7799
−1%
0.99


12790
Metabolite - 3108
50
0.9880
0.7799
 1%
1.01


16512
Metabolite - 4275
50
0.9908
0.7799
 0%
1.00


17330
Metabolite - 4769
50
0.9910
0.7799
 0%
1.00


19363
Metabolite - 6227
50
0.9929
0.7799
 0%
1.00


63
cholesterol
50
0.9929
0.7799
 0%
1.00


10782
Metabolite - 2486
61
0.9967
0.7812
 0%
1.00


22548
Metabolite - 9026
50
0.9988
0.7812
 0%
1.00









Recursive partitioning statistical analysis of all the metabolites in cardiac tissue between normal and DCM subjects identified Metabolite-3808 (Metabolite-3808) as a compound that separated both groups of subjects perfectly (LogWorth=6.61). Specifically, all subjects with DCM phenotype had levels of Metabolite-3808 above the cutoff value of 629275 while all the subjects with a normal phenotype had Metabolite-3808 levels below the cutoff value (FIG. 20). The cutoff value in the graph in FIG. 20 is indicated by a broken line.
To evaluate the biomarkers discovered in the mouse model of DCM, analysis of human subjects was performed. Biomarkers were discovered by (1) analyzing plasma samples from different groups of human subjects to determine the levels of metabolites in the samples and then (2) statistically analyzing the results to determine those metabolites that were differentially present in the two groups.
Two groups of subjects were used. One group consisted of 39 subjects (18 male, 21 female) with dilated cardiomyopathy (DCM). The second group consisted of 31 healthy control subjects (14 male, 17 male). Subjects were balanced for age and gender; mean age of control females was 50.1+/−10.1 and DCM females was 50.0+/−11.3 while the mean age of control males was 42.7+/−11.3 and DCM males was 45.8+/−10.9.
T-tests (Table 23) were used to determine differences in the mean levels of metabolites between the two populations (i.e., Dilated Cardiomyopathy, DCM vs. Healthy control). Classification analysis was carried out using random forest analyses to uncover the biomarkers that can best differentiate the two groups. The results of the Random forest analysis are shown in Table 24 and the most important biomarkers useful to classify subjects as healthy or DCM are listed in Table 25.
Biomarkers:
As listed below in Table 23, biomarkers were discovered that were differentially present between plasma samples collected from dilated cardiomyopathy subjects and healthy subjects.
Table 23 includes, for each listed biomarker, the p-value and q-value determined in the statistical analysis of the data concerning the biomarkers and an indication of the percentage difference in the dilated cardiomyopathy mean level as compared to the healthy mean level in plasma. “ID” refers to the compound identification number used as a primary key for that compound in the in-house chemical database. “Library” indicates the chemical library that was used to identify the compounds. The number 50 refers to the GC library and the numbers 200 and 201 refer to the LC library. “Mouse” indicates the compounds that were also biomarkers discovered in the mouse model of DCM.







TABLE 23







Biomarkers for DCM


















% Change



ID
Library
Compound
p-value
q-value
in DCM
Mouse
















32873
201
Metabolite - 11556
5.943E−07
0.0001
−10% 



1558
200
4-acetamidobutanoate
6.339E−06
0.0005
 7%


32709
200
Metabolite - 03056
1.925E−05
0.001
73%


33510
200
Metabolite - 12095
2.318E−05
0.001
−9%


33442
200
pseudouridine
3.734E−05
0.0012
 1%


32425
201
dehydroisoandrosterone sulfate
0.0001
0.0012
 4%




(DHEA-S)


32519
200
Metabolite - 11205
0.0001
0.0012
14%


32637
201
Metabolite - 11320
0.0001
0.0012
 6%


16866
50
Metabolite - 04523
0.0001
0.0015
 9%


1114
201
deoxycholate
0.0001
0.0017
11%


1284
200
threonine
0.0001
0.0017
 1%
Yes


32675
200
Metabolite - 03951
0.0001
0.0017
12%


15506
200
choline
0.0002
0.002
29%
Yes


32652
200
Metabolite - 11335
0.0002
0.0029
−31% 


32197
201
3-(4-hydroxyphenyl)lactate
0.0003
0.0035
−4%
Yes


33515
200
Metabolite - 12100
0.0003
0.0036
31%


18929
50
Metabolite - 05907
0.0004
0.004
−3%


32405
200
3-indolepropionate
0.0004
0.004
−13% 


19934
50
myo-inositol
0.0006
0.0051
17%
Yes


33453
50
alpha-ketoglutarate
0.0006
0.0055
 2%


1712
200
cortisol
0.0007
0.006
−53% 


32807
201
Metabolite - 11490
0.0012
0.0094
−12% 


25607
50
Metabolite - 10437
0.0013
0.0097
 4%


25459
50
Metabolite - 10395
0.0015
0.0104
−3%


33477
50
erythronate*
0.0016
0.0106
−25% 


32635
201
Metabolite - 11318
0.0018
0.0117
17%


57
50
glutamate
0.002
0.0126
−5%
Yes


18254
200
paraxanthine
0.0021
0.0126
−3%


33973
201
epiandrosterone sulfate
0.0021
0.0126
51%


32560
201
Metabolite - 07765
0.0025
0.0142
−4%


20699
50
erythritol
0.0026
0.0144
−19% 
Yes


32590
201
Metabolite - 11273
0.003
0.0154
−18% 


32619
201
Metabolite - 11302
0.003
0.0154
−11% 


22602
50
Metabolite - 09045
0.0033
0.0161
−6%


32829
200
Metabolite - 03653
0.0033
0.0161
−4%


32910
201
Metabolite - 11593
0.0035
0.0168
−8%


32599
201
Metabolite - 11282
0.0037
0.017
 3%


16653
50
Metabolite - 04361
0.004
0.0176
 2%


18232
50
Metabolite - 05403
0.004
0.0176
30%


15677
201
3-methylhistidine
0.0048
0.0206
120% 
Yes


12770
50
Metabolite - 03090
0.0055
0.0225
13%


31591
201
androsterone sulfate
0.0055
0.0225
−78% 


584
50
mannose
0.0063
0.0248
16%
Yes


15140
200
kynurenine
0.0067
0.0258
22%
Yes


33206
201
Metabolite - 11861
0.0068
0.0258
−17% 


33144
200
Metabolite - 11799
0.007
0.026
−13% 


33507
200
Metabolite - 12092
0.0076
0.0278
−3%


32740
201
Metabolite - 11423
0.0078
0.0278
−27% 


33652
201
Metabolite - 12230
0.008
0.0279
−3%


53
200
glutamine
0.0082
0.028
 2%
Yes


32808
201
Metabolite - 11491
0.0087
0.0289
39%


33516
200
Metabolite - 12101
0.0089
0.0289
80%


3147
50
xanthine
0.009
0.0289
20%


12753
50
Metabolite - 03074
0.0099
0.0309
−5%


1303
50
malate
0.0101
0.0309
 3%
Yes


32762
201
Metabolite - 11445
0.0101
0.0309
 4%


2734
200
gamma-glutamyltyrosine
0.0115
0.0339
−4%
Yes


32718
200
Metabolite - 01342
0.0116
0.0339
26%


32644
200
Metabolite - 11327
0.0117
0.0339
−25% 


32654
200
Metabolite - 11337
0.012
0.0341
 9%


33937
201
alpha-hydroxyisovalerate
0.0127
0.0355
38%


12017
200
3-methoxytyrosine
0.0134
0.0369
 7%


32753
201
Metabolite - 09789
0.0154
0.0418
−9%


32587
201
Metabolite - 02249
0.016
0.0427
44%


59
201
histidine
0.0179
0.047
−1%
Yes


22548
50
Metabolite - 09026
0.0188
0.0486
28%


16959
50
Metabolite - 04595
0.0191
0.0488
21%


33422
200
gammaglutamylphenylalanine
0.0197
0.0494
30%


64
200
phenylalanine
0.0211
0.0522
−4%
Yes


32110
50
Metabolite - 11086
0.0227
0.055
 8%


33132
200
Metabolite - 11787
0.0229
0.055
132% 


32859
200
Metabolite - 11542
0.0236
0.0557
−19% 


32672
200
Metabolite - 02546
0.024
0.0557
−5%


21044
50
2-hydroxybutyrate (AHB)
0.0244
0.0557
 3%
Yes


1356
201
nonadecanoate (19:0)
0.025
0.0557
−1%


32198
200
acetylcarnitine
0.025
0.0557
−44% 
Yes


32830
200
Metabolite - 11513
0.0251
0.0557
 1%


599
50
pyruvate
0.0261
0.0571
−7%


1358
201
stearate (18:0)
0.0269
0.0576
−13% 
Yes


32701
200
urate*
0.027
0.0576
−2%
Yes


32393
200
glutamylvaline
0.0276
0.0576
11%


24077
50
Metabolite - 09727
0.0277
0.0576
−12% 


24076
50
Metabolite - 09726
0.0285
0.0586
−31% 


1299
200
tyrosine
0.0288
0.0587
−3%
Yes


27718
200
creatine
0.0294
0.059
−29% 
Yes


12757
50
Metabolite - 03078
0.0299
0.0594
19%


1769
200
cortisone
0.0308
0.0604
33%


32836
200
HWESASXX*
0.0315
0.0612
11%


33028
200
Metabolite - 01497
0.0325
0.0624
−8%


33961
200
1-stearoylglycerophosphocholine
0.0329
0.0624
 4%


33157
200
Metabolite - 11812
0.0334
0.0624
27%


32606
201
bilirubin*
0.0336
0.0624
 2%


33939
201
N-acetylthreonine
0.0358
0.0659
 7%


25609
50
Metabolite - 10439
0.0363
0.066
75%


19368
50
Metabolite - 06267
0.0367
0.066
−7%


12789
50
Metabolite - 03107
0.0384
0.0683
17%
Yes


33441
200
isobutyrylcarnitine
0.0391
0.0684
−13% 


34035
201
linolenate [alpha or gamma; (18:3(n-3
0.0394
0.0684
−2%




or 6))]


33363
200
gamma-glutamylmethionine*
0.0396
0.0684
−17% 


30257
50
Metabolite - 10729
0.0417
0.0712
 3%


33821
200
Metabolite - 12393
0.0431
0.0724
 0%


33389
201
Metabolite - 12038
0.0432
0.0724
 3%


21047
201
3-methyl-2-oxobutyrate
0.0464
0.077
15%
Yes


18349
200
indolelactate
0.0472
0.0775
−55% 
Yes


12110
200
isocitrate
0.0478
0.0775
11%


33405
200
Metabolite - 12053
0.0481
0.0775
 0%


32497
201
10c-undecenoate
0.0496
0.0792
17%


33738
201
Metabolite - 12316
0.0502
0.0794
−14% 


19402
50
Metabolite - 06346
0.0518
0.0812
53%


33964
200
[H]HWESASLLR[OH]
0.0523
0.0813
26%


12795
50
Metabolite - 03113
0.0535
0.0824
 6%


32754
201
Metabolite - 11437
0.0542
0.0827
−13% 


18497
201
taurocholate
0.055
0.0831
−9%


1508
200
pantothenate
0.0565
0.0847
25%
Yes


32625
201
Metabolite - 11308
0.0609
0.0904
−50% 


32729
200
Metabolite - 11412
0.0621
0.0915
−26% 


31555
201
pyridoxate
0.0648
0.0946
18%


33960
200
1-oleoylglycerophosphocholine
0.0664
0.0961
 9%


1642
201
caprate (10:0)
0.068
0.0976
−5%


31454
50
cystine
0.0697
0.0985
−11% 


18477
200
glycodeoxycholate
0.07
0.0985
−44% 


32850
201
Metabolite - 11533
0.0711
0.0985
155% 


22895
50
Metabolite - 09299
0.0717
0.0985
−15% 


31618
50
Metabolite - 10964
0.0718
0.0985
−25% 


34007
50
Metabolite - 12502
0.072
0.0985
−12% 


1638
200
arginine
0.0748
0.1014
−14% 
Yes


33852
200
Metabolite - 12424
0.0767
0.1031
−20% 


33420
50
gamma-tocopherol*
0.0798
0.1058
−17% 


32398
200
sebacate
0.0799
0.1058
−9%


33403
200
Metabolite - 12051
0.082
0.1077
−10% 


33957
200
1-heptadecanoylglycerophosphocholine
0.0838
0.1085
−1%


32518
200
Metabolite - 11204
0.0849
0.1091
27%


1361
201
pentadecanoate (15:0)
0.0887
0.1131
69%


1645
201
laurate (12:0)
0.0906
0.1141
10%


32620
201
Metabolite - 11303
0.0908
0.1141
 3%


15990
200
glycerophosphorylcholine (GPC)
0.0928
0.1158
−9%
Yes


27531
201
hyodeoxycholate
0.0956
0.1184
 2%


1105
201
linoleate (18:2(n-6))
0.0969
0.1184
66%
Yes


33140
200
Metabolite - 11795
0.097
0.1184
−1%


16308
50
Metabolite - 04147
0.099
0.1193
38%


33927
200
Metabolite - 12481
0.0994
0.1193
18%


19363
50
Metabolite - 06227
0.0998
0.1193
16%


31509
50
Metabolite - 10931
0.1008
0.1196
31%


32561
201
Metabolite - 11244
0.1052
0.1239
−11% 


32846
201
Metabolite - 11529
0.1091
0.1257
253% 


21630
50
Metabolite - 08402
0.1098
0.1257
 2%


32550
201
Metabolite - 02272
0.1102
0.1257
21%


1107
50
allantoin
0.1104
0.1257
−13% 
Yes


32867
201
Metabolite - 11550
0.1104
0.1257
 5%


32549
201
Metabolite - 02269
0.1157
0.1309
 8%


32786
200
Metabolite - 11469
0.1181
0.1321
 8%


32501
201
dihomo-alpha-linolenate (20:3(n-3))
0.1188
0.1321
24%


21128
50
octadecanol
0.1191
0.1321
11%


16819
50
Metabolite - 04496
0.12
0.1323
−7%


33209
201
Metabolite - 11864
0.121
0.1324
−21% 


32778
200
Metabolite - 11461
0.1216
0.1324
−6%


32839
201
Metabolite - 11522
0.1284
0.1389
20%


32868
201
glycocholate*
0.1304
0.1401
−28% 


33969
201
stearidonate (18:4(n-3))
0.1388
0.146
89%


12783
50
Metabolite - 03101
0.1405
0.146
21%


31453
50
cysteine
0.141
0.146
16%


33103
50
Metabolite - 11758
0.1417
0.146
26%


32758
201
Metabolite - 11441
0.1421
0.146
24%


33935
200
piperine
0.1422
0.146
−26% 


33472
200
Metabolite - 12085
0.1426
0.146
 3%


32978
200
Metabolite - 11656
0.1427
0.146
−26% 


32504
201
n-3 DPA (22:5(n-3))
0.1448
0.1463
−3%


32877
201
Metabolite - 11560
0.1463
0.1463
47%


27273
50
Metabolite - 10506
0.1465
0.1463
36%


19370
50
Metabolite - 06268
0.149
0.1472
 2%


1572
50
glycerate
0.1506
0.1472
 1%
Yes


32346
201
glycochenodeoxycholate
0.1506
0.1472
13%


32769
201
Metabolite - 11452
0.1513
0.1472
−17% 


11777
50
glycine
0.1564
0.1508
−3%
Yes


32759
201
Metabolite - 11442
0.1572
0.1508
79%


513
200
creatinine
0.161
0.1537
−2%
Yes


32452
200
propionylcarnitine
0.163
0.1547
 9%


20675
201
1,5-anhydroglucitol (1,5-AG)
0.1674
0.1573
 5%


22600
50
Metabolite - 09043
0.1677
0.1573
56%


33380
201
Metabolite - 12029
0.1692
0.1573
−5%


25532
50
Metabolite - 10413
0.1695
0.1573
−7%


15335
50
mannitol
0.173
0.1598
 2%


32952
201
Metabolite - 02277
0.1747
0.1605
−13% 


27275
50
Metabolite - 10507
0.1777
0.1617
−8%


25522
50
Metabolite - 10407
0.178
0.1617
37%


18335
50
quinate
0.1831
0.1648
17%


1670
50
urea
0.1833
0.1648
−2%
Yes


31266
50
fructose
0.1853
0.1652
11%


32401
200
trigonelline (N′-methylnicotinate)
0.1856
0.1652
30%


33228
200
Metabolite - 11883
0.1941
0.1718
−4%


32776
200
Metabolite - 11459
0.1997
0.1754
−13% 


1121
201
margarate (17:0)
0.2008
0.1754
12%
Yes


33955
200
1-palmitoylglycerophosphocholine
0.2012
0.1754
−25% 


11438
50
phosphate
0.2028
0.1759
−7%
Yes


32756
201
Metabolite - 02276
0.205
0.1769
−23% 


21127
50
1-palmitoylglycerol (1-monopalmitin)
0.212
0.182
−40% 
Yes


1359
201
oleate(18:1(n-9))
0.2134
0.1823
−20% 


16665
50
Metabolite - 04364
0.2221
0.1888
63%


33662
200
Metabolite - 12240
0.2242
0.1896
17%


32572
200
Metabolite - 11255
0.2263
0.1899
12%


32814
201
Metabolite - 11497
0.2274
0.1899
16%


12774
50
Metabolite - 03094
0.2288
0.1899
−11% 


33774
201
Metabolite - 12349
0.2289
0.1899
33%


33386
50
Metabolite - 12035
0.233
0.1905
10%


33415
201
Metabolite - 12063
0.2331
0.1905
21%


33846
200
indoleacetate*
0.2365
0.1909
−16% 


21049
50
1,6-anhydroglucose
0.2368
0.1909
−27% 


16650
50
Metabolite - 04360
0.2369
0.1909
−40% 


15365
50
glycerol 3-phosphate (G3P)
0.2384
0.1913
12%
Yes


22189
200
palmitoylcarnitine
0.2412
0.1926
22%


30821
50
Metabolite - 10812
0.2425
0.1927
−47% 


33620
200
Metabolite - 12199
0.2445
0.1934
16%


12129
200
beta-hydroxyisovalerate
0.2499
0.1967
−3%
Yes


33408
200
Metabolite - 12056
0.2568
0.1992
 7%


31373
50
Metabolite - 10878
0.2571
0.1992
−10% 


32792
201
Metabolite - 11475
0.2573
0.1992
−17% 


18392
200
theobromine
0.2577
0.1992
−10% 


19323
201
docosahexaenoate (DHA; 22:6(n-3))
0.2666
0.2052
−14% 
Yes


18394
201
theophylline
0.2846
0.218
−6%


32795
201
Metabolite - 11478
0.2895
0.2208
−10% 


32698
200
Metabolite - 11381
0.2935
0.2219
−26% 


32412
200
butyrylcarnitine
0.2945
0.2219
 0%


32800
201
Metabolite - 11483
0.296
0.2219
 0%


33198
201
Metabolite - 11853
0.2979
0.2219
−50% 


33254
201
Metabolite - 11909
0.2988
0.2219
−26% 


22842
200
cholate
0.2993
0.2219
38%


33390
201
Metabolite - 12039
0.3001
0.2219
−22% 


12626
50
Metabolite - 03003
0.3038
0.2237
13%


12261
201
taurodeoxycholic acid
0.3087
0.2264
−46% 


32578
200
Metabolite - 11261
0.3152
0.2301
−18% 


18868
50
Metabolite - 05847
0.3218
0.2328
−1%


32735
200
Metabolite - 01911
0.3229
0.2328
−12% 


27719
50
galactonic acid
0.3253
0.233
−14% 


15122
50
glycerol
0.3269
0.233
16%
Yes


33204
201
Metabolite - 11859
0.3274
0.233
 3%


32328
200
hexanoylcarnitine
0.3294
0.2334
−17% 


1898
200
proline
0.3309
0.2334
−41% 
Yes


21421
50
Metabolite - 08214
0.3322
0.2334
 2%


32813
201
Metabolite - 11496
0.335
0.2334
−4%


32697
200
Metabolite - 11380
0.3352
0.2334
74%


22320
50
Metabolite - 08889
0.3383
0.2334
48%


32634
201
Metabolite - 11317
0.3387
0.2334
41%


33194
201
Metabolite - 11849
0.3388
0.2334
−34% 


527
50
lactate
0.3402
0.2335
95%
Yes


33154
200
Metabolite - 11809
0.3458
0.2356
−18% 


32492
201
caprylate (8:0)
0.3467
0.2356
 4%


32838
200
Metabolite - 11521
0.3511
0.2371
−6%


32616
201
Metabolite - 11299
0.3571
0.2402
−26% 


22154
200
bradykinin
0.3614
0.2422
−4%
Yes


32875
200
Metabolite - 11558
0.3753
0.2495
−33% 


32971
200
Metabolite - 11654
0.3753
0.2495
−11% 


16634
50
Metabolite - 04357
0.3868
0.2551
 2%


19576
50
Metabolite - 06627
0.3898
0.2562
−1%


33570
200
Metabolite - 12154
0.3919
0.2565
99%


2137
200
biliverdin
0.3952
0.2577
27%
Yes


32854
200
Metabolite - 11537
0.4062
0.2638
−3%


17747
200
sphingosine
0.4125
0.2669
−20% 


1365
201
myristate (14:0)
0.4169
0.2683
66%
Yes


32511
201
EDTA*
0.4192
0.2683
63%


32767
201
Metabolite - 11450
0.4192
0.2683
 7%


32847
201
Metabolite - 11530
0.4303
0.2743
10%


17805
201
dihomolinolenate (20:2(n-6))
0.4331
0.2743
15%


32793
200
Metabolite - 11476
0.4346
0.2743
88%


12781
50
Metabolite - 03099
0.4351
0.2743
64%


1648
50
serine
0.4384
0.2753
−10% 
Yes


32557
201
Metabolite - 06126
0.4398
0.2753
−3%


1301
50
lysine
0.4438
0.2767
24%
Yes


1126
50
alanine
0.4497
0.2786
 2%
Yes


569
200
caffeine
0.4501
0.2786
25%


32732
201
Metabolite - 11415
0.4532
0.2788
−2%


33089
50
Metabolite - 11744
0.4537
0.2788
64%


21184
200
oleoylglycerol (monoolein)
0.4564
0.2795
150% 


22481
50
Metabolite - 08988
0.4648
0.2828
−24% 


18369
200
gamma-glutamylleucine
0.4651
0.2828
−9%
Yes


20299
50
Metabolite - 07266
0.4717
0.2858
14%


33882
201
Metabolite - 12440
0.4802
0.2895
13%


1336
201
palmitate (16:0)
0.4822
0.2895
 6%
Yes


20489
50
glucose
0.4829
0.2895
46%


12764
50
Metabolite - 03084
0.4883
0.2907
−66% 


1493
200
ornithine
0.4906
0.2911
−6%
Yes


32595
200
Metabolite - 08893
0.4954
0.2929
40%


33968
201
5-dodecenoate (12:1(n-7))
0.4994
0.2942
−1%


12761
50
Metabolite - 03081
0.5076
0.298
20%


19374
50
Metabolite - 06270
0.5136
0.2988
14%


22116
201
4-methyl-2-oxopentanoate
0.5162
0.2988
−52% 


33447
201
palmitoleate (16:1(n-7))
0.5171
0.2988
−12% 


32656
201
Metabolite - 11339
0.5186
0.2988
−3%


32669
200
Metabolite - 11352
0.5188
0.2988
−11% 


542
200
3-hydroxybutyrate (BHBA)
0.5194
0.2988
 1%
Yes


31401
50
Metabolite - 10892
0.5325
0.3049
16%


32319
50
trans-4-hydroxyproline
0.5336
0.3049
137% 


1302
200
methionine
0.5401
0.3068
142% 
Yes


32855
201
Metabolite - 11538
0.543
0.3068
10%


12785
50
Metabolite - 03103
0.5441
0.3068
18%


32553
201
Metabolite - 03832
0.5469
0.3073
 0%


32869
200
Metabolite - 11552
0.5508
0.308
103% 


12782
50
Metabolite - 03100
0.553
0.308
38%


2730
200
gamma-glutamylglutamine
0.5534
0.308
71%


1564
50
citrate
0.5579
0.3089
18%
Yes


32761
201
Metabolite - 11444
0.5587
0.3089
15%


32632
200
Metabolite - 11315
0.561
0.3092
37%


1605
201
ursodeoxycholate
0.5703
0.3124
 2%


12593
50
Metabolite - 02973
0.5707
0.3124
44%


32885
200
Metabolite - 11568
0.5732
0.3124
−4%


32564
201
Metabolite - 11247
0.5743
0.3124
−9%


3127
200
hypoxanthine
0.5759
0.3124
28%


1444
200
pipecolate
0.5811
0.3138
−7%


1644
201
heptanoate
0.5823
0.3138
−11% 


33227
201
Metabolite - 11882
0.5902
0.3171
13%


54
200
tryptophan
0.598
0.3203
−7%
Yes


32418
201
myristoleate (14:1(n-5))
0.6043
0.3215
95%


15753
201
hippurate
0.6049
0.3215
 0%
Yes


32774
200
Metabolite - 11457
0.6122
0.3215
−49% 


32648
201
Metabolite - 11331
0.6136
0.3215
−4%


27710
50
N-acetylglycine
0.615
0.3215
−19% 


606
201
uridine
0.6154
0.3215
−4%
Yes


32797
201
Metabolite - 11480
0.6159
0.3215
35%


31787
201
3-carboxy-4-methyl-5-propyl-2-
0.6206
0.3215
−60% 




furanpropanoate (CMPF)


32586
200
Metabolite - 01327
0.6213
0.3215
−1%


32348
200
2-aminobutyrate
0.6217
0.3215
17%


31489
50
Metabolite - 10914
0.6229
0.3215
 8%


32748
201
Metabolite - 11431
0.6286
0.3235
−21% 


32815
201
Metabolite - 11498
0.6324
0.3235
−9%


33138
200
Metabolite - 11793
0.6362
0.3245
32%


12790
50
Metabolite - 03108
0.6404
0.3249
−8%


12035
201
pelargonate (9:0)
0.6409
0.3249
28%
Yes


27722
50
erythrose
0.6483
0.3277
−6%


33901
201
Metabolite - 12456
0.6545
0.3298
−12% 


15500
200
carnitine
0.6744
0.3389
136% 
Yes


33195
201
Metabolite - 11850
0.6796
0.3405
−9%


594
201
nicotinamide
0.6834
0.3406
−26% 


33638
201
Metabolite - 12217
0.6867
0.3406
31%


32593
200
Metabolite - 02036
0.6873
0.3406
−13% 


16511
50
Metabolite - 04274
0.6879
0.3406
−1%


17627
50
Metabolite - 04986
0.6934
0.3409
−3%


12767
50
Metabolite - 03087
0.696
0.3409
−5%


20694
50
oxalate (ethanedioate)
0.6974
0.3409
−8%
Yes


27672
201
3-indoxyl sulfate
0.6979
0.3409
82%


15676
201
3-methyl-2-oxovalerate
0.6983
0.3409
−9%


1561
50
alpha-tocopherol
0.7092
0.3442
−6%
Yes


32458
200
oleamide
0.7112
0.3442
 8%


32342
200
adenosine 5′-monophosphate (AMP)
0.7171
0.3458
−12% 


33131
200
Metabolite - 11786
0.7186
0.3458
 4%


33941
200
decanoylcarnitine
0.7205
0.3458
19%


27278
50
Metabolite - 10510
0.7254
0.3472
 3%


32970
201
Metabolite - 11653
0.7333
0.35
−12% 


32562
201
Metabolite - 11245
0.736
0.3503
27%


21631
50
Metabolite - 08403
0.7389
0.3507
−20% 


33230
200
Metabolite - 11885
0.7422
0.3513
−24% 


587
50
gluconate
0.7493
0.3526
−38% 


16508
50
Metabolite - 04272
0.7548
0.3526
−8%


33587
201
eicosenoate [9 or 11, cis or trans]
0.7575
0.3526
1446% 


24074
50
Metabolite - 09706
0.7579
0.3526
45%


15737
50
glycolate (hydroxyacetate)
0.7599
0.3526
15%
Yes


32489
201
caproate (6:0)
0.7624
0.3526
−24% 


32636
201
Metabolite - 11319
0.7652
0.3526
24%


33833
201
Metabolite - 12405
0.7667
0.3526
52%


32863
201
Metabolite - 11546
0.77
0.3526
55%


27738
50
threonate
0.7742
0.3526
23%


63
50
cholesterol
0.7753
0.3526
104% 
Yes


33402
200
Metabolite - 12050
0.7784
0.3526
40%


32651
200
Metabolite - 11334
0.7794
0.3526
37%


33265
200
Metabolite - 11920
0.7797
0.3526
 1%


32757
201
Metabolite - 11440
0.78
0.3526
71%


512
50
asparagine
0.7878
0.3551
65%
Yes


32857
200
Metabolite - 11540
0.7901
0.3552
54%


31617
50
Metabolite - 10963
0.7929
0.3555
61%


32738
200
Metabolite - 11421
0.8008
0.3571
−28% 


27256
50
Metabolite - 10500
0.8059
0.3571
−4%


32558
201
p-cresol sulfate*
0.8079
0.3571
 2%


27447
201
linoleoylglycerol (monolinolein)
0.808
0.3571
20%


1125
200
isoleucine
0.8081
0.3571
−11% 
Yes


16837
50
Metabolite - 04507
0.8101
0.3571
−42% 


60
200
leucine
0.8141
0.3572
−5%
Yes


1494
200
5-oxoproline
0.8154
0.3572
31%
Yes


33520
200
Metabolite - 12105
0.819
0.3579
−2%


1110
201
arachidonate (20:4(n-6))
0.8245
0.3594
−14% 
Yes


33972
201
10-nonadecenoate (19:1(n-9))
0.8354
0.3613
17%


16666
50
Metabolite - 04365
0.8692
0.3741
 5%


33971
201
10-heptadecenoate (17:1(n-7))
0.8763
0.3761
−16% 


27264
50
Metabolite - 10503
0.8865
0.3786
−14% 


22570
50
Metabolite - 09033
0.8891
0.3788
 2%


12771
50
Metabolite - 03091
0.8928
0.3794
74%


19490
50
Metabolite - 06488
0.8965
0.3801
108% 


32548
201
Metabolite - 11231
0.9079
0.383
 2%


21188
50
stearoylglycerol (monostearin)
0.909
0.383
49%
Yes


33488
50
lathosterol
0.9118
0.383
24%


15630
200
N-acetylornithine
0.9125
0.383
−5%


21011
50
Metabolite - 07888
0.9175
0.3837
−5%


32848
201
Metabolite - 11531
0.9202
0.3837
−11% 


33163
200
Metabolite - 11818
0.9209
0.3837
−11% 


15996
50
aspartate
0.9274
0.3855
−15% 
Yes


1649
200
valine
0.9428
0.391
61%
Yes


2132
200
citrulline
0.9557
0.3939
31%
Yes


25602
50
Metabolite - 10432
0.9562
0.3939
−21% 


32760
201
Metabolite - 11443
0.9578
0.3939
 9%


33936
200
octanoylcarnitine
0.9591
0.3939
−2%


33369
50
Metabolite - 12023
0.9705
0.3976
−35% 


3141
200
betaine
0.9845
0.4022
−18% 


32520
200
Metabolite - 11206
0.9863
0.4022
−21% 









Pathway trend analysis showed strong differentiation of DCM patients in energy and lipid pathways, suggesting TCA cycle inhibition, glucogenic amino acid mobilization, and β-oxidation increases. Adrenergic steroids (cortisol, cortisone) were increased, consistent with general stress, and androgen metabolites (DHEA-S) were strongly diminished in DCM patients, resulting in an apparent metabolic “feminization” of DCM males.
Comparison to the previous transgenic mouse DCM model plasma study showed that eight compounds, including urate, malate, tyrosine, phenylalanine, erythritol, and others exhibited similar responses and were strongly significant in both studies. Another 16 that were strongly significant in the human study trended in a similar manner in the mouse study. These included α-ketoglutarate, isocitrate, pantothenate, myo-inositol, and glutamate. The data confirm that metabolomic profiles of plasma reflect the disease state in human DCM patients, and that the transgenic mouse model shares many of the biomarker alterations associated with human disease.
T-tests are used to determine if the population means are different, but do not tell us about individual observations. Random Forest analysis is a multivariate technique for identifying compounds that distinguish the Groups. Random forests are used to classify individuals. Random forests are based on a consensus of a large number of decision trees; it is an extremely effective multivariate technique, being resistant to outliers, insensitive to method of normalization, and possesses highly predictive ability for new samples. Shown in Table 24 are results of using the biomarkers listed in Table 23 to classify the subjects as “Healthy” or “DCM”. The subjects are correctly classified as Healthy (Control) 81% of the time and correctly classified as having DCM 72% of the time. Subjects are correctly classified with >75% accuracy overall.







TABLE 24







Random Forest Classification of DCM and Healthy subjects











Control
DCM
error















Control
25
6
19%



DCM
11
28
28%





OOB estimate of error rate: 24.29%






The biomarkers that are most important to correctly classify subjects are shown in Table 25 and the Importance plot is shown in FIG. #.







TABLE 25





Important DCM biomarkers

















Metabolite-11556



4-acetamidobutanoate



Metabolite-03951



Choline



Metabolite-03056



Metabolite-11335



Metabolite-4523



erythronate



Metabolite-11593



pseudouridine



Metabolite-10395



Metabolite-12095



myo-inositol



3-indolepropionate



deoxycholate



Metabolite-11320



Metabolite-3090



Metabolite-5907



3-(4-hydroxyphenyl)lactate



Metabolite-11490



paraxanthine



Metabolite-11542



cortisol



Metabolite-4361



creatine



Metabolite-03653



Metabolite-11282



kynurenine









3D: Biomarkers of Obesity; Metabolites that are Differentially Present in Lean Compared to Obese Subjects

Biomarkers were discovered by (1) analyzing blood samples drawn from different groups of human subjects to determine the levels of metabolites in the samples and then (2) statistically analyzing the results to determine those metabolites that were differentially present in the two groups.
The plasma samples used for the analysis were from 40 lean subjects (BMI<25) and 40 obese subjects (BMI>30) that had been matched for age and gender. After the levels of metabolites were determined, the data was analyzed using univariate T-tests (i.e., Welch's T-test).
T-tests were used to determine differences in the mean levels of metabolites between the two populations (i.e., Obese vs. Lean).
Biomarkers:
As listed below in Table 26, biomarkers were discovered that were differentially present between samples from obese subjects and lean subjects.
Table 26 includes, for each listed biomarker, the p-value and q-value determined in the statistical analysis of the data concerning the biomarkers and an indication of the obese mean level, lean mean level, and the ratio of obese mean level to lean mean level (Table 26). The term “Isobar” as used in the table indicates the compounds that could not be distinguished from each other on the analytical platform used in the analysis (i.e., the compounds in an isobar elute at nearly the same time and have similar (and sometimes exactly the same) quant ions, and thus cannot be distinguished). Comp_ID refers to the compound identification number used as a primary key for that compound in the in-house chemical database. Library indicates the chemical library that was used to identify the compounds. The number 50 refers to the GC library and the number 61 refers to the LC library.







TABLE 26







Metabolite biomarkers that are differentially present in obese compared to lean subjects.



















Obese/
Mean
Mean


COMP_ID
Library
COMPOUND
p-value
q-value
Lean
LEAN
OBESE

















584
50
mannose
<0.0001
<0.0001
1.873
0.71
1.33


20489
50
D-glucose
<0.0001
<0.0001
1.500
0.78
1.17


18369
61
gamma-glu-leu
<0.0001
<0.0001
1.407
0.86
1.21


20675
50
1-5-anhydro-D-glucitol
<0.0001
<0.0001
0.629
1.24
0.78


1494
50
5-oxoproline
<0.0001
<0.0001
0.433
1.57
0.68


15365
50
sn-glycerol-3-phosphate
<0.0001
<0.0001
0.330
1.85
0.61


527
50
lactate
<0.0001
<0.0001
0.263
1.86
0.49


22803
61
Isobar-66-includes-
<0.0001
1.00E−04
0.207
3.92
0.81


3127
61
hypoxanthine
<0.0001
1.00E−04
0.166
2.9
0.48


25402
50
Metabolite - 10360
<0.0001
<0.0001
2.544
0.57
1.45


5652
61
Metabolite - 1090
<0.0001
<0.0001
0.200
1.7
0.34


7650
61
Metabolite - 1834
<0.0001
<0.0001
0.311
1.8
0.56


8959
61
Metabolite - 2100
<0.0001
<0.0001
0.202
5.09
1.03


10087
61
Metabolite - 2249
<0.0001
<0.0001
1.841
0.82
1.51


11053
61
Metabolite - 2567
<0.0001
<0.0001
1.457
0.81
1.18


12667
50
Metabolite - 3034
<0.0001
<0.0001
0.524
1.03
0.54


12969
61
Metabolite - 3135
<0.0001
1.00E−04
0.197
4.01
0.79


15278
61
Metabolite - 3843
<0.0001
<0.0001
1.547
0.75
1.16


16655
50
Metabolite - 4362
<0.0001
<0.0001
2.185
0.65
1.42


16848
50
Metabolite - 4511
<0.0001
<0.0001
0.460
1.13
0.52


17028
50
Metabolite - 4611
<0.0001
<0.0001
0.782
1.1
0.86


18871
61
Metabolite - 5848
<0.0001
<0.0001
0.380
2.21
0.84


21701
61
Metabolite - 8454
<0.0001
<0.0001
5.638
0.47
2.65


21107
61
5-sulfosalicylate
<0.0001
<0.0001
4.667
0.24
1.12


15686
50
beta-hydroxypyruvate
<0.0001
<0.0001
1.506
0.77
1.16


541
61
4-hydroxyphenylacetate
<0.0001
<0.0001
0.730
1.15
0.84


1303
50
malate
<0.0001
<0.0001
0.407
1.94
0.79


8649
61
Metabolite - 2053
<0.0001
<0.0001
1.407
0.81
1.14


10433
61
Metabolite - 2293
<0.0001
<0.0001
10.000
0.13
1.3


11094
61
Metabolite - 2589
<0.0001
<0.0001
8.727
0.11
0.96


15000
61
Metabolite - 3758
<0.0001
<0.0001
14.091
0.11
1.55


16821
50
Metabolite - 4498
<0.0001
<0.0001
0.613
0.93
0.57


17667
61
Metabolite - 5026
<0.0001
<0.0001
259.000
0.01
2.59


18010
61
Metabolite - 5231
<0.0001
<0.0001
0.425
1.53
0.65


19291
61
Metabolite - 6132
<0.0001
<0.0001
3.920
0.25
0.98


19377
50
Metabolite - 6272
<0.0001
<0.0001
0.532
1.26
0.67


19508
61
Metabolite - 6549
<0.0001
<0.0001
4.781
0.32
1.53


19969
50
Metabolite - 6931
<0.0001
<0.0001
1.605
0.76
1.22


21586
50
Metabolite - 8359
<0.0001
<0.0001
2.200
0.5
1.1


21644
61
Metabolite - 8406
<0.0001
<0.0001
12.917
0.12
1.55


21648
61
Metabolite - 8407
<0.0001
<0.0001
11.636
0.11
1.28


21650
61
Metabolite - 8409
<0.0001
<0.0001
10.500
0.12
1.26


21651
61
Metabolite - 8410
<0.0001
<0.0001
22.600
0.05
1.13


21652
61
Metabolite - 8411
<0.0001
<0.0001
50.400
0.05
2.52


21653
61
Metabolite - 8412
<0.0001
<0.0001
249.000
0.02
4.98


21657
61
Metabolite - 8416
<0.0001
<0.0001
15.000
0.09
1.35


21731
61
Metabolite - 8474
<0.0001
1.00E−04
11.929
0.14
1.67


22880
50
Metabolite - 9286
<0.0001
<0.0001
1.329
0.82
1.09


2150
61
pyridoxamine
1.00E−04
1.00E−04
1.274
0.84
1.07


24285
61
Metabolite - 10026
1.00E−04
2.00E−04
1.333
0.87
1.16


5702
61
Metabolite - 1114
1.00E−04
1.00E−04
0.466
1.16
0.54


21630
50
Metabolite - 8402
1.00E−04
1.00E−04
1.422
0.83
1.18


22590
61
Metabolite - 9040
1.00E−04
2.00E−04
3.783
0.69
2.61


25459
50
Metabolite - 10395
1.00E−04
1.00E−04
0.671
1.4
0.94


10049
61
Metabolite - 2238
1.00E−04
2.00E−04
6.308
0.39
2.46


12109
61
Metabolite - 2853
1.00E−04
1.00E−04
0.475
1.62
0.77


14117
61
Metabolite - 3441
1.00E−04
2.00E−04
0.364
2.64
0.96


16506
50
Metabolite - 4271
1.00E−04
1.00E−04
0.382
1.57
0.6


17151
61
Metabolite - 4656
1.00E−04
1.00E−04
5.833
0.24
1.4


21654
61
Metabolite - 8413
1.00E−04
1.00E−04
>100
0.001
1.92


2832
61
adenosine-5-monophosphate
2.00E−04
2.00E−04
0.291
2.23
0.65


1670
50
urea
4.00E−04
5.00E−04
1.311
0.9
1.18


20769
61
maltotriitol
4.00E−04
4.00E−04
0.454
2.18
0.99


10299
61
Metabolite - 2274
4.00E−04
4.00E−04
6.146
0.41
2.52


63
50
cholesterol
6.00E−04
5.00E−04
1.161
0.93
1.08


1110
50
arachidonic acid
6.00E−04
5.00E−04
0.685
1.27
0.87


19405
50
Metabolite - 6347
6.00E−04
6.00E−04
1.679
0.81
1.36


9016
61
Metabolite - 2109
7.00E−04
6.00E−04
1.695
0.95
1.61


1577
50
2-amino-butyrate
8.00E−04
8.00E−04
1.418
0.91
1.29


12625
50
Metabolite - 3002
8.00E−04
8.00E−04
1.482
0.83
1.23


5800
61
Metabolite - 1188
0.001
9.00E−04
0.271
2.47
0.67


19397
50
Metabolite - 6326
0.001
9.00E−04
1.295
0.95
1.23


12726
50
Metabolite - 3058
0.001
9.00E−04
0.752
1.21
0.91


6161
61
Phthalate-possible
0.0011
0.001
1.781
0.96
1.71


15683
50
4-methyl-2-oxopentanoate
0.0012
0.0011
1.283
0.92
1.18


18232
50
Metabolite - 5403
0.0013
0.0011
1.250
0.92
1.15


18882
61
taurodeoxycholic acid
0.0018
0.0015
0.305
2.46
0.75


8509
61
Metabolite - 2041
0.0018
0.0015
1.140
0.93
1.06


21188
50
1-stearoyl-rac-glycerol
0.0019
0.0016
1.753
0.77
1.35


19490
50
Metabolite - 6488
0.0019
0.0016
2.033
0.6
1.22


12644
50
Metabolite - 3016
0.002
0.0016
1.242
0.91
1.13


25548
50
Metabolite - 10419
0.0021
0.0017
0.865
1.11
0.96


12459
61
Isobar-10-includes-glutamine-
0.0028
0.0022
1.168
0.95
1.11




H-beta-ala-gly-OH-1-




methylguanine-H-Gly-Sar-




OH-lysine


1413
61
3-hydroxyphenylacetate
0.003
0.0024
1.198
0.91
1.09


21047
61
3-methyl-2-oxobutyrate
0.0032
0.0025
1.519
0.81
1.23


16903
61
Metabolite - 4547
0.0033
0.0025
1.505
0.97
1.46


22132
61
DL-alpha-hydroxyisocaproic
0.0037
0.0028
0.566
1.22
0.69




acid


21011
50
Metabolite - 7888
0.0039
0.0029
1.508
0.65
0.98


10825
61
Metabolite - 2546
0.0041
0.003
0.565
1.84
1.04


12912
61
Metabolite - 3129
0.0046
0.0033
1.589
0.95
1.51


16893
61
Metabolite - 4530
0.0046
0.0034
0.508
1.3
0.66


599
61
pyruvate
0.0052
0.0037
0.494
1.78
0.88


1604
61
uric acid
0.0054
0.0038
1.095
0.95
1.04


17068
61
Metabolite - 4627
0.0056
0.0039
0.286
2.69
0.77


17614
50
Metabolite - 4966
0.006
0.0042
1.454
0.97
1.41


19934
50
inositol
0.0062
0.0043
0.831
1.18
0.98


12673
50
Metabolite - 3040
0.0063
0.0043
0.592
1.79
1.06


10551
61
Metabolite - 2347
0.007
0.0048
0.235
3.27
0.77


22189
61
palmitoyl-carnitine
0.0076
0.0051
0.725
1.31
0.95


22020
50
Metabolite - 8749
0.0077
0.0052
0.406
2.34
0.95


9491
61
Metabolite - 2185
0.0081
0.0054
1.277
0.94
1.2


22602
50
Metabolite - 9045
0.0097
0.0064
0.673
1.07
0.72


16071
50
Metabolite - 4020
0.0103
0.0067
0.826
1.21
1


15677
61
3-methyl-L-histidine
0.0107
0.007
1.289
0.9
1.16


18476
61
glycocholic acid
0.0107
0.007
0.223
3.37
0.75


16496
50
Metabolite - 4251
0.0122
0.0078
0.798
1.19
0.95


597
61
phosphoenolpyruvate
0.0127
0.008
0.735
1.02
0.75


6851
61
Metabolite - 1497
0.0128
0.008
1.242
0.91
1.13


15650
61
1-methyladenosine
0.015
0.0093
1.117
0.94
1.05


22026
50
1-methylguanidine-
0.0151
0.0093
1.158
0.95
1.1




hydrochloride


12774
50
Metabolite - 3094
0.0173
0.0106
0.842
1.14
0.96


7944
61
Metabolite - 1915
0.0176
0.0107
2.514
1.38
3.47


14837
61
Metabolite - 3707
0.0179
0.0108
1.911
1.12
2.14


13589
61
Metabolite - 3327
0.0182
0.0109
0.374
2.97
1.11


9905
61
Metabolite - 2231
0.0183
0.0109
1.195
0.87
1.04


12648
50
Metabolite - 3020
0.0192
0.0115
0.745
1.41
1.05


10604
61
Metabolite - 2370
0.0231
0.0136
0.719
1.35
0.97


17389
50
Metabolite - 4796
0.0238
0.014
0.332
2.65
0.88


14715
61
Stachydrine-possible
0.0257
0.015
0.345
3.54
1.22


1574
61
histamine
0.0264
0.0152
0.841
1.07
0.9


15113
61
Metabolite - 3783
0.0268
0.0153
1.268
0.97
1.23


19514
61
Metabolite - 6553
0.0272
0.0155
1.620
0.5
0.81


20842
61
Metabolite - 7765
0.0288
0.0162
0.513
2.32
1.19


20194
61
Metabolite - 7147
0.0289
0.0162
0.887
1.06
0.94


5577
61
Metabolite - 1065
0.0323
0.0179
2.106
1.41
2.97


15227
61
Metabolite - 3816
0.0323
0.0179
0.767
1.29
0.99


15140
61
L-kynurenine
0.0329
0.0182
1.143
0.98
1.12


9748
61
Metabolite - 2212
0.034
0.0187
0.723
1.41
1.02


5765
61
5-hydroxypentanoate-or-beta-
0.036
0.0197
0.562
2.01
1.13




hydroxyisovaleric acid-




possible


19372
50
Metabolite - 6269
0.038
0.0207
0.857
0.98
0.84


11111
61
Metabolite - 2592
0.0404
0.0216
2.600
0.9
2.34


20166
61
Metabolite - 7091
0.0415
0.0219
1.443
1.06
1.53


22133
61
DL-hexanoyl-carnitine
0.0429
0.0224
1.240
1
1.24


10629
61
Metabolite - 2386
0.0467
0.0242
1.289
0.83
1.07


15122
50
glycerol
0.048
0.0248
1.175
0.97
1.14


1643
50
fumarate
0.051
0.0262
0.903
1.03
0.93


22337
61
Metabolite - 8893
0.0518
0.0265
1.153
0.98
1.13


8469
61
Metabolite - 2036
0.0521
0.0265
6.559
1.11
7.28


5687
61
Metabolite - 1110
0.0547
0.0274
1.648
1.25
2.06


15500
61
carnitine
0.0591
0.0294
0.821
1.17
0.96


17048
61
Metabolite - 4617
0.0614
0.0303
1.083
0.96
1.04


1105
50
Linoleic acid
0.0647
0.0316
0.882
1.1
0.97


20699
50
meso-erythritol
0.0703
0.0341
0.901
1.11
1


21763
61
Metabolite - 8507
0.0712
0.0342
0.779
1.04
0.81


7941
61
Metabolite - 1914
0.0724
0.0346
1.549
0.82
1.27


1336
50
n-hexadecanoic acid
0.0795
0.0379
0.885
1.13
1


9130
61
Metabolite - 2139
0.0815
0.0386
1.212
0.99
1.2


12720
61
Metabolite - 3056
0.0891
0.0421
0.847
1.18
1


17783
61
trans-2-3-4-
0.0906
0.0425
1.525
0.8
1.22




trimethoxycinnamic acid


16226
61
Isobar-28-includes-L-
0.0908
0.0425
0.871
1.16
1.01




threonine-L-allothreonine-L-




homoserine-S-4-amino-2-




hydroxybutyric acid


1358
50
octadecanoic acid
0.099
0.0461
0.916
1.07
0.98


542
50
3-hydroxybutanoic acid
0.1004
0.0466
0.624
1.97
1.23


6266
61
Metabolite - 1286
0.1007
0.0466
1.052
0.97
1.02


18392
61
theobromine
0.1072
0.0494
1.629
1.05
1.71


20950
50
Metabolite - 7846
0.1136
0.0521
0.579
2.35
1.36


18349
61
DL-indole-3-lactic acid
0.1144
0.0522
0.857
1.19
1.02


10245
61
Metabolite - 2269-
0.1162
0.0522
1.487
1.13
1.68


17304
61
Metabolite - 4759
0.1162
0.0522
1.233
0.9
1.11


18034
61
Metabolite - 5234
0.1162
0.0522
1.295
1.12
1.45


22001
61
3-hydroxyoctanoate
0.1208
0.0541
0.773
1.32
1.02


1572
50
glyceric acid
0.126
0.0555
0.874
1.11
0.97


12856
61
Metabolite - 3123
0.1261
0.0555
2.924
0.79
2.31


6497
61
Metabolite - 1374
0.1277
0.056
1.402
1.02
1.43


16650
50
Metabolite - 4360
0.1292
0.0565
0.713
1.29
0.92


10286
61
Metabolite - 2272
0.1333
0.0578
0.625
1.68
1.05


18657
61
Metabolite - 5726
0.1363
0.0588
0.816
1.14
0.93


15681
61
4-Guanidinobutanoic acid
0.1368
0.0588
0.896
1.06
0.95


8176
61
Metabolite - 1974
0.1394
0.0595
1.260
0.96
1.21


21418
61
Isobar-56-includes-DL-
0.1529
0.0643
0.768
1.64
1.26




pipecolic acid-1-amino-1-




cyclopentanecarboxylic acid


12710
61
Metabolite - 3052
0.1538
0.0644
0.920
1
0.92


13545
61
Metabolite - 3322
0.1626
0.0673
0.730
1.78
1.3


10715
61
Metabolite - 2395
0.163
0.0673
1.527
1.29
1.97


17478
61
Metabolite - 4873
0.1635
0.0673
1.481
0.81
1.2


19097
61
Metabolite - 5969
0.1709
0.0698
1.174
0.92
1.08


18963
61
Metabolite - 5918
0.1718
0.0699
2.337
1.01
2.36


24076
50
Metabolite - 9726
0.1767
0.0714
1.110
1
1.11


17093
61
Metabolite - 4642
0.1812
0.0729
1.146
0.96
1.1


10746
61
Isobar-6-includes-valine-
0.1889
0.0757
1.148
0.88
1.01




betaine


6362
61
p-cresol-sulfate
0.1896
0.0758
0.822
1.18
0.97


10092
61
Metabolite - 2250
0.1908
0.0759
0.636
2.06
1.31


22261
61
Isobar-60-includes-s-2-
0.1987
0.0785
1.288
0.73
0.94




hydroxybutyrate-2-




hydroxyisobutyrate


5733
61
Metabolite - 1127
0.201
0.0792
0.884
1.12
0.99


20830
61
Metabolite - 7762
0.2051
0.0802
1.357
0.98
1.33


10700
61
Metabolite - 2393
0.2053
0.0802
0.928
1.11
1.03


22120
61
6-gamma-gamma-
0.2065
0.0802
0.951
1.03
0.98




dimethylallyl-amino-purine


16518
50
Metabolite - 4276
0.2065
0.0802
1.154
0.91
1.05


18829
61
phenylalanine
0.2149
0.0832
0.952
1.05
1


14439
61
Metabolite - 3498
0.2271
0.0876
1.072
0.97
1.04


8300
61
Metabolite - 1988
0.2284
0.0878
1.165
1.03
1.2


6421
61
Metabolite - 1345
0.2299
0.088
1.341
1.23
1.65


1508
61
pantothenic acid
0.2315
0.0884
1.167
1.02
1.19


15121
61
Metabolite - 3786
0.2338
0.0889
0.763
1.18
0.9


18394
61
theophylline
0.2362
0.0893
1.420
1.19
1.69


12626
50
Metabolite - 3003
0.2364
0.0893
0.901
1.01
0.91


6492
61
Metabolite - 1371
0.2409
0.0907
1.369
1.11
1.52


6413
61
phenylacetylglutamine-or-
0.2441
0.0916
0.836
1.28
1.07




formyl-N-acetyl-5-




methoxykynurenamine-




possible


16662
61
Metabolite - 4363
0.2586
0.0967
0.787
1.41
1.11


24077
50
Metabolite - 9727
0.2726
0.1016
0.789
1.47
1.16


22259
61
Isobar-59-includes-N-6-
0.281
0.1041
1.106
0.94
1.04




trimethyl-L-lysine-H-




homoarg-OH


15676
50
3-methyl-2-oxovaleric acid
0.283
0.1041
0.912
0.91
0.83


13208
61
Metabolite - 3181
0.2918
0.107
0.904
1.04
0.94


10787
61
Metabolite - 2507
0.2989
0.1092
1.308
1.04
1.36


18705
61
Metabolite - 5768
0.3012
0.1097
0.822
1.35
1.11


16865
50
Metabolite - 4522
0.314
0.1133
1.041
0.97
1.01


12756
50
Metabolite - 3077
0.3211
0.1151
1.059
1.01
1.07


16909
61
Metabolite - 4549
0.3323
0.1179
0.716
1.41
1.01


18702
61
Metabolite - 5767
0.3381
0.1196
1.118
0.93
1.04


569
61
caffeine
0.3393
0.1196
0.683
3.82
2.61


1507
50
palmitoleic acid
0.34
0.1196
0.826
1.21
1


20248
61
Metabolite - 7177
0.3483
0.1217
1.317
1.01
1.33


15253
61
Metabolite - 3832-possible-
0.3576
0.1246
1.341
1.64
2.2




phenol-sulfate


1645
50
n-dodecanoate
0.3717
0.1291
0.922
1.16
1.07


22577
50
Metabolite - 9035
0.384
0.1329
0.897
1.16
1.04


20267
61
Metabolite - 7187
0.3923
0.1352
1.296
1.15
1.49


7933
61
Metabolite - 1911
0.4027
0.1377
0.702
2.25
1.58


17066
61
Metabolite - 4626
0.4028
0.1377
0.935
1.08
1.01


15529
61
Metabolite - 3951
0.4084
0.1391
0.962
1.05
1.01


513
61
creatinine
0.413
0.1399
0.971
1.04
1.01


8072
61
Metabolite - 1958
0.4193
0.1416
0.971
1.02
0.99


1564
50
citric acid
0.4489
0.1507
0.912
1.13
1.03


15737
50
hydroxyacetic acid
0.4522
0.1514
0.953
1.06
1.01


18015
61
Metabolite - A-3113
0.4749
0.1567
0.879
1.07
0.94


13142
61
Metabolite - 3165
0.4763
0.1567
0.962
1.05
1.01


24233
61
Metabolite - 9855
0.4766
0.1567
0.760
2.04
1.55


15663
61
2-3-dihydroxybenzoic acid
0.4858
0.1583
0.875
1.36
1.19


21421
50
Metabolite - 8214
0.487
0.1583
1.040
1.01
1.05


16070
50
Metabolite - 4019
0.487
0.1583
0.960
1.01
0.97


12478
61
Metabolite - 2898
0.4959
0.16
1.384
1.51
2.09


17271
61
Metabolite - 4746
0.4968
0.16
1.052
0.97
1.02


1417
61
Kynurenic acid
0.5074
0.162
1.066
1.06
1.13


11438
50
phosphate
0.5236
0.1667
0.980
1
0.98


14840
61
Metabolite - 3708
0.5402
0.171
0.887
1.15
1.02


17665
61
p-hydroxybenzaldehyde
0.5451
0.1711
1.020
1
1.02


54
61
tryptophan
0.5459
0.1711
0.981
1.03
1.01


15017
61
Metabolite - 3761
0.5464
0.1711
0.934
1.06
0.99


13179
61
Metabolite - 3176-possible-
0.5563
0.1732
0.936
1.1
1.03




creatine


14961
61
Metabolite - 3752
0.5662
0.1758
1.064
0.94
1


17298
61
Metabolite - 4756
0.571
0.1768
1.071
0.98
1.05


22053
61
3-hydroxydecanoic acid
0.5741
0.1773
0.917
1.08
0.99


10317
61
Metabolite - 2279
0.5777
0.1779
0.849
1.39
1.18


7029
61
Metabolite - 1597
0.5816
0.1786
1.020
1
1.02


16244
61
Isobar-21-includes-gamma-
0.5964
0.1816
0.952
1.05
1




aminobutyryl-L-histidine-L-




anserine


21044
50
Metabolite - s-2-
0.6118
0.1857
1.064
1.09
1.16




hydroxybutyric acid


10501
61
Metabolite - 2321
0.6132
0.1857
1.099
1.21
1.33


19787
61
Metabolite - 6746
0.62
0.187
0.981
1.05
1.03


1301
50
lysine
0.6207
0.187
0.922
1.29
1.19


16939
61
Metabolite - 4586
0.6303
0.1889
1.032
0.93
0.96


19906
61
Metabolite - 6827
0.6345
0.1896
1.071
1.12
1.2


10304
61
Metabolite - 2276
0.6399
0.1897
1.164
1.34
1.56


22145
61
O-acetyl-L-carnitine-
0.6448
0.19
1.029
1.04
1.07




hydrochloride


5809
61
3-indoxyl-sulfate
0.646
0.19
0.936
1.1
1.03


18706
61
Metabolite - 5769
0.6599
0.1935
0.963
1.08
1.04


12604
50
Metabolite - 2981
0.6621
0.1936
1.020
0.99
1.01


17488
61
Metabolite - 4887
0.6822
0.1975
0.966
0.89
0.86


1299
61
tyrosine
0.7135
0.2055
1.020
1.01
1.03


22154
61
bradykinin
0.7224
0.2069
0.925
1.46
1.35


606
61
uridine
0.7239
0.2069
1.020
1.01
1.03


12035
50
nonanate
0.7322
0.2087
0.990
0.99
0.98


6144
61
Metabolite - 1215
0.7562
0.2145
0.861
3.88
3.34


21762
61
Metabolite - 8506
0.785
0.222
0.939
1.65
1.55


1506
61
orotidine-5-phosphate
0.789
0.2226
0.971
1.03
1


13038
61
Metabolite - 3143
0.7911
0.2227
0.943
1.23
1.16


2734
61
gamma-L-glutamyl-L-
0.8158
0.2262
1.021
0.97
0.99




tyrosine


12924
61
Metabolite - 3131
0.8273
0.2288
1.033
1.22
1.26


1642
50
decanoic acid
0.8396
0.2305
1.022
0.93
0.95


22895
50
Metabolite - 9299
0.8504
0.2323
1.023
0.86
0.88


16016
61
Metabolite - 3994
0.8777
0.2388
1.024
0.84
0.86


15255
61
Metabolite - 3833
0.8785
0.2388
1.040
1.24
1.29


13146
61
Metabolite - 3166
0.8821
0.2392
0.970
0.99
0.96


17033
61
Metabolite - 4613
0.8939
0.2412
1.021
0.95
0.97


15753
61
hippuric acid
0.9065
0.2441
1.022
1.38
1.41


15612
61
Metabolite - 3972
0.9499
0.2546
1.000
0.97
0.97


18254
61
1-7-dimethylxanthine
0.9503
0.2546
1.026
1.94
1.99


594
61
niacinamide
0.9552
0.2548
1.024
0.83
0.85


15326
61
Metabolite - 3879
0.9567
0.2548
0.985
1.31
1.29


15765
61
ethylmalonic acid
0.9909
0.2611
1.000
0.96
0.96


1570
50
oleic acid
0.9978
0.2623
1.000
1.03
1.03









3E: Algorithms (Models) for Diagnosing Metabolic Syndrome and Pre-Disposition to Metabolic Syndrome (Insulin Sensitivity).

Models were developed to test the ability to predict insulin sensitivity (Rd) and metabolic syndrome using the biomarker metabolites alone and/or in combination with clinical measures of metabolic syndrome (e.g. BMI, Rd). The plasma samples used for the analysis were from subjects with various rates of glucose disposal (Rd).
Algorithms for determining insulin sensitivity were developed by multiple iterations of regression analysis of glucose utilization rate (i.e. Rd) in combination with measurements of metabolite biomarkers. The samples were divided into two groups. The first group was used as a ‘training’ set and the second group was used as a ‘test’ set. Then a model was developed using the training set and the predictive power of the resulting model was determined using the test set. Several models were developed to identify the most important biomarker metabolites for predicting insulin sensitivity and thereby demonstrating the utility of this approach and these biomarker metabolites.
A model was developed to predict insulin sensitivity (i.e. Rd) using plasma samples collected from a cohort with varying levels of insulin sensitivity and BMI less than 27.9 and BMI greater than 27.9. For this model the training group included half of the plasma samples and was balanced for BMI and Rd. The model was then tested using a test group that included the other half of the samples and was also balanced for BMI and Rd. The results of this analysis showed that the best model for predicting insulin sensitivity includes: BMI and the biomarker metabolites glucose, 3-methyl-2-oxobutyric acid, 1,5-anhydroglucitol and metabolite-6268.
Another model was developed to predict insulin sensitivity (i.e. Rd) using plasma samples collected from a cohort with varying levels of insulin sensitivity and BMI less than 27.9. For this model the training group included half of the plasma samples and was balanced for insulin sensitivity (Rd). The model was then tested using a test group that included the other half of the plasma samples and was also balanced for Rd. The results of this analysis showed that the best model for predicting insulin sensitivity includes the biomarker metabolites: glucose, metabolite-2546, metabolite-2853, metabolite-2370 and metabolite-2386.
Yet another model was developed to predict insulin sensitivity (i.e. Rd) using plasma samples collected from a cohort with varying levels of insulin sensitivity and BMI greater than 27.9. For this model the training group included half of the plasma samples and was balanced for insulin sensitivity (Rd). The model was then tested using a test group that included the other half of the plasma samples and was also balanced for Rd. The results of this analysis showed that the best model for predicting insulin sensitivity includes the biomarker metabolites: 3-methyl-2-oxobutyric, metabolite-3097, metabolite-4020, metabolite-3056 and metabolite-1831.
The model: BMI and the biomarker metabolites glucose, 3-methyl-2-oxobutyric acid, 1,5-anhydroglucitol and metabolite-6268; was used on a new cohort to predict insulin sensitivity (Rd). The model was developed to predict insulin sensitivity (i.e. Rd) using plasma samples collected from a cohort with varying levels of insulin sensitivity and BMI less than 27.9 and BMI greater than 27.9, as described above. The samples used to test the model for this analysis were obtained from 19 Caucasian males aged 18-39, average age of 25.6, that had been diagnosed with metabolic syndrome and 19 healthy, age-matched, Caucasian males. Plasma samples and serum samples were evaluated. The results of this analysis show that the model could correctly predict insulin sensitivity in this new cohort using either plasma (FIG. 21) or serum (FIG. 22) samples.
Example 4
Treatment Response Biomarkers
Biomarkers that are predictive of response to treatment were identified through comparisons of subjects that were ‘non-responders’ (i.e. those with little or no change (<15%) in Rd between baseline and 12 weeks post-treatment) and those subjects that were responsive to the treatment (i.e. ‘responders’). Biomarkers that were predictive of subjects as treatment responders or non-responders were based on compound levels at baseline only. The responders were defined either as those subjects with either a Rd change of 35% or higher or as those with a Rd change of 15% or higher. Data was analyzed by comparing Non-responders with both classes of Responders. Both analyses (i.e. Non-responder with Rd change under 15% vs Responder with Rd change over 35%, Non-responder with Rd change under 15% vs Responder with Rd change over 15%) were then combined and those biomarkers with a p value of <0.1 in EITHER of the 2 analyses were identified. The biomarkers are listed in Table 27. The biomarker measurements before treatment were predictive of thiazolidinedione (TZD) response, and thus can be used to select patients for treatment with TZD drugs. Experiments are planned to evaluate the biomarkers as predictive to other treatments for insulin sensitivity, pre-diabetes and diabetes control such as other therapeutic agents (e.g. metformin, etc.), weight loss, nutrition and other lifestyle modifications. This group of predictive biomarkers provide an extremely valuable tool for personalized medicine.







TABLE 27







Biomarkers to Classify Responders or Non-Responders of Treatment













P value





(Responder





vs Non-


COMPOUND
LIB_ID
COMP_ID
Responder)













Metabolite-11737
200
33082
0.0007


Metabolite-11849
201
33194
0.0013


inositol
50
19934
0.0041


glycerophosphorylcholine (GPC)
200
15990
0.0043


phenylalanine
200
64
0.0045


acetylcarnitine
200
32198
0.0049


linoleate (18:2(n-6))
201
32673
0.0079


Metabolite-11845
201
33190
0.0098


tryptophan
200
54
0.0107


Metabolite-10407
50
25522
0.0113


Metabolite-11379
201
32696
0.0117


Metabolite-9727
50
24077
0.0136


Metabolite-11205
200
32519
0.0151


Metabolite-11883
200
33228
0.0168


Metabolite-10954_200
200
32734
0.0178


glycerol
50
15122
0.0182


gondoate-20-1-n-9-
201
32402
0.0182


Metabolite-03832_201
201
32553
0.0209


oleate (18:1(n-9))
201
32630
0.0225


Metabolite-11793
200
33138
0.0225


gamma-glutamylphenylalanine-
200
33362
0.0231


Metabolite-11560
201
32877
0.0242


Metabolite-11247
201
32564
0.0273


Metabolite-11887
201
33232
0.0317


Metabolite-11206
200
32520
0.0317


tyrosine
200
1299
0.0317


Metabolite-12064
201
33416
0.0317


EDTA*
201
32511
0.0317


Metabolite-11790
200
33135
0.0338


3-hydroxybutyrate (BHBA)
50
542
0.0339


palmitoleate (16:1(n-7))
201
32628
0.0412


lysine
50
1301
0.0468


Metabolite-11314
200
32631
0.0468


Metabolite-11204
200
32518
0.0468


Metabolite-11437
201
32754
0.0491


alpha linolenate (18:3(n-3))
201
32416
0.0499


peptide-
200
31548
0.0531


DSGEGDFXAEGGGVR





Metabolite-11874
201
33219
0.0531


palmitate (16:0)
201
1336
0.0547


methionine
201
1302
0.0566


glycerol 3-phosphate (G3P)
50
15365
0.0566


Metabolite-2800
50
16287
0.0593


Metabolite-11522
201
32839
0.0611


Metabolite-11421
200
32738
0.062


Metabolite-11881
201
33226
0.0637


4-methyl-2-oxopentanoate
201
22116
0.0637


quinate
50
18335
0.0649


pelargonate-9-0-
201
12035
0.0676


creatinine
200
513
0.0676


3-methyl-2-oxobutyrate
201
21047
0.0676


Metabolite-11235
201
32552
0.0692


Metabolite-6272
50
19377
0.071


Metabolite-10360
50
25402
0.0726


saccharin
201
21151
0.0734


Metabolite-11593
201
32910
0.0742


ornithine
50
1493
0.076


cholate
201
22842
0.0775


1,6-anhydroglucose
50
21049
0.0787


Metabolite-11435
201
32752
0.0829


Metabolite-11832
201
33177
0.0891


deoxycholate
201
1114
0.0902


Metabolite-11880
201
33225
0.0902


docosahexaenoate (DHA)
50
19323
0.0927


Metabolite-12056
200
33408
0.0981


Metabolite-3075
50
12754
0.0987









Recursive pardoning analysis was carried out on the subjects. Baseline levels (i.e. prior to treatment) of the biomarker compounds were determined in the Responders (subjects with a post-treatment increase in Rd ≧35%, N=28) and the Non-Responders (subjects with a post-treatment Rd increase of <15%, N=14). The results of this analysis showed that the subjects were classified with an AUC of 0.8214. The analysis further identified “Metabolite-11737” as a biomarker that is particularly important in the classification of responders and non-responders. Using the baseline level of only this biomarker, 22 of 28 Responders were correctly classified and 12 of 14 Non-Responders were correctly classified. This marker alone had a sensitivity of 78.6% and a specificity of 85.7%. The Positive Predictive Value (PPV) was 91.7% and the Negative Predictive Value (NPV) was 66.7%.
Biomarkers that are pharmacodynamic (PD) biomarkers of treatment effectiveness were identified through comparisons of non-responders (i.e. those with little or no change (<15%) in Rd between baseline and 12 weeks post-treatment) vs responders. The PD biomarkers were based on the difference between baseline and 12 weeks post-treatment. Biomarkers were identified that showed changes from baseline levels upon TZD treatment and tracked with the change in insulin sensitivity in those subjects. These biomarkers are listed in Table 28.







TABLE 28







Pharmacodynamic Biomarkers of Treatment Response













P-Value





(Responder vs


COMPOUND
COMP_ID
LIB_ID
Non-Responder)













Metabolite - 11737
33082
200
0.0007


Metabolite - 11849
33194
201
0.0013


palmitoylglycerol
21127
50
0.0041


(monopalmitin)





glutamate
12751
50
0.0041


glycerophosphorylcholine
15990
200
0.0043


(GPC)





acetylcarnitine
32198
200
0.0049


gamma-
33362
200
0.0052


glutamylphenylalanine





alpha-tocopherol
1561
50
0.0053


glucose
20488
50
0.0062


phenylalanine
64
200
0.0071


inositol
19934
50
0.0075


Metabolite - 9727
24077
50
0.0079


Metabolite - 11385
32702
200
0.0083


Metabolite - 11845
33190
201
0.0098


tryptophan
54
200
0.0107


Metabolite - 10407
25522
50
0.0113


erythritol
20699
50
0.0136


Metabolite - 11205
32519
200
0.0151


Metabolite - 11883
33228
200
0.0168


Metabolite - 10954
32734
200
0.0178


methionine
1302
201
0.0196


Metabolite - 03832
32553
201
0.0209


5-oxoproline
1494
200
0.0242


Metabolite - 4611
17028
50
0.0242


linoleate (18:2(n-6))
32673
201
0.0258


galactonic acid
27719
50
0.0259


Metabolite - 11247
32564
201
0.0273


dipalmitin
27392
50
0.0297


tyrosine
1299
200
0.0317


Metabolite - 11379
32696
201
0.035


Metabolite - 11560
32877
201
0.0387


Metabolite - 11437
32754
201
0.0491


Metabolite - 11206
32520
200
0.0492


Metabolite - 11475
32792
201
0.0564


Metabolite - 11254
32571
200
0.0564


Metabolite - 10610
27889
50
0.0564


glycerol 3-phosphate (G3P)
15365
50
0.0566


gondoate-20-1-n-9
32402
201
0.0566


Metabolite - 2800
16287
50
0.0593


Metabolite - 11386
32703
200
0.06


Metabolite - 4055
16120
50
0.06


Metabolite - 11421
32738
200
0.062


Metabolite - 11244
32561
201
0.0637


Metabolite - 6486
19487
50
0.0637


Metabolite - 7846
20950
50
0.0637


quinate
18335
50
0.0649


Metabolite - 11881
33226
201
0.0649


3-methyl-2-oxovalerate
15676
201
0.0676


Metabolite - 9045
22602
50
0.0676


Metabolite - 4360
16650
50
0.0676


Metabolite - 11788
33133
200
0.0676


Metabolite - 6272
19377
50
0.071


3-hydroxybutyrate (BHBA)
542
50
0.071


oleate (18:1(n-9))
32630
201
0.071


Metabolite - 10360
25402
50
0.0726


saccharin
21151
201
0.0734


Metabolite - 11593
32910
201
0.0742


Metabolite - 11790
33135
200
0.0749


kynurenine
15140
200
0.076


Metabolite - 4357
16634
50
0.076


Metabolite - 3100
12782
50
0.076


cholate
22842
201
0.0775


1,6-anhydroglucose
21049
50
0.0787


Metabolite - 11887
33232
201
0.081


glycerol
15122
50
0.081


Metabolite - 06126
32557
201
0.0829


Metabolite - 4986
17627
50
0.0853


Metabolite - 3099
12781
50
0.0902


Metabolite - 11314
32631
200
0.0921


docosahexaenoate (DHA)
19323
50
0.0927


Metabolite - 11522
32839
201
0.0954


Metabolite - 01142
32747
201
0.0954


Metabolite - 3075
12754
50
0.0987









Recursive pardoning analysis was carried out on the subjects. Baseline levels (i.e. prior to treatment) and post-treatment levels of the biomarker compounds were determined in the Responders (subjects with a post-treatment increase in Rd≧35%, N=28) and the Non-Responders (subjects with a post-treatment Rd increase of <15%, N=14). The results of this analysis showed that the subjects were classified with an AUC of 0.7679. Additional experiments are planned to evaluate the biomarkers as PD biomarkers for other insulin sensitivity, pre-diabetes and diabetes therapeutic agents (e.g. metformin, etc.) as well as treatments involving modification to diet (e.g. weight loss, nutrition) and lifestyle (e.g. exercise).
Example 5
Analytical Characterization of Unnamed Biomarkers Compounds
Table 29 below includes analytical characteristics of each of the unnamed metabolites listed in the Tables above. The table includes, for each listed Metabolite, the retention time (RT), retention index (RI), mass, quant mass, and polarity obtained using the analytical methods described above. “Mass” refers to the mass of the C12 isotope of the parent ion used in quantification of the compound. “Polarity” indicates the polarity of the quantitative ion as being either positive (+) or negative (−).







TABLE 29







Analytical characteristics of biomarker metabolites.













COMP_ID
COMPOUND
LIBRARY
RT
RI
MASS
POLARITY
















25532
Metabolite - 10413
50
12.53
2042.7
204.1
+L


25602
Metabolite - 10432
50
12.29
2031.5
204
+L


27256
Metabolite - 10500
50
5.3
1229.9
211
+L


27264
Metabolite - 10503
50
7.28
1452.4
244
+L


27889
Metabolite - 10610
50
11.93
1987
204
+L


30288
Metabolite - 10750
50
5.51
1265
102.9
+L


30290
Metabolite - 10752
50
6.07
1323.7
231
+L


30832
Metabolite - 10814
50
12.84
2094
204.1
+L


31373
Metabolite - 10878
50
8.22
1583
334.1
+L


31509
Metabolite - 10931
50
12.02
1984
174.1
+L


31518
Metabolite - 10933
50
11.99
1979
318.1
+L


12781
Metabolite - 3099
50
11.77
2002.3
204
+L


12782
Metabolite - 3100
50
11.85
2010.7
204
+L


12795
Metabolite - 3113
50
12.73
2111.4
406.2
+L


16120
Metabolite - 4055
50
12.04
2021.4
304.1
+L


16138
Metabolite - 4080 -
50
14.02
2266.9
299
+L



retired for 1-palmitoyl-








sn-glycero-3-








phosphocholine







16509
Metabolite - 4273
50
10.34
1844.2
457.2
+L


16511
Metabolite - 4274
50
10.37
1854.7
158.1
+L


16512
Metabolite - 4275 -
50
10.68
1884.6
345.2
+L



retired-part of X-








3078







16518
Metabolite - 4276 -
50
13.92
2261
223.1
+L



retired for gamma-








tocopherol*







16634
Metabolite - 4357
50
8
1540.5
216
+L


16650
Metabolite - 4360
50
9.15
1678.4
347.2
+L


16665
Metabolite - 4364-
50
10.66
1852.1
232
+L



retired for L-








asparagine - 3







16666
Metabolite - 4365
50
11.05
1893.1
204
+L


16829
Metabolite - 4503
50
8.39
1589.3
227.2
+L


17028
Metabolite - 4611-
50
8.07
1546.9
292.1
+L



retired for erythronic








acid*







17330
Metabolite - 4769
50
11.3
1916.6
156
+L



retired for glutamine - 2







17389
Metabolite - 4796
50
3.53
1043.2
117
+L


17690
Metabolite - 5207
50
7.41
1493.6
151
+L


18120
Metabolite - 5348
50
9.25
1681.5
217.9
+L


19462
Metabolite - 6446
50
3.49
1021.1
204.1
+L


19478
Metabolite - 6467
50
11.09
1893.4
320.1
+L


19487
Metabolite - 6486
50
11.6
1949.8
217
+L


19576
Metabolite - 6627
50
11.96
1990.7
304.2
+L


19983
Metabolite - 6955
50
11.82
1979.1
306.1
+L


12162
Metabolite - A-2339
50
3.86
1109.8
221.0
+L


12222
Metabolite - A-2374
50
7.35
1510.9
188.0
+L


12803
Metabolite - A-2441
50
13.94
2270.9
129.0
+L


16074
Metabolite - A-2758
50
8.22
1597.1
211.0
+L


16285
Metabolite - A-2798
50
3.44
1005.8
163.0
+L


16287
Metabolite - A2800
50
3.53
1015.5
191.1
+L


24360
Metabolite -10206
50
9.04
1639.0
243.0
+L


25402
Metabolite -10360
50
10.23
1780.0
204.0
+L


25429
Metabolite -10369
50
10.92
1859.0
333.0
+L


25459
Metabolite -10395
50
9.94
1768.9
156.0
+L


25522
Metabolite -10407
50
9.94
1748.0
217.1
+L


25548
Metabolite -10419
50
16.29
2527.0
311.3
+L


25584
Metabolite -10425
50
7.52
1488.8
123.9
+L


25597
Metabolite -10427
50
11.21
1911.1
183.0
+L


25598
Metabolite -10428
50
11.31
1922.0
156.0
+L


25599
Metabolite -10429
50
11.60
1953.5
265.0
+L


25607
Metabolite -10437
50
8.43
1596.0
331.1
+L


25609
Metabolite -10439
50
8.84
1643.3
331.1
+L


25649
Metabolite -10450
50
17.00
2643.0
371.3
+L


27137
Metabolite -10498
50
12.06
1991.8
299.1
+L


27271
Metabolite -10504
50
9.94
1763.0
348.2
+L


27272
Metabolite -10505
50
10.82
1862.0
457.3
+L


27273
Metabolite -10506
50
11.30
1914.0
218.1
+L


27275
Metabolite -10507
50
11.97
1988.0
370.2
+L


27278
Metabolite -10510
50
15.77
2470.0
297.2
+L


27279
Metabolite -10511
50
17.12
2645.0
309.3
+L


27288
Metabolite -10517
50
10.16
1775.0
419.2
+L


27326
Metabolite -10527
50
11.71
1950.0
221.1
+L


27678
Metabolite -10584 -
50
10.15
1779.0
217.0
+L



retired for glucose-3







27841
Metabolite -10595
50
4.14
1101.0
151.0
+L


27888
Metabolite -10609
50
11.70
1961.0
348.2
+L


27890
Metabolite -10611
50
12.03
1998.0
369.1
+L


28059
Metabolite -10650
50
10.26
1800.6
345.1
+L


29817
Metabolite -10683
50
5.12
1213.8
171.0
+L


30265
Metabolite -10732
50
12.22
2024.0
204.0
+L


30273
Metabolite -10736
50
10.03
1814.0
342.1
+L


30282
Metabolite -10744
50
15.75
2503.0
311.2
+L


12533
Metabolite -2915
50
3.77
1099.0
174.0
+L


12593
Metabolite -2973
50
4.74
1213.4
281.0
+L


12604
Metabolite -2981
50
5.21
1265.2
211.0
+L


12609
Metabolite -2986
50
5.56
1304.3
201.0
+L


12625
Metabolite -3002
50
6.74
1440.8
296.0
+L


12626
Metabolite -3003
50
6.79
1446.6
218.0
+L


12638
Metabolite -3011
50
7.08
1479.2
174.0
+L


12639
Metabolite -3012
50
7.17
1489.8
232.0
+L


12644
Metabolite -3016
50
7.58
1537.5
186.0
+L


12645
Metabolite -3017
50
7.61
1541.4
246.0
+L


12647
Metabolite -3019
50
7.74
1556.4
260.0
+L


12648
Metabolite -3020
50
7.81
1564.1
292.0
+L


12650
Metabolite -3022
50
7.98
1584.9
142.0
+L


12656
Metabolite -3025
50
8.11
1600.3
274.0
+L


12658
Metabolite -3026
50
8.17
1606.1
274.0
+L


12663
Metabolite -3030
50
8.62
1659.7
320.0
+L


12666
Metabolite -3033
50
8.88
1689.4
117.0
+L


12667
Metabolite -3034
50
8.92
1694.9
299.0
+L


12673
Metabolite -3040
50
9.27
1735.7
274.0
+L


12726
Metabolite -3058
50
9.70
1786.9
335.0
+L


12742
Metabolite -3067
50
10.02
1824.2
132.0
+L


12751
Metabolite -3073
50
10.17
1838.8
362.0
+L


12753
Metabolite -3074
50
10.22
1844.5
204.0
+L


12754
Metabolite -3075
50
10.36
1857.9
204.0
+L


12756
Metabolite -3077
50
10.44
1866.2
308.0
+L


12757
Metabolite -3078
50
10.65
1887.0
203.0
+L


12767
Metabolite -3087
50
11.19
1942.0
174.0
+L


12768
Metabolite -3088
50
11.23
1946.1
372.0
+L


12770
Metabolite -3090
50
11.31
1955.0
243.0
+L


12771
Metabolite -3091
50
11.41
1966.2
232.0
+L


12773
Metabolite -3093
50
11.50
1975.6
204.0
+L


12774
Metabolite -3094
50
11.55
1980.6
299.0
+L


12777
Metabolite -3097
50
11.64
1990.4
204.0
+L


12780
Metabolite -3098
50
11.75
2003.0
308.0
+L


12783
Metabolite -3101
50
11.93
2022.2
290.0
+L


12785
Metabolite -3103
50
12.09
2039.8
290.0
+L


12789
Metabolite -3107
50
12.21
2053.2
204.0
+L


12790
Metabolite -3108
50
12.24
2056.5
246.0
+L


12791
Metabolite -3109
50
12.56
2092.6
202.0
+L


12796
Metabolite -3114
50
12.79
2120.6
204.0
+L


16028
Metabolite -3998
50
5.22
1252.7
171.0
+L


16044
Metabolite -4005
50
6.53
1401.3
86.0
+L


16060
Metabolite -4014
50
7.17
1474.9
252.0
+L


16070
Metabolite -4019
50
7.68
1534.5
174.0
+L


16071
Metabolite -4020
50
7.91
1561.5
220.0
+L


16116
Metabolite -4051
50
11.56
1970.2
357.0
+L


16290
Metabolite -4133
50
4.35
1108.9
198.0
+L


16308
Metabolite -4147
50
10.07
1767.1
290.0
+L


16496
Metabolite -4251
50
4.09
1130.7
217.0
+L


16506
Metabolite -4271
50
9.69
1777.4
419.0
+L


16653
Metabolite -4361
50
9.40
1706.2
232.0
+L


16655
Metabolite -4362
50
10.02
1779.9
319.0
+L


16819
Metabolite -4496
50
6.76
1398.2
204.0
+L


16821
Metabolite -4498
50
7.06
1434.9
103.0
+L


16831
Metabolite -4504
50
8.46
1597.1
244.0
+L


16843
Metabolite -4510
50
9.70
1740.1
254.0
+L


16848
Metabolite -4511
50
10.09
1788.4
206.0
+L


16859
Metabolite -4516
50
11.00
1886.5
217.0
+L


16860
Metabolite -4517
50
11.06
1892.7
217.0
+L


16865
Metabolite -4522
50
12.26
2025.4
217.0
+L


16983
Metabolite -4598
50
6.69
1392.2
170.0
+L


16984
Metabolite -4599
50
7.42
1471.1
113.0
+L


17064
Metabolite -4624
50
10.01
1779.1
342.0
+L


17083
Metabolite -4634
50
11.00
1884.3
333.0
+L


17327
Metabolite -4767
50
8.77
1626.2
117.0
+L


17359
Metabolite -4791
50
10.29
1796.4
366.5
+L


17390
Metabolite -4806
50
4.20
1122.8
105.0
+L


17614
Metabolite -4966
50
9.66
1749.4
218.0
+L


17627
Metabolite -4986
50
11.56
1956.4
204.0
+L


17971
Metabolite -5210
50
8.47
1616.4
254.0
+L


17975
Metabolite -5211
50
8.77
1652.1
326.0
+L


17978
Metabolite -5213
50
8.97
1675.3
111.0
+L


17987
Metabolite -5228
50
6.97
1442.5
181.0
+L


18118
Metabolite -5346
50
8.33
1573.0
202.0
+L


18122
Metabolite -5349
50
10.10
1782.2
312.0
+L


18146
Metabolite -5366
50
12.49
2044.7
204.0
+L


18147
Metabolite -5367
50
12.77
2079.3
171.0
+L


18232
Metabolite -5403
50
5.92
1300.2
319.0
+L


18273
Metabolite -5420
50
9.09
1669.0
417.0
+L


18384
Metabolite -5487
50
7.01
1426.3
204.0
+L


18388
Metabolite -5491
50
8.30
1575.9
129.0
+L


18868
Metabolite -5847
50
12.35
2040.0
288.2
+L


18929
Metabolite -5907
50
8.69
1643.2
229.1
+L


19110
Metabolite -5978
50
7.52
1468.9
232.1
+L


19362
Metabolite -6226
50
4.38
1137.4
154.0
+L


19363
Metabolite -6227
50
5.00
1210.5
196.1
+L


19364
Metabolite -6246
50
6.94
1428.2
160.1
+L


19367
Metabolite -6266
50
9.15
1683.5
240.2
+L


19368
Metabolite -6267
50
9.32
1704.5
257.1
+L


19370
Metabolite -6268
50
9.91
1773.8
271.1
+L


19372
Metabolite -6269 -
50
10.88
1880.9
217.1
+L



retired for inositol-3







19374
Metabolite -6270
50
11.35
1929.6
320.2
+L


19377
Metabolite -6272
50
12.60
2069.6
131.0
+L


19383
Metabolite -6286
50
16.36
2553.7
311.3
+L


19397
Metabolite -6326
50
7.66
1510.9
144.1
+L


19402
Metabolite -6346
50
8.00
1550.8
263.2
+L


19405
Metabolite -6347
50
8.16
1568.7
244.1
+L


19414
Metabolite -6350
50
11.41
1937.2
169.0
+L


19490
Metabolite -6488
50
12.25
2021.7
204.1
+L


19494
Metabolite -6506
50
12.81
2084.7
204.1
+L


19596
Metabolite -6647
50
9.13
1696.7
197.1
+L


19597
Metabolite -6648
50
9.17
1702.1
313.2
+L


19599
Metabolite -6649
50
11.47
1955.4
299.2
+L


19623
Metabolite -6671
50
9.65
1738.4
229.0
+L


19955
Metabolite -6907
50
9.22
1686.9
337.1
+L


19961
Metabolite -6913
50
9.53
1726.0
171.0
+L


19968
Metabolite -6930
50
10.32
1809.4
331.2
+L


19969
Metabolite -6931
50
10.35
1819.6
267.1
+L


20299
Metabolite -7266
50
7.82
1517.6
115.0
+L


20950
Metabolite -7846
50
5.10
1208.1
145.1
+L


21011
Metabolite -7888
50
15.96
2513.3
311.3
+L


21012
Metabolite -7889
50
16.83
2629.4
311.3
+L


21013
Metabolite -7890
50
17.76
2752.2
129.0
+L


21415
Metabolite -8209
50
14.77
2338.0
456.5
+L


21421
Metabolite -8214
50
17.13
2646.6
311.2
+L


21586
Metabolite -8359
50
7.14
1457.8
253.0
+L


21630
Metabolite -8402
50
15.27
2424.0
283.1
+L


21631
Metabolite -8403
50
15.96
2516.6
309.2
+L


22020
Metabolite -8749 -
50
9.74
1763.0
204.1
+L



retired for fructose-4







22032
Metabolite -8766
50
12.22
2034.0
315.1
+L


22054
Metabolite -8792
50
17.61
2737.0
129.0
+L


22320
Metabolite -8889
50
8.62
1635.0
521.2
+L


22480
Metabolite -8987
50
7.02
1449.7
160.1
+L


22494
Metabolite -8994
50
10.76
1879.0
447.2
+L


22507
Metabolite -9010 -
50
12.98
2126.2
217.1
+L



retired for adenosine - 1







22548
Metabolite -9026
50
8.45
1600.0
156.0
+L


22555
Metabolite -9027
50
8.50
1605.0
357.2
+L


22570
Metabolite -9033
50
9.61
1736.4
217.1
+L


22572
Metabolite -9034
50
9.63
1739.1
241.1
+L


22577
Metabolite -9035
50
9.82
1760.7
285.1
+L


22600
Metabolite -9043
50
11.75
1974.1
204.1
+L


22601
Metabolite -9044
50
13.38
2169.0
204.1
+L


22602
Metabolite -9045
50
13.91
2239.0
450.3
+L


22609
Metabolite -9047
50
8.06
1574.0
243.2
+L


22649
Metabolite -9108
50
11.20
1896.0
156.0
+L


22880
Metabolite -9286
50
8.77
1617.5
221.0
+L


22895
Metabolite -9299
50
10.54
1827.5
305.1
+L


22993
Metabolite -9448
50
14.86
2352.5
343.2
+L


24074
Metabolite -9706
50
4.39
1107.0
190.0
+L


24076
Metabolite -9726
50
4.91
1167.0
245.0
+L


24077
Metabolite -9727
50
5.24
1204.0
177.0
+L


10737
Isobar 01
61
1.45
1481.0
225.0
−i


10743
Isobar 04
61
1.52
1567.0
195.0
−i


10746
Isobar 06
61
2.13
2160.0
118.0
+i


10750
Isobar 08
61
10.04
10116.0
138.0
+i


12459
Isobar 10
61
1.40
1527.0
147.0
+i


16233
Isobar 13
61
1.40
1530.0
193.0
−i


16232
Isobar 17
61
1.49
1620.0
175.0
+i


16235
Isobar 19
61
1.55
1700
199
−i


16244
Isobar 21
61
1.59
1620.0
241.0
+i


16228
Isobar 22
61
1.55
1635.0
148.0
+i


16229
Isobar 24
61
1.43
1545.0
153.0
+i


16226
Isobar 28
61
1.46
1525.0
120.0
+i


18829
Isobar 45
61
8.38
8475.0
166.2
+i


18882
Isobar 47
61
15.51
15700.0
498.4
−i


21404
Isobar 48
61
1.50
1550.0
106.1
+i


21410
Isobar 52
61
1.55
1650.0
134.1
+i


21418
Isobar 56
61
2.45
2850.0
130.1
+i


22258
Isobar 58
61
1.38
1620.0
164.0
+i


22259
Isobar 59
61
1.82
1700.0
189.1
+i


22261
Isobar 60
61
4.26
4725.0
148.9
−i


22262
Isobar 61
61
9.30
9675.0
174.8
−i


22803
Isobar 66
61
15.06
15500.0
450.2
+i


27773
Isobar 71
61
1.57
1700.0
206.9
−i


32718
Metabolite -
200
2.8
2848
265.1
+i



01342_200







32735
Metabolite -
200
4.26
4275
464.1
+i



01911_200







32596
Metabolite -
200
5.14
5158
286.2
+i



02250_200 - retired








for piperine







32672
Metabolite -
200
0.75
764
129.2
+i



02546_200







32829
Metabolite -
200
0.82
826
144.2
+i



03653_200 - retired








for stachydrine







32595
Metabolite -
200
5.19
5200
431.9
+i



08893_200







32734
Metabolite -
200
4.14
4229
288.2
+i



10954_200 - retired








for (+/−) octanoyl








carnitine







32514
Metabolite - 11200 -
200
5.62
5637
496.4
+i



retired for 1-palmitoyl-








sn-glycero-3-








phosphocholine







32516
Metabolite - 11202 -
200
5.8
5823
524.4
+i



retired for 1-stearoyl-








sn-glycero-3-








phosphocholine







32517
Metabolite - 11203 -
200
5.65
5665
522.4
+i



retired for 1-Oleoyl-








sn-glycero-3-








phosphocholine







32518
Metabolite - 11204
200
5.26
5263
229.2
+i


32519
Metabolite - 11205 -
200
5.55
5558
520.4
+i



retired for 1-linoleoyl








GPC







32520
Metabolite - 11206
200
0.59
575
138.8
+i


32578
Metabolite - 11261
200
3.69
3600
286.2
+i


32631
Metabolite - 11314
200
0.64
634
243
+i


32632
Metabolite - 11315
200
1.19
1210
130.2
+i


32644
Metabolite - 11327
200
5.16
5176
269.2
+i


32652
Metabolite - 11335
200
0.97
991
229.2
+i


32654
Metabolite - 11337
200
1
1020
160.2
+i


32671
Metabolite - 11354
200
0.76
770
146.2
+i


32738
Metabolite - 11421
200
4.54
4575
314.2
+i


32786
Metabolite - 11469
200
3.82
3874
239.1
+i


32793
Metabolite - 11476
200
4.52
4525
189.1
+i


32875
Metabolite - 11558
200
5.64
5606
420.2
+i


32971
Metabolite - 11654 -
200
2.53
2500
246.2
+i



retired for isovaleryl-








L-carnitine







33080
Metabolite - 11735
200
2.51
2584
207.2
+i


33081
Metabolite - 11736
200
2.58
2639
379.4
+i


33082
Metabolite - 11737
200
2.7
2747
235.2
+i


33132
Metabolite - 11787
200
1.13
1126
148.1
+i


33135
Metabolite - 11790
200
3.4
3472
823.3
+i


33138
Metabolite - 11793
200
3.57
3634
601.1
+i


33228
Metabolite - 11883
200
5.54
5524
544.3
+i


33323
Metabolite - 11977
200
3.21
3287
270.1
+i


33403
Metabolite - 12051
200
5.83
5739
456.4
+i


33408
Metabolite - 12056
200
1.12
1129
156.2
+i


33531
Metabolite - 12116
200
1.6
1640
286.1
+i


33587
Isobar: cis-9, cis-11,
201
6.13
5955
309.4
−i



trans-11 eicosenoate







32747
Metabolite -
201
1.19
1176
117.2
−i



01142_201 - retired








for 2-hydroxy-3-








methylbutyric acid







32588
Metabolite -
201
4.25
4242
583.2
−i



01327_201







32609
Metabolite -
201
4.9
4887
369.2
−i



01345_201 - retired








for epiandrosterone








sulfate







32587
Metabolite -
201
4.03
4025
267.2
−i



02249_201







32550
Metabolite -
201
1.97
1958
189
−i



02272_201







32756
Metabolite -
201
3.35
3339
199.1
−i



02276_201







32553
Metabolite -
201
2.2
2199
173.1
−i



03832_201







32557
Metabolite -
201
2.69
2684
203.1
−i



06126_201







32753
Metabolite -
201
2.62
2613
153.1
−i



09789_201







32548
Metabolite - 11231
201
1.47
1471
330
−i


32561
Metabolite - 11244
201
3.78
3771
224.2
−i


32564
Metabolite - 11247
201
3.94
3932
213.1
−i


32616
Metabolite - 11299
201
4.9
4893
507.2
−i


32619
Metabolite - 11302
201
5.01
4998
397.3
−i


32625
Metabolite - 11308
201
5.15
5133
365.3
−i


32635
Metabolite - 11318
201
5.81
5699
476.3
−i


32637
Metabolite - 11320 -
201
5.85
5740
593.9
−i



retired duplicate of X-








12528







32648
Metabolite - 11331 -
201
0.69
686
164.2
−i



retired EDTA ions







32656
Metabolite - 11339 -
201
0.69
689
156.2
−i



retired-EDTA ions







32682
Metabolite - 11365 -
201
5.61
5527
303.3
−i



retired for arachidonic








acid







32696
Metabolite - 11379 -
201
5.65
5566
267.3
−i



retired for cis-10-








heptadecenoic acid







32732
Metabolite - 11415 -
201
0.69
692
313.1
−i



retired - EDTA ions







32748
Metabolite - 11431
201
1.58
1575
330
−i


32754
Metabolite - 11437
201
2.89
2888
231
−i


32757
Metabolite - 11440
201
3.58
3571
246.3
−i


32760
Metabolite - 11443
201
3.92
3910
225.3
−i


32761
Metabolite - 11444
201
3.99
3983
541.2
−i


32769
Metabolite - 11452
201
4.12
4109
352.1
−i


32792
Metabolite - 11475
201
4.25
4240
383.2
−i


32795
Metabolite - 11478
201
4.3
4286
165.2
−i


32807
Metabolite - 11490
201
4.77
4762
279.8
−i


32813
Metabolite - 11496
201
5.58
5508
271.3
−i


32839
Metabolite - 11522
201
4.76
4754
313.2
−i


32848
Metabolite - 11531
201
4.86
4850
391.3
−i


32850
Metabolite - 11533
201
4.91
4904
243.2
−i


32855
Metabolite - 11538
201
4.93
4920
311.3
−i


32877
Metabolite - 11560
201
5.29
5245
295.3
−i


32910
Metabolite - 11593
201
0.79
790
189.2
−i


32970
Metabolite - 11653
201
5.82
5686
331.3
−i


33172
Metabolite - 11827
201
1.56
1575
312.1
−i


33177
Metabolite - 11832
201
1.95
1962
216.1
−i


33185
Metabolite - 11840
201
2.56
2574
135.2
−i


33190
Metabolite - 11845
201
2.87
2891
615
−i


33194
Metabolite - 11849
201
3.2
3229
266.2
−i


33198
Metabolite - 11853
201
3.59
3602
187.1
−i


33210
Metabolite - 11865
201
5.04
5037
456.2
−i


33219
Metabolite - 11874 -
201
5.23
5199
197.3
−i



retired for cis-5-








Dodecenoic acid







33225
Metabolite - 11880
201
5.44
5378
537.4
−i


33226
Metabolite - 11881
201
5.48
5414
380.3
−i


33227
Metabolite - 11882 -
201
5.52
5445
301.3
−i



retired for cis-








5,8,11,14,17-








eicosapentaenoic








acid







33232
Metabolite - 11887 -
201
5.85
5736
307.4
−i



retired for cis-11,14-








eicosadienoic acid







33237
Metabolite - 11892
201
0.71
710
367.1
−i


33380
Metabolite - 12029
201
0.68
683
329.1
−i


33388
Metabolite - 12037 -
201
5.88
5795
295.4
−i



retired for cis-10-








nonadecenoic acid







33389
Metabolite - 12038
201
5.82
5736
245.3
−i


33415
Metabolite - 12063
201
4.82
4822
427.2
−i


33416
Metabolite - 12064
201
1
999
101.3
−i









While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

## Claims

1. A method of monitoring the progression or regression of insulin resistance in a subject, the method comprising: analyzing a biological sample from a subject to determine a level of linoleoyl lysophosphatidylcholine (LPC) in the sample; and comparing the level of linoleoyl LPC in the sample to insulin resistance progression and/or insulin resistance-regression reference levels of linoleoyl LPC in order to monitor the progression or regression of insulin resistance in a subject.

2. The method of claim 1, wherein the method further comprises analyzing the biological sample to determine the level(s) of one or more additional biomarkers selected from the group consisting of, 2-hydroxybutyrate, oleoyl LPC, oleate, linolenate, linoleate, glycerophosphorylcholine (GPC), and stearate; and comparing the level(s) of the one or more additional biomarkers in the sample to insulin resistance progression and/or insulin resistance-regression reference levels of the one or more additional biomarkers in order to monitor the progression or regression of insulin resistance in a subject.

3. A method of monitoring the efficacy of insulin resistance treatment, the method comprising: analyzing a first biological sample from a subject to determine a level of linoleoyl LPC, the first sample obtained from the subject at a first time point; treating the subject for insulin resistance; analyzing a second biological sample from the subject to determine the level of linoleoyl LPC, the second sample obtained from the subject at a second time point after treatment; comparing the level linoleoyl LPC in the first sample to the level of linoleoyl LPC in the second sample to assess the efficacy of the treatment for treating insulin resistance.

4. The method of claim 3, wherein the treatment comprises administering a therapeutic agent to the subject.

5. The method of claim 4, wherein the therapeutic agent is an insulin sensitizer.

6. The method of claim 5, wherein the insulin sensitizer is a thiazolidinedione.

7. The method of claim 3, wherein the treatment comprises a lifestyle modification of the subject.

8. The method of claim 7, wherein the lifestyle modification includes a modification of the nutrition, diet, or exercise routine of the subject.

9. The method of claim 3, wherein the method further comprises analyzing the first and second biological samples to determine the level(s) of one or more additional biomarkers selected from the group consisting of 2-hydroxybutyrate, oleoyl LPC, oleate, linolenate, linoleate, glycerophosphorylcholine (GPC), and stearate; and comparing the level(s) of the one or more additional biomarkers in the first sample to the level(s) of the one or more additional biomarkers in the second sample to assess the efficacy of the treatment for treating insulin resistance.

10. The method of claim 3, wherein the method further comprises determining the subject's measurements of fasting plasma insulin, fasting plasma glucose, fasting plasma pro-insulin, fasting free fatty acids, HDL-cholesterol, LDL-cholesterol, C-peptide, adiponectin, peptide YY, hemoglobin A1C, waist circumference, body weight, or body mass index.

11. A method of determining insulin sensitivity in a subject, the method comprising: predicting a glucose disposal rate in a subject by analyzing a biological sample from a subject to determine a level of linoleoyl LPC in the sample; and comparing the level of linoleoyl LPC in the sample to glucose disposal reference levels of linoleoyl LPC in order to determine insulin sensitivity in the subject.

12. The method of claim 10, wherein the method further comprises analyzing the biological sample to determine the level(s) of one or more additional biomarkers selected from the group consisting of, 2-hydroxybutyrate, oleoyl LPC, oleate, linolenate, linoleate, glycerophosphorylcholine (GPC), and stearate; and comparing the level(s) of the one or more additional biomarkers in the sample to glucose disposal reference levels of the one or more additional biomarkers in order to determine insulin sensitivity in the subject.

13. The method of claim 10, wherein the method further comprises determining the subject's measurements of fasting plasma insulin, fasting plasma glucose, fasting plasma pro-insulin, fasting free fatty acids, HDL-cholesterol, LDL-cholesterol, C-peptide, adiponectin, peptide YY, hemoglobin A1C, waist circumference, body weight, or body mass index.

14. The method of claim 13, wherein the method further comprises analyzing the subject and a biological sample from the subject using a mathematical model comprising linoleoyl LPC and one or more additional biomarkers or measurements selected from the group consisting of, 2-hydroxybutyrate, oleoyl LPC, oleate, linolenate, linoleate, glycerophosphorylcholine (GPC), stearate, fasting plasma insulin, fasting plasma glucose, fasting plasma pro-insulin, fasting free fatty acids, HDL-cholesterol, LDL-cholesterol, C-peptide, adiponectin, peptide YY, hemoglobin A1C, waist circumference, body weight, and body mass index.

15. The method of claim 10, wherein the method further comprises analyzing the biological sample to determine the level of 2-hydroxybutyrate, oleoyl LPC, oleate, linolenate, linoleate, glycerophosphorylcholine (GPC), and stearate.

16. The method of claim 10, wherein the biological sample is a plasma sample.