HK1216189B - Biomarkers for fatty liver disease and methods using the same - Google Patents
Biomarkers for fatty liver disease and methods using the same Download PDFInfo
- Publication number
- HK1216189B HK1216189B HK16104033.3A HK16104033A HK1216189B HK 1216189 B HK1216189 B HK 1216189B HK 16104033 A HK16104033 A HK 16104033A HK 1216189 B HK1216189 B HK 1216189B
- Authority
- HK
- Hong Kong
- Prior art keywords
- metabolite
- biomarkers
- steatohepatitis
- subject
- level
- Prior art date
Links
Description
The invention generally relates to biomarkers for fatty liver disease and methods based on the same biomarkers.
Fatty change in the liver results from excessive accumulation of lipids within hepatocytes. Fatty liver is the accumulation of triglycerides and other fats in the liver cells. Fatty liver disease can range from fatty liver alone (simple fatty liver, steatosis) to fatty liver associated with hepatic inflammation (steatohepatitis). Although having fat in the liver is not normal, by itself it probably causes little harm or permanent damage. Steatosis is generally believed to be a benign condition, with rare progression to chronic liver disease. In contrast, steatohepatitis may progress to liver fibrosis and cirrhosis, can be associated with hepatocellular carcinoma and may result in liver-related morbidity and mortality.
Steatosis can occur with the use of alcohol (alcohol-related fatty liver) or in the absence of alcohol (nonalcoholic fatty liver disease, NAFLD). Steatohepatitis may be related to alcohol-induced hepatic damage or may be unrelated to alcohol. If steatohepatitis is present but a history of alcohol use is not, the condition is termed nonalcoholic steatohepatitis (NASH).
In the absence of alcohol the main risk factors for simple fatty liver (NAFLD) and NASH are obesity, diabetes, and high triglyceride levels. In NASH, fat builds up in the liver and eventually causes scar tissue. This type of hepatitis appears to be associated with diabetes, protein malnutrition, obesity, coronary artery disease, and treatment with corticosteroid medications. Fibrosis or cirrhosis in the liver is present in 15-50% of patients with NASH. Approximately 30% of patients with fibrosis develop cirrhosis after 10 years.
Fatty liver disease is now the most common cause for elevated liver function tests in the United States. It is now probably the leading reason for mild elevations of transaminases. Steatosis affects approximately 25-35% of the general population. NAFLD is found in over 80% of patients who are obese. NASH affects 2 to 5 percent of Americans and has been detected in 1.2-9% of patients undergoing routine liver biopsy. Over 50% of patients undergoing bariatric surgery have NASH. The disease strikes males and females; early studies report >70% of cases were in females but recent studies report 50% of patients are females. Fatty liver occurs in all age groups. In the United States NASH is the most common liver disease among adolescents and is the third most common cause of chronic liver disease in adults (after hepatitis C and alcohol).
Both NASH and NAFLD are becoming more common, possibly because of the greater number of Americans with obesity. In the past 10 years, the rate of obesity has doubled in adults and tripled in children. Obesity also contributes to diabetes and high blood cholesterol, which can further complicate the health of someone with NASH. Diabetes and high blood cholesterol are also becoming more common among Americans.
NASH is usually a silent disease with few or no symptoms. Patients generally feel well in the early stages and only begin to have symptoms-such as fatigue, weight loss, and weakness-once the disease is more advanced or cirrhosis develops. The progression of NASH can take years, even decades. The process can stop and, in some cases, reverse on its own without specific therapy. Or NASH can slowly worsen, causing scarring or "fibrosis" to appear and accumulate in the liver. As fibrosis worsens, cirrhosis develops; the liver becomes seriously scarred, hardened, and unable to function normally. Not every person with NASH develops cirrhosis, but once serious scarring or cirrhosis is present, few treatments can halt the progression. A person with cirrhosis experiences fluid retention, muscle wasting, bleeding from the intestines, and liver failure. Liver transplantation is the only treatment for advanced cirrhosis with liver failure, and transplantation is increasingly performed in people with NASH. NASH ranks as one of the major causes of cirrhosis in America, behind hepatitis C and alcoholic liver disease.
NASH is usually first suspected in a person who is found to have elevations in liver tests that are included in routine blood test panels, such as alanine aminotransferase (ALT) or aspartate aminotransferase (AST). When further evaluation shows no apparent reason for liver disease (such as medications, viral hepatitis, or excessive use of alcohol) and when x-rays or imaging studies of the liver show fat, NASH is suspected. The only means of proving a diagnosis of NASH and separating it from simple fatty liver is a liver biopsy. A liver biopsy requires a needle to be inserted through the skin and the removal of a small piece of the liver. If the tissue shows fat without inflammation and damage, simple fatty liver or NAFLD is diagnosed. NASH is diagnosed when microscopic examination of the tissue shows fat along with inflammation and damage to liver cells. A biopsy is required to determine whether scar tissue has developed in the liver. Currently, no blood tests or scans can reliably provide this information. Therefore there exists a need for a less invasive diagnostic method (i.e. a method that would not require a biopsy).
Palekar et al, Liver International 2006:26: 151-156 describes a clinical model for distinguishing NASH from simple steatosis in patients with nonalcoholic fatty liver disease.
Bragoszewski et al, Acta Biochimica Polonica 2007: 2: 341-348 describes the use of expression of genes encoding mitochondrial proteins to distinguish nonalcoholic steatosis from steatohepatitis.
Pagani et al, Min.Med.1984:75:99-107 describes the determination of serum bile acids in hepatobiliary diseases.
Biffl et al, Acta Medica Austriaca 1979:6(4): 113-120 describes measurement of cholylglycin and sulfolithocholylglycine in patients with various liver and bile diseases.
Puri et al Hepatoloy, 2007:46:1081-1090 describes a lipidomic analysis of nonalcoholic fatty liver disease.
Paxton et al, Biochem. J. 1986: 234: 295-303 describes the role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.
In a first aspect, the present invention provides a method of distinguishing NAFLD from NASH in a subject having liver disease or disorder, comprising: analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for NAFLD and/or NASH in the sample, where the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, and Figures 1 , 2 , 3 ; and comparing the level(s) of the one or more biomarkers in the sample to NAFLD-positive reference levels that distinguish over NASH and/or to NASH-positive reference levels that distinguish over NAFLD in order to determine whether the subject has NASH.
In an embodiment of the first aspect, the method comprises determining the level of 3-methyl-2-oxobutyrate and 3-methyl-2-oxovalerate.
In addition, the present invention provides as a second aspect a method of diagnosing whether a subject has NASH, comprising: analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for NASH in the sample, where the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, and/or Figure 2 and/or Figure 3 and comparing the level(s) of the one or more biomarkers in the sample to NASH-positive and/or NASH-negative reference levels of the one or more biomarkers in order to diagnose whether the subject has NASH.
The present invention provides as a third aspect a method of determining whether a subject is predisposed to developing steatohepatitis, comprising: analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for steatohepatitis in the sample, where the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, and/or Figure 2 and/or 3; and comparing the level(s) of the one or more biomarkers in the sample to steatohepatitis-positive and/or steatohepatitis-negative reference levels of the one or more biomarkers in order to determine whether the subject is predisposed to developing steatohepatitis.
In an embodiment of the third aspect said method is a method of determining whether a subject is predisposed to developing non-alcoholic steatohepatitis and wherein said one or more additional biomarkers are selected from Tables 1, 4B, 6B, 10, 11, 12, 13, and/or Figure 2 and/or 3
In a fourth aspect, the present invention provides a method of monitoring progression/regression of steatohepatitis in a subject comprising: analyzing a first biological sample from a subject to determine the level(s) of one or more biomarkers for steatohepatitis in the sample, where the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, and/or Figure 2 and/or 3 and the first sample is obtained from the subject at a first time point; analyzing a second biological sample from a subject to determine the level(s) of the one or more biomarkers, where the second sample is obtained from the subject at a second time point; and 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 steatohepatitis in the subject.
In an embodiment of the fourth aspect, the method further comprises comparing the level(s) of one or more biomarkers in the first sample, the level(s) of one or more biomarkers in the second sample, and/or the results of the comparison of the level(s) of the one or more biomarkers in the first and second samples to steatohepatitis-positive and/or steatohepatitis-negative reference levels of the one or more biomarkers.
In the fourth aspect, said method may optionally be a method of monitoring progression/regression of non-alcoholic steatohepatitis (NASH).
In any of the methods of the invention, the sample may be analyzed using one or more techniques selected from the group consisting of mass spectrometry, enzymatic or biochemicalreactions, clinical chemistry, ELISA, and antibody linkage.
- Figure 1 provides one embodiment of biomarker compounds that are important in distinguishing steatosis subjects and Control subjects.
- Figure 2 provides one embodiment of biomarker compounds that are important in distinguishing steatohepatitis subjects from control subjects.
- Figure 3 provides an embodiment of biomarker compounds that are important in distinguishing steatosis, steatohepatitis and control subjects.
- Figure 4 provides one example of recursive partitioning results showing the classification of Control and steatosis subjects using the biomarkers identified in Table 8.
- Figure 5 provides an example of recursive partitioning results showing the classification of steatosis and steatohepatitis (NASH, SH) using the biomarkers listed in Table 9.
- Figure 6 provides an example of recursive partitioning results showing the classification of control and steatohepatitis using the biomarkers listed in Table 10.
- Figure 7 provides an example of recursive partitioning results showing the use of the biomarkers to classify steatosis, steatohepatitis and control subjects using the biomarkers listed in Table 11.
The present invention relates to biomarkers of steatosis and steatohepatitis, methods for diagnosis (or assisting in the diagnosis) of non-alcoholic steatohepatitis, methods of distinguishing between NAFLD and NASH, methods of determining predisposition to steatohepatitis, and methods of monitoring progression/regression of steatohepatitis. Prior to describing the invention in further detail, however, the following terms will first be defined.
"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 (i.e., 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).
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" 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, tissue, blood, blood plasma, urine, or cerebral spinal fluid (CSF).
"Subject" means any animal, but is preferably a mammal, such as, for example, a human, monkey, non-human primate, mouse, or rabbit.
A "reference level" of a biomarker means a level of the biomarker that is indicative of a particular disease state, phenotype, or predisposition to developing a particular disease state or phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or predisposition to developing a particular disease state or phenotype, 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 "NASH-positive reference level" of a biomarker means a level of a biomarker that is indicative of a positive diagnosis of NASH in a subject, and a "NASH-negative reference level" of a biomarker means a level of a biomarker that is indicative of a negative diagnosis of NASH in a subject. 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 or gender-matched so that comparisons may be made between biomarker levels in samples from subjects of a certain age or gender and reference levels for a particular disease state, phenotype, or lack thereof in a certain age or gender 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.
"Steatosis" refers to fatty liver disease without the presence of inflammation. The condition can occur with the use of alcohol or in the absence of alcohol use.
"Non-alcoholic fatty liver disease" (NAFLD) refers to fatty liver disease (steatosis) that occurs in subjects even in the absence of consumption of alcohol in amounts considered harmful to the liver.
"Steatohepatitis" refers to fatty liver disease that is associated with inflammation. Steatohepatitis can progress to cirrhosis and can be associated with hepatocellular carcinoma. The condition can occur with the use of alcohol or in the absence of alcohol use.
"Non-alcoholic steatohepatitis" (NASH) refers to steatohepatitis that occurs in subjects even in the absence of consumption of alcohol in amounts considered harmful to the liver. NASH can progress to cirrhosis and can be associated with hepatocellular carcinoma.
The NAFLD and NASH 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. Patent No. 7,005,255 and U.S. Patent Application Nos. 11/357,732 , 10/695,265 (Publication No. 2005/0014132 ), 11/301,077 (Publication No. 2006/0134676 ), 11/301,078 (Publication No. 2006/0134677 ), 11/301,079 (Publication No. 2006/0134678 ), and 11/405,033 .
Generally, metabolic profiles were determined for biological samples from human subjects diagnosed with steatosis as well as from one or more other groups of human subjects (e.g., healthy control subjects not diagnosed with steatosis), as well as from human subjects diagnosed with NASH. The metabolic profile for biological samples from a subject having steatosis was compared to the metabolic profile for biological samples from the one or more other groups of subjects. 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 steatosis as compared to another group (e.g., healthy control subjects not diagnosed with steatosis) were identified as biomarkers to distinguish those groups. In addition, 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 steatosis as compared to NASH were also identified as biomarkers to distinguish those groups.
The biomarkers are discussed in more detail herein. The biomarkers that were discovered correspond with the following group(s):
- Biomarkers for distinguishing subjects having steatosis vs. control subjects not diagnosed with liver disease (see Tables 2, 4B, 5B, 8, 11, 12, 13, Figure 1(gamma-glutamyltyrosine, taurocholate, butyrylcarnitine, Metabolite - 11235, Tyrosine, uridine, glutamate, Metabolite - 11304, Metabolite - 4611, Metabolite - 6488, 10c-undecenoate, Metabolite - 11431, Metabolite - 3107, Glutamylvaline, Metabolite - 11230, Metabolite - 3083, Metabolite - 11491, Metabolite - 10432, Metabolite - 11422, Metabolite - 11728, Metabolite - 10914, Metabolite - 11432, Metabolite - 11314, Metabolite - 12035, Metabolite - 11242, Metabolite - 11529, Metabolite - 11897, gamma-glutamylphenylalanine-, Metabolite - 11628, Metabolite - 3108, Alanine, glycocholate, Isobar 47 (taurochenodeoxycholic acid, taurodeoxycholic acid), Isobar 66 (glycochenodeoxycholic acid, glychodeoxycholic acid), Lactate, mannose, Metabolite-10026, Metabolite-10951, Metabolite-2347, Metabolite-2821, Metabolite-3016, Metabolite-3019, Metabolite-3025, Metabolite-3026, Metabolite-3073, Metabolite-3077, Metabolite-3098, Metabolite-3165, Metabolite-3330, Metabolite-4167, Metabolite-4361, Metabolite-4759, Metabolite-4806, Metabolite-5346, Metabolite-5769, Metabolite-8506, Thyroxine, urate), and Figure 3 (gamma-glutamyltyrosine, glutamate, Metabolite - 11728, butyrylcarnitine, taurocholate, glutamylvaline tyrosine, Metabolite - 11422, kynurenine, glycocholate, gamma-tocopherol, Metabolite - 11431, creatine, Metabolite - 10914, Metabolite - 11235, hippurate, Metabolite - 11529, Metabolite - 4611, Metabolite - 01911, Metabolite - 11491, glycerophosphorylcholine (GPC), urate-, gamma-glutamylphenylalanine-, Metabolite - 11230, 10c-undecenoate, Metabolite - 11315, histidine, Metabolite - 11304, Metabolite - 11432, Metabolite - 11242, alanine, isocitrate, isoleucine, lactate, leucine, mannose, Metabolite - 10026, Metabolite - 10812, Metabolite - 1496, Metabolite - 1911, Metabolite - 2395, Metabolite - 3016, Metabolite - 3026, Metabolite - 3073, Metabolite - 3098, Metabolite - 3330, Metabolite - 4274, Metabolite - 5769, Metabolite - 7187, Metabolite - 8506, Metabolite - 9855, theobromine, thryoxine, urate, valine, xanthine));
- Biomarkers for distinguishing subjects having NASH vs. control subjects not diagnosed with liver disease (see Tables 1, 6B, 10, 11, 12, 13, Figure 2 (Metabolite - 11728, Kynurenine, glycocholate, butyrylcarnitine, glutamate, gamma-glutamyltyrosine, hippurate, Metabolite - 10914, Metabolite - 11422, gamma-tocopherol-, Metabolite - 11564, Glutamylvaline, Metabolite - 11205, Metabolite - 11431, urate, Metabolite - 02272, Tyrosine, Metabolite - 11231, gamma-glutamylphenylalanine, mannose, Metabolite - 03951, Metabolite - 11315, Metabolite - 11529, Metabolite - 4611, 10c-undecenoate, Metabolite - 11432, Metabolite - 4147, Metabolite - 11227, Taurocholate, Metabolite - 11380, 3-methyl-2-oxobutyric acid, Alanine, glutamine, isocitrate, isoleucine, leucine, meso-erythritol, Metabolite-10026, Metabolite-10812, Metabolite-1086, Metabolite-1110, Metabolite-1335, Metabolite-1496, Metabolite-2041, Metabolite-2272, Metabolite-2395, Metabolite-3073, Metabolite-3087, Metabolite-3098, Metabolite-4274, Metabolite-5769, Metabolite-7187, Valine, xanthine));
Biomarkers for distinguishing subjects having steatohepatitis (NASH) vs. subjects having steatosis (see Tables 3, 4B, 9, 11, 12, 13, and Figure 3 (gamma-glutamyltyrosine, glutamate, Metabolite - 11728, butyrylcarnitine, taurocholate, glutamylvalinetyrosine, Metabolite - 11422, kynurenine, glycocholate, gamma-tocopherol, Metabolite - 11431, creatine, Metabolite - 10914, Metabolite - 11235, hippurate, Metabolite - 11529, Metabolite - 4611, Metabolite - 01911, Metabolite - 11491, glycerophosphorylcholine (GPC), urate-, gamma-glutamylphenylalanine-, Metabolite - 11230, 10c-undecenoate, Metabolite - 11315, histidine, Metabolite - 11304, Metabolite - 11432, Metabolite - 11242, alanine, isocitrate, isoleucine, lactate, leucine, mannose, Metabolite - 10026, Metabolite - 10812, Metabolite - 1496, Metabolite - 1911, Metabolite - 2395, Metabolite - 3016, Metabolite - 3026, Metabolite - 3073, Metabolite - 3098, Metabolite - 3330, Metabolite - 4274, Metabolite - 5769, Metabolite - 7187, Metabolite - 8506, Metabolite - 9855, theobromine, thryoxine, urate, valine, xanthine));
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.
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., wherein the biomarkers at least include 3-methyl-2-oxobutyrate and optionally include a combination of all of the biomarkers in each or all of Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 11, 12, 13, Figure 1 , Figure 3 or any fraction or combination thereof which includes 3-methyl-2-oxobutyrate. 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.
In addition, the methods disclosed herein using the biomarkers listed in the tables may be used in combination with clinical diagnostic measures of fatty liver diseases. 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 fatty liver diseases).
The identification of biomarkers for steatosis allows for the diagnosis of (or for aiding in the diagnosis of) steatosis in subjects presenting one or more symptoms of liver dysfunction. A method of diagnosing (or aiding in diagnosing) whether a subject has steatosis comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of steatosis in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to steatosis-positive and/or steatosis-negative reference levels of the one or more biomarkers in order to diagnose (or aid in the diagnosis of) whether the subject has steatosis. The one or more biomarkers that are used are selected from Tables 2, 3, 4B, 5B, 8, 9, 11, 12, 13, Figure 1 , Figure 3 and combinations thereof. When such a method is used to aid in the diagnosis of steatosis, 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 steatosis.
In one method for the diagnosis of (or for aiding in the diagnosis of) steatosis, the method comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of steatosis in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to steatosis-positive and/or steatosis-negative reference levels of the one or more biomarkers in order to diagnose (or aid in the diagnosis of) whether the subject has steatosis, and the one or more biomarkers that are used are selected from Tables 2, 3, 4B, 5B, 8, 9, 11, 12, 13, Figure 1 , Figure 3 and combinations thereof.
In one method for the diagnosis of (or for aiding in the diagnosis of) steatohepatitis, the method comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of steatohepatitis in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to steatohepatitis-positive and/or steatohepatitis-negative reference levels of the one or more biomarkers in order to diagnose (or aid in the diagnosis of) whether the subject has steatohepatitis, and the one or more biomarkers used include 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, Figure 2 , Figure 3 and combinations thereof.
In the diagnosis of (or for aiding in the diagnosis of) NASH, the method comprises (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of NASH in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to NASH-positive and/or NASH-negative reference levels of the one or more biomarkers in order to diagnose (or aid in the diagnosis of) whether the subject has NASH, and the one or more biomarkers that are used comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, Figure 2 , Figure 3 and combinations thereof.
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), enzymatic or biochemical reactions, clinical chemistry, 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.
The levels of 3-methyl-2-oxobutyrate and one or more of the biomarkers of Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 may be determined in the methods of diagnosing and methods of aiding in diagnosing whether a subject has steatosis or steatohepatitis. For example, 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, etc., including a combination of all of the biomarkers in Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 or any fraction thereof, wherein one of the biomarkers is 3-methyl-2-oxobutyrate, may be determined and used in such methods. Determining levels of combinations of the biomarkers may allow greater sensitivity and specificity in diagnosing steatosis and aiding in the diagnosis of steatosis, and may allow better differentiation of steatosis from other liver disorders (e.g. fibrosis, cirrhosis, liver cancer, etc.) that may have similar or overlapping biomarkers to steatosis (as compared to a subject not having steatosis). For example, ratios of the levels of certain biomarkers (and non-biomarker compounds) in biological samples may allow greater sensitivity and specificity in diagnosing steatosis and aiding in the diagnosis of steatosis and may allow better differentiation of steatosis from other liver disorders that may have similar or overlapping biomarkers to steatosis (as compared to a subject not having steatosis).
One or more biomarkers that are specific for diagnosing steatosis or steatohepatitis (or aiding in diagnosing steatosis or steatohepatitis) in a certain type of sample (e.g., liver tissue sample, liver biopsy, urine sample, or blood plasma sample) may also be used. For example, when the biological sample is plasma, 3-methyl-2-oxobutyrate, and optionally one or more biomarkers listed in Tables 2, 4B, 5B, 8, 11, 12, 13, Figure 1 , Figure 3 may be used to diagnose (or aid in diagnosing) whether a subject has steatosis.
After the level(s) of the one or more biomarkers in the sample are determined, the level(s) are compared to steatosis-positive and/or steatosis-negative reference levels (or steatohepatitis-positive and/or steatohepatitis-negative reference levels) to aid in diagnosing or to diagnose whether the subject has steatosis (or steatohepatitis). Levels of the one or more biomarkers in a sample matching the steatosis-positive reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of a diagnosis of steatosis in the subject. Levels of the one or more biomarkers in a sample matching the steatosis-negative reference levels (e.g., levels that are the same as the reference levels, substantially the same as the reference levels, above and/or below the minimum and/or maximum of the reference levels, and/or within the range of the reference levels) are indicative of a diagnosis of no steatosis in the subject. 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 steatosis-negative reference levels are indicative of a diagnosis of steatosis in the subject. 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 steatosis-positive reference levels are indicative of a diagnosis of no steatosis in the subject.
The level(s) of the one or more biomarkers may be compared to steatosis-positive and/or steatosis-negative and/or steatohepatitis-positive and/or steatohepatitis-negative reference levels using various techniques, including a simple comparison (e.g., a manual comparison) of the level(s) of the one or more biomarkers in the biological sample to steatosis-positive and/or steatosis-negative and/or steatohepatitis-positive and/or steatohepatitis-negative reference levels. The level(s) of the one or more biomarkers in the biological sample may also be compared to steatosis-positive and/or steatosis-negative and/or steatohepatitis-positive and/or steatohepatitis-negative reference levels using one or more statistical analyses (e.g., t-test, Welch's T-test, Wilcoxon's rank sum test, ANOVA, recursive partitioning, random forest).
In addition, the biological samples may be analyzed to determine the level(s) of one or more non-biomarker compounds. The level(s) of such non-biomarker compounds may also allow differentiation of steatosis from other liver disorders that may have similar or overlapping biomarkers to steatosis and/or steatohepatitis (as compared to a subject not having a liver disorder). For example, a known non-biomarker compound present in biological samples of subjects having steatosis and subjects not having steatosis could be monitored to verify a diagnosis of steatosis as compared to a diagnosis of another liver disorder when biological samples from subjects having the liver disorder do not have the non-biomarker compound.
The methods of diagnosing (or aiding in diagnosing) whether a subject has steatosis may also be conducted specifically to diagnose (or aid in diagnosing) whether a subject has NAFLD and/or steatohepatitis (e.g. NASH). Such methods comprise (1) analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers of NAFLD (and/or NASH) in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to NAFLD-positive and/or NAFLD-negative reference levels (or NASH-positive and/or NASH-negative reference levels) in order to diagnose (or aid in the diagnosis of) whether the subject has NAFLD (or NASH). Biomarker specific for NAFLD are listed in Tables 2, 4B, 5B, 8, 11, 12, 13, Figure 1 , Figure 3 and biomarkers specific for NASH are listed in Tables 1, 4B, 6B, 10, 11, 12, 13, Figure 2 , Figure 3 .
The identification of biomarkers for distinguishing NAFLD versus NASH allows NAFLD and NASH to be distinguished in patients. A method of distinguishing NAFLD from NASH in a subject having steatosis comprises (1) analyzing a biological sample from a subject to determine the level(s) in the sample of one or more biomarkers of NAFLD that distinguish over NASH and/or one or more biomarkers of NASH that distinguish over NAFLD, and (2) comparing the level(s) of the one or more biomarkers in the sample to NAFLD-positive reference levels that distinguish over NASH and/or NASH-positive reference levels that distinguish over NAFLD of the one or more biomarkers in order to determine whether the subject has NAFLD or NASH. The biomarkers that are used include 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 and combinations thereof.
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), enzymatic or biochemical reactions, clinical chemistry, 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.
The levels of 3-methyl-2-oxobutyrate and optionally one or more of the biomarkers of Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 may be determined in the methods of diagnosing and methods of aiding in diagnosing whether a subject has steatohepatitis. For example, 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, etc., including a combination of all of the biomarkers in Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 or any fraction thereof including at least 3-methyl-2-oxobutyrate, may be determined and used in such methods. Determining levels of combinations of the biomarkers may allow greater sensitivity and specificity in distinguishing between NAFLD and NASH.
One or more biomarkers that are specific for distinguishing between NAFLD and NASH in a certain type of sample (e.g., liver tissue sample, urine sample, or blood plasma sample) may also be used. For example, when the biological sample is blood plasma, 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers listed in Tables 1, 2, 3, 4B, 5B, 6B,8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 may be used.
After the level(s) of the one or more biomarkers in the sample are determined, the level(s) are compared to NAFLD-positive reference levels that distinguish over NASH-negative and/or NASH-positive reference levels that distinguish over NAFLD of the one or more biomarkers in order to determine whether the subject has NAFLD or NASH. Levels of the one or more biomarkers in a sample matching the NAFLD-positive reference levels that distinguish over NASH (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 NAFLD in the subject. Levels of the one or more biomarkers in a sample matching the NASH-positive reference levels that distinguish over NAFLD (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 NASH in the subject. If the level(s) of the one or more biomarkers are more similar to the NAFLD-positive reference levels that distinguish over NASH (or less similar to the NASH-positive reference levels), then the results are indicative of NAFLD in the subject. If the level(s) of the one or more biomarkers are more similar to the NASH-positive reference levels that distinguish over NAFLD (or less similar to the NAFLD-positive reference levels), then the results are indicative of NASH in the subject.
The level(s) of the one or more biomarkers may be compared to NAFLD-positive reference levels that distinguish over NASH and/or NASH-positive reference levels that distinguish over NAFLD using various techniques, including a simple comparison (e.g., a manual comparison) of the level(s) of the one or more biomarkers in the biological sample to NAFLD-positive and/or NASH-positive reference levels. The level(s) of the one or more biomarkers in the biological sample may also be compared to NAFLD-positive reference levels that distinguish over NASH and/or NASH-positive reference levels that distinguish over NAFLD using one or more statistical analyses (e.g., t-test, Welch's T-test, Wilcoxon's rank sum test, ANOVA, recursive partitioning, random forest).
In addition, the biological samples may be analyzed to determine the level(s) of one or more non-biomarker compounds. The level(s) of such non-biomarker compounds may also allow differentiation of NAFLD from NASH.
The identification of biomarkers for steatosis and steatohepatitis also allows for the determination of whether a subject having no symptoms of steatohepatitis or steatosis is predisposed to developing steatohepatitis or steatosis. For example, a method of determining whether a subject having no symptoms of steatohepatitis is predisposed to developing steatohepatitis comprises (1) analyzing a biological sample from a subject to determine the level(s) of 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers listed in Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, Figure 2 , Figure 3 in the sample and (2) comparing the level(s) of the one or more biomarkers in the sample to steatohepatitis-positive and/or steatohepatitis-negative reference levels of the one or more biomarkers in order to determine whether the subject is predisposed to developing steatohepatitis. 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 steatohepatitis.
As described above in connection with methods of diagnosing (or aiding in the diagnosis of) steatohepatitis, 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.
As with the methods of diagnosing (or aiding in the diagnosis of) steatohepatitis or steatosis described above, 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, etc., including a combination of all of the biomarkers, for example, in Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, Figure 2 , Figure 3 or any fraction thereof including 3-methyl-2-oxobutyrate, may be determined and used in methods of determining whether a subject having no symptoms of steatohepatitis is predisposed to developing steatohepatitis.
After the level(s) of the one or more biomarkers in the sample are determined, the level(s) are compared to, for example, steatohepatitis-positive and/or steatohepatitis-negative reference levels in order to predict whether the subject is predisposed to developing steatohepatitis. Levels of the one or more biomarkers in a sample matching the steatohepatitis-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 steatohepatitis. Levels of the one or more biomarkers in a sample matching the steatohepatitis-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 steatohepatitis. 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 steatohepatitis-negative reference levels are indicative of the subject being predisposed to developing steatohepatitis. 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 steatohepatitis-positive reference levels are indicative of the subject not being predisposed to developing steatohepatitis.
Furthermore, it may also be possible to determine reference levels specific to assessing whether or not a subject that does not have steatohepatitis is predisposed to developing steatohepatitis. 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 steatohepatitis. Such reference levels could be used for comparison to the levels of the one or more biomarkers in a biological sample from a subject.
As with the methods described above, the level(s) of the one or more biomarkers may be compared to steatohepatitis-positive and/or steatohepatitis-negative reference levels using various techniques, including a simple comparison, one or more statistical analyses, and combinations thereof.
As with the methods of diagnosing (or aiding in diagnosing) whether a subject has steatohepatitis and/or steatosis, the methods of determining whether a subject having no symptoms of steatohepatitis or steatosis is predisposed to developing steatosis or steatohepatitis may further comprise analyzing the biological sample to determine the level(s) of one or more non-biomarker compounds.
The methods of determining whether a subject having no symptoms of steatohepatitis is predisposed to developing steatohepatitis may also be conducted specifically to determine whether a subject having no symptoms of steatohepatitis is predisposed to developing NAFLD and/or NASH. Biomarker specific for NAFLD are listed in Tables 2, 4B, 5B, 8, 11, 12, 13, Figure 1 , Figure 3 and biomarkers specific for NASH are listed in Tables 1, 4B, 6B, 10, 11, 12, 13, Figure 2 , Figure 3 .
In addition, methods of determining whether a subject having NAFLD is predisposed to developing NASH may be conducted using one or more biomarkers selected from Tables 1, 4B, 6B, 10, 11, 12, 13, Figure 2 , Figure 3 .
The identification of biomarkers for steatosis and steatohepatitis also allows for monitoring progression/regression of steatosis and/or steatohepatitis in a subject. For example, a method of monitoring the progression/regression of steatosis in a subject comprises (1) analyzing a first biological sample from a subject to determine the level(s) of one or more biomarkers for steatosis selected from Tables 2, 3, 4B, 6B, 8, 9, 11, 12, 13, Figure 1 , Figure 3 , 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 steatosis in the subject. The results of the method are indicative of the course of steatosis (i.e., progression or regression, if any change) in the subject.
The change (if any) in the level(s) of the one or more biomarkers over time may be indicative of progression or regression of steatosis in the subject. In order to characterize the course of steatosis 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 steatosis-positive, steatosis-negative, NAFLD-positive, NAFLD-negative, high-grade steatosis-positive, and/or NASH-negative reference levels as well as NAFLD-positive reference levels that distinguish over NASH and/or NASH-positive reference levels that distinguish over NAFLD. 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 steatosis-positive reference levels (or less similar to the steatosis-negative reference levels), to the NASH reference levels, or, when the subject initially has NAFLD, to the NASH-positive reference levels that distinguish over NAFLD, then the results are indicative of steatosis 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 steatosis-negative reference levels (or less similar to the steatosis-positive reference levels), or, when the subject initially has NASH, to NAFLD reference levels and/or to NAFLD-positive reference levels that distinguish over NASH, then the results are indicative of steatosis regression.
As with the other methods described herein, the comparisons made in the methods of monitoring progression/regression of steatosis 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 steatosis in a subject.
As described above in connection with methods of diagnosing (or aiding in the diagnosis of) steatosis, 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) of one or more biomarkers, including a combination of all of the biomarkers in Tables 2, 4B, 5B, 8, 11, 12, 13, Figure 1 , Figure 3 or any fraction thereof, may be determined and used in methods of monitoring progression/regression of steatosis in a subject.
Such methods could be conducted to monitor the course of steatosis in subjects having steatosis or could be used in subjects not having steatosis (e.g., subjects suspected of being predisposed to developing steatosis) in order to monitor levels of predisposition to steatosis.
Such methods could be conducted to monitor the course of steatohepatitis in subjects having steatohepatitis or could be used in subjects not having steatohepatitis (e.g., subjects suspected of being predisposed to developing steatohepatitis) in order to monitor levels of predisposition to steatohepatitis. The levels(s) of 3-methyl-2-oxobutyrate and optionally one or more biomarkers, including a combination of all of the biomarkers in Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, Figure 2 and/or Figure 3 , or any fraction thereof including at least 3-methyl-2-oxobutyrate, may be determined and used in methods of monitoring progression/regression of steatohepatitis in a subject.
It is believed that some of the biomarkers for steatosis and steatohepatitis described herein may also be biomarkers for liver disorders (e.g. liver fibrosis, cirrhosis, liver cancer, etc.) in general. Therefore, it is believed that at least some of the steatosis biomarkers or steatohepatitis biomarkers may be used in the methods described herein for liver disorders in general. That is, the methods described herein with respect to steatosis and/or steatohepatitis may also be used for diagnosing (or aiding in the diagnosis of) a liver disorder, methods of monitoring progression/regression of a liver disorder, methods of assessing efficacy of compositions for treating a liver disorder, methods of screening a composition for activity in modulating biomarkers associated with a liver disorder, methods of identifying potential drug targets for liver disorder, and methods of treating a liver disorder. Such methods could be conducted as described herein with respect to steatosis.
The biomarkers discussed herein may be used in other methods. For example, the methods described in U.S. Patent No. 7,005,255 and U.S. Patent Application No. 10/695,265 may be conducted using a small molecule profile comprising one or more of the biomarkers disclosed herein.
In methods listed herein, the biomarkers used include at least 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from those biomarkers in Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figures 1 , 2 , and 3 , having p-values of less than 0.05 and/or those biomarkers in Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figures 1 , 2 , and 3 having q-values of less than 0.10. The biomarkers that are used in methods described herein may also be selected from those biomarkers in Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figures 1 , 2 , and 3 that are decreased in steatosis and/or steatohepatitis (as compared to the control) or that are decreased in remission (as compared to control or steatosis and/or steatohepatitis) 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 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, Figure 1 , Figure 2 , Figure 3 that are increased in steatosis and/or steatohepatitis (as compared to the control or remission) or that are increased in remission (as compared to the control or steatosis or steatohepatitis) 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.
The invention will be further explained by the following illustrative examples that are intended to be non-limiting.
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.
The data was analyzed using statistical tests of significance to identify molecules (either known, named metabolites or unnamed metabolites) present at differential levels in a definable population or subpopulation (e.g., biomarkers for steatosis biological samples compared to control biological samples or compared to patients in remission from steatosis) useful for distinguishing between the definable populations (e.g., steatosis (NAFLD) and control, steatohepatitis (NASH) and control, NAFLD and NASH). Other molecules (either known, named metabolites or unnamed metabolites) in the definable population or subpopulation were also identified. For significance tests, Analysis of variance (ANOVA), analysis of covariance (ANCOVA) and Wilcoxon signed rank test were used to statistically analyze the data. For classification tests, Random Forest and Recursive Partitioning were used to analyze the data.
Various peaks identified in the analyses (e.g. GC-MS, LC-MS, MS-MS), including those identified as statistically significant, were subjected to a mass spectrometry based chemical identification process.
Biomarkers were discovered by (1) analyzing blood 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.
The plasma samples used for the analysis were 25 control samples that were from healthy subjects, 11 samples from patients with steatosis and 24 samples from patients with NASH. Subjects for all groups included males and females. After the levels of metabolites were determined, the data was analyzed using significance tests (ANOVA, ANCOVA, Wilcoxon).
ANOVA was used to identify significant differences in the mean levels of metabolites between two populations (i.e., Steatosis vs. Control, NASH vs. Control, Steatosis vs. NASH).
As listed below in Tables 1, 2 and 3, biomarkers were discovered that were differentially present between plasma samples from steatosis patients and Control subjects, biomarkers that were discovered that were differentially present between plasma samples from patients with NASH and from Control subjects and biomarkers that were discovered that were differentially present between plasma samples from steatohepatitis (NASH) and plasma samples from subjects with steatosis.
Tables 1, 2 and 3 include, for each listed biomarker, the p-value and q-value determined in the statistical analysis of the data concerning the biomarkers in the steatosis mean level as compared to the control mean level, the NASH mean level as compared to the control mean level, and the steatohepatitis (NASH) mean level as compared to the steatosis mean level. Library indicates the chemical library that was used to identify the compounds. The number 50 refer to the GC library and the numbers 61, 200 and 201 refer to the LC library. Comp ID refers to the identification number for the compound in our internal chemical compound database. The data is presented as a percent change based upon the ratio of means and indicates biomarkers that increase or decrease in NASH relative to control (Table 1), in steatosis relative to control (Table 2) and/or in NASH relative to steatosis (Table 3).
Table 1. Biomarkers from subjects with steatohepatitis (NASH) compared to Control subjects
Table 2. Biomarkers from subjects with Steatosis (NAFLD) compared to Control subjects.
Table 3. Biomarkers from subjects with Steatosis compared to Steatohepatitis subjects.
| 12751 | Metabolite-3073 | 50 | 1.36E-07 | 1.08E-05 | 129% |
| 32322 | glutamate | 50 | 1.52E-07 | 3.11 E-05 | 137% |
| 1113 | isocitrate | 61 | 3.94E-07 | 0.000021 | 85% |
| 1125 | isoleucine | 50 | 6.7E-07 | 2.27E-05 | 52% |
| 18706 | Metabolite-5769 | 61 | 7.12E-07 | 2.27E-05 | 64% |
| 33073 | Metabolite-11728 | 200 | 1.36E-06 | 0.0001 | -57% |
| 60 | leucine | 50 | 3.12E-06 | 0.0001 | 11% |
| 16511 | Metabolite-4274 | 50 | 3.22E-06 | 0.0001 | 61% |
| 32868 | glycocholate* | 201 | 8.56E-06 | 0.0005 | 331% |
| 32393 | glutamylvaline | 200 | 8.94E-06 | 0.0005 | 33% |
| 1126 | alanine | 50 | 0.000011 | 0.0002 | 40% |
| 1299 | tyrosine | 61 | 1.77E-05 | 0.0003 | 30% |
| 24285 | Metabolite-10026 | 61 | 2.22E-05 | 0.0003 | 52% |
| 2734 | gamma-glutamyltyrosine | 200 | 2.23E-05 | 0.0009 | 29% |
| 15140 | kynurenine | 61 | 2.96E-05 | 0.0004 | 33% |
| 31489 | Metabolite-10914 | 50 | 4.45E-05 | 0.0014 | -45% |
| 1647 | glutamine | 50 | 0.0001 | 0.0007 | 37% |
| 8509 | Metabolite-2041 | 61 | 0.0001 | 0.0008 | 34% |
| 12780 | Metabolite-3098 | 50 | 0.0001 | 0.0007 | 40% |
| 17068 | Metabolite-4627 | 61 | 0.0001 | 0.0009 | 250% |
| 20267 | Metabolite-7187 | 61 | 0.0001 | 0.001 | 85% |
| 32701 | urate- | 200 | 0.0001 | 0.0023 | 22% |
| 33362 | gamma-glutamylphenylalanine- | 200 | 0.0001 | 0.0023 | 29% |
| 33420 | gamma-tocopherol- | 50 | 0.0001 | 0.0023 | 87% |
| 12774 | Metabolite-3094 | 50 | 0.0001 | 0.0023 | 52% |
| 16865 | Metabolite-4522 | 50 | 0.0001 | 0.0011 | 30% |
| 20699 | erythritol | 50 | 0.0001 | 0.0007 | 36% |
| 30821 | Metabolite-10812 | 50 | 0.0001 | 0.0007 | 21% |
| 2761 | thyroxine | 61 | 0.0002 | 0.0013 | 46% |
| 6847 | Metabolite-1496 | 61 | 0.0002 | 0.0014 | 50% |
| 13600 | Metabolite-3330 | 61 | 0.0002 | 0.0014 | 362% |
| 18882 | Isobar-47-includes-taurochenodeoxycholic acid-and-taurodeoxycholic acid | 61 | 0.0002 | 0.0014 | 268% |
| 32497 | 10c-undecenoate | 201 | 0.0002 | 0.003 | -9% |
| 32675 | Metabolite-03951 | 200 | 0.0002 | 0.003 | 25% |
| 32881 | Metabolite-11564 | 201 | 0.0002 | 0.003 | 33% |
| 584 | mannose | 50 | 0.0002 | 0.0012 | 29% |
| 1336 | palmitate (16:0) | 50 | 0.0002 | 0.0012 | 10% |
| 16650 | Metabolite-4360 | 50 | 0.0002 | 0.0012 | 213% |
| 5628 | Metabolite-1086 | 61 | 0.0003 | 0.0016 | 105% |
| 24233 | Metabolite-9855 | 61 | 0.0003 | 0.0017 | 146% |
| 18497 | taurocholate | 201 | 0.0003 | 0.0042 | 303% |
| 22570 | Metabolite-9033 | 50 | 0.0003 | 0.0017 | 1% |
| 5687 | Metabolite-1110 | 61 | 0.0004 | 0.0021 | -71% |
| 59 | histidine | 201 | 0.0004 | 0.0042 | -14% |
| 32863 | Metabolite-11546 | 201 | 0.0004 | 0.0042 | 223% |
| 33133 | Metabolite-11788 | 200 | 0.0004 | 0.0042 | 20% |
| 64 | phenylalanine | 61 | 0.0004 | 0.002 | 13% |
| 31595 | Metabolite-10951 | 61 | 0.0005 | 0.0022 | 60% |
| 12783 | Metabolite-3101 | 50 | 0.0005 | 0.0042 | 61% |
| 15990 | glycerophosphorylcholine (GPC) | 200 | 0.0005 | 0.0042 | -28% |
| 17028 | Metabolite-4611 | 50 | 0.0005 | 0.0046 | 35% |
| 32632 | Metabolite-11315 | 200 | 0.0005 | 0.0042 | -32% |
| 12767 | Metabolite-3087 | 50 | 0.0006 | 0.0025 | 46% |
| 12791 | Metabolite-3109 | 50 | 0.0006 | 0.0025 | -28% |
| 32564 | Metabolite-11247 | 201 | 0.0006 | 0.0048 | -67% |
| 32808 | Metabolite-11491 | 201 | 0.0006 | 0.0048 | 130% |
| 527 | lactate | 50 | 0.0006 | 0.0025 | 26% |
| 21047 | 3-methyl-2-oxobutyrate | 61 | 0.0006 | 0.0025 | 10% |
| 7933 | Metabolite-1911 | 61 | 0.0007 | 0.0027 | 112% |
| 31510 | Metabolite-10932 | 61 | 0.0007 | 0.0028 | 34% |
| 32735 | Metabolite-01911 | 200 | 0.0007 | 0.0052 | 99% |
| 1358 | stearate (18:0) | 50 | 0.0008 | 0.0029 | -1% |
| 3147 | xanthine | 61 | 0.0009 | 0.0031 | 91% |
| 27801 | Metabolite-10589 | 61 | 0.0009 | 0.0031 | 64% |
| 1494 | 5-oxoproline | 50 | 0.0009 | 0.0031 | 0% |
| 32552 | Metabolite-11235 | 201 | 0.001 | 0.0065 | 90% |
| 32739 | Metabolite-11422 | 201 | 0.001 | 0.0066 | 31% |
| 599 | pyruvate | 50 | 0.001 | 0.0065 | 44% |
| 25602 | Metabolite-10432 | 50 | 0.001 | 0.0065 | 73% |
| 10551 | Metabolite-2347 | 61 | 0.0011 | 0.0036 | 214% |
| 16016 | Metabolite-3994 | 61 | 0.0011 | 0.0037 | -45% |
| 32846 | Metabolite-11529 | 201 | 0.0011 | 0.0071 | 151% |
| 13296 | Metabolite-3230 | 61 | 0.0013 | 0.004 | 25% |
| 32519 | Metabolite-11205 | 200 | 0.0013 | 0.0077 | -28% |
| 32549 | Metabolite-02269 | 201 | 0.0014 | 0.0077 | -67% |
| 32550 | Metabolite-02272 | 201 | 0.0014 | 0.0077 | -28% |
| 8210 | Metabolite-1981 | 61 | 0.0015 | 0.0048 | -41% |
| 15753 | hippurate | 201 | 0.0016 | 0.0085 | -32% |
| 32656 | Metabolite-11339 | 201 | 0.0017 | 0.0087 | -10% |
| 13214 | Metabolite-3183 | 61 | 0.0018 | 0.0055 | 30% |
| 32544 | Metabolite-11227 | 201 | 0.0019 | 0.0096 | 13% |
| 10286 | Metabolite-2272 | 61 | 0.002 | 0.006 | -26% |
| 22803 | Isobar-66-includes-glycochenodeoxycholic acid-glycodeoxycholic acid | 61 | 0.002 | 0.006 | 195% |
| 10245 | Metabolite-2269- | 61 | 0.0021 | 0.006 | -62% |
| 32548 | Metabolite-11231 | 201 | 0.0022 | 0.0108 | 82% |
| 32559 | Metabolite-11242 | 201 | 0.0022 | 0.0108 | 60% |
| 1110 | arachidonate-20-4-n-6- | 50 | 0.0023 | 0.0064 | 11% |
| 32637 | Metabolite-11320 | 201 | 0.0024 | 0.0115 | -27% |
| 10715 | Metabolite-2395 | 61 | 0.0025 | 0.0068 | 107% |
| 32813 | Metabolite-11496 | 201 | 0.0027 | 0.0122 | -18% |
| 32412 | butyrylcarnitine | 200 | 0.0029 | 0.0129 | 27% |
| 10672 | Metabolite-2390 | 61 | 0.0031 | 0.0084 | 35% |
| 15529 | Metabolite-3951 | 61 | 0.0034 | 0.009 | 27% |
| 16308 | Metabolite-4147 | 50 | 0.0034 | 0.015 | 43% |
| 32641 | Metabolite-11324 | 201 | 0.0037 | 0.0159 | -25% |
| 32786 | Metabolite-11469 | 200 | 0.0039 | 0.0163 | -68% |
| 32346 | glycochenodeoxycholate | 201 | 0.004 | 0.0165 | 102% |
| 20488 | glucose | 50 | 0.004 | 0.0105 | 18% |
| 607 | urocanate | 200 | 0.0041 | 0.0165 | -20% |
| 7941 | Metabolite-1914 | 61 | 0.0043 | 0.0112 | -69% |
| 32748 | Metabolite-11431 | 201 | 0.0045 | 0.0179 | 73% |
| 21421 | Metabolite-8214 | 50 | 0.0051 | 0.013 | 30% |
| 15122 | glycerol | 50 | 0.0053 | 0.0131 | 18% |
| 15996 | aspartate | 50 | 0.0055 | 0.021 | 40% |
| 18657 | Metabolite-5726 | 61 | 0.0057 | 0.0137 | 65% |
| 32910 | Metabolite-11593 | 201 | 0.0057 | 0.021 | 23% |
| 33131 | Metabolite-11786 | 200 | 0.0057 | 0.021 | -34% |
| 17627 | Metabolite-4986 | 50 | 0.0057 | 0.0137 | -26% |
| 22600 | Metabolite-9043 | 50 | 0.0057 | 0.0137 | 28% |
| 30728 | Metabolite-10797 | 61 | 0.0064 | 0.015 | 72% |
| 32547 | Metabolite-11230 | 201 | 0.0065 | 0.0232 | 70% |
| 32854 | Metabolite-11537 | 200 | 0.0066 | 0.0232 | -47% |
| 32752 | Metabolite-11435 | 201 | 0.0067 | 0.0233 | 68% |
| 12644 | Metabolite-3016 | 50 | 0.0072 | 0.0164 | 14% |
| 18118 | Metabolite-5346 | 50 | 0.0072 | 0.0164 | 31% |
| 27710 | N-acetylglycine | 50 | 0.0076 | 0.017 | -32% |
| 32749 | Metabolite-11432 | 201 | 0.0079 | 0.027 | 71% |
| 18291 | 3-4-5-trimethoxycinnamic acid | 61 | 0.008 | 0.0178 | -64% |
| 19462 | Metabolite-6446 | 50 | 0.0083 | 0.0181 | 24% |
| 3141 | Betaine | 200 | 0.0084 | 0.0283 | -16% |
| 10414 | Metabolite-2291 | 61 | 0.0086 | 0.0186 | -79% |
| 33386 | Metabolite-12035 | 50 | 0.0086 | 0.0284 | 42% |
| 16496 | Metabolite-4251 | 50 | 0.0087 | 0.0186 | 36% |
| 1769 | Cortisone | 201 | 0.0089 | 0.0287 | -22% |
| 15500 | carnitine | 200 | 0.009 | 0.0287 | 10% |
| 18369 | gamma-glutamylleucine | 200 | 0.0093 | 0.0294 | 19% |
| 32848 | Metabolite-11531 | 201 | 0.0095 | 0.0296 | 78% |
| 16518 | Metabolite-4276 | 50 | 0.0098 | 0.0205 | 79% |
| 32545 | Metabolite-11228 | 201 | 0.0099 | 0.0303 | 79% |
| 19323 | docosahexaenoate-DHA- | 50 | 0.0101 | 0.0209 | -27% |
| 19490 | Metabolite-6488 | 50 | 0.0102 | 0.0308 | 54% |
| 32684 | Metabolite-11367 | 201 | 0.0105 | 0.0313 | -37% |
| 27738 | threonate | 50 | 0.0106 | 0.0216 | -25% |
| 1301 | lysine | 50 | 0.0107 | 0.0216 | 12% |
| 33242 | Metabolite-11897 | 201 | 0.011 | 0.0318 | 61% |
| 9905 | Metabolite-2231 | 61 | 0.0112 | 0.0223 | 17% |
| 27288 | Metabolite-10517 | 50 | 0.0114 | 0.0325 | 39% |
| 1431 | p-hydroxyphenyllactate-HPLA- | 50 | 0.0115 | 0.0226 | 34% |
| 25522 | Metabolite-10407 | 50 | 0.0116 | 0.0226 | 25% |
| 32515 | valine* | 200 | 0.0117 | 0.0327 | 10% |
| 22154 | bradykinin | 200 | 0.0118 | 0.0327 | 1226% |
| 32751 | Metabolite-11434 | 201 | 0.0125 | 0.0341 | 33% |
| 10247 | Metabolite-2270 | 61 | 0.0127 | 0.0244 | -68% |
| 19397 | Metabolite-6326 | 50 | 0.0132 | 0.0251 | 24% |
| 15958 | phenylacetate | 201 | 0.0134 | 0.0362 | -23% |
| 15506 | choline | 61 | 0.0138 | 0.026 | -3% |
| 10737 | Isobar-1-includes-mannose-fructose-glucose-galactose-alpha-L-sorbopyranose-I nos itol-D-al lose-D--altrose-D-psicone-L--gulose-allo-inositol | 61 | 0.014 | 0.026 | 9% |
| 587 | gluconate | 50 | 0.0142 | 0.0261 | 22% |
| 33154 | Metabolite-11809 | 200 | 0.0145 | 0.0387 | -18% |
| 12782 | Metabolite-3100 | 50 | 0.0152 | 0.0276 | 64% |
| 33227 | Metabolite-11882 | 201 | 0.0154 | 0.0406 | -41% |
| 31787 | 3-carboxyl-4-methyl-5-propyl-2-furanpropanoic acid | 201 | 0.017 | 0.0436 | -43% |
| 32197 | 3-(4-hydroxyphenyl)lactate | 201 | 0.0178 | 0.0449 | 25% |
| 9130 | Metabolite-2139 | 61 | 0.0198 | 0.0355 | 26% |
| 33087 | peptide-RPPGFSPF | 200 | 0.0212 | 0.0516 | 818% |
| 32682 | Metabolite-11365 | 201 | 0.0216 | 0.0521 | -17% |
| 32746 | Metabolite-11429 | 201 | 0.0221 | 0.0526 | 18% |
| 16337 | Metabolite-4167 | 61 | 0.0222 | 0.0394 | 32% |
| 5689 | Metabolite-1111 | 61 | 0.0225 | 0.0395 | 66% |
| 27727 | glutathione, oxidized (GSSG) | 200 | 0.0228 | 0.0536 | -69% |
| 11923 | Metabolite-2821 | 61 | 0.025 | 0.0432 | 21% |
| 14837 | Metabolite-3707 | 61 | 0.0251 | 0.0432 | -39% |
| 13142 | Metabolite-3165 | 61 | 0.0263 | 0.0446 | 14% |
| 32769 | Metabolite-11452 | 201 | 0.0267 | 0.0622 | 51% |
| 606 | uridine | 61 | 0.0274 | 0.0461 | -8% |
| 18591 | Metabolite-5646 | 61 | 0.0278 | 0.0463 | -34% |
| 10087 | Metabolite-2249 | 61 | 0.029 | 0.0477 | 34% |
| 32551 | Metabolite-11234 | 201 | 0.0302 | 0.069 | 26% |
| 33228 | Metabolite-11883 | 200 | 0.0303 | 0.069 | -24% |
| 12656 | Metabolite-3025 | 50 | 0.0308 | 0.0502 | 13% |
| 22261 | Isobar-60-includes-s-2-hydroxybutyric acid-2-hydroxyisobutyric acid | 61 | 0.0312 | 0.0502 | 55% |
| 32405 | 3-indolepropionate | 200 | 0.0313 | 0.0704 | -36% |
| 10156 | Metabolite-2259 | 61 | 0.0314 | 0.0502 | 73% |
| 7650 | Metabolite-1834 | 61 | 0.0328 | 0.0518 | 47% |
| 32741 | Metabolite-11424 | 200 | 0.0329 | 0.0732 | 15% |
| 32757 | Metabolite-11440 | 201 | 0.0339 | 0.074 | 39% |
| 33012 | Metabolite-11674 | 200 | 0.0339 | 0.074 | 13% |
| 12658 | Metabolite-3026 | 50 | 0.0343 | 0.0537 | 15% |
| 32110 | Metabolite-11086 | 50 | 0.0344 | 0.0742 | -14% |
| 1284 | threonine | 50 | 0.0352 | 0.0546 | -7% |
| 32648 | Metabolite-11331 | 201 | 0.0356 | 0.0758 | -4% |
| 32517 | Metabolite-11203 | 200 | 0.0359 | 0.0758 | -17% |
| 10092 | Metabolite-2250 | 61 | 0.0363 | 0.0557 | 55% |
| 63 | cholesterol | 50 | 0.0373 | 0.0561 | 12% |
| 1507 | palmitoleate | 50 | 0.0376 | 0.0561 | 28% |
| 12768 | Metabolite-3088 | 50 | 0.0376 | 0.0561 | -18% |
| 32578 | Metabolite-11261 | 200 | 0.0389 | 0.0806 | 26% |
| 8796 | Metabolite-2074 | 61 | 0.0391 | 0.0578 | 90% |
| 33140 | Metabolite-11795 | 200 | 0.04 | 0.082 | 24% |
| 21127 | 1-palmitoylglycerol-1-monopalmitin- | 50 | 0.0424 | 0.0621 | 13% |
| 27256 | Metabolite-10500 | 50 | 0.0432 | 0.0871 | 15% |
| 13024 | Metabolite-3139 | 61 | 0.0433 | 0.0628 | 33% |
| 32599 | Metabolite-11282 | 201 | 0.0434 | 0.0871 | 39% |
| 30633 | Metabolite-10785 | 61 | 0.044 | 0.0633 | -22% |
| 9491 | Metabolite-2185 | 61 | 0.0445 | 0.0634 | 25% |
| 32880 | Metabolite-11563 | 201 | 0.0449 | 0.0893 | -16% |
| 594 | nicotinamide | 61 | 0.045 | 0.0636 | -13% |
| 12663 | Metabolite-3030 | 50 | 0.0457 | 0.0638 | -11% |
| 30281 | Metabolite-10743 | 50 | 0.047 | 0.0644 | -17% |
| 17390 | Metabolite-4806 | 50 | 0.0478 | 0.0644 | 21% |
| 12790 | Metabolite-3108 | 50 | 0.048 | 0.0946 | 24% |
| 19370 | Metabolite-6268 | 50 | 0.048 | 0.0644 | 4% |
| 6413 | Metabolite-1342 | 61 | 0.0481 | 0.0644 | -13% |
| 18015 | Metabolite-3113 | 61 | 0.0487 | 0.0644 | -15% |
| 17033 | Metabolite-4613 | 61 | 0.049 | 0.0644 | 53% |
| 12645 | Metabolite-3017 | 50 | 0.0499 | 0.0644 | -13% |
| 17304 | Metabolite-4759 | 61 | 0.0502 | 0.0644 | 26% |
| 22895 | Metabolite-9299 | 50 | 0.0503 | 0.0644 | -9% |
| 12647 | Metabolite-3019 | 50 | 0.0513 | 0.0646 | 12% |
| 16045 | Metabolite-4006 | 50 | 0.0513 | 0.0646 | 26% |
| 24077 | Metabolite-9727 | 50 | 0.0519 | 0.0647 | 19% |
| 27275 | Metabolite-10507 | 50 | 0.0527 | 0.0653 | -11% |
| 32315 | serine | 50 | 0.0532 | 0.1028 | -15% |
| 31401 | Metabolite-10892 | 50 | 0.0543 | 0.1029 | 36% |
| 32452 | propionylcarnitine | 200 | 0.055 | 0.1029 | 18% |
| 21631 | Metabolite-8403 | 50 | 0.0552 | 0.1029 | 19% |
| 32348 | 2-aminobutyrate | 200 | 0.0561 | 0.1036 | -13% |
| 16019 | Metabolite-3995 | 61 | 0.0584 | 0.0716 | -24% |
| 32776 | Metabolite-11459 | 200 | 0.0584 | 0.1065 | 19% |
| 10629 | Metabolite-2386 | 61 | 0.0587 | 0.0716 | 26% |
| 1572 | Glycerate | 50 | 0.0588 | 0.1065 | -16% |
| 12035 | pelargonate-9-0- | 50 | 0.0592 | 0.0716 | -1% |
| 31529 | Metabolite-10941 | 61 | 0.0597 | 0.0717 | 18% |
| 32762 | Metabolite-11445 | 201 | 0.06 | 0.1078 | -55% |
| 32691 | Metabolite-11374 | 200 | 0.0607 | 0.108 | 19% |
| 528 | alpha-keto-glutarate | 61 | 0.0625 | 0.0744 | 28% |
| 32792 | Metabolite-11475 | 201 | 0.0631 | 0.1115 | 54% |
| 32572 | Metabolite-11255 | 200 | 0.0644 | 0.112 | -51% |
| 6398 | Metabolite-1335 | 61 | 0.0645 | 0.0763 | 41% |
| 27718 | creatine | 200 | 0.0652 | 0.112 | 17% |
| 16666 | Metabolite-4365 | 50 | 0.0656 | 0.112 | -22% |
| 18335 | quinate | 50 | 0.0656 | 0.112 | -52% |
| 12666 | Metabolite-3033 | 50 | 0.0683 | 0.08 | -11% |
| 1366 | trans-4-hydroxyproline | 50 | 0.0699 | 0.0809 | 35% |
| 32385 | Metabolite-11180 | 50 | 0.0702 | 0.1172 | -10% |
| 18477 | glycodeoxycholate | 201 | 0.0704 | 0.1172 | 247% |
| 10501 | Metabolite-2321 | 61 | 0.0709 | 0.0815 | -25% |
| 32328 | hexanoylcarnitine | 200 | 0.0713 | 0.1177 | 17% |
| 22133 | DL-hexanoyl-carnitine | 61 | 0.0719 | 0.082 | 28% |
| 32596 | Metabolite-02250 | 200 | 0.0722 | 0.1178 | 36% |
| 32740 | Metabolite-11423 | 201 | 0.0725 | 0.1178 | 13% |
| 12785 | Metabolite-3103 | 50 | 0.0743 | 0.1195 | 42% |
| 32697 | Metabolite-11380 | 200 | 0.0747 | 0.1195 | 13% |
| 13557 | Metabolite-3323 | 61 | 0.0779 | 0.0883 | 71% |
| 32761 | Metabolite-11444 | 201 | 0.0805 | 0.1278 | 31% |
| 32952 | Metabolite-02277 | 201 | 0.0819 | 0.1286 | -75% |
| 32945 | Metabolite-11628 | 201 | 0.0822 | 0.1286 | 38% |
| 32338 | glycine | 50 | 0.084 | 0.1292 | -15% |
| 1712 | hydrocortisone | 201 | 0.0854 | 0.1292 | -19% |
| 21418 | Isobar-56-includes-DL-pipecolic acid-1-amino-1-cyclopentanecarboxylic acid | 61 | 0.0856 | 0.0958 | -24% |
| 1105 | linoleate | 50 | 0.0858 | 0.0958 | 11% |
| 32638 | Metabolite-11321 | 201 | 0.0867 | 0.1296 | -12% |
| 12919 | Metabolite-3130 | 61 | 0.0889 | 0.0978 | 67% |
| 33203 | Metabolite-11858 | 201 | 0.0893 | 0.1321 | -63% |
| 33366 | Metabolite-12020 | 200 | 0.0896 | 0.1321 | -27% |
| 22842 | cholate | 201 | 0.0996 | 0.144 | 125% |
| 32814 | Metabolite-11497 | 201 | 0.1001 | 0.144 | -7% |
| 32743 | bradykinin, hydroxyproline form- | 200 | 0.1007 | 0.144 | 158% |
| 32671 | Metabolite-11354 | 200 | 0.1009 | 0.144 | -13% |
| 25459 | Metabolite-10395 | 50 | 0.1012 | 0.144 | -14% |
| 12067 | undecanoate | 201 | 0.1035 | 0.1452 | -5% |
| 18868 | Metabolite-5847 | 50 | 0.1035 | 0.1452 | 70% |
| 21762 | Metabolite-8506 | 61 | 0.1759 | 0.1569 | -16% |
| 18706 | Metabolite-5769 | 61 | 2.09E-06 | 0.0004 | 80% |
| 12644 | Metabolite-3016 | 50 | 2.08E-05 | 0.0021 | 30% |
| 12751 | Metabolite-3073 | 50 | 0.0001 | 0.0043 | 102% |
| 24285 | Metabolite-10026 | 61 | 0.0001 | 0.0031 | 64% |
| 1125 | isoleucine | 50 | 0.0002 | 0.0076 | 48% |
| 18882 | Isobar-47-includes-taurochenodeoxycholic acid-and-taurodeoxycholic acid | 61 | 0.0003 | 0.0076 | 309% |
| 18476 | glycocholate | 61 | 0.0004 | 0.0092 | 248% |
| 32322 | glutamate | 50 | 0.0005 | 0.0384 | -91% |
| 32497 | 10c-undecenoate | 201 | 0.0005 | 0.0384 | 11% |
| 31489 | Metabolite-10914 | 50 | 0.0006 | 0.0384 | 47% |
| 33073 | Metabolite-11728 | 200 | 0.0006 | 0.0384 | 53% |
| 12658 | Metabolite-3026 | 50 | 0.0007 | 0.0136 | 30% |
| 33386 | Metabolite-12035 | 50 | 0.0007 | 0.0384 | -80% |
| 584 | mannose | 50 | 0.001 | 0.0177 | -25% |
| 1299 | tyrosine | 200 | 0.0011 | 0.0499 | -27% |
| 17068 | Metabolite-4627 | 61 | 0.0012 | 0.0203 | 263% |
| 12780 | Metabolite-3098 | 50 | 0.0015 | 0.0208 | 34% |
| 60 | leucine | 50 | 0.0015 | 0.0208 | -13% |
| 18118 | Metabolite-5346 | 50 | 0.0016 | 0.0208 | 42% |
| 32393 | glutamylvaline | 200 | 0.0016 | 0.0577 | -28% |
| 2734 | gamma-glutamyltyrosine | 200 | 0.0018 | 0.0577 | -24% |
| 11923 | Metabolite-2821 | 61 | 0.0019 | 0.0227 | 38% |
| 27801 | Metabolite-10589 | 61 | 0.002 | 0.0231 | 80% |
| 32846 | Metabolite-11529 | 201 | 0.002 | 0.0577 | -172% |
| 32701 | urate- | 200 | 0.0021 | 0.0577 | -23% |
| 10672 | Metabolite-2390 | 61 | 0.0023 | 0.0245 | 57% |
| 1649 | valine | 50 | 0.0024 | 0.0247 | 37% |
| 32749 | Metabolite-11432 | 201 | 0.003 | 0.0722 | -106% |
| 13142 | Metabolite-3165 | 61 | 0.0031 | 0.029 | 25% |
| 7650 | Metabolite-1834 | 61 | 0.0035 | 0.029 | 144% |
| 10715 | Metabolite-2395 | 61 | 0.0035 | 0.029 | 661% |
| 10551 | Metabolite-2347 | 61 | 0.0036 | 0.029 | 154% |
| 22133 | DL-hexanoyl-carnitine | 61 | 0.0036 | 0.029 | 317% |
| 59 | histidine | 201 | 0.0036 | 0.0804 | 14% |
| 16337 | Metabolite-4167 | 61 | 0.0038 | 0.029 | 66% |
| 22803 | Isobar-66-includes-glycochenodeoxycholic acid-glycodeoxycholic acid | 61 | 0.0039 | 0.029 | 194% |
| 10737 | Isobar-1-includes-mannose-fructose-glucose-galactose-alpha-L-sorbopyranose-Inos itol-D-allose-D--altrose-D-psicone-L--gulose-allo-inositol | 61 | 0.004 | 0.029 | 13% |
| 25602 | Metabolite-10432 | 50 | 0.0044 | 0.0867 | -62% |
| 12770 | Metabolite-3090 | 50 | 0.0045 | 0.0867 | -67% |
| 9491 | Metabolite-2185 | 61 | 0.0048 | 0.0338 | 50% |
| 606 | uridine | 201 | 0.0049 | 0.0882 | 22% |
| 12656 | Metabolite-3025 | 50 | 0.0052 | 0.0354 | 22% |
| 32776 | Metabolite-11459 | 200 | 0.006 | 0.0975 | -40% |
| 21047 | 3-methyl-2-oxobutyric acid | 61 | 0.0062 | 0.0409 | 20% |
| 32748 | Metabolite-11431 | 201 | 0.0062 | 0.0975 | -82% |
| 32641 | Metabolite-11324 | 201 | 0.0065 | 0.0975 | 29% |
| 18369 | gamma-glutamylleucine | 61 | 0.0065 | 0.0414 | -18% |
| 22600 | Metabolite-9043 | 50 | 0.0068 | 0.0414 | -51% |
| 1126 | alanine | 50 | 0.0071 | 0.0414 | 26% |
| 32412 | butyrylcarnitine | 200 | 0.0073 | 0.1038 | -38% |
| 16511 | Metabolite-4274 | 50 | 0.0089 | 0.0489 | 39% |
| 17304 | Metabolite-4759 | 61 | 0.0094 | 0.0503 | 48% |
| 17028 | Metabolite-4611 | 50 | 0.0095 | 0.1279 | -31% |
| 32548 | Metabolite-11231 | 201 | 0.0102 | 0.1281 | -75% |
| 13214 | Metabolite-3183 | 61 | 0.0109 | 0.0524 | 28% |
| 30821 | Metabolite-10812 | 50 | 0.0112 | 0.0524 | -20% |
| 16016 | Metabolite-3994 | 61 | 0.0113 | 0.0524 | -45% |
| 21762 | Metabolite-8506 | 61 | 0.0118 | 0.0536 | 50% |
| 33362 | gamma-glutamylphenylalanine- | 200 | 0.0122 | 0.1281 | -23% |
| 9130 | Metabolite-2139 | 61 | 0.0126 | 0.0557 | 40% |
| 13600 | Metabolite-3330 | 61 | 0.013 | 0.0557 | 139% |
| 31401 | Metabolite-10892 | 50 | 0.0132 | 0.1281 | -64% |
| 32552 | Metabolite-11235 | 201 | 0.0134 | 0.1281 | -53% |
| 64 | phenylalanine | 61 | 0.0134 | 0.0557 | -9% |
| 32564 | Metabolite-11247 | 201 | 0.0135 | 0.1281 | 62% |
| 19490 | Metabolite-6488 | 50 | 0.0139 | 0.1281 | -47% |
| 32578 | Metabolite-11261 | 200 | 0.0144 | 0.1281 | -54% |
| 16308 | Metabolite-4147 | 50 | 0.0145 | 0.1281 | -48% |
| 32754 | Metabolite-11437 | 201 | 0.0148 | 0.1281 | 50% |
| 32547 | Metabolite-11230 | 201 | 0.0151 | 0.1281 | -83% |
| 32808 | Metabolite-11491 | 201 | 0.0154 | 0.1281 | -86% |
| 12783 | Metabolite-3101 | 50 | 0.0158 | 0.1281 | -39% |
| 1769 | cortisone | 201 | 0.0162 | 0.1281 | 28% |
| 32945 | Metabolite-11628 | 201 | 0.0179 | 0.1339 | -68% |
| 2761 | thyroxine | 61 | 0.018 | 0.0734 | 31% |
| 12647 | Metabolite-3019 | 50 | 0.0184 | 0.0734 | 17% |
| 12763 | Metabolite-3083 | 50 | 0.0186 | 0.1339 | -36% |
| 32881 | Metabolite-11564 | 201 | 0.0186 | 0.1339 | -26% |
| 15500 | carnitine | 200 | 0.0189 | 0.1339 | -11% |
| 16865 | Metabolite-4522 | 50 | 0.0192 | 0.075 | 22% |
| 1494 | 5-oxoproline | 50 | 0.0197 | 0.075 | 20% |
| 30805 | Metabolite-10810 | 50 | 0.0198 | 0.1349 | -75% |
| 32753 | Metabolite-09789 | 201 | 0.0201 | 0.1349 | 51% |
| 1642 | caprate-10-0- | 201 | 0.0219 | 0.1425 | 7% |
| 32854 | Metabolite-11537 | 200 | 0.0223 | 0.1425 | 46% |
| 32863 | Metabolite-11546 | 201 | 0.0257 | 0.1608 | -116% |
| 9905 | Metabolite-2231 | 61 | 0.0262 | 0.0921 | 18% |
| 12790 | Metabolite-3108 | 50 | 0.0276 | 0.1669 | -26% |
| 527 | lactate | 50 | 0.0276 | 0.0952 | -20% |
| 32926 | Metabolite-11609 | 201 | 0.028 | 0.1669 | -44% |
| 8509 | Metabolite-2041 | 61 | 0.0298 | 0.0996 | 22% |
| 8210 | Metabolite-1981 | 61 | 0.0309 | 0.1016 | -35% |
| 32519 | Metabolite-11205 | 200 | 0.0312 | 0.1764 | 22% |
| 32813 | Metabolite-11496 | 201 | 0.0347 | 0.1875 | 16% |
| 32698 | Metabolite-11381_200 | 200 | 0.036 | 0.1875 | -22% |
| 32631 | Metabolite-11314 | 200 | 0.0365 | 0.1875 | -11% |
| 32517 | Metabolite-11203 | 200 | 0.037 | 0.1875 | 26% |
| 32807 | Metabolite-11490 | 201 | 0.04 | 0.1972 | -125% |
| 18497 | taurocholate | 201 | 0.0404 | 0.1972 | -115% |
| 15140 | L-kynurenine | 61 | 0.0407 | 0.1275 | 24% |
| 12782 | Metabolite-3100 | 50 | 0.0422 | 0.1978 | -49% |
| 27727 | glutathione, oxidized (GSSG) | 200 | 0.0426 | 0.1978 | 72% |
| 32621 | Metabolite-11304 | 200 | 0.0427 | 0.1978 | -43% |
| 13557 | Metabolite-3323 | 61 | 0.0447 | 0.135 | 68% |
| 31595 | Metabolite-10951 | 61 | 0.0451 | 0.135 | 27% |
| 20699 | erythritol | 50 | 0.0452 | 0.2057 | -23% |
| 32878 | Metabolite-11561 | 201 | 0.0477 | 0.2137 | -32% |
| 1113 | isocitrate | 61 | 0.0497 | 0.144 | 34% |
| 32910 | Metabolite-11593 | 201 | 0.0507 | 0.2233 | -20% |
| 17390 | Metabolite-4806 | 50 | 0.0519 | 0.1462 | 27% |
| 32752 | Metabolite-11435 | 201 | 0.0533 | 0.2263 | -41% |
| 1647 | glutamine | 50 | 0.0534 | 0.147 | 21% |
| 33242 | Metabolite-11897 | 201 | 0.0544 | 0.2263 | -38% |
| 32452 | propionylcarnitine | 200 | 0.0545 | 0.2263 | -27% |
| 32978 | Metabolite-11656 | 200 | 0.0547 | 0.2263 | 6% |
| 27710 | N-acetylglycine | 50 | 0.0581 | 0.155 | -29% |
| 12785 | Metabolite-3103 | 50 | 0.0581 | 0.2331 | -48% |
| 33228 | Metabolite-11883 | 200 | 0.0581 | 0.2331 | 18% |
| 1336 | palmitate | 50 | 0.0586 | 0.155 | 14% |
| 594 | nicotinamide | 200 | 0.0598 | 0.2337 | 16% |
| 15996 | aspartate | 50 | 0.0606 | 0.2337 | -29% |
| 10245 | Metabolite-2269- | 61 | 0.0622 | 0.1602 | -42% |
| 33131 | Metabolite-11786 | 200 | 0.0626 | 0.2367 | 33% |
| 32559 | Metabolite-11242 | 201 | 0.0641 | 0.2372 | -27% |
| 15529 | Metabolite-3951 | 61 | 0.0658 | 0.1647 | 22% |
| 5687 | Metabolite-1110 | 61 | 0.0702 | 0.1672 | -43% |
| 19370 | Metabolite-6268 | 50 | 0.0707 | 0.1672 | 25% |
| 32549 | Metabolite-02269 | 201 | 0.0733 | 0.2653 | 44% |
| 12789 | Metabolite-3107 | 50 | 0.0741 | 0.2653 | -59% |
| 528 | alpha-keto-glutarate | 61 | 0.0742 | 0.1717 | -27% |
| 31510 | Metabolite-10932 | 61 | 0.0764 | 0.1746 | 21% |
| 19985 | Metabolite-6957 | 50 | 0.0799 | 0.2678 | -20% |
| 10247 | Metabolite-2270 | 61 | 0.0802 | 0.1773 | -64% |
| 18868 | Metabolite-5847 | 50 | 0.0804 | 0.2678 | -67% |
| 32675 | Metabolite-03951_200 | 200 | 0.0812 | 0.2678 | -15% |
| 20488 | glucose | 50 | 0.0819 | 0.1773 | 11% |
| 18335 | quinate | 50 | 0.0822 | 0.2678 | 20% |
| 15676 | 3-methyl-2-oxovalerate | 201 | 0.0827 | 0.2678 | -17% |
| 25601 | Metabolite-10431 | 50 | 0.0828 | 0.2678 | -41% |
| 12593 | Metabolite-2973 | 50 | 0.0852 | 0.2678 | -22% |
| 32838 | Metabolite-11521 | 200 | 0.0856 | 0.2678 | -35% |
| 19397 | Metabolite-6326 | 50 | 0.086 | 0.1815 | 19% |
| 31787 | 3-carboxyl-4-methyl-5-propyl-2-furanpropanoic acid | 201 | 0.0861 | 0.2678 | 53% |
| 12604 | Metabolite-2981 | 50 | 0.0864 | 0.2678 | -15% |
| 21631 | Metabolite-8403 | 50 | 0.0885 | 0.2678 | -26% |
| 1515 | salicylic acid | 50 | 0.0887 | 0.2678 | -140% |
| 1301 | lysine | 200 | 0.09 | 0.2687 | -14% |
| 32511 | EDTA* | 201 | 0.0942 | 0.2759 | -7% |
| 32971 | Metabolite-11654 | 200 | 0.0945 | 0.2759 | -36% |
| 12767 | Metabolite-3087 | 50 | 0.096 | 0.1954 | 35% |
| 32795 | Metabolite-11478 | 201 | 0.0982 | 0.2796 | -35% |
| 32793 | Metabolite-11476 | 200 | 0.0988 | 0.2796 | 6% |
| 21762 | Metabolite-8506 | 61 | 0.0006 | 0.1449 | -44% |
| 528 | alpha-keto-glutarate | 61 | 0.0017 | 0.1921 | 75% |
| 6847 | Metabolite-1496 | 61 | 0.0025 | 0.1921 | 49% |
| 16650 | Metabolite-4360 | 50 | 0.0033 | 0.1921 | -144% |
| 18591 | Metabolite-5646 | 61 | 0.0041 | 0.1921 | -53% |
| 27718 | creatine | 200 | 0.0071 | 1 | -50% |
| 599 | pyruvate | 61 | 0.0087 | 0.253 | -42% |
| 32621 | Metabolite-11304 | 200 | 0.0124 | 1 | 31% |
| 1113 | isocitrate | 61 | 0.0151 | 0.3249 | 38% |
| 15506 | choline | 61 | 0.0163 | 0.3249 | -28% |
| 32729 | Metabolite-11412 | 200 | 0.0166 | 1 | -14% |
| 10501 | Metabolite-2321 | 61 | 0.0183 | 0.3249 | -40% |
| 12644 | Metabolite-3016 | 50 | 0.0186 | 0.3249 | -13% |
| 32735 | Metabolite-01911 | 200 | 0.0199 | 1 | -99% |
| 15753 | hippurate | 201 | 0.0255 | 1 | 44% |
| 30805 | Metabolite-10810 | 50 | 0.0302 | 1 | 37% |
| 12768 | Metabolite-3088 | 50 | 0.0305 | 0.4178 | -25% |
| 10629 | Metabolite-2386 | 61 | 0.0335 | 0.4178 | 25% |
| 33209 | Metabolite-11864 | 201 | 0.0376 | 1 | 45% |
| 32855 | Metabolite-11538 | 201 | 0.0429 | 1 | 34% |
| 32416 | alpha linolenate (18:3(n-3)) | 201 | 0.0486 | 1 | 32% |
| 16518 | Metabolite-4276 | 50 | 0.0499 | 0.481 | 63% |
| 20267 | Metabolite-7187 | 61 | 0.0521 | 0.481 | 37% |
| 19462 | Metabolite-6446 | 50 | 0.0552 | 0.481 | 22% |
| 33420 | gamma-tocopherol- | 50 | 0.0556 | 1 | -54% |
| 1515 | salicylic acid | 50 | 0.0569 | 1 | 60% |
| 32567 | Metabolite-11250 | 200 | 0.0573 | 1 | 40% |
| 32769 | Metabolite-11452 | 201 | 0.0608 | 1 | -79% |
| 12774 | Metabolite-3094 | 50 | 0.0625 | 1 | -24% |
| 32632 | Metabolite-11315 | 200 | 0.0651 | 1 | 21% |
| 32741 | Metabolite-11424 | 200 | 0.0655 | 1 | -13% |
| 12658 | Metabolite-3026 | 50 | 0.0658 | 0.525 | -12% |
| 32110 | Metabolite-11086 | 50 | 0.0659 | 1 | 17% |
| 30728 | Metabolite-10797 | 61 | 0.0693 | 0.525 | 65% |
| 32625 | Metabolite-11308 | 201 | 0.0697 | 1 | 27% |
| 15140 | kynurenine | 200 | 0.0702 | 1 | -14% |
| 12645 | Metabolite-3017 | 50 | 0.0709 | 0.525 | -22% |
| 33154 | Metabolite-11809 | 200 | 0.0711 | 1 | 19% |
| 12067 | undecanoate | 201 | 0.072 | 1 | 6% |
| 5628 | Metabolite-1086 | 61 | 0.0723 | 0.525 | 45% |
| 19402 | Metabolite-6346 | 50 | 0.077 | 1 | -20% |
| 32631 | Metabolite-11314 | 200 | 0.0779 | 1 | 8% |
| 7933 | Metabolite-1911 | 61 | 0.0792 | 0.5308 | 75% |
| 32797 | Metabolite-11480 | 201 | 0.0818 | 1 | -213% |
| 18254 | paraxanthine | 200 | 0.0824 | 1 | -69% |
| 32912 | Metabolite-11595 | 201 | 0.0834 | 1 | 25% |
| 32717 | Metabolite-11400 | 200 | 0.0855 | 1 | 39% |
| 33133 | Metabolite-11788 | 200 | 0.0862 | 1 | -11% |
| 22261 | Isobar-60-includes-s-2-hydroxybutyric acid-2-hydroxyisobutyric acid | 61 | 0.0869 | 0.5308 | 42% |
| 19374 | Metabolite-6270 | 50 | 0.09 | 1 | 27% |
| 6398 | Metabolite-1335 | 61 | 0.0908 | 0.5308 | 28% |
| 12770 | Metabolite-3090 | 50 | 0.0919 | 1 | 28% |
| 18392 | theobromine | 200 | 0.0956 | 1 | -118% |
| 32978 | Metabolite-11656 | 200 | 0.0961 | 1 | -6% |
| 32761 | Metabolite-11444 | 201 | 0.0962 | 1 | -11% |
| 31548 | peptide-DSGEGDFXAEGGGVR | 200 | 0.0981 | 1 | -323% |
| 3141 | betaine | 200 | 0.0986 | 1 | 14% |
| 606 | uridine | 201 | 0.0987 | 1 | -17% |
| 24233 | Metabolite-9855 | 61 | 0.0996 | 0.5308 | 99% |
Random forest analyses were used for classification of samples into groups (e.g. disease or healthy, steatosis or healthy, steatohepatitis or healthy, steatosis or NASH). 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.
Random forest results show that the samples can be classified correctly with varying degrees of accuracy using the biomarkers listed in Tables 1, 2, and/or 3. The confusion matrices demonstrate that using plasma samples steatosis subjects, steatohepatitis subjects and control subjects can be distinguished (Table4), steatosis subjects can be distinguished from control subjects (Table 5), steatohepatitis subjects can be distinguished from control subjects (Table 6) and steatosis subjects can be distinguished from steatohepatitis subjects (Table 7). 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 steatosis or a control subject).
Table 4. Confusion Matrix showing Control subjects, Steatosis subjects and NASH subjects can be distinguished from each other.
| Control_ Predicted | Steatosis_ Predicted | NASH_ Predicted | Error | |
| Control | 23 | 0 | 2 | 8% |
| Steatosis | 3 | 9 | 12 | 63% |
| NASH | 2 | 4 | 5 | 55% |
| Out_of_bag_error | 38% | |||
Based on the OOB Error rate of 38%, the Random Forest model that was created could be used to predict whether a subject has steatosis, steatohepatitis, or no fatty liver disease with about 62% accuracy from analysis of the levels of the biomarkers in samples from the subject. The biomarkers that are the most important biomarkers for distinguishing the groups are listed in Table 4B and displayed in Figure 3 .
Table 4B. Most important biomarkers to distinguish control, steatosis and steatohepatitis subjects from Random Forest Importance Plot shown in Figure 3.
Table 5. Confusion Matrix showing Control subjects can be distinguished from Steatosis subjects.
| 2734 | gamma-glutamyltyrosine |
| 32322 | glutamate |
| 33073 | Metabolite - 11728 |
| 32412 | butyrylcarnitine |
| 18497 | taurocholate |
| 32393 | glutamylvaline |
| 1299 | tyrosine |
| 32739 | Metabolite - 11422 |
| 15140 | kynurenine |
| 32868 | glycocholate* |
| 33420 | gamma-tocopherol- |
| 32748 | Metabolite - 11431 |
| 27718 | creatine |
| 31489 | Metabolite - 10914 |
| 32552 | Metabolite - 11235 |
| 15753 | hippurate |
| 32846 | Metabolite - 11529 |
| 17028 | Metabolite - 4611 |
| 32735 | Metabolite - 01911_200 |
| 32808 | Metabolite - 11491 |
| 15990 | glycerophosphorylcholine (GPC) |
| 32701 | urate- |
| 33362 | gamma-glutamylphenylalanine- |
| 32547 | Metabolite - 11230 |
| 32497 | 10c-undecenoate |
| 32632 | Metabolite - 11315 |
| 59 | histidine |
| 32621 | Metabolite - 11304 |
| 32749 | Metabolite - 11432 |
| 32559 | Metabolite - 11242 |
| 1126 | alanine |
| 1113 | isocitrate |
| 1125 | isoleucine |
| 527 | lactate |
| 60 | leucine |
| 584 | mannose |
| 24285 | Metabolite - 10026 |
| 30821 | Metabolite - 10812 |
| 6847 | Metabolite - 1496 |
| 7933 | Metabolite - 1911 |
| 10715 | Metabolite - 2395 |
| 12644 | Metabolite - 3016 |
| 12658 | Metabolite - 3026 |
| 12751 | Metabolite - 3073 |
| 12780 | Metabolite - 3098 |
| 13600 | Metabolite - 3330 |
| 16511 | Metabolite - 4274 |
| 18706 | Metabolite - 5769 |
| 20267 | Metabolite - 7187 |
| 21762 | Metabolite - 8506 |
| 24233 | Metabolite - 9855 |
| 18392 | theobromine |
| 2761 | thryoxine |
| 1604 | urate |
| 1649 | valine |
| 3147 | xanthine |
| Control-Predicted | Steatosis-Predicted | Error | |
| Control | 22 | 3 | 12% |
| Steatosis | 1 | 10 | 9% |
| Out_of_bag_error | 11% | ||
Based on the OOB Error rate of 11%, the Random Forest model that was created could be used to predict whether a subject has steatosis or no fatty liver disease with about 89% accuracy from analysis of the levels of the biomarkers in samples from the subject. The biomarkers that are the most important biomarkers for distinguishing the groups are listed in Table 5B and displayed in Figure 1 .
Table 5 B. Most important biomarkers from Random Forest Importance Plot shown in Figure 1.
Table 6. Confusion Matrix showing Control subjects can be distinguished from NASH subjects.
| 2734 | gamma-glutamyltyrosine |
| 18497 | taurocholate |
| 32412 | butyrylcarnitine |
| 32552 | Metabolite - 11235 |
| 1299 | tyrosine |
| 606 | uridine |
| 32322 | glutamate |
| 32621 | Metabolite - 11304 |
| 17028 | Metabolite - 4611 |
| 19490 | Metabolite - 6488 |
| 32497 | 10c-undecenoate |
| 32748 | Metabolite - 11431 |
| 12789 | Metabolite - 3107 |
| 32393 | glutamylvaline |
| 32547 | Metabolite - 11230 |
| 12763 | Metabolite - 3083 |
| 32808 | Metabolite - 11491 |
| 25602 | Metabolite - 10432 |
| 32739 | Metabolite - 11422 |
| 33073 | Metabolite - 11728 |
| 31489 | Metabolite - 10914 |
| 32749 | Metabolite - 11432 |
| 32631 | Metabolite - 11314 |
| 33386 | Metabolite - 12035 |
| 32559 | Metabolite - 11242 |
| 32846 | Metabolite - 11529 |
| 33242 | Metabolite - 11897 |
| 33362 | gamma-glutamylphenylalanine- |
| 32945 | Metabolite - 11628 |
| 12790 | Metabolite - 3108 |
| 1126 | alanine |
| 18476 | glycocholate |
| Isobar 47 (taurochenodeoxycholic acid, taurodeoxycholic acid) | |
| Isobar 66 (glycochenodeoxycholic acid, glychodeoxycholic acid) | |
| 57 | lactate |
| 584 | mannose |
| 24285 | Metabolite-10026 |
| 31595 | Metabolite-10951 |
| 10551 | Metabolite-2347 |
| 11923 | Metabolite-2821 |
| 12644 | Metabolite-3016 |
| 12647 | Metabolite-3019 |
| 12656 | Metabolite-3025 |
| 12658 | Metabolite-3026 |
| 12751 | Metabolite-3073 |
| Metabolite-3077 | |
| 12780 | Metabolite-3098 |
| 13142 | Metabolite-3165 |
| 13600 | Metabolite-3330 |
| 16337 | Metabolite-4167 |
| Metabolite-4361 | |
| 17304 | Metabolite-4759 |
| 17390 | Metabolite-4806 |
| 18118 | Metabolite-5346 |
| 18706 | Metabolite-5769 |
| 21762 | Metabolite-8506 |
| 2761 | thyroxine |
| 1604 | urate |
| Control-Predicted | NASH-Predicted | Error | |
| Control | 23 | 2 | 8% |
| NASH | 2 | 22 | 8% |
| Out_of_bag_error | 8% | ||
Based on the OOB Error rate of 8%, the Random Forest model that was created could be used to predict whether a subject has steatohepatitis or no fatty liver disease with about 92% accuracy from analysis of the levels of the biomarkers in samples from the subject. The biomarkers that are the most important biomarkers for distinguishing the groups are listed in Table 6B and displayed in the importance plot in Figure 2 .
Table 6B. Most important biomarkers from Random Forest Importance Plot shown in Figure 2.
Table 7. Confusion Matrix showing Steatosis subjects can be distinguished from steatohepatitis (NASH) subjects.
| 33073 | Metabolite - 11728 |
| 15140 | kynurenine |
| 32868 | glycocholate* |
| 32412 | butyrylcarnitine |
| 32322 | glutamate |
| 2734 | gamma-glutamyltyrosine |
| 15753 | hippurate |
| 31489 | Metabolite - 10914 |
| 32739 | Metabolite - 11422 |
| 33420 | gamma-tocopherol- |
| 32881 | Metabolite - 11564 |
| 32393 | glutamylvaline |
| 32519 | Metabolite - 11205 |
| 32748 | Metabolite - 11431 |
| 32701 | urate- |
| 32550 | Metabolite - 02272_201 |
| 1299 | tyrosine |
| 32548 | Metabolite - 11231 |
| 33362 | gamma-glutamylphenylalanine- |
| 584 | mannose |
| 32675 | Metabolite - 03951_200 |
| 32632 | Metabolite - 11315 |
| 32846 | Metabolite - 11529 |
| 17028 | Metabolite - 4611 |
| 32497 | 10c-undecenoate |
| 32749 | Metabolite - 11432 |
| 16308 | Metabolite - 4147 |
| 32544 | Metabolite - 11227 |
| 18497 | taurocholate |
| 32697 | Metabolite - 11380 |
| 21047 | 3-methyl-2-oxobutyric acid |
| 1126 | alanine |
| 1647 | glutamine |
| 1113 | isocitrate |
| 1125 | isoleucine |
| 60 | leucine |
| 20699 | meso-erythritol |
| 24285 | Metabolite-10026 |
| 30821 | Metabolite-10812 |
| 5628 | Metabolite-1086 |
| 5687 | Metabolite-1110 |
| 6398 | Metabolite-1335 |
| 6847 | Metabolite-1496 |
| 8509 | Metabolite-2041 |
| 10286 | Metabolite-2272 |
| 10715 | Metabolite-2395 |
| 12751 | Metabolite-3073 |
| 12767 | Metabolite-3087 |
| 12780 | Metabolite-3098 |
| 16511 | Metabolite-4274 |
| 18706 | Metabolite-5769 |
| 20267 | Metabolite-7187 |
| 1649 | valine |
| 3147 | xanthine |
| Steatosis-Predicted | NASH-Predicted | Error | |
| Steatosis | 10 | 14 | 58% |
| NASH | 5 | 6 | 45% |
| Out_of_bag_error | 54% | ||
Based on the OOB Error rate of 54%, the Random Forest model that was created could be used to predict whether a subject has steatosis or steatohepatitis with about 46% accuracy from analysis of the levels of the biomarkers in samples from the subject.
The compounds that are the most important biomarkers for distinguishing the groups are shown in the importance plots in Figures 1 , 2 , 3 . Figure 1 lists the biomarker compounds that are most important in distinguishing steatosis subjects and Control subjects. Listed in Figure 2 are the biomarker compounds that are most important in distinguishing steatohepatitis subjects from control subjects. Listed in Figure 3 are the biomarker compounds that are most important in distinguishing steatosis, steatohepatitis and control subjects.
Recursive partitioning was performed to uncover the biomarkers that can best differentiate the 2 groups of subjects. It relates a 'dependent' variable (e.g. Group or Y) to a collection of independent ('predictor') variables (e.g. metabolites or 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 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 log10 of a raw p-value.
The recursive partitioning results showing the classification of Control and steatosis subjects is shown in Figure 4 . The biomarkers that contribute to the classification are listed in Table 8. The recursive partitioning results showing the classification of steatosis and steatohepatitis (NASH, SH) are shown in Figure 5 and the biomarkers used in the classification are listed in Table 9. The recursive partitioning results showing the classification of control and steatohepatitis are shown in Figure 6 and the biomarkers are listed in Table 10. The recursive partitioning results showing the use of the biomarkers to classify steatosis, steatohepatitis and control subjects are shown in Figure 7 and the biomarkers are listed in Table 11.
The sensitivity and specificity of a given biomarker was determined by the Receiver Operating Characteristic curve (or ROC curve), which plots the true positive rate against the false positive rate for the different possible cutpoints of the specific biomarker. The area under the curve is a measure of test accuracy. An area of 1 represents a perfect test while an area of 0.5 represents a worthless test. A rough guide for classifying the accuracy of a diagnostic test is the traditional academic point system:
- 90-1 = excellent
- 80-.90 = good
- 70-.80 = fair
- 60-.70 = poor
- 50-.60 = fail
ROC curves are shown in Figures 4 , 5 , 6 and 7 . The control subjects are separated from steatosis subjects with an accuracy of >90 (Figure 4 ). The steatosis and steatohepatitis subjects can be distinguished with >82% accuracy (Figure 5 ). The Control and steatohepatitis subjects are classified >87% accuracy (Figure 6 ). The three groups can be classified using the biomarkers with >70% (NASH), >77% (Steatosis), >86% accuracy (control) (Figure 7 ). Table 8. Recursive Partitioning Biomarkers, Steatosis vs control.
Table 9. RP Biomarker Candidates, steatosis vs. steatohepatitis.
Table 10. RP Biomarkers of steatohepatitis (NASH) vs Control.
Table 11. RP Biomarkers of Steatosis vs Steatohepatitis (NASH) Vs Control.
| 2734 | gamma glutamyltyrosine | 200 | 7.2110 | 29.7676 |
| 18497 | taurocholate | 201 | 4.5487 | 20.2589 |
| 32599 | Metabolite - 11282 | 201 | 3.4916 | 17.2822 |
| 20699 | erythritol | 50 | 3.4916 | 17.2822 |
| 32552 | Metabolite - 11235 | 201 | 3.3649 | 16.7903 |
| 32322 | glutamate | 50 | 3.3649 | 16.7903 |
| 32393 | glutamylvaline | 200 | 3.0618 | 15.2513 |
| 12763 | Metabolite - 3083 | 50 | 3.0147 | 15.2513 |
| 32497 | 10c undecenoate | 201 | 2.9895 | 15.3210 |
| 19490 | Metabolite - 6488 | 50 | 2.9743 | 15.2513 |
| 1299 | tyrosine | 200 | 2.9718 | 15.2513 |
| 33242 | Metabolite - 11897 | 201 | 2.9718 | 15.2513 |
| 32748 | Metabolite - 11431 | 201 | 2.9601 | 15.2052 |
| 32621 | Metabolite - 11304 | 200 | 2.8266 | 14.6774 |
| 606 | uridine | 201 | 2.8266 | 14.6774 |
| 17028 | Metabolite - 4611 | 50 | 2.8266 | 14.6774 |
| 12789 | Metabolite - 3107 | 50 | 2.4680 | 12.6627 |
| 32808 | Metabolite - 11491 | 201 | 2.3944 | 12.6627 |
| 32412 | butyrylcarnitine | 200 | 2.3372 | 12.6627 |
| 32547 | Metabolite - 11230 | 201 | 2.3242 | 12.6627 |
| 12790 | Metabolite - 3108 | 50 | 2.2779 | 12.4746 |
| 33073 | Metabolite - 11728 | 200 | 2.2201 | 12.1291 |
| 32739 | Metabolite - 11422 | 201 | 2.1932 | 12.1291 |
| 30805 | Metabolite - 10810 | 50 | 2.1932 | 12.1291 |
| 32776 | Metabolite - 11459 | 200 | 2.1488 | 11.9121 |
| 32697 | Metabolite - 11380 | 200 | 2.1443 | 11.9121 |
| 32701 | urate | 200 | 2.1403 | 11.9121 |
| 32559 | Metabolite - 11242 | 201 | 2.1279 | 11.8611 |
| 32631 | Metabolite - 11314 | 200 | 2.1279 | 11.8611 |
| 32945 | Metabolite - 11628 | 201 | 2.1279 | 11.8611 |
| 33386 | Metabolite - 12035 | 50 | 2.0266 | 11.2117 |
| 27718 | creatine | 200 | 2.8020 | 14.5688 |
| 12067 | undecanoate | 201 | 2.7871 | 14.5095 |
| 32729 | Metabolite - 11412 | 200 | 2.5472 | 13.5499 |
| 32855 | Metabolite - 11538 | 201 | 2.5472 | 13.5499 |
| 32735 | Metabolite - 01911_200 | 200 | 2.4708 | 13.0756 |
| 32621 | Metabolite - 11304 | 200 | 2.1940 | 12.1162 |
| 27275 | Metabolite - 10507 | 50 | 2.0579 | 11.5561 |
| 33073 | Metabolite - 11728 | 200 | 7.3726 | 31.8519 |
| 32868 | glycocholate | 201 | 6.5169 | 28.4230 |
| 15140 | kynurenine | 200 | 6.4082 | 27.7714 |
| 2734 | gamma glutamyltyrosine | 200 | 5.5940 | 24.5076 |
| 32412 | butyrylcarnitine | 200 | 5.4888 | 24.6395 |
| 15753 | hippurate | 201 | 5.2301 | 23.5921 |
| 32322 | glutamate | 50 | 4.5652 | 21.1976 |
| 33420 | gamma tocopherol | 50 | 4.5330 | 21.1976 |
| 32748 | Metabolite - 11431 | 201 | 4.3254 | 20.3263 |
| 32739 | Metabolite - 11422 | 201 | 4.2214 | 19.9467 |
| 32393 | glutamylvaline | 200 | 3.9888 | 19.1924 |
| 32550 | Metabolite - 02272 | 201 | 3.9860 | 19.0839 |
| 31489 | Metabolite - 10914 | 50 | 3.8423 | 18.5637 |
| 32519 | Metabolite - 11205 | 200 | 3.8117 | 18.4417 |
| 33133 | Metabolite - 11788 | 200 | 3.6919 | 17.9988 |
| 32632 | Metabolite - 11315 | 200 | 3.6913 | 17.9988 |
| 32548 | Metabolite - 11231 | 201 | 3.6260 | 17.7540 |
| 16308 | Metabolite - 4147 | 50 | 3.6255 | 17.7540 |
| 32881 | Metabolite - 11564 | 201 | 3.5761 | 17.5685 |
| 32675 | Metabolite - 03951 | 200 | 3.5752 | 17.5685 |
| 32697 | Metabolite - 11380 | 200 | 3.4581 | 17.1290 |
| 32863 | Metabolite - 11546 | 201 | 3.4182 | 16.9876 |
| 17028 | Metabolite - 4611 | 50 | 3.3802 | 16.8375 |
| 584 | mannose | 50 | 3.3005 | 16.5389 |
| 33362 | gamma glutamylphenylalanine | 200 | 3.1967 | 16.1427 |
| 32749 | Metabolite - 11432 | 201 | 3.1952 | 16.1427 |
| 32701 | urate | 200 | 3.1486 | 15.9669 |
| 32846 | Metabolite - 11529 | 201 | 3.1224 | 15.8433 |
| 1299 | tyrosine | 200 | 3.1159 | 15.8433 |
| 32544 | Metabolite - 11227 | 201 | 3.1019 | 15.7905 |
| 32564 | Metabolite - 11247 | 201 | 2.9040 | 15.0281 |
| 32517 | Metabolite - 11203 | 200 | 2.9012 | 15.0281 |
| 32808 | Metabolite - 11491 | 201 | 2.8993 | 14.9830 |
| 33012 | Metabolite - 11674 | 200 | 2.8909 | 14.9830 |
| 31787 | 3-carboxyl-4-methyl-5-propyl-2-furanpropanoic acid | 201 | 2.8894 | 14.9830 |
| 20699 | erythritol | 50 | 2.8894 | 14.9830 |
| 32110 | Metabolite - 11086 | 50 | 2.8894 | 14.9830 |
| 12783 | Metabolite - 3101 | 50 | 2.8894 | 14.9830 |
| 19402 | Metabolite - 6346 | 50 | 2.8894 | 14.9830 |
| 18497 | taurocholate | 201 | 2.6891 | 13.9546 |
| 59 | histidine | 201 | 2.6508 | 14.0671 |
| 12774 | Metabolite - 3094 | 50 | 2.6508 | 14.0671 |
| 32682 | Metabolite - 11365 | 201 | 2.6217 | 13.9546 |
| 33227 | Metabolite - 11882 | 201 | 2.5727 | 13.7651 |
| 15990 | glycerophosphorylcholine (GPC) | 200 | 2.5636 | 13.7298 |
| 32547 | Metabolite - 11230 | 201 | 2.5131 | 13.5334 |
| 32497 | X10c undecanoate | 201 | 2.4846 | 13.4226 |
| 32549 | Metabolite - 02269 | 201 | 2.4738 | 13.3217 |
| 32552 | Metabolite - 11235 | 201 | 2.4690 | 13.3619 |
| 32385 | Metabolite - 11180 | 50 | 2.4078 | 13.0513 |
| 32621 | Metabolite - 11304 | 200 | 2.3922 | 13.0617 |
| 20488 | glucose | 50 | 2.3922 | 13.0617 |
| 16865 | Metabolite - 4522 | 50 | 2.3036 | 12.7022 |
| 64 | phenylalanine | 200 | 2.2026 | 12.3145 |
| 32637 | Metabolite - 11320 | 201 | 2.1916 | 12.2709 |
| 32813 | Metabolite - 11496 | 201 | 2.1916 | 12.2709 |
| 32854 | Metabolite - 11537 | 200 | 2.1916 | 12.2709 |
| 27256 | Metabolite - 10500 | 50 | 2.1916 | 12.2709 |
| 32346 | glycochenodeoxycholate | 201 | 2.1443 | 11.9681 |
| 32656 | Metabolite - 11339 | 201 | 2.0482 | 11.6993 |
| 16650 | Metabolite - 4360 | 50 | 2.0204 | 11.5103 |
| 607 | urocanate | 200 | 2.0135 | 10.2237 |
| 32559 | Metabolite - 11242 | 201 | 2.0012 | 11.5103 |
| 2734 | gamma glutamyltyrosine | 200 | 8.7610 | 39.6669 |
| 33073 | Metabolite - 11728 | 200 | 6.9137 | 33.1250 |
| 32868 | glycocholate | 201 | 5.6594 | 28.4476 |
| 32412 | butyrylcarnitine | 200 | 5.5221 | 27.9670 |
| 15140 | kynurenine | 200 | 5.4648 | 27.7794 |
| 32322 | glutamate | 50 | 5.4202 | 27.6254 |
| 18497 | taurocholate | 201 | 4.8465 | 25.6695 |
| 32748 | Metabolite - 11431 | 201 | 4.5458 | 24.5681 |
| 32393 | glutamylvaline | 200 | 4.4325 | 24.1102 |
| 1299 | tyrosine | 200 | 4.4170 | 24.1102 |
| 15753 | hippurate | 201 | 4.2727 | 23.5947 |
| 32739 | Metabolite - 11422 | 201 | 3.9941 | 22.5914 |
| 17028 | Metabolite - 4611 | 50 | 3.7736 | 21.7885 |
| 33420 | gamma tocopherol | 50 | 3.6961 | 21.4058 |
| 20699 | erythritol | 50 | 3.4424 | 20.5670 |
| 31489 | Metabolite - 10914 | 50 | 3.4147 | 20.4362 |
| 32552 | Metabolite - 11235 | 201 | 3.4135 | 20.4592 |
| 32697 | Metabolite - 11380 | 200 | 3.2207 | 19.7221 |
| 32519 | Metabolite - 11205 | 200 | 3.2148 | 19.7148 |
| 32749 | Metabolite - 11432 | 201 | 3.1735 | 19.5592 |
| 33362 | gamma glutamylphenylalanine | 200 | 3.1317 | 19.2619 |
| 32846 | Metabolite - 11529 | 201 | 3.1175 | 19.1830 |
| 32550 | Metabolite - 02272_201 | 201 | 3.0781 | 19.1976 |
| 32808 | Metabolite - 11491 | 201 | 2.9873 | 18.6852 |
| 32547 | Metabolite - 11230 | 201 | 2.9438 | 18.6852 |
| 16308 | Metabolite - 4147 | 50 | 2.8966 | 18.4875 |
| 32548 | Metabolite - 11231 | 201 | 2.8923 | 18.4875 |
| 33133 | Metabolite - 11788 | 200 | 2.8602 | 18.3636 |
| 32735 | Metabolite - 01911_200 | 200 | 2.8564 | 18.1394 |
| 32863 | Metabolite - 11546 | 201 | 2.8379 | 18.1709 |
| 32881 | Metabolite - 11564 | 201 | 2.8103 | 18.1709 |
| 33242 | Metabolite - 11897 | 201 | 2.8094 | 18.1676 |
| 32675 | Metabolite - 03951_200 | 200 | 2.7808 | 18.0072 |
| 59 | histidine | 201 | 2.7802 | 18.0542 |
| 32632 | Metabolite - 11315 | 200 | 2.7704 | 17.9993 |
| 27718 | creatine | 200 | 2.6480 | 17.5391 |
| 32701 | urate | 200 | 2.6462 | 17.5321 |
| 584 | mannose | 50 | 2.6462 | 17.5321 |
| 32599 | Metabolite - 11282 | 201 | 2.5826 | 17.2822 |
| 32559 | Metabolite - 11242 | 201 | 2.5799 | 17.2717 |
| 32497 | 10c undecenoate | 201 | 2.5001 | 16.9560 |
| 606 | uridine | 201 | 2.4079 | 16.5890 |
| 32621 | Metabolite - 11304 | 200 | 2.4027 | 16.5683 |
| 12783 | Metabolite - 3101 | 50 | 2.4027 | 16.5683 |
| 32564 | Metabolite - 11247 | 201 | 2.3935 | 16.4772 |
| 32517 | Metabolite - 11203 | 200 | 2.3800 | 16.4772 |
| 12763 | Metabolite - 3083 | 50 | 2.3715 | 16.2338 |
| 32544 | Metabolite - 11227 | 201 | 2.3539 | 16.3727 |
| 19490 | Metabolite - 6488 | 50 | 2.3240 | 16.2338 |
| 12067 | undecanoate | 201 | 2.3176 | 16.2264 |
| 32854 | Metabolite - 11537 | 200 | 2.2206 | 15.7775 |
| 32813 | Metabolite - 11496 | 201 | 2.2068 | 15.7775 |
| 32945 | Metabolite - 11628 | 201 | 2.1937 | 15.7238 |
| 32110 | Metabolite - 11086 | 50 | 2.1659 | 15.6103 |
| 19402 | Metabolite - 6346 | 50 | 2.1659 | 15.6103 |
| 31787 | 3-carboxyl-4-methyl-5-propyl-2-furanpropanoic acid | 201 | 2.1292 | 15.4023 |
| 32729 | Metabolite - 11412 | 200 | 2.1279 | 15.4545 |
| 32549 | Metabolite - 02269 | 201 | 2.0837 | 15.0750 |
| 15990 | glycerophosphorylcholine GPC | 200 | 2.0516 | 15.1396 |
| 33012 | Metabolite - 11674 | 200 | 2.0280 | 14.9832 |
| 32385 | Metabolite - 11180 | 50 | 2.0268 | 14.8693 |
Biomarkers were discovered by (1) analyzing blood plasma samples from different groups of female human subjects and from different groups of male 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.
For males, the plasma samples used for the analysis were 7 control samples that were from healthy subjects, 6 samples from patients with steatosis and 8 samples from patients with steatohepatitis. For females, the plasma samples used for the analysis were 18 control samples that were from healthy subjects, 5 samples from patients with steatosis and 16 samples from patients with steatohepatitis. In both the male and the female studies, after the levels of metabolites were determined, the data was analyzed using significance tests (ANOVA, ANCOVA, Wilcoxon).
ANOVA was used to identify significant differences in the mean levels of metabolites between two populations (i.e., Steatosis vs. Control, Steatohepatitis vs. Control, Steatosis vs. Steatohepatitis).
As listed below in Tables 12 (male subjects) and 13 (female subjects), biomarkers were discovered that were differentially present between plasma samples from steatosis patients and Control subjects, biomarkers that were discovered that were differentially present between plasma samples from patients with steatohepatitis and from Control subjects and biomarkers that were discovered that were differentially present between plasma samples from steatosis and plasma samples from subjects with steatohepatitis (i.e. NASH).
Tables 12 and 13 include, for each listed biomarker, the p-value and the q-value determined in the statistical analysis of the data concerning the biomarkers in the steatosis mean level as compared to the control mean level, the NASH mean level as compared to the control mean level, and the steatosis mean level as compared to the NASH mean level. 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. Comp ID refers to the identification number for the compound in our internal chemical compound database.
Table 12 lists biomarkers for males, including, for each biomarker, the p-value and q-value for the specified comparisons of groups as described above.
Table 12. Biomarkers from male subjects with Steatosis compared to Control subjects, steatohepatitis (NASH) compared to Control subjects and Steatosis compared to steatohepatitis (NASH) subjects.
| p-value | q-value | p-value | q-value | p-value | q-value | |||
| stearate | 1358 | 50 | 4.00E-04 | 0.0633 | 0.0128 | 0.2286 | 0.3457 | 0.7893 |
| palmitate | 1336 | 50 | 9.00E-04 | 0.0633 | 0.0091 | 0.1836 | 0.5612 | 0.8289 |
| isoleucine | 1125 | 50 | 0.0011 | 0.0633 | 0.0025 | 0.1264 | 0.9729 | 0.8893 |
| glutamate | 57 | 50 | 0.0011 | 0.0633 | 0.0305 | 0.2368 | 0.3124 | 0.7803 |
| Metabolite-9033 | 22570 | 50 | 0.0021 | 0.0872 | 0.2901 | 0.66 | 0.0507 | 0.6206 |
| Metabolite-3087 | 12767 | 50 | 0.0031 | 0.0872 | 0.1491 | 0.4771 | 0.1507 | 0.7628 |
| valine | 1649 | 50 | 0.0034 | 0.0872 | 0.0243 | 0.2368 | 0.5868 | 0.832 |
| isoleucine | 18118 | 50 | 0.0036 | 0.0872 | 0.0019 | 0.1264 | 0.6472 | 0.8475 |
| glutamate | 12751 | 50 | 0.0036 | 0.0872 | 0.0472 | 0.2804 | 0.4112 | 0.8022 |
| Metabolite-4274 | 16511 | 50 | 0.0038 | 0.0872 | 0.106 | 0.3961 | 0.2349 | 0.7803 |
| linoleate | 1105 | 50 | 0.0049 | 0.0872 | 0.051 | 0.2862 | 0.4508 | 0.8145 |
| leucine | 60 | 50 | 0.0054 | 0.0872 | 0.0175 | 0.2368 | 0.8025 | 0.8527 |
| Metabolite-5769 | 18706 | 61 | 0.0054 | 0.0872 | 0.0025 | 0.1264 | 0.626 | 0.8421 |
| isocitrate | 1113 | 61 | 0.0055 | 0.0872 | 0.094 | 0.3845 | 0.3151 | 0.7803 |
| Metabolite-2041 | 8509 | 61 | 0.0059 | 0.0872 | 0.0536 | 0.2922 | 0.4791 | 0.8182 |
| Metabolite-2270 | 10247 | 61 | 0.0061 | 0.0872 | 0.0206 | 0.2368 | 0.7827 | 0.8519 |
| Metabolite-10932 | 31510 | 61 | 0.007 | 0.0872 | 0.0338 | 0.2455 | 0.6578 | 0.8475 |
| alanine | 1126 | 50 | 0.0072 | 0.0872 | 0.0298 | 0.2368 | 0.7063 | 0.8475 |
| tyrosine | 12780 | 50 | 0.0073 | 0.0872 | 0.0272 | 0.2368 | 0.7393 | 0.8519 |
| Metabolite-8214 | 21421 | 50 | 0.0085 | 0.0948 | 0.0925 | 0.3845 | 0.398 | 0.8022 |
| glucose | 20488 | 50 | 0.0095 | 0.0948 | 0.0911 | 0.3845 | 0.4236 | 0.8022 |
| Metabolite-10439 | 25609 | 50 | 0.0098 | 0.0948 | 0.0341 | 0.2455 | 0.7445 | 0.8519 |
| Metabolite-1496 | 6847 | 61 | 0.0098 | 0.0948 | 0.9827 | 0.8655 | 0.0124 | 0.348 |
| choline | 15506 | 61 | 0.01 | 0.0948 | 0.6398 | 0.7767 | 0.0429 | 0.6206 |
| glutamine | 1647 | 50 | 0.0116 | 0.1058 | 0.1325 | 0.4409 | 0.3566 | 0.7893 |
| Metabolite-9855 | 24233 | 61 | 0.0121 | 0.1061 | 0.8754 | 0.8551 | 0.0105 | 0.3457 |
| Metabolite-7187 | 20267 | 61 | 0.0145 | 0.1218 | 0.4129 | 0.698 | 0.1218 | 0.7385 |
| Metabolite-5646 | 18591 | 61 | 0.0157 | 0.1272 | 0.4187 | 0.698 | 0.0021 | 0.2874 |
| 3-methyl-2-oxobutyric acid | 21047 | 61 | 0.0164 | 0.1281 | 0.1039 | 0.3961 | 0.5063 | 0.8289 |
| Metabolite-4096 | 16186 | 61 | 0.0169 | 0.1281 | 0.1525 | 0.4771 | 0.3895 | 0.8005 |
| Metabolite-9727 | 24077 | 50 | 0.0185 | 0.1342 | 0.0298 | 0.2368 | 0.9767 | 0.8893 |
| Metabolite-1911 | 7933 | 61 | 0.0189 | 0.1342 | 0.6182 | 0.7691 | 0.0064 | 0.2874 |
Table 13 lists biomarkers for females, including, for each biomarker, the p-value and the q-value for the specified comparisons of groups as described above.
Table 13. Biomarkers from female subjects with Steatosis compared to Control subjects, steatohepatitis (NASH) compared to Control subjects and Steatosis compared to steatohepatitis (NASH) subjects.
| p-value | q-value | p-value | q-value | p-value | q-value | |||
| glutamate | 57 | 50 | 1.73E-08 | 3.22E-06 | 0.0023 | 0.0904 | 0.2067 | 0.6864 |
| urate | 1604 | 50 | 3.32E-07 | 3.08E-05 | 0.0278 | 0.2958 | 0.1005 | 0.6474 |
| glutamate | 12751 | 50 | 8.91E-07 | 1.00E-04 | 0.0036 | 0.0977 | 0.4745 | 0.6864 |
| Metabolite-5769 | 18706 | 61 | 4.24E-06 | 2.00E-04 | 8.00E- | 0.0661 | 0.9332 | 0.7199 |
| 04 | ||||||||
| isocitrate | 1113 | 61 | 8.14E-06 | 3.00E-04 | 0.255 | 0.4932 | 0.0342 | 0.4905 |
| Metabolite-10026 | 24285 | 61 | 1.94E-05 | 6.00E-04 | 0.0231 | 0.2804 | 0.4083 | 0.6864 |
| glycocholate | 18476 | 61 | 2.91E-05 | 8.00E-04 | 0.0172 | 0.2557 | 0.5271 | 0.6864 |
| L-kynurenine | 15140 | 61 | 1.00E-04 | 0.0013 | 0.9893 | 0.73 | 0.0051 | 0.432 |
| Metabolite-3330 | 13600 | 61 | 1.00E-04 | 0.0016 | 0.1335 | 0.3923 | 0.1878 | 0.6864 |
| leucine | 60 | 50 | 1.00E-04 | 0.0016 | 0.104 | 0.3726 | 0.241 | 0.6864 |
| valine | 1649 | 50 | 1.00E-04 | 0.0019 | 0.0938 | 0.3726 | 0.288 | 0.6864 |
| isoleucine | 1125 | 50 | 1.00E-04 | 0.0013 | 0.0574 | 0.3465 | 0.3262 | 0.6864 |
| Metabolite-1110 | 5687 | 61 | 1.00E-04 | 0.0016 | 0.0032 | 0.0977 | 0.8124 | 0.6937 |
| Metabolite-10951 | 31595 | 61 | 2.00E-04 | 0.0026 | 0.2368 | 0.4917 | 0.1503 | 0.6474 |
| Metabolite-4522 | 16865 | 50 | 2.00E-04 | 0.0021 | 0.0689 | 0.359 | 0.3877 | 0.6864 |
| Metabolite-4274 | 16511 | 50 | 2.00E-04 | 0.0021 | 0.0431 | 0.3397 | 0.516 | 0.6864 |
| Isobar-47-includes-taurochenodeoxy cholic acid-and-taurodeoxycholic acid | 18882 | 61 | 2.00E-04 | 0.0021 | 0.032 | 0.3148 | 0.5941 | 0.6868 |
| tyrosine | 1299 | 61 | 3.00E-04 | 0.0026 | 0.0548 | 0.3465 | 0.492 | 0.6864 |
| alanine | 1126 | 50 | 4.00E-04 | 0.0036 | 0.1507 | 0.414 | 0.2717 | 0.6864 |
| thyroxine | 2761 | 61 | 5.00E-04 | 0.0043 | 0.2645 | 0.5014 | 0.1712 | 0.6644 |
| Metabolite-2347 | 10551 | 61 | 5.00E-04 | 0.0048 | 0.0022 | 0.0904 | 0.4853 | 0.6864 |
| Metabolite-10812 | 30821 | 50 | 7.00E-04 | 0.0058 | 0.1583 | 0.421 | 0.3247 | 0.6864 |
| Metabolite-2269- | 10245 | 61 | 7.00E-04 | 0.0056 | 0.0025 | 0.0904 | 0.4809 | 0.6864 |
| glutamine | 1647 | 50 | 9.00E-04 | 0.0067 | 0.1091 | 0.3726 | 0.458 | 0.6864 |
| meso-erythritol | 20699 | 50 | 0.001 | 0.0067 | 0.1995 | 0.463 | 0.2975 | 0.6864 |
| Metabolite-3183 | 13214 | 61 | 0.001 | 0.0067 | 0.106 | 0.3726 | 0.4805 | 0.6864 |
| docosahexaenoat e-DHA- | 19323 | 50 | 0.001 | 0.0067 | 0.0405 | 0.3397 | 0.7884 | 0.6896 |
| Metabolite-4627 | 17068 | 61 | 0.001 | 0.0067 | 0.0268 | 0.2958 | 0.9307 | 0.7199 |
| Isobar-66-includes-glycochenodeoxy cholic acid-glycodeoxycholi c acid | 22803 | 61 | 0.0011 | 0.0067 | 0.0519 | 0.3465 | 0.7198 | 0.6896 |
| 5-oxoproline | 1494 | 50 | 0.0012 | 0.0076 | 0.1022 | 0.3726 | 0.5195 | 0.6864 |
| Metabolite-10589 | 27801 | 61 | 0.0013 | 0.0078 | 0.2598 | 0.4988 | 0.2575 | 0.6864 |
| mannose | 584 | 50 | 0.0013 | 0.0078 | 0.0435 | 0.3397 | 0.8177 | 0.6937 |
| Metabolite-4360 | 16650 | 50 | 0.0015 | 0.0083 | 0.6433 | 0.6655 | 0.0796 | 0.5826 |
| gamma-L-glutamyl-L-tyrosine | 2734 | 61 | 0.0016 | 0.009 | 0.3615 | 0.5823 | 0.1942 | 0.6864 |
| Metabolite-1086 | 5628 | 61 | 0.0017 | 0.0092 | 0.6934 | 0.6655 | 0.0743 | 0.5826 |
| phenylalanine | 64 | 61 | 0.0019 | 0.0095 | 0.1746 | 0.4408 | 0.4085 | 0.6864 |
| lactate | 527 | 50 | 0.0019 | 0.0095 | 0.1018 | 0.3726 | 0.586 | 0.6868 |
| arachidonate | 1110 | 50 | 0.002 | 0.0098 | 0.8148 | 0.6934 | 0.0194 | 0.4834 |
| Metabolite-7187 | 20267 | 61 | 0.0021 | 0.0098 | 0.747 | 0.6811 | 0.0705 | 0.5824 |
| Metabolite-1914 | 7941 | 61 | 0.0021 | 0.0098 | 0.0075 | 0.1505 | 0.5693 | 0.6868 |
| Metabolite- 3109 | 12791 | 50 | 0.0024 | 0.0109 | 0.783 | 0.6934 | 0.0195 | 0.4834 |
| tyrosine | 12780 | 50 | 0.0031 | 0.0138 | 0.0321 | 0.3148 | 0.9228 | 0.7184 |
| xanthine | 3147 | 61 | 0.0032 | 0.0138 | 0.2695 | 0.5014 | 0.3371 | 0.6864 |
| Metabolite-1496 | 6847 | 61 | 0.0035 | 0.0149 | 0.3752 | 0.5823 | 0.005 | 0.432 |
| Metabolite-2041 | 8509 | 61 | 0.0037 | 0.0154 | 0.3041 | 0.5383 | 0.3177 | 0.6864 |
| 3-4-5-trimethoxycinna mic acid | 18291 | 61 | 0.0045 | 0.0183 | 0.0182 | 0.2557 | 0.6793 | 0.6896 |
| Metabolite-3951 | 15529 | 61 | 0.0049 | 0.0192 | 0.0586 | 0.3465 | 0.9515 | 0.7253 |
| Metabolite-3994 | 16016 | 61 | 0.005 | 0.0192 | 0.0249 | 0.2887 | 0.7558 | 0.6896 |
| Metabolite-3094 | 12774 | 50 | 0.0055 | 0.0209 | 0.6697 | 0.6655 | 0.0218 | 0.4834 |
| Metabolite-9855 | 24233 | 61 | 0.0058 | 0.0216 | 0.124 | 0.3816 | 0.7024 | 0.6896 |
| Metabolite-5726 | 18657 | 61 | 0.006 | 0.022 | 0.7399 | 0.6781 | 0.1182 | 0.6474 |
| glycerol | 15122 | 50 | 0.0065 | 0.0229 | 0.0928 | 0.3726 | 0.8347 | 0.6975 |
| Metabolite-2395 | 10715 | 61 | 0.0066 | 0.0229 | 0.0016 | 0.0904 | 0.1741 | 0.6654 |
| Metabolite-4986 | 17627 | 50 | 0.0067 | 0.0229 | 0.1963 | 0.4608 | 0.5502 | 0.6868 |
| 3-methyl-2-oxobutyric acid | 21047 | 61 | 0.0069 | 0.0234 | 0.0928 | 0.3726 | 0.8462 | 0.6991 |
| Metabolite-2259 | 10156 | 61 | 0.0071 | 0.0235 | 0.6427 | 0.6655 | 0.1628 | 0.6474 |
| Metabolite-3230 | 13296 | 61 | 0.0072 | 0.0235 | 0.6656 | 0.6655 | 0.1545 | 0.6474 |
| palmitate | 1336 | 50 | 0.0075 | 0.0237 | 0.3757 | 0.5823 | 0.3312 | 0.6864 |
| Metabolite-1911 | 7933 | 61 | 0.0075 | 0.0237 | 0.1736 | 0.4408 | 0.6183 | 0.6868 |
| Metabolite-2291 | 10414 | 61 | 0.0079 | 0.0245 | 0.0098 | 0.1787 | 0.4316 | 0.6864 |
| pyruvate | 599 | 61 | 0.0118 | 0.0359 | 0.9667 | 0.7259 | 0.0792 | 0.5826 |
| Isobar-1-includes-mannose-fructose-glucose-galactose-alpha-L-sorbopyranose-Inositol-D-allose-D--altrose-D-psicone-L-gulose-allo-inositol | 10737 | 61 | 0.0126 | 0.0375 | 0.055 | 0.3465 | 0.8389 | 0.6975 |
| Metabolite-2272 | 10286 | 61 | 0.0128 | 0.0375 | 0.0731 | 0.359 | 0.94 | 0.7208 |
| lysine | 1301 | 50 | 0.0132 | 0.0384 | 0.271 | 0.5014 | 0.5375 | 0.6864 |
| uridine | 606 | 61 | 0.0139 | 0.0397 | 0.1016 | 0.3726 | 0.9509 | 0.7253 |
| Metabolite-2390 | 10672 | 61 | 0.0143 | 0.0402 | 0.0557 | 0.3465 | 0.8179 | 0.6937 |
| Metabolite-9043 | 22600 | 50 | 0.0154 | 0.0427 | 0.0118 | 0.2006 | 0.375 | 0.6864 |
| Metabolite-2329 | 10544 | 61 | 0.0157 | 0.0429 | 0.3917 | 0.5823 | 0.4157 | 0.6864 |
| Metabolite-9033 | 22570 | 50 | 0.0163 | 0.0437 | 0.0719 | 0.359 | 0.883 | 0.7052 |
| Metabolite-4251 | 16496 | 50 | 0.018 | 0.0478 | 0.7335 | 0.6781 | 0.1998 | 0.6864 |
| Metabolite-4276 | 16518 | 50 | 0.0183 | 0.0479 | 0.3795 | 0.5823 | 0.4523 | 0.6864 |
| Metabolite-10932 | 31510 | 61 | 0.02 | 0.0515 | 0.6694 | 0.6655 | 0.2414 | 0.6864 |
| Metabolite-3323 | 13557 | 61 | 0.0221 | 0.0561 | 0.4812 | 0.657 | 0.3823 | 0.6864 |
| Metabolite-3016 | 12644 | 50 | 0.0229 | 0.0575 | 0.0038 | 0.0977 | 0.1636 | 0.6474 |
| Metabolite-1981 | 8210 | 61 | 0.0248 | 0.0613 | 0.2371 | 0.4917 | 0.7138 | 0.6896 |
| Metabolite-3088 | 12768 | 50 | 0.0272 | 0.0663 | 0.8115 | 0.6934 | 0.0842 | 0.5956 |
| Metabolite-3087 | 12767 | 50 | 0.0295 | 0.0712 | 0.2356 | 0.4917 | 0.7535 | 0.6896 |
| Metabolite-10797 | 30728 | 61 | 0.0309 | 0.0735 | 0.7815 | 0.6934 | 0.2311 | 0.6864 |
| Metabolite-3100 | 12782 | 50 | 0.0316 | 0.0742 | 0.0624 | 0.3465 | 0.6928 | 0.6896 |
| 2-amino butyrate | 12645 | 50 | 0.033 | 0.0756 | 0.3666 | 0.5823 | 0.5717 | 0.6868 |
| threonine | 1284 | 50 | 0.033 | 0.0756 | 0.1048 | 0.3726 | 0.8734 | 0.7052 |
| Metabolite-3707 | 14837 | 61 | 0.0338 | 0.0761 | 0.019 | 0.2557 | 0.3591 | 0.6864 |
| Metabolite-1834 | 7650 | 61 | 0.0343 | 0.0761 | 4.00E-04 | 0.0646 | 0.0289 | 0.4905 |
| Metabolite-10785 | 30633 | 61 | 0.0344 | 0.0761 | 0.0612 | 0.3465 | 0.6678 | 0.6896 |
| Metabolite-2139 | 9130 | 61 | 0.0349 | 0.0762 | 0.4999 | 0.6647 | 0.4382 | 0.6864 |
| Metabolite-4613 | 17033 | 61 | 0.0375 | 0.0808 | 0.4934 | 0.663 | 0.4563 | 0.6864 |
| gluconate | 587 | 50 | 0.039 | 0.083 | 0.9105 | 0.7087 | 0.1966 | 0.6864 |
| N-acetylglycine | 27710 | 50 | 0.0396 | 0.083 | 0.1303 | 0.3923 | 0.9192 | 0.7177 |
| Metabolite-10507 | 27275 | 50 | 0.0398 | 0.083 | 0.8432 | 0.6996 | 0.1135 | 0.6474 |
| threonate | 27738 | 50 | 0.0404 | 0.0834 | 0.5847 | 0.6655 | 0.3906 | 0.6864 |
| p-hydroxyphenylla ctate-HPLA- | 1431 | 50 | 0.0411 | 0.0837 | 0.7669 | 0.6934 | 0.0954 | 0.6428 |
| palmitoleate | 1507 | 50 | 0.0427 | 0.0854 | 0.6825 | 0.6655 | 0.3285 | 0.6864 |
| Metabolite-3995 | 16019 | 61 | 0.0428 | 0.0854 | 0.6036 | 0.6655 | 0.3849 | 0.6864 |
| Metabolite-2231 | 9905 | 61 | 0.0463 | 0.0913 | 0.3818 | 0.5823 | 0.6207 | 0.6868 |
| Metabolite-3165 | 13142 | 61 | 0.0481 | 0.0939 | 0.0439 | 0.3397 | 0.4996 | 0.6864 |
| stearate | 1358 | 50 | 0.0486 | 0.0939 | 0.7225 | 0.6781 | 0.3214 | 0.6864 |
| Metabolite-9726 | 24076 | 50 | 0.0504 | 0.0954 | 0.6042 | 0.6655 | 0.0688 | 0.5824 |
| serine | 12663 | 50 | 0.0504 | 0.0954 | 0.578 | 0.6655 | 0.4331 | 0.6864 |
| Metabolite-5774 | 18723 | 61 | 0.0514 | 0.0964 | 0.8537 | 0.6996 | 0.254 | 0.6864 |
| Metabolite-10933 | 31518 | 50 | 0.0578 | 0.1062 | 0.2685 | 0.5014 | 0.0197 | 0.4834 |
| Metabolite-2321 | 10501 | 61 | 0.0918 | 0.1468 | 0.3393 | 0.5814 | 0.0402 | 0.4905 |
| Isobar-60-includes-s-2-hydroxybutyric acid-2-hydroxyisobutyri c acid | 22261 | 61 | 0.1198 | 0.1696 | 0.2887 | 0.5228 | 0.0389 | 0.4905 |
| threonine | 12666 | 50 | 0.131 | 0.18 | 0.3057 | 0.5383 | 0.0454 | 0.5204 |
| Metabolite-2821 | 11923 | 61 | 0.1533 | 0.1987 | 5.00E-04 | 0.0646 | 0.0098 | 0.4834 |
| creatine | 27718 | 61 | 0.1942 | 0.2211 | 0.1218 | 0.3816 | 0.0183 | 0.4834 |
| paraxanthine | 18254 | 61 | 0.2253 | 0.247 | 0.0823 | 0.3726 | 0.013 | 0.4834 |
| Metabolite-3443 | 14125 | 61 | 0.2434 | 0.258 | 0.1778 | 0.4408 | 0.0371 | 0.4905 |
| fructose | 577 | 50 | 0.2708 | 0.2732 | 0.2032 | 0.4674 | 0.0487 | 0.5204 |
| theophylline | 18394 | 61 | 0.3262 | 0.301 | 0.15 | 0.414 | 0.0399 | 0.4905 |
| alpha-keto-glutarate | 528 | 61 | 0.364 | 0.3232 | 0.0187 | 0.2557 | 0.004 | 0.432 |
| Metabolite-6346 | 19402 | 50 | 0.4153 | 0.3451 | 0.0919 | 0.3726 | 0.029 | 0.4905 |
| Metabolite-8506 | 21762 | 61 | 0.4328 | 0.3491 | 0.0711 | 0.359 | 0.0227 | 0.4834 |
| Metabolite-1667 | 7132 | 61 | 0.4557 | 0.3628 | 0.1102 | 0.3726 | 0.0398 | 0.4905 |
| caffeine | 569 | 61 | 0.462 | 0.3658 | 0.036 | 0.3397 | 0.0114 | 0.4834 |
| Metabolite-4238 | 16471 | 61 | 0.9278 | 0.51 | 0.0226 | 0.2804 | 0.0282 | 0.4905 |
Table 14 below includes analytical characteristics of each of the isobars and the unnamed metabolites listed in Tables 1-3 and 8-13 above. The table includes, for each listed Metabolite, the Metabolite ID (ID), the retention time (RT), retention index (RI), mass, library, and polarity obtained using the analytical methods described above. "Metabolite ID" refers to the unique identifier for that compound in our internal chemical library database. "Mass" refers to the mass of the C12 isotope of the parent ion used in quantification of the compound. The values for "Library" give an indication of the analytical method used for quantification: "50" indicates GC-MS and "61", "200" and "201" indicate LC-MS. "Polarity" indicates the polarity of the quantitative ion as being either positive (+) or negative (-).
Table 14. Analytical Characteristics of Unnamed Metabolites.
| 1882 | Isobar 47 includes taurochenodeoxycholic acid and taurodeoxycholic acid | 61 | 15.51 | 15700.0 | 498.4 | - |
| 5628 | Metabolite - 1086 | 61 | 4.56 | 4811.0 | 294.1 | + |
| 6398 | Metabolite - 1335 | 61 | 8.74 | 9162.2 | 367.2 | + |
| 6847 | Metabolite - 1496 | 61 | 1.53 | 1562.0 | 133.0 | - |
| 7933 | Metabolite - 1911 | 61 | 11.42 | 11799.6 | 464.1 | + |
| 10501 | Metabolite - 2321 | 61 | 13.44 | 12940.0 | 314.3 | + |
| 10629 | Metabolite - 2386 | 61 | 11.94 | 12150.0 | 539.2 | - |
| 10737 | Isobar 1 includes mannose, fructose, glucose, galactose, alpha-L-sorbopyranose, Inositol, D-allose,D-(+)-altrose,D-psicone,L-(+)-gulose, allo-nositol | 61 | 1.45 | 1481.0 | 215.0 | - |
| 11923 | Metabolite - 2821 | 61 | 6.80 | 7980.0 | 119.1 | + |
| 12593 | Metabolite-2973 | 50 | 4.74 | 1213.4 | 281 | + |
| 12604 | Metabolite-2981 | 50 | 5.21 | 1265.2 | 210.9 | + |
| 12626 | Metabolite-3003 | 50 | 6.79 | 1446.6 | 218.1 | + |
| 12754 | Metabolite-3075 | 50 | 10.36 | 1857.9 | 204 | + |
| 12757 | Metabolite-3078 | 50 | 10.65 | 1887 | 203.1 | + |
| 12761 | Metabolite-3081 | 50 | 10.89 | 1911.5 | 204 | + |
| 12763 | Metabolite-3083 | 50 | 10.94 | 1916.1 | 204 | + |
| 12769 | Metabolite-3089 | 50 | 11.28 | 1951.5 | 116.9 | + |
| 12770 | Metabolite-3090 | 50 | 11.31 | 1955 | 243.1 | + |
| 12771 | Metabolite-3091 | 50 | 11.41 | 1966.2 | 232.1 | + |
| 12774 | Metabolite-3094 | 50 | 11.55 | 1980.6 | 299 | + |
| 12781 | Metabolite-3099 | 50 | 11.77 | 2005.2 | 204 | + |
| 12782 | Metabolite-3100 | 50 | 11.85 | 2013.2 | 204 | + |
| 12783 | Metabolite-3101 | 50 | 11.93 | 2022.2 | 290 | + |
| 12785 | Metabolite-3103 | 50 | 12.09 | 2039.8 | 290.1 | + |
| 12789 | Metabolite-3107 | 50 | 12.21 | 2053.2 | 204.1 | + |
| 12790 | Metabolite-3108 | 50 | 12.24 | 2056.5 | 246 | + |
| 13142 | Metabolite - 3165 | 61 | 8.38 | 8472.2 | 265.0 | + |
| 13214 | Metabolite - 3183 | 61 | 9.37 | 9441.0 | 295.2 | + |
| 13296 | Metabolite - 3230 | 61 | 3.10 | 3043.2 | 245.0 | + |
| 13557 | Metabolite - 3323 | 61 | 14.47 | 14696.5 | 624.4 | - |
| 13600 | Metabolite - 3330 | 61 | 14.06 | 14292.2 | 514.3 | - |
| 15529 | Metabolite - 3951 | 61 | 8.41 | 8705.4 | 367.1 | + |
| 16016 | Metabolite - 3994 | 61 | 1.63 | 1640.4 | 427.0 | + |
| 16019 | Metabolite - 3995 | 61 | 2.19 | 2230.0 | 207.7 | - |
| 16138 | Metabolite-4080 | 50 | 14.02 | 2270.2 | 299 | + |
| 16308 | Metabolite-4147 | 50 | 10.07 | 1767.1 | 290.2 | + |
| 16337 | Metabolite - 4167 | 61 | 11.03 | 10920.4 | 286.2 | + |
| 16650 | Metabolite-4360 | 50 | 9.15 | 1678.2 | 347.2 | + |
| 16665 | Metabolite-4364 | 50 | 10.66 | 1852.4 | 232 | + |
| 16666 | Metabolite-4365 | 50 | 11.05 | 1892.9 | 204 | + |
| 16819 | Metabolite-4496 | 50 | 6.76 | 1398.2 | 204 | + |
| 16829 | Metabolite-4503 | 50 | 8.39 | 1589 | 227.2 | + |
| 16865 | Metabolite-4522 | 50 | 12.26 | 2025.4 | 217.1 | + |
| 16959 | Metabolite-4595 | 50 | 5.65 | 1274.4 | 130 | + |
| 17028 | Metabolite-4611 | 50 | 8.07 | 1546.6 | 292.1 | + |
| 17033 | Metabolite - 4613 | 61 | 12.73 | 12778.0 | 541.4 | - |
| 17068 | Metabolite - 4627 | 61 | 10.84 | 11034.9 | 591.3 | + |
| 17304 | Metabolite - 4759 | 61 | 11.80 | 11906.0 | 310.2 | + |
| 17627 | Metabolite-4986 | 50 | 11.56 | 1956.4 | 204.1 | + |
| 18232 | Metabolite-5403 | 50 | 5.92 | 1300.2 | 319 | + |
| 18591 | Metabolite - 5646 | 61 | 8.96 | 8750.0 | 228.0 | - |
| 18657 | Metabolite - 5726 | 61 | 14.05 | 13757.0 | 417.3 | - |
| 18706 | Metabolite - 5769 | 61 | 11.12 | 10753.2 | 485.2 | - |
| 18868 | Metabolite-5847 | 50 | 12.35 | 2040 | 288.2 | + |
| 18929 | Metabolite-5907 | 50 | 8.69 | 1643.2 | 229.1 | + |
| 19363 | Metabolite-6227 | 50 | 5 | 1210.5 | 196.1 | + |
| 19368 | Metabolite-6267 | 50 | 9.32 | 1704.5 | 257.1 | + |
| 19370 | Metabolite-6268 | 50 | 9.91 | 1773.8 | 271.1 | + |
| 19374 | Metabolite-6270 | 50 | 11.35 | 1929.6 | 320.2 | + |
| 19402 | Metabolite-6346 | 50 | 8 | 1550.8 | 263.2 | + |
| 19490 | Metabolite-6488 | 50 | 12.25 | 2021.7 | 204.1 | + |
| 19985 | Metabolite-6957 | 50 | 11.71 | 1966 | 290.1 | + |
| 20267 | Metabolite - 7187 | 61 | 8.38 | 8500.0 | 328.0 | + |
| 20950 | Metabolite-7846 | 50 | 5.1 | 1208.1 | 145.1 | + |
| 21418 | Isobar 56 - pipecolate; aminocyclopentane carboxylic acid | 61 | 2.45 | 2850.0 | 130.1 | + |
| 21762 | Metabolite - 8506 | 61 | 8.92 | 8811.0 | 475.1 | + |
| 22261 | Isobar 60 includes (s)-2-hydroxybutyric acid,2-hydroxyisobutyric acid | 61 | 4.26 | 4725.0 | 148.9 | - |
| 24233 | Metabolite - 9855 | 61 | 11.85 | 11605.3 | 462.2 | - |
| 24285 | Metabolite - 10026 | 61 | 9.47 | 9300.3 | 259.1 | - |
| 27801 | Metabolite - 10589 | 61 | 15.43 | 17536.8 | 619.3 | - |
| 30633 | Metabolite - 10785 | 61 | 11.49 | 12073.4 | 363.0 | + |
| 30728 | Metabolite - 10797 | 61 | 10.92 | 11563.3 | 352.2 | - |
| 31510 | Metabolite - 10932 | 61 | 11.54 | 11928.0 | 190.0 | + |
| 31529 | Metabolite - 10941 | 61 | 3.60 | 4101.0 | 191.8 | + |
| 32514 | Metabolite-11200 | 200 | 5.62 | 5637 | 496.4 | + |
| 32516 | Metabolite-11202 | 200 | 5.8 | 5823 | 524.4 | + |
| 32517 | Metabolite-11203 | 200 | 5.65 | 5665 | 522.4 | + |
| 32518 | Metabolite-11204 | 200 | 5.26 | 5263 | 229.2 | + |
| 32519 | Metabolite-11205 | 200 | 5.55 | 5558 | 520.4 | + |
| 32520 | Metabolite-11206 | 200 | 0.59 | 575 | 138.8 | + |
| 32545 | Metabolite-11228 | 201 | 1.15 | 1136 | 278.1 | - |
| 32547 | Metabolite-11230 | 201 | 1.44 | 1438 | 278.1 | - |
| 32548 | Metabolite-11231 | 201 | 1.47 | 1471 | 330 | - |
| 32549 | Metabolite-02269 | 201 | 1.55 | 1551 | 255.1 | - |
| 32550 | Metabolite-02272 | 201 | 1.97 | 1958 | 189 | - |
| 32551 | Metabolite-11234 | 201 | 2.01 | 2001 | 334.1 | - |
| 32552 | Metabolite-11235 | 201 | 2.04 | 2033 | 326.1 | - |
| 32553 | Metabolite-03832 | 201 | 2.2 | 2199 | 173.1 | - |
| 32557 | Metabolite-06126 | 201 | 2.69 | 2684 | 203.1 | - |
| 32559 | Metabolite-11242 | 201 | 2.96 | 2958 | 360 | - |
| 32560 | Metabolite-07765 | 201 | 3.71 | 3705 | 245.1 | - |
| 32561 | Metabolite-11244 | 201 | 3.78 | 3771 | 224.2 | - |
| 32562 | Metabolite-11245 | 201 | 3.91 | 3902 | 238.3 | - |
| 32563 | Metabolite-11246 | 201 | 3.94 | 3930 | 143.2 | - |
| 32564 | Metabolite-11247 | 201 | 3.94 | 3932 | 213.1 | - |
| 32566 | Metabolite-11249 | 200 | 1.24 | 1271 | 280.1 | + |
| 32567 | Metabolite-11250 | 200 | 1.3 | 1339 | 150.2 | + |
| 32571 | Metabolite-11254 | 200 | 2.05 | 2095 | 328.1 | + |
| 32572 | Metabolite-11255 | 200 | 2.38 | 2442 | 247.1 | + |
| 32575 | Metabolite-11258 | 200 | 2.89 | 2941 | 362 | + |
| 32578 | Metabolite-11261 | 200 | 3.69 | 3732 | 286.2 | + |
| 32587 | Metabolite-02249 | 201 | 4.03 | 4025 | 267.2 | - |
| 32588 | Metabolite-01327 | 201 | 4.25 | 4242 | 583.2 | - |
| 32590 | Metabolite-11273 | 201 | 4.56 | 4552 | 369.2 | - |
| 32593 | Metabolite-02036 | 200 | 4.94 | 4985 | 616.2 | + |
| 32596 | Metabolite-02250 | 200 | 5.14 | 5158 | 286.2 | + |
| 32599 | Metabolite-11282 | 201 | 4.77 | 4763 | 254.8 | - |
| 32602 | Metabolite-11285 | 200 | 5.28 | 5260 | 288.1 | + |
| 32609 | Metabolite-01345 | 201 | 4.9 | 4887 | 369.2 | - |
| 32616 | Metabolite-11299 | 201 | 4.9 | 4893 | 507.2 | - |
| 32619 | Metabolite-11302 | 201 | 5.01 | 4998 | 397.3 | - |
| 32620 | Metabolite-11303 | 201 | 5.02 | 5015 | 512.3 | - |
| 32621 | Metabolite-11304 | 200 | 0.8 | 801 | 217.1 | + |
| 32625 | Metabolite-11308 | 201 | 5.15 | 5133 | 365.3 | - |
| 32631 | Metabolite-11314 | 200 | 0.64 | 634 | 243 | + |
| 32632 | Metabolite-11315 | 200 | 1.19 | 1210 | 130.2 | + |
| 32634 | Metabolite-11317 | 201 | 5.81 | 5703 | 219.3 | - |
| 32635 | Metabolite-11318 | 201 | 5.81 | 5699 | 476.3 | - |
| 32636 | Metabolite-11319 | 201 | 5.81 | 5700 | 269.4 | - |
| 32637 | Metabolite-11320 | 201 | 5.85 | 5740 | 593.9 | - |
| 32638 | Metabolite-11321 | 201 | 6.15 | 5997 | 569.9 | - |
| 32641 | Metabolite-11324 | 201 | 6.24 | 6075 | 596 | - |
| 32643 | Metabolite-11326 | 201 | 6.35 | 6160 | 213.2 | - |
| 32644 | Metabolite-11327 | 200 | 5.16 | 5176 | 269.2 | + |
| 32648 | Metabolite-11331 | 201 | 0.69 | 686 | 164.2 | - |
| 32651 | Metabolite-11334 | 200 | 0.96 | 982 | 259.1 | + |
| 32652 | Metabolite-11335 | 200 | 0.97 | 991 | 229.2 | + |
| 32654 | Metabolite-11337 | 200 | 1 | 1020 | 160.2 | + |
| 32656 | Metabolite-11339 | 201 | 0.69 | 689 | 156.2 | - |
| 32670 | Metabolite-11353 | 200 | 0.8 | 811 | 203.2 | + |
| 32671 | Metabolite-11354 | 200 | 0.76 | 770 | 146.2 | + |
| 32672 | Metabolite-02546 | 200 | 0.75 | 764 | 129.2 | + |
| 32675 | Metabolite-03951 | 200 | 1.87 | 1912 | 367.1 | + |
| 32682 | Metabolite-11365 | 201 | 5.61 | 5527 | 303.3 | - |
| 32689 | Metabolite-11372 | 201 | 5.35 | 5303 | 467.4 | - |
| 32691 | Metabolite-11374 | 200 | 0.69 | 690 | 370.9 | + |
| 32692 | Metabolite-11375 | 200 | 0.73 | 732 | 309.1 | + |
| 32694 | Metabolite-11377 | 200 | 0.77 | 770 | 249.2 | + |
| 32696 | Metabolite-11379 | 201 | 5.65 | 5566 | 267.3 | - |
| 32697 | Metabolite-11380 | 200 | 1.04 | 1053 | 339.8 | + |
| 32698 | Metabolite-11381 | 200 | 1.11 | 1126 | 186.2 | + |
| 32699 | Metabolite-11382 | 200 | 1.16 | 1180 | 250.8 | + |
| 32702 | Metabolite-11385 | 200 | 1.38 | 1430 | 312.1 | + |
| 32703 | Metabolite-11386 | 200 | 1.47 | 1525 | 344.1 | + |
| 32704 | Metabolite-11387 | 200 | 1.65 | 1699 | 294.1 | + |
| 32707 | Metabolite-11390 | 200 | 2 | 2041 | 100.2 | + |
| 32709 | Metabolite-03056 | 200 | 2.21 | 2264 | 185.2 | + |
| 32710 | Metabolite-11393 | 200 | 2.34 | 2403 | 367.1 | + |
| 32717 | Metabolite-11400 | 200 | 2.81 | 2855 | 710.1 | + |
| 32718 | Metabolite-01342 | 200 | 2.8 | 2848 | 265.1 | + |
| 32722 | Metabolite-11405 | 200 | 3.02 | 3075 | 264.8 | + |
| 32729 | Metabolite-11412 | 200 | 3.78 | 3836 | 204.2 | + |
| 32732 | Metabolite-11415 | 201 | 0.69 | 692 | 313.1 | - |
| 32734 | Metabolite-10954 | 200 | 4.14 | 4229 | 288.2 | + |
| 32735 | Metabolite-01911 | 200 | 4.26 | 4354 | 464.1 | + |
| 32738 | Metabolite-11421 | 200 | 4.54 | 4634 | 314.2 | + |
| 32739 | Metabolite-11422 | 201 | 0.89 | 884 | 151.1 | - |
| 32740 | Metabolite-11423 | 201 | 1.05 | 1038 | 260.1 | - |
| 32741 | Metabolite-11424 | 200 | 4.58 | 4664 | 227.1 | + |
| 32744 | Metabolite-11427 | 201 | 1.16 | 1145 | 181.1 | - |
| 32746 | Metabolite-11429 | 201 | 1.16 | 1151 | 245.1 | - |
| 32747 | Metabolite-01142 | 201 | 1.19 | 1176 | 117.2 | - |
| 32748 | Metabolite-11431 | 201 | 1.58 | 1575 | 330 | - |
| 32749 | Metabolite-11432 | 201 | 1.89 | 1886 | 292.1 | - |
| 32751 | Metabolite-11434 | 201 | 2.11 | 2107 | 370.1 | - |
| 32752 | Metabolite-11435 | 201 | 2.14 | 2131 | 365.1 | - |
| 32753 | Metabolite-09789 | 201 | 2.62 | 2613 | 153.1 | - |
| 32754 | Metabolite-11437 | 201 | 2.89 | 2888 | 231 | - |
| 32755 | Metabolite-11438 | 201 | 3.1 | 3092 | 241.2 | - |
| 32756 | Metabolite-02276 | 201 | 3.35 | 3339 | 199.1 | - |
| 32757 | Metabolite-11440 | 201 | 3.58 | 3571 | 246.3 | - |
| 32758 | Metabolite-11441 | 201 | 3.78 | 3773 | 331.1 | - |
| 32759 | Metabolite-11442 | 201 | 3.91 | 3902 | 331.1 | - |
| 32760 | Metabolite-11443 | 201 | 3.92 | 3910 | 225.3 | - |
| 32761 | Metabolite-11444 | 201 | 3.99 | 3983 | 541.2 | - |
| 32762 | Metabolite-11445 | 201 | 4.01 | 3995 | 239.3 | - |
| 32764 | Metabolite-11447 | 200 | 0.98 | 989 | 278.1 | + |
| 32767 | Metabolite-11450 | 201 | 4.11 | 4103 | 224.2 | - |
| 32769 | Metabolite-11452 | 201 | 4.12 | 4109 | 352.1 | - |
| 32776 | Metabolite-11459 | 200 | 2.49 | 2545 | 246.2 | + |
| 32786 | Metabolite-11469 | 200 | 3.82 | 3874 | 239.1 | + |
| 32787 | Metabolite-11470 | 201 | 4.16 | 4151 | 525.2 | - |
| 32792 | Metabolite-11475 | 201 | 4.25 | 4240 | 383.2 | - |
| 32793 | Metabolite-11476 | 200 | 4.52 | 4616 | 189.1 | + |
| 32795 | Metabolite-11478 | 201 | 4.3 | 4286 | 165.2 | - |
| 32797 | Metabolite-11480 | 201 | 4.42 | 4406 | 229 | - |
| 32800 | Metabolite-11483 | 201 | 4.45 | 4443 | 505.2 | - |
| 32802 | Metabolite-11485 | 201 | 4.49 | 4478 | 378.2 | - |
| 32807 | Metabolite-11490 | 201 | 4.77 | 4762 | 279.8 | - |
| 32808 | Metabolite-11491 | 201 | 4.85 | 4846 | 567.3 | - |
| 32813 | Metabolite-11496 | 201 | 5.58 | 5508 | 271.3 | - |
| 32814 | Metabolite-11497 | 201 | 5.37 | 5324 | 233.3 | - |
| 32815 | Metabolite-11498 | 201 | 5.78 | 5674 | 500.3 | - |
| 32822 | Metabolite-11505 | 201 | 1.61 | 1614 | 101.2 | - |
| 32825 | Metabolite-11508 | 201 | 2.97 | 2987 | 173.2 | - |
| 32827 | Metabolite-11510 | 201 | 3.92 | 3925 | 385.2 | - |
| 32829 | Metabolite-03653 | 200 | 0.82 | 826 | 144.2 | + |
| 32838 | Metabolite-11521 | 200 | 3.71 | 3755 | 286.2 | + |
| 32839 | Metabolite-11522 | 201 | 4.76 | 4754 | 313.2 | - |
| 32845 | Metabolite-11528 | 200 | 4.74 | 4832 | 316.3 | + |
| 32846 | Metabolite-11529 | 201 | 4.85 | 4845 | 624.3 | - |
| 32847 | Metabolite-11530 | 201 | 4.87 | 4866 | 313.2 | - |
| 32848 | Metabolite-11531 | 201 | 4.86 | 4850 | 391.3 | - |
| 32854 | Metabolite-11537 | 200 | 5.14 | 5160 | 366.3 | + |
| 32855 | Metabolite-11538 | 201 | 4.93 | 4920 | 311.3 | - |
| 32858 | Metabolite-11541 | 201 | 4.99 | 4984 | 399 | - |
| 32863 | Metabolite-11546 | 201 | 5.02 | 5015 | 448.4 | - |
| 32865 | Metabolite-11548 | 201 | 5.09 | 5081 | 391.3 | - |
| 32866 | Metabolite-11549 | 201 | 5.11 | 5093 | 339.3 | - |
| 32878 | Metabolite-11561 | 201 | 1.26 | 1252 | 267.1 | - |
| 32879 | Metabolite-11562 | 201 | 0.98 | 972 | 197.1 | - |
| 32880 | Metabolite-11563 | 201 | 0.98 | 971 | 115.2 | - |
| 32881 | Metabolite-11564 | 201 | 1.2 | 1188 | 177.1 | - |
| 32910 | Metabolite-11593 | 201 | 0.79 | 790 | 189.2 | - |
| 32912 | Metabolite-11595 | 201 | 0.92 | 919 | 665 | - |
| 32914 | Metabolite-11597 | 201 | 1 | 991 | 665 | - |
| 32926 | Metabolite-11609 | 201 | 1.48 | 1490 | 310.1 | - |
| 32945 | Metabolite-11628 | 201 | 3.14 | 3133 | 360.1 | - |
| 32952 | Metabolite-02277 | 201 | 3.61 | 3604 | 201.1 | - |
| 32954 | Metabolite-06132 | 201 | 3.66 | 3655 | 321.1 | - |
| 32965 | Metabolite-11648 | 201 | 4.84 | 4819 | 534.3 | - |
| 32970 | Metabolite-11653 | 201 | 5.82 | 5686 | 331.3 | - |
| 32971 | Metabolite-11654 | 200 | 2.53 | 2500 | 246.2 | + |
| 32978 | Metabolite-11656 | 200 | 0.6 | 612 | 227 | + |
| 33012 | Metabolite-11674 | 200 | 1.14 | 1151 | 189.1 | + |
| 33072 | Metabolite-11727 | 200 | 0.71 | 709 | 241 | + |
| 33073 | Metabolite-11728 | 200 | 0.8 | 804 | 427 | + |
| 33084 | Metabolite-11739 | 200 | 3.15 | 3239 | 769 | + |
| 33131 | Metabolite-11786 | 200 | 0.87 | 864 | 136 | + |
| 33132 | Metabolite-11787 | 200 | 1.13 | 1126 | 148.1 | + |
| 33133 | Metabolite-11788 | 200 | 1.17 | 1161 | 245 | + |
| 33138 | Metabolite-11793 | 200 | 3.57 | 3634 | 601.1 | + |
| 33140 | Metabolite-11795 | 200 | 1.46 | 1457 | 148.1 | + |
| 33154 | Metabolite-11809 | 200 | 5.23 | 5248 | 381.5 | + |
| 33159 | Metabolite-11814 | 200 | 2.39 | 2372 | 152.1 | + |
| 33163 | Metabolite-11818 | 200 | 2.56 | 2535 | 146.1 | + |
| 33169 | Metabolite-11824 | 201 | 1.4 | 1402 | 326.1 | - |
| 33171 | Metabolite-11826 | 201 | 1.48 | 1489 | 194.1 | - |
| 33173 | Metabolite-11828 | 201 | 1.69 | 1703 | 246.1 | - |
| 33174 | Metabolite-11829 | 201 | 1.8 | 1813 | 230 | - |
| 33178 | Metabolite-11833 | 201 | 1.97 | 1977 | 260.1 | - |
| 33183 | Metabolite-11838 | 201 | 2.3 | 2314 | 276 | - |
| 33188 | Metabolite-11843 | 201 | 2.69 | 2710 | 230.1 | - |
| 33190 | Metabolite-11845 | 201 | 2.87 | 2891 | 615 | - |
| 33192 | Metabolite-11847 | 201 | 3.05 | 3074 | 259.2 | - |
| 33194 | Metabolite-11849 | 201 | 3.2 | 3229 | 266.2 | - |
| 33198 | Metabolite-11853 | 201 | 3.59 | 3602 | 187.1 | - |
| 33203 | Metabolite-11858 | 201 | 4.42 | 4400 | 437.1 | - |
| 33206 | Metabolite-11861 | 201 | 4.63 | 4617 | 229.2 | - |
| 33209 | Metabolite-11864 | 201 | 5.02 | 5012 | 280.9 | - |
| 33216 | Metabolite-11871 | 200 | 5.25 | 5249 | 370.3 | + |
| 33219 | Metabolite-11874 | 201 | 5.23 | 5199 | 197.3 | - |
| 33225 | Metabolite-11880 | 201 | 5.44 | 5378 | 537.4 | - |
| 33227 | Metabolite-11882 | 201 | 5.52 | 5445 | 301.3 | - |
| 33228 | Metabolite-11883 | 200 | 5.54 | 5524 | 544.3 | + |
| 33232 | Metabolite-11887 | 201 | 5.85 | 5736 | 307.4 | - |
| 33237 | Metabolite-11892 | 201 | 0.71 | 710 | 367.1 | - |
| 33242 | Metabolite-11897 | 201 | 1.69 | 1664 | 292.1 | - |
| 33248 | Metabolite-11903 | 201 | 2.53 | 2484 | 283.1 | - |
| 33250 | Metabolite-11905 | 201 | 4.44 | 4401 | 283.2 | - |
| 33252 | Metabolite-11907 | 201 | 5.06 | 5036 | 313.3 | - |
| 33254 | Metabolite-11909 | 201 | 5.3 | 5272 | 297.3 | - |
| 33258 | Metabolite-11913 | 201 | 5.43 | 5402 | 275.3 | - |
| 33265 | Metabolite-11920 | 200 | 0.68 | 686 | 268.1 | + |
| 33323 | Metabolite-11977 | 200 | 3.21 | 3287 | 270.1 | + |
| 33366 | Metabolite-12020 | 200 | 5.15 | 5178 | 424.4 | + |
| 33380 | Metabolite-12029 | 201 | 0.68 | 683 | 329.1 | - |
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 scope of the invention.
Claims (9)
- A method of distinguishing non-alcoholic steatosis (NAFLD) from non-alcoholic steatohepatitis(NASH) in a subject having liver disease or disorder, comprising:analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for NAFLD and/or NASH in the sample, wherein the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 2, 3, 4B, 5B, 6B, 8, 9, 10, 11, 12, 13, and Figures 1, 2, 3; andcomparing the level(s) of the one or more biomarkers in the sample to NAFLD-positive reference levels that distinguish over NASH and/or to NASH-positive reference levels that distinguish over NAFLD in order to determine whether the subject has NASH.
- The method according to claim 1, wherein said method comprises determining the level of 3-methyl-2-oxobutyrate and 3-methyl-2-oxovalerate.
- A method of diagnosing whether a subject has non-alcoholic steatohepatitis (NASH), comprising:analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for non-alcoholic steatohepatitis in the sample, wherein the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, and/or Figure 2 and/or 3; andcomparing the level(s) of the one or more biomarkers in the sample to non-alcoholic steatohepatitis-positive and/or non-alcoholic steatohepatitis-negative reference levels of the one or more biomarkers in order to diagnose whether the subject has non-alcoholic steatohepatitis.
- A method of determining whether a subject is predisposed to developing steatohepatitis, comprising:analyzing a biological sample from a subject to determine the level(s) of one or more biomarkers for steatohepatitis in the sample, wherein the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected from Tables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, and/or Figure 2 and/or 3; andcomparing the level(s) of the one or more biomarkers in the sample to steatohepatitis-positive and/or steatohepatitis-negative reference levels of the one or more biomarkers in order to determine whether the subject is predisposed to developing steatohepatitis.
- The method according to claim 4, wherein said method is a method of determining whether a subject is predisposed to developing non-alcoholic steatohepatitis and wherein said one or more additional biomarkers are selected from Tables 1, 4B, 6B, 10, 11, 12, 13, and/or Figure 2 and/or 3.
- A method of monitoring progression/regression of steatohepatitis in a subject comprising: analyzing a first biological sample from a subject to determine the level(s) of one or more biomarkers for steatohepatitis in the sample, wherein the one or more biomarkers comprise 3-methyl-2-oxobutyrate and optionally one or more additional biomarkers selected fromTables 1, 3, 4B, 6B, 9, 10, 11, 12, 13, and/or Figure 2 and/or 3 and the first sample is obtained from the subject at a first time point; analyzing a second biological sample from a subject to determine the level(s) of the one or more biomarkers, wherein the second sample is obtained from the subject at a second time point; and 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 steatohepatitis in the subject.
- The method of claim 6, wherein the method further comprises comparing the level(s) of one or more biomarkers in the first sample, the level(s) of one or more biomarkers in the second sample, and/or the results of the comparison of the level(s) of the one or more biomarkers in the first and second samples to steatohepatitis-positive and/or steatohepatitis-negative reference levels of the oneor more biomarkers.
- The method according to claim 6 or claim 7, wherein said method is a method of monitoring progression/regression of non-alcoholic steatohepatitis (NASH).
- The method of any of the preceding claims, wherein the sample is analyzed using one or more techniques selected from the group consisting of mass spectrometry, enzymatic or biochemicalreactions, clinical chemistry, ELISA, and antibody linkage.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US98494207P | 2007-11-02 | 2007-11-02 | |
| US984942P | 2007-11-02 | ||
| US4245908P | 2008-04-04 | 2008-04-04 | |
| US42459P | 2008-04-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1216189A1 HK1216189A1 (en) | 2016-10-21 |
| HK1216189B true HK1216189B (en) | 2019-01-04 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2930512B1 (en) | Biomarkers for fatty liver disease and methods using the same | |
| AU2008279778B2 (en) | Method for Determining Insulin Sensitivity with Biomarkers | |
| JP6595984B2 (en) | Biomarkers associated with renal function and methods of using the same | |
| US20120003158A1 (en) | Biomarkers for Inflammatory Bowel Disease and Methods Using the Same | |
| WO2011036117A1 (en) | Method for the diagnosis of non-alcoholic steatohepatitis based on a metabolomic profile | |
| WO2009026153A1 (en) | Biomarkers for alzheimer's disease and methods using the same | |
| WO2019097089A1 (en) | Methods for prediction and early detection of diabetes | |
| Semmler et al. | Metabolomic profiles differentiate between porto-sinusoidal vascular disorder, cirrhosis, and healthy individuals | |
| FI20216291A1 (en) | Method for determining whether a subject is at risk of developing a musculoskeletal and/or connective tissue disease | |
| US8563318B2 (en) | Method for the diagnosis of non-alcoholic steatohepatitis based on a metabolomic profile | |
| HK1216189B (en) | Biomarkers for fatty liver disease and methods using the same | |
| EP4431945A1 (en) | Biomarkers discriminating the discordant phenotypes of x-adrenoleukodystrophy | |
| EP4679082A1 (en) | Method for improved determination of the acylcarnitine profile in biological material | |
| KR102519776B1 (en) | Biomarkers for diagnosing and predicting rheumatoid arthritis | |
| AU2016206265B2 (en) | Method for Determining Insulin Sensitivity with Biomarkers | |
| WO2024030680A1 (en) | Metabolic vulnerability analyzed by nmr | |
| CN119470683A (en) | Biomarkers, systems and applications for predicting ACLF | |
| Coen | Rabiya Zia | |
| AU2014216035B2 (en) | Method for Determining Insulin Sensitivity with Biomarkers |