2-hydroxyglutarate as a biomarker for chronic hypoxia

Abstract

The present invention provides biomarkers for sensitive, specific, accurate and quantitative diagnosis and assessment of chronic hypoxia. In particular, the present invention provides 2-hydroxyglutarate as a biomarker that is differentially produced in chronic hypoxia. Furthermore, embodiments of the invention are able to differentiate between chronic and acute hypoxia. Assays for levels of 2-hydroxyglutarate may be used alone or in conjunction with additional biomarkers of hypoxia to increase the precision of analysis. In particular embodiments of the invention, the level of 2-hydroxyglutarate and at least one second biomarker may be assayed to generate a hypoxic profile that can be compared to a reference or control profile, thereby diagnosing a subject as normoxic, chronically hypoxic, or acutely hypoxic.

1 . A method of diagnosing chronic hypoxia in a subject, comprising steps of:
 measuring a level of at least one biomarker in a sample from the subject, wherein the at least one biomarker comprises 2-hydroxyglutarate; and   comparing the measured level with a control level representative of acute hypoxia or normoxia, where chronic hypoxia is diagnosed when the measured level shows a significant difference from the control level.   
     
     
         2 . The method of  claim 1 , further comprising a step of managing treatment of the subject based on the determination of chronic hypoxia. 
     
     
         3 . The method of  claim 2 , further comprising a step of measuring the at least one biomarker after the step of managing. 
     
     
         4 . The method of  claim 1 , wherein the step of measuring comprises:
 measuring levels of at least two biomarkers in a sample from the subject, wherein one of the at least two biomarkers is 2-hydroxyglutarate; and   the method further comprises a step of comparing the measured levels with control levels representative of acute hypoxia or normoxia, where chronic hypoxia is diagnosed when the measure level shows a significant difference from the control levels.   
     
     
         5 . The method of  claim 1 , wherein the step of measuring biomarker comprises measuring by using mass spectrometry. 
     
     
         6 . The method of  claim 5 , wherein the mass spectrometry comprises gas-chromatography-mass spectrometry. 
     
     
         7 . The method of  claim 1 , wherein the control level is obtained from a normoxic individual. 
     
     
         8 . The method of  claim 7 , wherein the normoxic individual is the subject. 
     
     
         9 . The method of  claim 1 , wherein the control level is obtained from a population of normoxic subjects. 
     
     
         10 . The method of  claim 1 , wherein the control level is obtained from an acutely hypoxic individual. 
     
     
         11 . The method of  claim 1 , wherein the control level is obtained from a population of acutely hypoxic subjects. 
     
     
         12 . The method of  claim 9 , wherein the difference between the levels of the one or more biomarkers measure in the sample and the control level correlates with the severity of chronic hypoxia in the subject. 
     
     
         13 . The method of  claim 1 , wherein the at least one biomarker consists of 2-hydroxyglutarate. 
     
     
         14 . The method of  claim 1 , wherein the sample is selected from the group consisting of urine, blood, serum, cerebrospinal fluid, and combinations thereof. 
     
     
         15 . The method of  claim 1 , wherein the at least one biomarker is selected from the group consisting of, 2-hydroxyglutarate, IDH1, IDH2, HIF1, HIF2, 2HG dehydrogenase, and combinations thereof. 
     
     
         16 . A method of generating a hypoxic profile in a subject, the method comprising steps of:
 obtaining a sample from the subject;   measuring the level of at least one first biomarker, wherein the at least one first biomarker comprises 2-hydroxyglutarate;   measuring the level of at least one second biomarker;   comparing the levels of the at least one first and second biomarkers to a reference profile comprised of levels of the same biomarkers obtained by identical methods from a population of control subjects; and   based on the comparison, generating a hypoxic prolife that indicates whether the subject is normoxic or has chronic or acute hypoxia.   
     
     
         17 . The method of  claim 16 , wherein the reference profile is obtained from a population of normoxic control subjects. 
     
     
         18 . The method of  claim 16 , wherein the reference profile is obtained from a population of subjects having chronic hypoxia. 
     
     
         19 . The method of  claim 16 , wherein the reference profile is obtained from a population of subjects having acute hypoxia. 
     
     
         20 . The method of  claim 16 , wherein the step of measuring comprises measuring by mass spectrometry. 
     
     
         21 . The method of  claim 16 , wherein the step of measuring comprises measuring protein expression level. 
     
     
         22 . The method of  claim 21 , wherein the protein expression level is measured by immunoassay using one or more antibodies that specifically bind to the at least one second biomarker. 
     
     
         23 . The method of  claim 16 , wherein the step of measuring comprises measuring of nucleic acid expression level. 
     
     
         24 . The method of  claim 23 , wherein the nucleic acid expression level is measured by Northern blotting or RT-PCR amplification. 
     
     
         25 . A method of treating chronic hypoxia or an underlying cause thereof, the method comprising steps of:
 measuring a level of 2-hydroxyglutarate in a sample from a subject;   determining whether the level of 2-hydroxyglutarate is elevated at least two-fold compared to a normoxic control sample; and   administering a treatment to the subject.   
     
     
         26 . The method of  claim 25 , wherein the treatment is directed to chronic hypoxia. 
     
     
         27 . The method of  claim 26 , wherein the treatment is selected from the group consisting of supplemental oxygen therapy, transfusion of packed red blood cells, caffeine, vitamin therapy, mechanical ventilation, positive pressure therapy, physical exercise, surgical intervention, and combinations thereof. 
     
     
         28 . The method of  claim 25 , wherein the treatment is directed towards an underlying cause of chronic hypoxia. 
     
     
         29 . The method of  claim 28 , wherein the underlying cause of chronic hypoxia is selected from the group consisting of chronic obstructive pulmonary disorder, airway obstruction, acute respiratory distress syndrome, pneumonia, pneumothorax, emphysema, congenital heart defects, pulmonary embolism, pulmonary edema, asthma, cystic fibrosis, and combinations thereof. 
     
     
         30 . The method of  claim 25 , wherein the level of 2-hydroxyglutarate is elevated at least three-fold. 
     
     
         31 . The method of  claim 25 , further comprising a step of determining the level of 2-hydroxyglutarate in a similar sample from the subject after the step of administering. 
     
     
         32 . The method of  claim 31 , wherein the treatment reduces the level of 2-hydroxyglutarate by at least one-fold compared to pretreatment levels. 
     
     
         33 . The method of  claim 31 , wherein the treatment reduces the level of 2-hydroxyglutarate by at least two-fold compared to pretreatment levels. 
     
     
         34 . The method of  claim 25 , wherein the level of 2-hydroxyglutarate is measured by gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry or enzymatic assay. 
     
     
         35 . A method of screening a tumor sample for susceptibility to treatment with hypoxia-inducible prodrugs, the method comprising a step of determining whether the level of 2-hydroxyglutarate is elevated at least two-fold compared to a control sample. 
     
     
         36 . The method of  claim 35 , wherein the control sample is obtained from tissue adjacent to the tumor. 
     
     
         37 . The method of  claim 36 , wherein the control sample is a mean or median measurement from one or more similar tissues in a population of healthy individuals. 
     
     
         38 . The method of  claim 1 , wherein the step of measuring comprises measuring NADH level.