METHODS OF QUANTIFYING N2-(1-CARBOXYETHYL)-2'-DEOXY-GUANOSINE (CEdG) AND SYNTHESIS OF OLIGONUCLEOTIDES CONTAINING CEdG

Abstract

Methods of quantifying a N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) levels in biological samples and comparing those levels to known normal levels can diagnose a number of disorders, including diabetes and cancer. Methods can also determine whether therapies for disorders are effective by measuring CEdG levels before and after treatment. Measurement of CEdG levels occurs using liquid chromatography electrospray ionization tandem mass spectrometry.

1 . A method of quantifying N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) in a tumor tissue sample, comprising:
 performing a liquid chromatography electrospray ionizing tandem mass spectrometry assay on a tumor tissue sample from a subject using a stable isotope dilution and an internal  15 N 5 -CEdG standard comprising oligonucleotides containing stereochemically pure (R) or (S)  15 N 5 -CEdG; 
 wherein the assay determines the quantity of CEdG in the tumor tissue sample. 
 
     
     
         2 . The method of  claim 1 , wherein the quantity of CEdG in the tumor tissue sample is compared to a quantity of CEdG in a normal adjacent tissue sample to determine whether the tumor tissue is cancerous. 
     
     
         3 . The method of  claim 2 , wherein the tumor tissue is likely to be cancerous and indicative of the presence of a glycolytic cancer when the quantity of CEdG in the tumor tissue sample is lower than the quantity of CEdG in the normal adjacent tissue. 
     
     
         4 . The method of  claim 3 , wherein the glycolytic cancer is breast cancer, lung cancer or kidney cancer. 
     
     
         5 . The method of  claim 1 , wherein the internal  15 N 5 -CEdG standard quantifies diastereomers CEdG (S) and CEdG (R).  
     
     
         6 . The method of  claim 4 , wherein elevated or depressed CEdG (S) levels, CEdG (R) levels, or both in a subject as compared to normal physiological CEdG (S) levels, CEdG (R) levels, or both indicates the presence of a cancer in the subject. 
     
     
         7 . The method of  claim 4 , wherein depressed CEdG (S) levels, CEdG (R) levels, or both in a subject as compared to normal physiological CEdG (S) levels, CEdG (R) levels, or both indicates the presence of a glycolytic cancer. 
     
     
         8 . The method of  claim 6 , wherein the glycolytic cancer is breast cancer, lung cancer or kidney cancer. 
     
     
         9 . A method of detecting cancer in a subject comprising:
 quantifying a CEdG level in a biological sample from a subject using liquid chromatography electrospray ionization tandem mass spectrometry and an internal CEdG standard comprising oligonucleotides containing stereochemically pure (R) or (S)  15 N 5 -CEdG;   wherein a lower level of CEdG as compared to a normal physiological CEdG level indicates that the subject has a glycolytic cancer.   
     
     
         10 . The method of  claim 9 , wherein the internal CEdG standard quantifies diastereomers CEdG (S) and CEdG (R). 
     
     
         11 . The method of  claim 9 , wherein the glycolytic cancer is resistant to chemotherapy.  
     
     
         12 . The method of  claim 11 , wherein the method further comprises, upon determining that a subject has a glycolytic cancer:
 administering a treatment for the glycolytic cancer to restore sensitivity to chemotherapy; and   after administration, repeating the steps in  claim 5  to determine whether the treatment is therapeutically effective;   wherein if the level of CEdG has been reduced or returned to normal, the treatment is therapeutically effective.   
     
     
         13 . The method of  claim 12 , wherein the treatment is chemotherapy or radiotherapy. 
     
     
         14 . The method of  claim 12 , wherein the treatment is an inhibitor of glycoxalase 1, aldose reductase, or both. 
     
     
         15 . The method of  claim 9 , wherein the glycolytic cancer is breast cancer, lung cancer or kidney cancer. 
     
     
         16 . The method of  claim 9 , further comprising preventing artifactual CEdG formation by adding aminoguanidine and/or D-penicillamine to the sample prior to quantifying the CEdG level in the sample.