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 an advanced glycation end product (AGE) in a sample, comprising:
 (a) obtaining a biological sample from a subject; and   (b) performing liquid chromatography electrospray ionizing tandem mass spectrometry assay on the sample using a stable isotope dilution and an internal standard;   wherein the assay determines the quantity of AGE in the sample.   
     
     
         2 . The method of  claim 1 , wherein the AGE is N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) and the internal standard is  15 N 5 -CEdG. 
     
     
         3 . The method of  claim 2 , wherein elevated or depressed CEdG levels in a subject as compared to normal physiological CEdG levels indicates the presence of a metabolic disorder in the subject. 
     
     
         4 . The method of  claim 2 , wherein elevated CEdG levels in a subject as compared to normal physiological CEdG levels indicates diabetes. 
     
     
         5 . The method of  claim 2 , wherein depressed CEdG levels in a subject as compared to normal physiological CEdG levels indicates cancer. 
     
     
         6 . The method of  claim 5 , wherein the cancer is breast cancer. 
     
     
         7 . The method of  claim 1 , wherein the biological sample is selected from the group consisting of nucleic acid, urine, and tissue. 
     
     
         8 . A method of detecting diabetes in a subject comprising:
 (a) obtaining a biological sample from the subject; and   (b) quantifying N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) level in the sample using liquid chromatography electrospray ionization tandem mass spectrometry and an internal CEdG standard;   wherein, an elevated level of CEdG indicates that the subject has diabetes.   
     
     
         9 . The method of  claim 8 , wherein the method further comprises, upon determining that a subject has diabetes,
 (c) administering a treatment for diabetes; and   (d) after administration, repeating steps (a) and (b) 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.   
     
     
         10 . The method of  claim 8 , wherein the biological sample is urine or nucleic acid. 
     
     
         11 . The method of  claim 10 , wherein the nucleic acid is double-stranded DNA. 
     
     
         12 . The method of  claim 8 , wherein the internal standard is  15 N 5 -CEdG. 
     
     
         13 . The method of  claim 8 , further comprising preventing artifactual CEdG formation by adding aminoguanidine and/or D-penicillamine to the sample between steps (a) and (b). 
     
     
         14 . A method of detecting a glycolytic cancer in a subject comprising:
 (a) obtaining a biological sample from the subject; and   (b) quantifying a N 2 -carboxyethyl-2′-deoxyguanosine (CEdG) level in the sample using liquid chromatography electrospray ionization tandem mass spectrometry and an internal standard of  15 N 5 -CEdG;   wherein, a depressed level of CEdG indicates that the subject has a glycolytic cancer.   
     
     
         15 . The method of  claim 14 , wherein the method further comprises, upon determining that a subject has a glycolytic cancer,
 (c) administering a treatment for the cancer; and   (d) after administration, repeating steps (a) and (b) to determine whether the treatment is therapeutically effective, wherein if the level of CEdG has been increased or returned to normal, the treatment is therapeutically effective.   
     
     
         16 . The method of  claim 15 , wherein the treatment for cancer is chemotherapy or radiotherapy. 
     
     
         17 . The method of  claim 14 , wherein glycolytic cancer is breast cancer and the biological sample is breast tissue. 
     
     
         18 . The method of  claim 14 , wherein the biological sample is nucleic acid. 
     
     
         19 . The method of  claim 18 , wherein the nucleic acid is double-stranded DNA.