Identification and quantification of (5 0 R)- and (5 0 S)-8,5 0 -cyclo-2 0 -deoxyadenosines in human urine as putative biomarkers of oxidatively induced damage to DNA Pawel Jaruga a,b, * , Miral Dizdaroglu a a Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA b Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland article info Article history: Received 29 April 2010 Available online 13 May 2010 Keywords: 8,5 0 -Cyclo-2 0 -deoxyadenosines Disease biomarkers Hydroxyl radical Liquid chromatography/tandem mass spectrometry Oxidatively induced DNA damage abstract Biomarkers of oxidatively induced DNA damage are of great interest and can potentially be used for the early detection of disease, monitoring the progression of disease and determining the efficacy of therapy. The present work deals with the measurement in human urine of (5 0 R)-8,5 0 -cyclo-2 0 -deoxyadenosine (R- cdA) and (5 0 S)-8,5 0 -cyclo-2 0 -deoxyadenosine (S-cdA). These modified nucleosides had hitherto not been considered or investigated to be present in urine as possible biomarkers of oxidatively induced DNA dam- age. Urine samples were collected from volunteers, purified and analyzed by LC-MS/MS with isotope- dilution. R-cdA and S-cdA were detected in urine and quantified. Creatinine levels were also measured. In addition, we measured 8-hydroxy-2 0 -deoxyguanosine that is commonly used as a biomarker. This study shows, for the first time, that R-cdA and S-cdA exist in human urine and can be identified and quan- tified by LC-MS/MS. We propose that R-cdA and S-cdA may be well-suited biomarkers for disease pro- cesses such as carcinogenesis. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Oxidatively induced DNA damage comprising numerous prod- ucts occurs in living organisms by endogenous and exogenous sources, and is implicated in a variety of disease processes includ- ing carcinogenesis and aging [1]. Among DNA lesions, 8,5 0 -cyclop- urine-2 0 -deoxynucleosides are unique in that they represent concomitant damage to both the sugar and base moieties of a nucleoside [2]. Most of the oxidatively induced DNA base lesions are repaired by base excision repair (BER). However, 8,5 0 -cyclopu- rine-2 0 -deoxynucleosides cannot be removed from DNA by DNA glycosylases and are subject to nucleotide excision repair (NER) [3–5]. These compounds exist in living tissues and accumulate due to lack of DNA repair and various pathological conditions [2]. Biomarkers of oxidatively induced DNA damage are of great interest because of the involvement of this type of damage in disease processes, including carcinogenesis. Products of DNA damage can potentially be used as biomarkers for the early detection of disease, monitoring the progression of disease and determining the efficacy of therapy. In addition, such biomarkers may be useful in establish- ing the effect of antioxidants and other drugs to reduce the risk of disease development. DNA damage can be measured in living tis- sues, mostly requiring invasive procedures. The use of urine instead as the measurement matrix offers an alternative non-invasive route that can readily be employed to assess DNA products as potential disease biomarkers. Among these products, 8-hydroxyguanine (8- OH-Gua) and 8-hydroxy-2 0 -deoxyguanosine (8-OH-dG) have re- ceived the most attention for measurement in DNA and especially in urine [1,6,7]. Techniques such as high-performance liquid chro- matography (HPLC)/electrochemical detection and enzyme-linked immunosorbent assay (ELISA) were used for their measurement in urine. Mass spectrometric techniques with isotope-dilution have also been applied for the measurement of these and some other le- sions [8–12]. In most studies, there has been a significant discrep- ancy between the results obtained with these techniques in different laboratories [7]. It is beyond the scope of the present work to review the vast literature on this subject. For this purpose, the reader is referred to some recent review articles [6,7,13]. In the present study, we hypothesized that there must be other modified nucleosides in human urine, resulting from oxidatively induced damage to DNA. To test this hypothesis, we decided to search for (5 0 R)-8,5 0 -cyclo-2 0 -deoxyadenosine (R-cdA) and (5 0 S)- 8,5 0 -cyclo-2 0 -deoxyadenosine (S-cdA) that had hitherto not been considered or investigated to be present in human urine as possi- ble biomarkers of oxidatively induced DNA damage. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.050 Abbreviations: R-cdA, (5 0 R)-8,5 0 -cyclo-2 0 -deoxyadenosine; S-cdA, (5 0 S)-8,5 0 - cyclo-2 0 -deoxyadenosine; BER, base excision repair; NER, nucleotide excision repair; LC-MS/MS, liquid chromatography/tandem mass spectrometry; 8-OH-Gua, 8-hydroxyguanine; 8-OH-dG, 8-hydroxy-2 0 -deoxyguanosine; 8-OH-dA, 8-hydroxy- 2 0 -deoxyadenosine; HPLC, high-performance liquid chromatography; SPE, solid phase extraction. * Corresponding author at: Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. Fax: +1 301 975 8505. E-mail address: pawel.jaruga@nist.gov (P. Jaruga). Biochemical and Biophysical Research Communications 397 (2010) 48–52 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc