8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine concentrations in various human body fluids – implications for their measurement and interpretation

Chiung-Wen Hu a, Marcus S. Cooke b,c, Yi-Hung Tsai d, Mu-Rong Chao d,e,*

a Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan

b Oxidative Stress Group, Department of Cancer Studies and Department Genetics, University of Leicester, Leicester, LE2 7LX, UK

c Department of Environmental & Occupational Health, Robert Stempel College of Public Health and Social Work, Florida International University, Miami, FL 33199, USA

d Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan

e Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan

* Corresponding author:

Supplementary Data V

Determination of native nucleobase (guanine, Gua) and 2′-deoxyribonucleoside (2′-deoxyguanosine, dGuo) in saliva, plasma and urine using online SPE LC-MS/MS

Four plasma samples, four saliva samples and four urine samples were obtained from healthy subjects. For plasma and saliva analysis, after addition of [15N5]-guanine ([15N5]-Gua) and [15N5]-2'-deoxyguanosine ([15N5]-dGuo), the samples were pretreated as described in the Materials and methods. Urine was simply diluted 10 times with a solution containing [15N5]-Gua and [15N5]-dGuo. Pretreated plasma and saliva or diluted urine samples were analyzed using two validated LC-MS/MS methods as previously reported by Chao et al. (2008) for dGuo analysis and Chao et al. (2007) for Gua analysis with a minor modification in chromatography gradient. The samples were analyzed in the positive ion multiple reaction monitoring mode, and the MRM transitions of the analytes were as follows: Gua (m/z 152→135 as quantifier ion and m/z 152→110 as qualifier ion), [15N5]-Gua (m/z 157→139 and m/z 157→114), dGuo (m/z 268→152 and m/z 268→117) and [15N5]-dGuo (m/z 273→157 and m/z 273→122). The results were shown in below Table S4. Concentration factors of Gua and dGuo were also calculated and shown in Fig. S5.

References:

Chao MR, Yen CC, Hu CW. (2008) Prevention of artifactual oxidation in determination of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction. Free Radic Biol Med 44:464-73

Chao MR, Wang CJ, Yen CC, Yang HH, Lu YC, Chang LW, Hu CW. (2007) Simultaneous determination of N7-alkylguanines in DNA by isotope-dilution LC-tandem MS coupled with automated solid-phase extraction and its application to a small fish model. Biochem J 402:483-90

Table S4 Gua and dGuo concentrations in plasma, saliva and urine

Variable / Concentrations (ng/mL)
In plasma (n=4),
Gua
dGuo
Gua/dGuoa / 16.0 ± 13
0.19 ± 0.08
147
In saliva (n=4),
Gua
dGuo
Gua/dGuo / 59 ± 42
1.9 ± 0.7
55
In urine (n=4),
Gua
dGuo
Gua/dGuo / 252 ± 97
5.2 ± 2.7
86

a The ratio expressed in mol/mol

Fig. S5 Mole ratio of Gua to dGuo in human body fluids and the concentration factor (CF) of Gua and dGuo for plasma-urine and plasma-saliva; the CF was obtained by dividing the mean Gua (or dGuo) concentration in urine (or saliva) by its mean concentration in plasma