Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.

Abstract

DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solidphase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dualelectrode high-efficiency electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell per day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates.Biological damage caused by reactive oxygen species such as°2 -, H202, -OH, and '02 (1, 2) has been proposed to contribute to aging as well as a number of degenerative processes associated with aging such as cancer, heart disease, and cataract formation (3, 4). Various methods for detecting damage caused by these reactive oxygen species in vitro have led to the identification of oxidatively modified DNA bases (5).To study the relationship between endogenous oxidative damage to DNA and degenerative diseases associated with aging, it is desirable to develop techniques that can be used for the routine quantitative analysis ofthese damage products in individuals. Analytical approaches such as GC/MS and immunochemical techniques have been shown to be effective methods for detecting a number of specific lesions caused by reactive oxygen species, including the modified bases 8-hydroxyguanine (oh8Gua), 8-hydroxyadenine, and thymine glycol (5, 6). In general, these methods are useful for detecting the effects of high levels of damage caused by irradiation but are less useful for measuring levels of oxidative damage generated by endogenous metabolic processes. Previous efforts aimed at quantitating endogenously produced oxidative DNA damage involved measuring the urinary levels of thymine glycol and thymidine glycol (7,8) by HPLC combined with UV detection. However, this method also has certain limitations, principally the time and difficulty required to perform the assay.The inherent problem associated with detecting low levels of oxidatively damaged bases of DNA has been resolved by the identification of an oxidized DNA adduct, 8-hydroxy-2'-deoxyguanosine (oh8dG) (9), which can be quantitated with a high degree of sensitivity and selectivity by electrochemical detection (EC) after separation of enzymatic hydrolyzates of DNA by reversed-phase HPLC (HPLC/EC) (10). With this sensitive and selective technique, endogenous levels of oh8dG, which are increased upon exposure to oxidative conditions, have been measured in DNA isolated from v...

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Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

“…When damaged DNA is repaired, 8-OHdG produced is excreted in urine without further metabolism [9,10]. Urinary 8-OHdG has been widely used as a sensitive marker of oxidative DNA damage and of the total systemic oxidative stress in vivo [11,12]. Recent reports [13][14][15] found an increased 8-OHdG level in mononuclear cells and urine in patients with insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM).…”