Assessing biomarkers of oxidative stress: analysis of guanosine and oxidized guanosine nucleotide triphosphates by high performance liquid chromatography with electrochemical detection

Abstract

Oxidation of the guanosine moiety in DNA has become a hallmark biomarker in assessing oxidative stress. The oxidation of guanosine in the nucleotide triphosphate pool has been overlooked due to the lack of a reliable methodology. This method describes a sample processing and high performance liquid chromatography with electrochemical detection protocol for the analysis of the cellular pool of guanosine triphosphates and oxidized guanosine triphosphates. Validation of this method is demonstrated along with evaluation of these analytes in control and oxidizing conditions in vitro and in HEK 293T cells. Oxidation of this triphosphate pool occurred independently of oxidation to DNA.

Figure 1

Chromatographic profiles for a standard containing GTP, dGTP, G, and 2-dG (A) detected in channels set at 700, 785, and 850 mV, a standard containing oxo⁸G and oxo⁸dG detected with a channel set at 250 mV (B), and standards of oxo⁸GTP and oxo⁸dGTP detected at 250 mV (C).

Figure 2

Solid lines are calibration curves built with known amounts of 2-dG(A), G (B), oxo⁸dG (C), and oxo⁸G (D). Dashed lines are calibration curves built with equivalent amounts of standards containing dGTP (A), GTP (B), oxo⁸dGTP (C), and oxo⁸GTP (D) after alkaline phosphatase treatment. (n = 2).

Figure 3

Typical chromatograhic profiles of cytosolic cellular extracts, after treatment with alkaline phosphatase, obtained from HEK 293T cells in control or MPA-exposed populations (2μM, 1 hour). Typical retention times for G and 2-dG generated are presented in 3A, and that for oxo⁸G in 3Bm (chromatograms reflect a 3D shift for display purposes). Although channels 700, 785, and 850 mV were used for quantitation of GTP and dGTP, only channel 785 mV shown in figure. Bar graphs show cellular levels of GTP (C), dGTP (D), and oxo⁸GTP (E) in control (black bar) and MPA treated (white bar) cells. Values expressed as nanomoles (dGTP and GTP) or picomoles (oxo⁸GTP) per 10⁶ cells. Data expressed as the mean ± SEM (n =3–4, * p < 0.05).

Figure 4

Chromatograhic profiles of dephosphorylated GTP (A) and oxo⁸GTP (B) after GTP exposure to control (1mM L-ascorbic acid) or oxidizing conditions (1mM L-ascorbic acid and 10μM cupric sulfate). Bar graph shows nanomoles of GTP (C) and picomoles oxo⁸GTP (D) in GTP samples exposed to control (black bar) or oxidizing conditions (white bar). Data expressed as the mean ± SEM (n =6–9, * p < 0.05).

Figure 5

Chromatograhic profiles of alkaline phosphatase treated cytosolic cellular extracts obtained from HEK 293T cells in control (1mM L-ascorbic acid) or oxidizing (1mM L-ascorbic acid, 10 μM cupric sulfate) conditions. Typical elution profiles for the generated G and 2-dG (A) and typical elution profile for the oxo⁸G generated (B). Bar graphs represent cellular levels of GTP (C), dGTP (D), and oxo⁸GTP (E) in control (black bar and treated cell populations (white bar). Data expressed as levels per 10⁶ cells. Data expressed as the mean ± SEM (n =3, * p < 0.05).

Figure 6

Levels of oxo⁸dG, as compared to the levels of 2-dG, in DNA isolated from HEK 293T cells exposed to 1mM L-ascorbic acid (control, black bar) or 1mM L-ascorbic acid, 10 μM cupric sulfate (oxidizing conditions, white bar). Data expressed femtomoles of oxo⁸dG per nanomoles of 2-dG in DNA. Bars represent mean ± SEM (n =4).