Association between Oxidative DNA Damage and Risk of Colorectal Cancer: Sensitive Determination of Urinary 8-Hydroxy-2'-deoxyguanosine by UPLC-MS/MS Analysis

Abstract

Oxidative DNA damage plays crucial roles in the pathogenesis of numerous diseases including cancer. 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the most representative product of oxidative modifications of DNA, and urinary 8-OHdG is potentially the best non-invasive biomarker of oxidative damage to DNA. Herein, we developed a sensitive, specific and accurate method for quantification of 8-OHdG in human urine. The urine samples were pretreated using off-line solid-phase extraction (SPE), followed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. By the use of acetic acid as an additive to the mobile phase, we improved the UPLC-MS/MS detection of 8-OHdG by 2.7-5.3 times. Using the developed strategy, we measured the contents of 8-OHdG in urine samples from 142 healthy volunteers and 84 patients with colorectal cancer (CRC). We observed increased levels of urinary 8-OHdG in patients with CRC and patients with tumor metastasis, compared to healthy controls and patients without tumor metastasis, respectively. Additionally, logistic regression analysis and receiver operator characteristic (ROC) curve analysis were performed. Our findings implicate that oxidative stress plays important roles in the development of CRC and the marked increase of urinary 8-OHdG may serve as a potential liquid biomarker for the risk estimation, early warning and detection of CRC.

Figure 1. Effect of mobile-phase additives on the MS detection sensitivity of 8-OHdG.

The mobile phase consisted of solvents A and B (pure methanol). An isocratic elution of 92.5% A and 7.5% B was used, and the flow was set at 0.25 mL/min. The concentration of 8-OHdG standard was 20 nM, and the injection volume was 5.0 μL.

Figure 2. Identification of 8-OHdG in urine sample by UPLC-MS/MS.

(a) Representative chromatograms from human urine sample displaying internal standard [¹⁵N₅]8-OHdG (m/z 289.1 > 173.0), 8-OHdG (m/z 284.1 > 168.0) and corresponding qualifier ion (m/z 284.1 > 117.0). (b) Representative chromatograms of internal standard and 8-OHdG standard.

Figure 3. Quantification and statistical analysis of 8-OHdG in human urine samples.

(a) 8-OHdG content in healthy volunteers and patients with CRC. (b) 8-OHdG content in patients from stage I to IV. (c) 8-OHdG content in patients without (stage I and II) and with (stage III and IV) tumor metastasis. (d) ROC curve for urinary 8-OHdG score.