Sensitive Detection of Urinary 8-OHdG as Colorectal Cancer Biomarker

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

Journal: Scientific Reports

Published: 2016

Background

Oxidative DNA damage, primarily caused by reactive oxygen species (ROS), plays a significant role in the development of many diseases, including cancer. 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a major oxidative DNA damage marker and a potential non-invasive biomarker detectable in urine. Colorectal cancer (CRC) is one of the most prevalent cancers globally, and early detection is essential for reducing its impact. Existing methods for 8-OHdG detection vary in sensitivity and specificity, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) offering advantages in accuracy. However, challenges in ionization efficiency have limited sensitivity. This study developed a highly sensitive method using UPLC-MS/MS and solid-phase extraction to quantify urinary 8-OHdG and explored its association with CRC risk, highlighting its potential use in early cancer detection and monitoring.

Materials & Methods

Chemicals:

High-performance liquid chromatography (HPLC) grade methanol, 8-hydroxy-2'-deoxyguanosine (8-OHdG), 2'-deoxyguanosine (dG), formic acid, acetic acid, ammonium formate, ammonium acetate, and ammonium bicarbonate were obtained from Sigma-Aldrich. Ultrapure water was prepared with a Milli-Q purification system. The isotopically labeled internal standard, [15N5]8-OHdG, was purchased from Cambridge Isotope Laboratories.

Sample Collection:

The study was approved by the IRB of the Second Affiliated Hospital, Zhejiang University School of Medicine (SAHZU). A total of 84 untreated colorectal cancer (CRC) patients and 142 healthy controls were recruited. All subjects gave informed consent. Morning midstream urine samples were collected, frozen immediately, and stored at −80 °C. Creatinine levels were also measured to normalize urine concentrations.

UPLC-ESI-MS/MS Analysis:

An Acquity UPLC system was used with a BEH C18 column. The mobile phase consisted of 0.1% acetic acid (A) and methanol (B), run isocratically (92.5% A, 7.5% B). Detection was performed using a 4000 QTRAP mass spectrometer in positive ESI mode. Quantification and confirmation of 8-OHdG and [15N5]8-OHdG were conducted using multiple reaction monitoring (MRM) with optimized ion source conditions.

Solid-Phase Extraction (SPE):

Urine was thawed, centrifuged, and mixed with internal standard. SPE was performed using Oasis HLB cartridges preconditioned with methanol and water. After sample loading and washing, elution was performed with methanol/water (1:1). Samples were dried and reconstituted in water before UPLC-MS/MS analysis.

Method Validation:

Selectivity: Assessed by MRM and inspection of chromatograms for interferences.
Linearity: Evaluated using standard curves (1–300 nM) with internal standard.
LOD/LOQ: Defined as signal-to-noise ratios of 3:1 and 10:1, respectively.
Precision & Accuracy: Assessed via intra- and inter-day tests using QC samples at 10, 50, and 200 nM.
Recovery: Determined by spiking known concentrations of 8-OHdG into urine and comparing results.
Matrix Effect: Evaluated by comparing calibration curves from standard-spiked urine extracts and pure standards.

Statistical Analysis:

Performed using SPSS 20.0. T-tests compared 8-OHdG levels between CRC patients and controls. One-way ANOVA evaluated differences across cancer stages. Logistic regression assessed associations with age, gender, and 8-OHdG levels. ROC analysis tested model performance. Significance was defined as p < 0.05.

Results

Optimization of Mobile Phase Additive for MS Detection of 8-OHdG

Among five volatile additives tested (CH₃COOH, CH₃COONH₄, HCOOH, HCOONH₄, and NH₄HCO₃), acetic acid (CH₃COOH) significantly enhanced the MS signal response for 8-OHdG, improving ionization efficiency by suppressing formation of sodium and potassium adducts. Compared to NH₄HCO₃, CH₃COOH increased the signal by 2.7–5.3 times and provided better signal-to-noise ratio and chromatographic separation from 2'-deoxyguanosine (dG), reducing potential interference.

Validation of Analytical Method

The method showed excellent selectivity, linearity, sensitivity, precision, and accuracy:

Linearity: R² = 0.9993 for 1.0–300.0 nM concentration range.
LOD/LOQ: 1 fmol and 3 fmol, respectively.
Precision: Intra-day RSD: 1.0–1.9%; Inter-day RSD: 0.9–1.4%.
Accuracy: 98.0–101.7%.
Recovery: 101.9–118.1% with RSD < 4.0%.
Matrix effect: Negligible (slope ratio: 91.5%).
System stability: Maintained over hundreds of injections.

Identification and Quantification in Urine Samples

The method was applied to urine samples from 142 healthy individuals and 84 CRC patients. Retention times and ion transitions confirmed accurate identification of 8-OHdG. Urinary 8-OHdG was normalized to creatinine levels:

Healthy controls: 0.24–2.47 nmol/mmol creatinine (avg: 1.07 ± 0.49).
CRC patients: 0.51–4.37 nmol/mmol creatinine (avg: 1.68 ± 0.85); significantly higher (p < 0.0001).

Correlation with Cancer Stage and Metastasis

Urinary 8-OHdG levels increased progressively with CRC stage:

Stage I: 1.30 ± 0.61
Stage II: 1.51 ± 0.78
Stage III: 1.78 ± 0.84
Stage IV: 2.29 ± 1.07 nmol/mmol creatinine

Patients with metastatic tumors (stage III/IV) showed significantly higher levels than non-metastatic (stage I/II) patients (p < 0.05).

Logistic Regression and ROC Analysis

Higher urinary 8-OHdG (>1.5 nmol/mmol) was associated with a 3.68-fold increased risk of CRC (p < 0.0001).

Age, not gender, also significantly correlated with CRC risk.

ROC analysis:

AUC (8-OHdG only): 0.722
AUC (8-OHdG + age): 0.853, indicating excellent diagnostic performance.

These results demonstrate that urinary 8-OHdG is a sensitive, non-invasive biomarker for CRC presence, progression, and metastatic status.

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

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.

Reference

Guo, Cheng, et al. "Association between oxidative DNA damage and risk of colorectal cancer: sensitive determination of urinary 8-hydroxy-2'-deoxyguanosine by UPLC-MS/MS analysis." Scientific reports 6.1 (2016): 1-9. https://doi.org/10.1038/srep32581

* For Research Use Only. Not for use in diagnostic procedures.

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