Assessment of oxidative damage to proteins and DNA in urine of newborn infants by a validated UPLC-MS/MS approach

Julia Kuligowski, Isabel Torres-Cuevas, Guillermo Quintás, Denise Rook, Johannes B van Goudoever, Elena Cubells, Miguel Asensi, Isabel Lliso, Antonio Nuñez, Máximo Vento, Javier Escobar, Julia Kuligowski, Isabel Torres-Cuevas, Guillermo Quintás, Denise Rook, Johannes B van Goudoever, Elena Cubells, Miguel Asensi, Isabel Lliso, Antonio Nuñez, Máximo Vento, Javier Escobar

Abstract

The assessment of oxidative stress is highly relevant in clinical Perinatology as it is associated to adverse outcomes in newborn infants. This study summarizes results from the validation of an Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method for the simultaneous quantification of the urinary concentrations of a set of endogenous biomarkers, capable to provide a valid snapshot of the oxidative stress status applicable in human clinical trials, especially in the field of Perinatology. The set of analytes included are phenylalanine (Phe), para-tyrosine (p-Tyr), ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-NO2-tyrosine (3NO2-Tyr), 3-Cl-tyrosine (3Cl-Tyr), 2'-deoxyguanosine (2dG) and 8-hydroxy-2'-deoxyguanosine (8OHdG). Following the FDA-based guidelines, appropriate levels of accuracy and precision, as well as adequate levels of sensitivity with limits of detection (LODs) in the low nanomolar (nmol/L) range were confirmed after method validation. The validity of the proposed UPLC-MS/MS method was assessed by analysing urine samples from a clinical trial in extremely low birth weight (ELBW) infants randomized to be resuscitated with two different initial inspiratory fractions of oxygen.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Typical chromatograms of the selected…
Figure 1. Typical chromatograms of the selected biomarkers extracted from the analysis of spiked urine sample.
Note: Spiking concentrations were 73 nmol/L for 8OhdG and m-Tyr, 182 nmol/L for 2-dG, o-Tyr, 3NO2-Tyr and 3Cl-Tyr and 23 μM for p-Tyr and Phe.
Figure 2. Non-zero concentrations found from the…
Figure 2. Non-zero concentrations found from the analysis of 222 urine samples of extremely low birth-weight infants included in the double-blinded randomized clinical study REOX (REOX 2012-2013, EUDRACT 2088-005047-42).
The percentages of concentrations2-Tyr, 68% for 3Cl-Tyr, 0.4% for 8OH-dG and 0.4% for 2dG. Boxes indicate the 1st and the 3rd quartiles, the median is shown as a black line, whiskers mark the 9th and 91st percentiles, red triangles represent mean concentrations and blue circles are outliers.
Figure 3. Boxplots of the ratios of…
Figure 3. Boxplots of the ratios of o-Tyr/Phe, m-Tyr/Phe, 3NO2-Tyr/p-Tyr, 3Cl-Tyr/p-Tyr and 8OHdG/2dG for the set of non-zero urine samples included in the REOX clinical study.
Boxes indicate the 1st and the 3rd quartiles, the median is shown as a black line, whiskers mark the 9th and 91st percentiles, red triangles represent mean concentrations and blue circles are outliers.

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