Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry

Abstract

Oxidative DNA damage, linked pathogenically to a variety of diseases such as cancer and ageing, can be investigated by measuring specific DNA repair products in urine. Within the last decade, since it was established that such products were excreted into urine, progress in their analysis in urine has been limited. Guanine is the DNA base most prone to oxidation. We present a method for determination of the urinary 8-hydroxylated species of guanine, based on direct injection of urine onto a high-performance liquid chromatography (HPLC)-tandem mass spectrometry system. The analysis covers the 8-hydroxylated base, ribonucleoside and deoxynucleoside, and the corresponding non-oxidised species. Without pre-treatment of urine the detection limits for the nucleobases are approximately 2 nM (50 fmol injected) and for the nucleosides approximately 0.5 nM (12.5 fmol injected). Previously, liquid chromatography of the nucleobases has been problematic but is made possible by low-temperature reverse-phase C18 chromatography, a method that increases retention on the column. In the case of the nucleosides, retention was almost total and provides a means for on-column concentration of larger urine samples and controlled high peak gradient elution. The total excretion of 8-hydroxylated guanine species was 212 nmol/24 h. The oxidised base accounted for 64%, the ribonucleoside for 23% and the deoxynucleoside for 13%, indicating substantial oxidation of RNA in humans. In rat urine, excretion of the oxidised base was more dominant, the percentages of the oxidised base, ribonucleoside and deoxynucleosides being 89, 8 and 3%. This finding is at odds with previous reports using immunoaffinity pre-purification and HPLC-electrochemical detection analysis. The developed method now makes it possible to measure oxidative nucleic acid stress to both RNA and DNA in epidemiological and intervention settings, and our findings indicate a substantial RNA oxidation in addition to DNA oxidation. The small volume needed also makes the method applicable to small experimental animals.

Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine

“…Urine is a very complex matrix, and therefore, the common challenge for all chromatographic techniques has been to clean-up the urine sufficiently to simplify analysis, which very often also extends instrument life. At its simplest, column switching has meant that, after chromatographic separation of the urine's constituents, only the fraction containing the compound of interest (e.g., 8-oxodG) is applied to the final separation column and detector, either ECD (19,20) or MS (21); the remainder is diverted to waste.…”

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

“…The loss of HNCO from the [M ϩ H] ϩ ion of [1,[3][4][5][6][7][8][9][10][11][12][13][14][15] N]cytosine, however, would require a rearrangement of cytosine component before the elimination. In this context, it is well-known that adenosine can undergo Dimroth rearrangement [22,23] to exchange the exocyclic N 6 and endocyclic N1.…”

“…It has been suggested that these modified nucleosides in body fluids may serve as biomarkers for various diseases [10 -12] and oxidative stress [13,14]. Mass spectrometry may offer an accurate and sensitive means for the identification and quantification of modified nucleosides, especially when coupled to gas or liquid chromatography (GC or LC) [15,16].…”

Low Melatonin Production During Adulthood - Phase 2: Association with Levels of Hydroxyl Radical Scavenging and DNA Damage

“…High-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) has been used to simultaneously quantitate urinary levels of . This assay appears superior to both the comet and the HPLC-ECD assays (Weimann et al 2002). Assays to estimate melatonin production were already performed in Phase 1.…”