Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection: a biomarker of oxidative DNA damageupon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissu e. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromato graphy and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluore scence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per mi urine, which corresponds to about half of the phys iological excretion level in healthy control persons. After kidney transpla ntation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an ex cretion rate of up to 7.2 g of total protein per mmol creatinine was also o bserved immediately after transplantation and declined within the first 24 h of allograft function (0.35 +/- 0.26 g/mmol creatinine). The protein excr etion pattern, based on separation of urinary proteins on sodium dodecyl su lphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tub ular damage. The increased excretion of thymidine glycol after kidney trans plantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.