DETERMINATION OF URINARY 8-HYDROXYDEOXYGUANOSINE BY AUTOMATED COUPLED-COLUMN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - A POWERFUL TECHNIQUE FOR ASSAYING IN-VIVO OXIDATIVE DNA-DAMAGE IN CANCER-PATIENTS

An automated analytical method has been developed for determination of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (80HdG) in human urine, based on coupled-column high performance liquid chromatography with electrochemical detection. Urine is concentrated on Bondelut CH by means of an automated sample processor, and the enriched sample injected on to a polymeric reversed phase column coupled in line with an electrochemical detector and a C-18 reversed phase column. By use of the electrochemical detector, a suitable retention time interval is set for collection of the fraction containing 80HdG from the chromatography on the first column; this fraction is collected in a 2 mL loop and injected onto the C-18 column. The system is operated by an automatic valve station controlled by an integrator. The method has a large sample capacity and measures 31.1, 15.7, and 7.43 n/mol 80HdG/L urine with variation coefficients of 8, 8 and 24% within series and 8, 11 and 23% between series. Normal healthy individuals were found to excrete 14.9 +/- 7.8 nmol 80HdG/24 h, or 1.11 +/- 0.62 mu mol 80HdG per mel creatinine, in their urine, whereas increased levels of 80HdG were found in 24 h collections from a variety of cancer patients, both in samples taken before onset of oncological therapy (1.84 +/- 1.12 mu mol/mol creatinine, P < 0.01 versus healthy individuals) and after therapy onset (2.18 +/- 1.44 mu mol/mol creatinine, P < 0.001 versus healthy individuals). Moreover, mean values of 80HdG in random urinary samples from cancer patients were significantly higher than from healthy individuals (2.42 +/- 2.28 versus 1.19 +/- 0.48 mu mol/mol creatinine, P < 0.001), both in samples taken before therapy onset(l.91 +/- 0.96, P < 0.001 versus healthy individuals) and after (2.57 +/- 2.46, P < 0.001 versus healthy individuals). High levels of urinary 80HdG were found in patients subjected to whole body irradiation, and in patients receiving chemotherapy with various cytostatic agents. The potential use of the method for detecting increased urinary 80HdG excretion and conditions associated with increased oxidative DNA damage in humans is discussed.