Quantitative determination of 8-hydroxy-2 '-deoxyguanosine in human urine by isotope dilution mass spectrometry: Normal levels in hemochromatosis

A previously developed method for quantitative determination of 8-hydroxygu anine by gas chromatography-mass spectrometry was modified to allow measure ment of 8-hydroxy-2'-deoxyguanosine in human urine. [4,5,6,8-C-13(4)]8-Hydr oxy-2'-deoxyguanosine was prepared by enzymatic coupling of [4,5,6,8-C-13(4 )]8-hydroxyguanine to deoxyribose-1-phosphate. Samples of human urine (2 mi ) were spiked with the labeled nucleoside (13 nmol) and subjected to solid phase extraction and reversed phase high performance liquid chromatography. The 8-hydroxy-2'-deoxyguanosine thus isolated was hydrolyzed by treatment with aqueous formic acid, and the resulting 8-hydroxyguanine was converted into its tetrakis-trimethylsilyl derivative: and subjected to gas-liquid ch romatographic-mass spectrometric analysis. Repeated determinations of 8-hyd roxy-2'-deoxyguanosine in pools of urine showed coefficients of variation o f 5 and 8% at concentrations of 8-hydroxy-2'-deoxyguanosine equal to 18 and 2 nM, respectively. Determination of 8-hydroxy-2'-deoxyguanosine in sample s of urine spiked with different amounts of the unlabeled nucleoside showed a mean recovery of 102%. Application of the analytical method to a group o f 11 apparently healthy subjects (mean age, 47 years) showed a mean level o f endogenously produced 8-hydroxy-2'-deoxyguanosine equal to 1.33 +/- 0.29 mu mol/mol creatinine. The level recorded for another group of 15 younger s ubjects (mean age, 28 years) was somewhat higher, that is, 1.58 +/- 0.84 mu mol/mol creatinine, corresponding to a 24-h production rate of 8-hydroxy-2 '-deoxyguanosine equal to 20.6 +/- 11.6 nmol (288 +/- 140 pmo1/24 h . kg bo dy weight). Hemochromatosis is a hereditary disease characterized by increa sed absorption of iron from the gastrointestinal tract and deposition of ir on in organs. Application of the analytical method to a group of 12 patient s with hereditary hemochromatosis who were under treatment with venesection s showed a mean level of urinary 8-hydroxy-2'-deoxyguanosine equal to 1.39 +/- 0.40 mu mol/mol creatinine. This value was not significantly different from those of healthy subjects. The fact that these patients had only sligh t or moderate iron overload at the time of urinary sample collection may ha ve influenced the urinary levels of 8-hydroxy-2'-deoxyguanosine in the pres ent study. (C) 1998 Elsevier Science Inc.