A simple and useful method for evaluation of oxidative stress in vivo by spectrofluorometric estimation of urinary pteridines

Abstract

Pteridine derivatives are intermediate metabolites of folic acid and its cofactors. Oxidized-form pteridines, but not reduced-form pteridines, are fluorescent substances. The purpose of this study was to clarify whether oxidized-form pteridine level in urine, estimated by spectrofluorometry, reflects oxidative stress in vivo. The subjects were healthy middle-aged men (n = 258). Urinary pteridine level was estimated by spectrofluorometry with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Relationships of urinary pteridines with oxidative stress markers (urinary DNA/RNA oxidation products and 15-isoprostane F_2t) and with smoking were analyzed. Concentrations of pteridines, DNA/RNA oxidation products and 15-isoprostane F_2t were used after logarithmic transformation in linear analyses. Pteridine levels were significantly correlated with levels of DNA/RNA oxidation products (Pearson's correlation coefficient: 0.626, p < 0.01) and 15-isoprostane F_2t (Pearson's correlation coefficient: 0.695, p < 0.01). These correlations were not confounded by age, body mass index, history of smoking and estimated glomerular filtration rate in multivariate analysis. The mean urinary pteridine level was significantly higher in heavy smokers (16 cigarettes or more per day) than in nonsmokers and light smokers (less than 16 cigarettes per day) and was higher in light smokers than in nonsmokers. Thus, urinary fluorometric pteridine levels were shown to be associated with known biomarkers of oxidative stress as well as smoking, which causes oxidative stress in vivo. We propose spectrofluorometrical estimation of urinary pteridines as a simple and useful method for evaluation of oxidative stress in vivo.

Figure 1

Fluorescence intensity of each pteridine derivative (1 μM) measured at different excitation wavelengths with an emission wavelength fixed at 450 nm (A) and at different emission wavelengths with an excitation wavelength fixed at 360 nm (B).

Figure 2

Histograms of urinary fluorometric pteridine levels before creatinine correction without (A) and with (B) logarithmic transformation and urinary fluorometric pteridine levels after creatinine correction without (C) and with (D) logarithmic transformation.

Figure 3

Scatter plots for correlations of log-transformed urinary pteridines with log-transformed urinary DNA/RNA oxidation products (A) and 15-isoprostane F_2t (B). Pearson’s correlation coefficients are shown in the figures.