A highly sensitive assay of human plasma xanthine oxidoreductase activity using stable isotope-labeled xanthine and LC/TQMS.

Abstract

Associations between elevated plasma xanthine oxidoreductase (XOR) activity and various pathologies have been widely reported. However, it has been difficult to accurately measure human plasma XOR activity because the XOR activity of humans is lower than that of animals such as mouse. We developed a highly sensitive assay for XOR activity utilizing a combination of [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. In the present study, we established and validated a novel human plasma XOR activity assay utilizing this technique. The calibration curve of [13C2,15N2]uric acid showed linearity over the range of 4-4000nM (r2>0.995) with a lower limit of quantitation of 4nM which corresponds to an XOR activity of 6.67pmol/h/mL plasma. Intra- and inter-assay coefficients of variation of pooled human plasma XOR activity were 6.5% and 9.1%, respectively. Plasma XOR activities of 20 healthy volunteers ranged from 32.8 to 227pmol/h/mL (mean±SD=89.1±55.1, n=20), which correlated with alanine transaminase (r=0.827), aspartate transaminase (r=0.487), and uric acid (r=0.502). The established assay is expected to be useful for investigating the function of XOR and the effect of its inhibitors in various diseases.