RESUMEN
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.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Xantina Deshidrogenasa/sangre , Activación Enzimática , Estabilidad de Enzimas , Femenino , Humanos , Hipoxantina/metabolismo , Marcaje Isotópico , Isótopos , Masculino , Ácido Úrico/metabolismo , Xantina/metabolismo , Xantina Deshidrogenasa/metabolismoRESUMEN
In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2) = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 µL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.
Asunto(s)
Radioisótopos de Carbono/química , Pruebas de Enzimas/métodos , Radioisótopos de Nitrógeno/química , Ácido Úrico/química , Ácido Úrico/metabolismo , Xantina Deshidrogenasa/metabolismo , Animales , Cromatografía Liquida , Humanos , Espectrometría de Masas , Ratones , Ácido Úrico/farmacología , Xantina Deshidrogenasa/sangreRESUMEN
Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter.