Simultaneous Measurement of Allantoin and Urate in Plasma: Analytical Evaluation and Potential Clinical Application in Oxidant:Antioxidant Balance Studies

Abstract

In humans, allantoin is formed by nonenzymatic oxidation of urate; it may, therefore, be useful in assessing oxidative stress(1)(2). Most published methods involve separate analysis of urate and allantoin and require extraction, hydrolysis, and derivatization procedures (1)(2)(3)(4)(5)(6). The primary aim of this study was to evaluate a slightly modified version of an HPLC assay described by Lux et al. (7) for the simultaneous measurement of urate and allantoin. A secondary aim was to explore the clinical utility of allantoin as a biomarker of oxidative stress, the hypothesis being that in disease associated with increased oxidative stress, allantoin increases because of an increased “oxidative turnover” of urate. The final aim of the study was to investigate the effect of age on urate and allantoin concentrations. Allantoin and uric acid were from Sigma; 1-heptanesulfonic acid, sodium salt monohydrate was from Sigma-Aldrich; potassium dihydrogen phosphate was from Merck; sodium hydroxide was from Riedel-de Haen; orthophosphoric acid was from BDH, and Moni-Trol Level 1 Chemistry Control Serum was from Dade International. MilliQ water (Millipore ultra-pure water system; Millipore) was used for preparation of all solutions. Aqueous stock solutions of allantoin (1000 μmol/L) and urate (2000 μmol/L) were prepared and stored at 4 °C. Because uric acid (urate) is more soluble at alkaline pH, sodium hydroxide (1 mol/L) was added dropwise until the pH was ∼9.0; at this pH, all urate was dissolved. Calibrators (10–100 μmol/L for allantoin; 50–1000 μmol/L for urate) were prepared in mobile phase from stock solutions: 25 μL of each calibrator was mixed with 25 μL of Moni-Trol control serum and 75 μL of mobile phase. Ultrafiltrates (see below) of diluted calibrators were used to construct daily calibration curves. For precision studies, we used 1-mL aliquots of pooled …