Fluorescence quantification of allantoin in biological samples by cap-immobilized allantoinase/resorcinol assay

Allantoin, the final product of urate degradation in non-hominoid mammals, forms in vivo through an enzyme-dependent pathway whose restoration has been proposed for the treatment of hyperuricemic conditions. Non-enzymatic urate oxidation to allantoin also occurs, making it a potential biomarker of oxidative stress in human physiology and pathology. This justifies the quest for fast and accurate methods for allantoin quantification overcoming the limitations of the current approaches, ranging from chromatographic to mass spectrometry techniques. We propose a simple fluorimetric assay that includes an enzymatic step for the conversion of S-allantoin to allantoate, followed by acid hydrolysis of the latter into glyoxylate in the presence of resorcinol. The lactone forming upon reaction of glyoxylate with resorcinol, in basic conditions, exhibits strong and stable fluorescence emission in the visible range. The assay proved to be specific for allantoin, with a linear response in the range of physiological values for several biological fluids, such as serum, urine, and saliva. We also verified the lack of reactivity by the most common interferents, including urate and its degradation intermediates. The assay, which does not require sophisticated instrumentation other than a conventional spectrofluorometer or a lab bench plate reader, was implemented on a solid support, exploiting enzyme encapsulation in a silica gel matrix, to allow reusability and improve stability over time.