Enzymatic assay of allantoin in serum using allantoinase and allantoate amidohydrolase

A new enzymatic assay for specifically measuring allantoin concentration in serum has been developed. The currently used methods for allantoin analysis are time consuming and nonspecific or depend on the use of expensive equipment. In our method, allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5). The allantoate produced is hydrolyzed to ureidoglycine and ammonia by the action of allantoate amidohydrolase (EC 3.5.3.9). Nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (EC 1.4.1.4) subsequently acts on the ammonia produced, resulting in a change in absorbance at 340 nm due to the consumption of reduced nicotinamide adenine dinucleotide phosphate. The amount of allantoin present is related to the change in the absorbance. The standard curve is linear up to at least 1 mM allantoin. The procedure is simple, rapid, and accurate. The method has been used to measure serum allantoin levels after oral administration of purine nucleotides to experimental animals, including rats that have uricase catalyzing the conversion of urate to allantoin.