The Role of Urea Cycle Functional Studies in Preclinical Research

Inborn errors of metabolism affecting the urea cycle are rare severe conditions caused by impaired nitrogen detoxification, leading to hyperammonemia and neurological morbidity across a broad clinical spectrum. Current biochemical diagnostics largely rely on quantification of metabolites. While indispensable for diagnosis and acute management, these snapshot readouts are highly dependent on external factors and often fail to reflect urea cycle function in metabolically compensated states, limiting their usefulness for disease stratification, therapeutic monitoring, and preclinical evaluation of treatments. Here, we reflect on the rationale, development, and application of a functional ureagenesis assay based on administration of [¹⁵N]-labeled ammonium chloride, enabling quantitative assessment of total in vivo urea cycle function. This assay development was prompted by discordant biochemical and phenotypic findings in a gene therapy-treated mouse model of ornithine transcarbamylase deficiency where supraphysiological enzyme activity did not translate into clinical improvement. We demonstrate that the [¹⁵N]-isotope based ureagenesis assay is applicable to in vivo models and enables detection of functional correction of the urea cycle. Importantly, we show its applicability in metabolically stable individuals affected by urea cycle disorders, where classical biochemical markers are often normal despite significant enzyme or transporter impairment. Together, these data establish the [¹⁵N]ammonium chloride-based functional ureagenesis assay as a robust and sensitive tool for quantifying urea cycle function. This approach offers clear advantages for preclinical research and translational studies, particularly in the evaluation of novel therapeutic strategies where traditional biomarkers lack discriminatory power.