Stable Isotope Assays for Urea Cycle Disorders

We quantitatively determine urea synthesis rates using sodium heparin plasma specimens collected after oral ingestion of [1-¹³C] sodium acetate (Yudkoff et al). Once a baseline ureagenesis rate is established, changes can be evaluated in subjects with urea cycle disorders such as ornithine transcarbamylase (OTC) deficiency, including during gene therapy or RNA-based therapeutic studies.

The ureagenesis assay provides a direct functional measure of urea cycle activity. It also enhances patient safety by offering an endpoint beyond ammonia and amino acid monitoring—supporting decisions around pathway engagement and eligibility for adjustments in disease management during clinical trials.

Ureagenesis requires measurement of both ¹³C-urea enrichment and total urea concentration. Only small plasma volumes (25–50 µL) are needed, enabling studies in preclinical rodent models as well as pediatric and adult clinical populations.

The glutamate → glutamine route of nitrogen waste clearance complements the urea cycle by providing distributed ammonia detoxification, nitrogen trafficking, and becomes upregulated when urea-cycle capacity is limited (e.g., liver disease, urea-cycle disorders). Glutamate is converted to glutamine by glutamine synthetase: glutamate + NH₃ + ATP → glutamine

Following administration of ¹⁵N-glutamate or ¹⁵N-ammonium chloride, we measure isotopic enrichment of ¹⁵N-glutamine—and its unlabeled counterpart—in plasma samples using gas chromatography–mass spectrometry (GC-MS) or gas chromatography–combustion isotope ratio mass spectrometry (GC-C-IRMS).

These measurements quantify GS activity alongside ureagenesis, providing complementary insight into total nitrogen waste clearance.