Development of an Analytical Approach to Quantify Ammonium Flux in Tissue and Biofluids
- AKI: Mechanisms - I
November 02, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
- Hahn, Oliver, Aarhus Universitet Institut for Biomedicin, Aarhus, Midtjylland, Denmark
- Jaegers, Johannes, Aarhus Universitet Institut for Biomedicin, Aarhus, Midtjylland, Denmark
- Rinschen, Markus M., Aarhus Universitet Institut for Biomedicin, Aarhus, Denmark
Group or Team Name
- Kidney Omics Lab.
Ammonia is a waste product of amino acids and other nitrogen-containing compounds, and its accumulation in the body can be toxic. The detection of abundance and flux is challenging because of its volatile nature, its conversion to ammonium and its fixation and excretion in other metabolic cycles such as the urea cycle. Here, we aimed to develop a method for quantifying changes in ammonia metabolism in multiple organs, urine, and plasma.
We modified the Berthelot method to derivatize ammonium into a measurable molecule called indophenol. Using ultra-high pressure liquid chromatography – triple quadrupole tandem mass spectrometry, this allows for quantifying both normal (14N) and heavy (15N) labeled ammonia through indophenol derivatization. The method was validated by measuring the pure indophenol and replicating the same peak when analyzing different dilutions of derivatized ammonium chloride standards.
Standard curves correlated with initial concentrations of the standards. We then used a porcine kidney injury model with unilateral IRI and infused heavy labeled 15N-ammonium chloride to trace the handling of ammonia in different organs. The 15N to 14N ratio was used to observe changes in the handling of the infused heavy ammonia in different organs during kidney injury, revealing strong alterations in the inner medulla.
The data also allowed for calculation of fluxes, revealing altered detoxification into urea. This method will be used in future studies to determine changes in waste product metabolism during kidney injury.