Optimization of a stable isotope-labelled substrate assay for measuring AGAT activity
2Genetics and Genome Biology, Hospital for Sick Children, Toronto, Canada
3Department of Paediatrics, Hospital for Sick Children, Toronto, Canada
4Clinical and Metabolic Genetics, Hospital for Sick Children, Toronto, Canada
Creatine is a molecule that facilitates the regeneration and distribution of ATP and acts to buffer imbalances in ATP levels to maintain its homeostasis. The synthesis of creatine occurs in a 2-step pathway that involves two enzymes: Arginine: Glycine Amidinotransferase (AGAT) and Guanidinoacetate N-Methyltransferase (GAMT).
Pathogenic variants in GATM, GAMT, or SLC6A8 cause the development of creatine deficiency syndromes (CDS). These diseases are characterized by a severe reduction of creatine within the brain and result in developmental delays and seizures. While CDS are primarily diagnosed through techniques such as biochemistry, MRS, and genetic sequencing, our goal is to develop a supplementary assay that can accurately measure AGAT activity in potential AGAT deficient patients. This can be done by measuring AGAT activity in healthy controls to establish a baseline activity and comparing those results to prospective patients.
To measure AGAT enzymatic activity, we adapted the stable-isotope method from Verhoeven et al. In this assay, 15N2 arginine and 13C215N glycine are used to generate penta-labelled 13C215N3 guanidinoacetate (GAA). We can then use LC-MS/MS to quantify the amount of penta-labelled GAA to determine specific AGAT activity.
We optimized the assay conditions by determining the optimal temperature, pH, substrate concentrations, and duration for the assay. We were able to quantify AGAT in all immortalized cells lines such as RH30, HepaRG, and HAP1 cells. With regards to patient samples, we chose to measure AGAT in lymphocytes, lymphoblasts, and fibroblasts as these cell types would be readily obtainable from blood and skin, respectively. AGAT activity was measurable in lymphocyte pellets and lymphoblasts, but not fibroblasts.
Our results indicate that this assay allows for reliable quantification of AGAT from cell lines and patient samples except fibroblasts. Moving forward, we aim to measure AGAT from additional human samples to establish a comprehensive baseline of AGAT activity.
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