Alex Edwin: “A Simple Fluorometric Assay for Distinguishing Creatine from Creatine Prodrugs”
ABSTRACT
Creatine Transporter Deficiency (CTD) is a rare metabolic disorder characterized, in part, by impaired transport of creatine into and throughout the brain, motivating the development of creatine prodrug therapeutics designed to enable transporter-independent cellular uptake. Accurate, high-throughput analytic methods capable of distinguishing creatine from structurally related prodrugs are therefore critical for therapeutic evaluation. Commercially available colorimetric and fluorometric creatine detection kits, while simple and sensitive, broadly detect creatine-containing molecules, limiting their utility in prodrug development. Ninhydrin-based creatine detection, originally developed in the late 1950s and recently introduced to the CTD field by the Schulze Lab, may offer greater specificity.
Here, we investigated the ability of the ninhydrin assay to distinguish creatine from creatine prodrugs and other guanidine-containing metabolites. Assay linearity and sensitivity were established using a two-fold serial dilution creatine standard curve. Reaction kinetics were characterized across multiple timepoints to define optimal incubation conditions. Compound specificity was assessed across a panel of guanidine-containing molecules, and quantum mechanical calculations of partial charge on guanidine nitrogen atoms were used to rationalize differential ninhydrin reactivity. The assay was then used to quantify intracellular creatine in HAP1 wild-type and SLC6A8 knockout cells treated with creatine or prodrug candidates, with fluorescence normalized to protein content.
The assay demonstrates robust linearity, predictable reaction kinetics, and clear discrimination between creatine and structurally related guanidine-containing metabolites, confirming its suitability as a practical screening tool for creatine prodrug development.