Creatine Exposure in <i>Caenorhabditis elegans</i> Induces Physiological and Morphological Changes without Metabolic Conversion to Creatinine: A Novel HPLC Analysis

Creatine, a dietary supplement widely used to enhance muscle performance, undergoes metabolic processing in vertebrates that leads to creatinine as a byproduct. However, its impact on noncreatine-metabolizing organisms remains unclear. In this study, we investigate the effects of creatine and creatinine exposure on <i>Caenorhabditis elegans</i> (<i>C. elegans</i>), an invertebrate model lacking creatine kinase and instead relying on arginine kinase for energy homeostasis. We exposed <i>C. elegans</i> to creatine and creatinine, observing significant physiological changes in creatine-treated animals, including increased body size, heightened pharyngeal pumping rates, and enhanced locomotion, all of which persisted beyond the initial exposure period. By contrast, creatinine exposure showed minimal effects on these measures, suggesting a distinctive biological response to creatine itself. To determine whether <i>C. elegans</i> could metabolize creatine in conditions of high creatine concentrations, we developed a novel high-performance liquid chromatography (HPLC) method to quantify creatinine formation. Our results indicate no detectable metabolic conversion of creatine to creatinine in <i>C. elegans</i>, with creatinine levels comparable to spontaneous breakdown in media. The observed physiological changes thus likely stem from noncanonical interactions of creatine with cellular pathways, possibly disrupting osmotic balance, neuromuscular signaling, or energy homeostasis without typical metabolic processing. These findings imply that creatine can exert xenobiotic effects in organisms that lack native pathways for its metabolism, challenging assumptions about creatine's inertness outside vertebrate systems. Our study highlights <i>C. elegans</i> as a valuable model for investigating the environmental and biomedical impacts of creatine exposure, providing foundational insights into how non-native compounds may interfere with cellular functions in diverse organisms.