The role of different organs in the hydroxylation of pumiliotoxin (+)-251D to allopumiliotoxin (+)-267A in the poison frog Adelphobates galactonotus

Chemical defense is a pivotal strategy for predator, pathogen, and parasite avoidance in amphibians. Although most defensive chemicals in amphibians are obtained through biosynthesis, poison frogs sequester dietary alkaloids, in some cases hydroxylating or N-methylating the ingested alkaloids to form more potent chemicals. Although more than 1200 lipophilic alkaloids have been isolated in anuran secretions, the mechanisms underlying alkaloid sequestration and biotransformation remain largely unknown. This study investigates the biochemical pathways employed by Adelphobates galactonotus to sequester and transform ingested alkaloids. Specifically, we focused on the hydroxylation of orally administered pumiliotoxin (+)-251D to form allopumiliotoxin (+)267A using ultra performance liquid chromatography - triple quadrupole mass spectrometry. We examined the distribution of these alkaloids across several organs, including the liver, skin, digestive tract, gallbladder, kidney, and fat bodies. Using a novel protocol for sample preparation via solid-phase extraction, our study elucidates the translocation patterns of these alkaloids, along with differences in their ratios. We detected pumiliotoxin (+)-251D, and its hydroxylated derivate in the liver, suggesting hepatic involvement in processing, while the skin was the primary site of storage for both alkaloids. Additionally, we report the presence of these alkaloids in the urine and fat bodies, suggesting a passive storage mechanism. This study advances our understanding of anuran chemical defense and metabolic adaptations.