Caffeine Biotransformation in Zebrafish Larvae: Integrated LC-MS/MS Quantification and FTIR Spatial Mapping.

Area of Science:

• Pharmacology
• Developmental Biology
• Metabolomics

Background:

• Caffeine (CAF) is a widely consumed psychoactive substance.
• Its metabolism is well-studied in humans and rodents, but limited in zebrafish larvae.
• Understanding CAF biotransformation in zebrafish is crucial for developmental toxicology and pharmacokinetics.

Purpose of the Study:

• To comprehensively characterize caffeine biotransformation in zebrafish larvae.
• To quantify caffeine metabolites using LC-MS/MS.
• To map the spatial distribution of caffeine and its metabolites using FTIR imaging.

Main Methods:

• Zebrafish larvae (4 dpf) were exposed to caffeine (15, 25, 50 mg/L) for 18 hours.
• Quantitative metabolite profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Spatially resolved tissue distribution mapping was conducted using Fourier-transform infrared (FTIR) imaging.

Main Results:

• Caffeine was dose-dependently metabolized to paraxanthine (up to 4.54%), theobromine (up to 1.32%), and theophylline (up to 0.27%).
• Metabolite ratios (PAR/CAF, THR/CAF) increased with caffeine concentration, while THY/CAF decreased.
• FTIR imaging confirmed the presence and tissue localization of caffeine, paraxanthine, and theobromine, but not theophylline.

Conclusions:

• This study provides the first comprehensive characterization of caffeine metabolism in zebrafish larvae.
• Low but detectable CYP450-mediated metabolic activity was observed prior to full hepatic maturation.
• Zebrafish larvae are validated as a suitable model for developmental pharmacokinetic studies.