Introduction: Zebrafish vhl mutants develop key aspects of the human disease condition, including activation of the hypoxia-inducible factor (HIF) signaling pathway, polycythemia, excessive neovascularization, macular edema, and pronephric abnormalities.1 Whether the human VHL-related pheochromocytoma/paraganglioma (PPGL) phenotype is replicated in vhl mutant zebrafish has not been investigated. Our aim was to develop a method to measure catecholamines and metanephrines in zebrafish and to investigate the presence of features of PPGL.
Methods: liquid chromatography/mass spectrometry was used to measure metanephrines in zebrafish of lysates of larvae and in (urine containing) medium they were swimming in. Concentrations of metanephrines were compared between vhl-/- and wild-type (wt) zebrafish.
Results: Protocols for optimal sample preparation and (nor)metanephrine assays were established for both fish lysates and swimming medium. In lysates of 30 5-days-post-fertilization wt zebrafish larvae, the mean levels of metanephrine and normetanephrine were 0.010±0.001 (SEM and 0.036±0.002 pmol/larva, respectively. In 30 vhl-/- zebrafish larvae, normetanephrine levels were 2-fold higher (0.056±0.01; P<0,01), whereas metanephrine levels were >2 (0.004±0.001; P<0,001). In the swimming water, metanephrins were undetectable, while in medium of vhl-/- larvae the normetanephrine levels were 2,5-fold increased () as compared to wt (1.093±0.115 versus 0.429±0.049 pmol/larva; P<0,01). No obvious tumors of the fish’s ‘adrenal’ gland were observed.
Conclusion: We have established a method for the quantification of (nor)metanephrines in zebrafish and Vhl knock-out larvae display a normetanephrine dominant phenotype despite absence of obvious PPGL-like tumors. As such, vhl zebrafish represent a unique in vivo model for studying human VHL genotype–phenotype correlations, including (features of) PPGL.