Measurement of Melanin Metabolism in Live Cells by [U-13C]-L-Tyrosine Fate Tracing Using Liquid Chromatography-Mass Spectrometry.

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography. Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U-13C] L-tyrosine using liquid chromatography-mass spectrometry. Using this method, we confirmed the previous reports of the differences in melanin synthesis between melanocytes derived from individuals with different skin colors and MC1R genotype and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-3,4-dihydroxyphenylalanine, an important metabolite of L-tyrosine, is differentially controlled by multiple factors. Because L-tyrosine fate tracing is compatible with untargeted liquid chromatography-mass spectrometry‒based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis.