Apr 06, 2023

Public workspaceChemical degradation and determination of pheomelanin and eumelanin markers

DOI
dx.doi.org/10.17504/protocols.io.36wgqjz43vk5/v1
  • Kazumasa Wakamatsu1
  • 1Institute for Melanin Chemistry, Fujita Health University, Toyoake, Japan
Daniel Choo
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DOI: dx.doi.org/10.17504/protocols.io.36wgqjz43vk5/v1
Protocol Citation: Kazumasa Wakamatsu 2023. Chemical degradation and determination of pheomelanin and eumelanin markers. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjz43vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 06, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 80142
Keywords: ASAPCRN
Funders Acknowledgement:
From cancer associations to altered immunity in the pathogenesis of Parkinson's disease
Grant ID: ASAP-000312
Abstract
This is protocol involving chemical oxidation and reduction methods followed by high performance liquid chromatography (HPLC) detection of markers for pheomelanin and eumelanin.
Homogenize samples in water with Ten-Broeck glass homogenizer at a concentration of 10 mg/mL (if samples were < 5 mg, use 0.5 mL of water).
100 µL aliquots are then subjected to the chemical reactions.
Oxidation: Oxidize samples with 1.5% H2O2/K2CO3. After termination of reaction, leave mixtures for 20 hours at 25ºC (inducing secondary production of PTCA, PDCA, and TTCA)
Reduction: Heat samples with 57% HI in presence of H3PO2 at 130ºC for 20 hours. Analyze products, 4-AHP and 4-AHPEA
Levels of 4-AHP, the degradative product of DOPA pheomelanin, and 4-AHPEA, the degradative product of DA pheomelanin were analyzed by HPLC-ECD.
Protocol references
Ito, S., Nakanishi, Y., Valenzuela, R. K., Brilliant, M. H., Kolbe, L., & Wakamatsu, K. (2011). Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples: application to chemical analysis of human hair melanins.Pigment cell & melanoma research,24(4), 605-613.

Mitra, D., Luo, X., Morgan, A., Wang, J., Hoang, M. P., Lo, J., ... & Fisher, D. E. (2012). An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background.Nature,491(7424), 449-453.

Wakamatsu, K., & Ito, S. (2002). Advanced chemical methods in melanin determination.Pigment Cell Research,15(3), 174-183.