Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent

et al. 2020

IJMS

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Residual melanins have been detected in multimillion-year-old animal body fossils; however, confident identification and characterization of these natural pigments remain challenging due to loss of chemical signatures during diagenesis. Here, we simulate this post-burial process through artificial maturation experiments using three synthetic and one natural eumelanin exposed to mild (100 °C/100 bar) and harsh (250 °C/200 bar) environmental conditions, followed by chemical analysis employing alkaline hydrogen peroxide oxidation (AHPO) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results show that AHPO is sensitive to changes in the melanin molecular structure already during mild heat and pressure treatment (resulting, e.g., in increased C-C cross-linking), whereas harsh maturation leads to extensive loss of eumelanin-specific chemical markers. In contrast, negative-ion ToF-SIMS spectra are considerably less affected by mild maturation conditions, and eumelanin-specific features remain even after harsh treatment. Detailed analysis of ToF-SIMS spectra acquired prior to experimental treatment revealed significant differences between the investigated eumelanins. However, systematic spectral changes upon maturation reduced these dissimilarities, indicating that intense heat and pressure treatment leads to the formation of a common, partially degraded, eumelanin molecular structure. Our findings elucidate the complementary nature of AHPO and ToF-SIMS during chemical characterization of eumelanin traces in fossilized organismal remains.

Section: Methodsmentioning

confidence: 99%

Chemical Evaluation of Eumelanin Maturation by ToF-SIMS and Alkaline Peroxide Oxidation HPLC Analysis

Jarenmark

Sjövall

et al. 2020

IJMS

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“…We developed the widely used microanalytical methods of alkaline H 2 O 2 oxidation (AHPO) and hydroiodic acid (HI) hydrolysis followed by high‐performance liquid chromatography (HPLC). AHPO generates pyrrole‐2,3,5‐tricarboxylic acid (PTCA), a EM marker, and thiazole‐2,4‐5‐tricarboxylic acid (TTCA), a PM marker (Ito et al., 2011, Ito, Del Bino et al, 2020; Ito & Fujita, 1985). PM can also be analyzed as 4‐amino‐3‐hydroxyphenylalanine (4‐AHP) generated by HI hydrolysis (Wakamatsu et al., 2002).…”

Section: Biochemical Control Of Mixed Melanogenesis By Tyrosinase Activity and Cysteine Concentrationmentioning

confidence: 99%

Chemical and biochemical control of skin pigmentation with special emphasis on mixed melanogenesis

Wakamatsu

Zippin

Pigment Cell Melanoma Res

2021

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Melanins are widely distributed in animals and plants; in vertebrates, most melanins are present on the body surface. The diversity of pigmentation in vertebrates is mainly attributed to the quantity and ratio of eumelanin and pheomelanin synthesis. Most natural melanin pigments in animals consist of both eumelanin and pheomelanin in varying ratios, and thus, their combined synthesis is called “mixed melanogenesis.” Gene expression is an established mechanism for controlling melanin synthesis; however, there are multiple factors that affect melanin synthesis besides gene expression. Due to the differential sensitivity of the eumelanin and pheomelanin synthetic pathways to pH, melanosomal pH likely plays a major role in mixed melanogenesis. Here, we focused on various factors affecting mixed melanogenesis including (1) chemical regulation of melanin synthesis, (2) melanosomal pH regulation during normal melanogenesis and effect on mixed melanogenesis, and (3) mechanisms of melanosomal pH control (proton pumps, channels, transporters, and signaling pathways).

“…Pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), and thiazole-2,4,5-tricarboxylic acid (TTCA), which are the markers of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA) eumelanin subunits, native and modified benzothiazole units of pheomelanin, respectively, were measured using alkaline H 2 O 2 oxidation (AHPO) as described elsewhere [ 21 ]. These markers were analyzed by the improved method of HPLC [ 43 ]. 4-Amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP), which are the markers of 5- S -cysteinyldopa and 2- S -cysteinyldopa, respectively, were measured using hydroiodic acid (HI) hydrolysis as described elsewhere [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

Photoreactivity of Hair Melanin from Different Skin Phototypes—Contribution of Melanin Subunits to the Pigments Photoreactive Properties

Mokrzyński

Wakamatsu

et al. 2021

IJMS

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Photoreactivity of melanin has become a major focus of research due to the postulated involvement of the pigment in UVA-induced melanoma. However, most of the hitherto studies were carried out using synthetic melanin models. Thus, photoreactivity of natural melanins is yet to be systematically analyzed. Here, we examined the photoreactive properties of natural melanins isolated from hair samples obtained from donors of different skin phototypes (I, II, III, and V). X-band and W-band electron paramagnetic resonance (EPR) spectroscopy was used to examine the paramagnetic properties of the pigments. Alkaline hydrogen peroxide degradation and hydroiodic acid hydrolysis were used to determine the chemical composition of the melanins. EPR oximetry and spin trapping were used to examine the oxygen photoconsumption and photo-induced formation of superoxide anion, and time-resolved near infrared phosphorescence was employed to determine the singlet oxygen photogeneration by the melanins. The efficiency of superoxide and singlet oxygen photogeneration was related to the chemical composition of the studied melanins. Melanins from blond and chestnut hair (phototypes II and III) exhibited highest photoreactivity of all examined pigments. Moreover, melanins of these phototypes showed highest quantum efficiency of singlet oxygen photogeneration at 332 nm and 365 nm supporting the postulate of the pigment contribution in UVA-induced melanoma.