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HPLC analysis of pheomelanin degradation products in human urine
Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan.
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2003 (English)In: Pigment Cell Research, ISSN 0893-5785, Vol. 16, no 5, 480-486 p.Article in journal (Refereed) Published
Abstract [en]

A sensitive and specific high performance liquid chromatography (HPLC) method was developed to quantify 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP) in urine. In degradation studies of melanin pigment, 4-AHP and 3-AHP are derived from benzothiazine units of pheomelanin and pheomelanin-related metabolites such as trichochromes. 5-S-Cysteinyldopa-derived benzothiazine products give 4-AHP while 2-S-cysteinyldopa-derived benzothiazine products give 3-AHP. 3-AHP is also derived from nitrotyrosine formed by nitration of tyrosine with reactive nitrogen species. For this reason, the influence of this biological process on the amount of 3-AHP found in biological material have been investigated. The method is based on hydriodic acid hydrolysis of the melanin polymer and reversed-phase HPLC with electrochemical detection of the degradation products 4-AHP and 3-AHP. The mobile phase consists of 25 mM ammonium acetate and sodium octanesulfonate as an ion-pairing reagent. The 4-AHP and 3-AHP peaks were well separated and the detector response was linear within the range 0-2 ng injected for both compounds. With the developed chromatographic system, 4-AHP and 3-AHP showed good separation in the biological samples. There was a strong correlation between 4-AHP and 3-AHP in the urine of 50 malignant melanoma patients and two healthy subjects (R0.977). The two compounds were also strongly correlated with 5-S-cysteinyldopa in urine, the correlation coefficients being 0.862 and 0.907, respectively. The method described is sensitive enough for analysis of pheomelanin in urine and in several other biological samples. The results indicate that 3-AHP in urine is not influenced by excreted 3-nitrotyrosine and the data indicate that pheomelanins are excreted in the urine of melanoma patients.

Place, publisher, year, edition, pages
2003. Vol. 16, no 5, 480-486 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-21494DOI: 10.1034/j.1600-0749.2003.00086.xPubMedID: 12950724OAI: diva2:241439
Available from: 2009-10-02 Created: 2009-10-02 Last updated: 2009-10-12Bibliographically approved
In thesis
1. Pheomelanin markers in melanoma with reference to their excretion into urine
Open this publication in new window or tab >>Pheomelanin markers in melanoma with reference to their excretion into urine
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Skin pigmentation is an important issue in most cultures. Until recently we have not understood the most important elements of pigmentation regarding detailed chemical structure. The synthesis of melanin is very complex, and although core enzymes, other important proteins, and parts of the melanin structure have been identified much information in this context awaits disclosure.

The function of the melanocyte and the deposition of melanin pigments into the keratinocytes are very important in the protection against UV light. Melanin pigments consist of high-molecular structures often described as brown to black eumelanin and yellow to red pheomelanin. Eumelanin is photoprotective, whereas pheomelanin is believed to be carcinogenic after UV radiation. There is strong evidence that people of fair complexion with freckles who tan poorly are at higher risk of developing melanoma. These people have a higher pheomelanin to eumelanin ratio in their skin.

Melanoma, one of the most widely spread cancers, is derived from melanocytes. There is accumulating evidence that pigment constitution is highly involved in the development of melanoma. We found that patients with advanced melanoma secrete substantial amounts of pigment structures into the urine, in particular those with diffuse melanosis. In subsequently performed experiments we purified these pigments and subjected the product to chemical degradation by either hydrogen peroxide oxidation or hydriodic hydrolysis. Several new chromatographic methods were developed for the structural analysis of these products. Structural analysis of new chromatographic peaks was performed. In conclusion, complex pheomelanin structures as well as low molecular weight pigments and free benzothiazoles have been identified in the urine of patients with melanoma and diffuse melanosis.

The present thesis provides new insight into melanogenesis and melanoma progression. This opens the doorway to further approaches to the investigation of melanins and can help to understand fundamental problems about the structure and biosynthesis of natural melanins.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 67 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1143
Pheomelanin, melanoma, benzothiazole, aminohydroxyphenylalanine, diffuse melanosis, HILIC
National Category
Other Clinical Medicine
urn:nbn:se:liu:diva-21486 (URN)978-91-7393-566-1 (ISBN)
Public defence
2009-10-23, Berzeliussalen, Hälsouniversitet, Campus US, Linköpings Universitet, Linköping, 13:00 (Swedish)
Available from: 2009-10-12 Created: 2009-10-02 Last updated: 2010-01-14Bibliographically approved

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