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Pheomelanin markers in melanoma with reference to their excretion into urine
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.
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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1143
Keyword [en]
Pheomelanin, melanoma, benzothiazole, aminohydroxyphenylalanine, diffuse melanosis, HILIC
National Category
Other Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-21486ISBN: 978-91-7393-566-1 (print)OAI: oai:DiVA.org:liu-21486DiVA: diva2:241887
Public defence
2009-10-23, Berzeliussalen, Hälsouniversitet, Campus US, Linköpings Universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2009-10-12 Created: 2009-10-02 Last updated: 2010-01-14Bibliographically approved
List of papers
1. HPLC analysis of pheomelanin degradation products in human urine
Open this publication in new window or tab >>HPLC analysis of pheomelanin degradation products in human urine
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2003 (English)In: Pigment Cell Research, ISSN 1755-1471, E-ISSN 1755-148X, 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-21494 (URN)10.1034/j.1600-0749.2003.00086.x (DOI)12950724 (PubMedID)
Available from: 2009-10-02 Created: 2009-10-02 Last updated: 2017-12-13Bibliographically approved
2. Hydrophilic interaction liquid chromatographic analysis of aminohydroxyphenylalanines from melanin pigments
Open this publication in new window or tab >>Hydrophilic interaction liquid chromatographic analysis of aminohydroxyphenylalanines from melanin pigments
2007 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1163, no 1-2, 70-79 p.Article in journal (Refereed) Published
Abstract [en]

Malignant melanomas are more often seen in subjects with light colored skin who tan poorly than in persons who tan more rapidly. This has been attributed to the structure of their pigment, pheomelanin, which differs markedly from the eumelanin of persons with darker skin. To study the hydrolysis products of pheomelanin pigments a new method was developed for analysis of 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Pheomelanin samples were hydrolyzed and extracted with solid-phase extraction columns using strong cation-exchange (SCX) cartridges. Separation of 4-AHP and 3-AHP was achieved on a ZIC-HILIC column (150 mm × 2.1 mm I.D.) with a mobile phase consisting of acetonitrile:0.1 M ammonium acetate buffer, pH 4.5 (82:18, v/v). Detection was performed with an electrochemical detector at +400 mV. Run time was 30 min. The limits of detection were 73 pg and 51 pg for 4-AHP and 3-AHP respectively, using 2 μl injections. Good linearity was found within the range 0.05-5.0 μg/ml. Absolute recovery was 70% and relative recovery was 100%. The AHPs were stable for 1 year in the hydrolyzed samples, for 4 days in the eluates from solid-phase sorbents stored in the refrigerator, and for 2 days diluted with mobile phase and stored in the autosampler at 10 °C. The within-day imprecision was <5% and the between-day imprecision was <7% for the two analytes. The method, applied to the analysis of pheomelanin in urine from human melanoma patients, allows the analysis of 30 samples in one set and is suitable for routine work with human hair and melanoma cells. By using the ZIC-HILIC stationary phase, ion-pairing reagents could be avoided, which makes the method suitable to further analysis of degradation products from pheomelanins using mass spectrometric detection. © 2007 Elsevier B.V. All rights reserved.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-39471 (URN)10.1016/j.chroma.2007.06.007 (DOI)48765 (Local ID)48765 (Archive number)48765 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
3. Gas chromatography-mass spectrometry analysis of pheomelanin degradation products
Open this publication in new window or tab >>Gas chromatography-mass spectrometry analysis of pheomelanin degradation products
2009 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 30, 5730-5739 p.Article in journal (Refereed) Published
Abstract [en]

Melanoma is most rapidly increasing in the white population and people with pheomelanin skin type are at high risk to develop melanoma. However, little is known about the pheomelanin structure and function, and further elucidation of this melanin is therefore an important task. A GC/MS method was developed based on hydriodic acid hydrolysis of pheomelanin in the urine. Derivatization was performed with ethyl chloroformate and ethanol:pyridine (4:1, v/v). N,O-Ethoxycarbonyl-ethyl esters were extracted with chloroform and analyzed by GC/MS. 4-Amino-3-hydroxyphenylaianine and 3-amino4-hydroxyphenylaianine together with one benzothiazinone and two benzothiazole compounds were detected and identified in hydrolyzed samples of synthetic pheomelanin and melanin from the urine of a patient with melanoma. These findings strongly suggest that heterocyclic pheomelanin-type units are incorporated in the pigment structures.

Keyword
Alkyl chloroformate; Aminohydroxyphenylalanine; Derivatization; Gas chromatography-mass spectrometry; Melanin; Melanoma; Pheomelanin; 7-(2-Amino-2-carboxyethyl)-5-hydroxy-3, 4-dihydro-2H-1, 4-benzothiazine-3-one 6-(2-Amino-2-carboxyethyl)-4-hydroxybenzothiazole 6-(2-Amino-2-carboxyethyl)-4-hydroxy-2-methyl-benzothiazole; Benzothiazine; Benzothiazole; Benzothiazinone
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20135 (URN)10.1016/j.chroma.2009.05.063 (DOI)
Available from: 2009-08-31 Created: 2009-08-31 Last updated: 2017-12-13Bibliographically approved
4. Pheomelanin-related benzothiazole isomers in the urine of patients with diffuse melanosis of melanoma
Open this publication in new window or tab >>Pheomelanin-related benzothiazole isomers in the urine of patients with diffuse melanosis of melanoma
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2010 (English)In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 411, no 17-18, 1195-1203 p.Article in journal (Refereed) Published
Abstract [en]

Currently used as structural markers for pheomelanin identification and quantitation, benzothiazole compounds derived from isomeric cysteinyldopas have been indicated by recent in vitro studies as new potential pheomelanogenesis intermediates. Prompted by previous reports on the occurrence of large amounts of 5-S-cysteinyldopa (5-S-CD) and trichochromes in urine of patient with diffuse melanosis of melanoma we investigated the presence of benzothiazole compounds in the urine of these patients.

Hydrophilic interaction liquid chromatography on zwitterionic stationary phase (ZIC-HILIC) and photo-diode array (PDA) detection was used for analysis of 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-5), and 7-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole-2-carboxylic acid (BTCA-2), derived from 5-S-CD and 2-S-cysteinyldopa (2-S-CD) isomers, respectively. Isocratic mobile phase with minimal sample preparation allowed efficient separation of the compounds, which were safely identified by their typical absorption features.

Among 21 melanoma patients examined three showed diffuse melanosis. The levels of urinary BTCAs were found to be highly associated with melanosis but more loosely to excreted 5-S-CD. Analysis of the pigmented fraction of urine following alkaline hydrogen peroxide degradation and quantitation of BTCAs provided evidence for the presence of pheomelanins at higher levels in patients with melanosis.

Place, publisher, year, edition, pages
lsevier Science B.V., Amsterdam, 2010
Keyword
Benzothiazole, benzothiazole-2-carboxylic acids, diffuse melanosis, melanoma, HILIC, pheomelanin, BTCA
National Category
Other Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-21817 (URN)10.1016/j.cca.2010.04.019 (DOI)000280033400005 ()
Available from: 2009-10-05 Created: 2009-10-05 Last updated: 2017-12-13Bibliographically approved

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Nezirevic Dernroth, Dzeneta

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