Skin Pigmentation and Its Analysis To demonstrate the activity of NADPH oxidase in biological tissues, the samples are incu-bated with a reaction medium containing cerium chloride, the substrate NADPH, and specific inhibitors of other enzymes potentially producing ROS, such as catalase or xanthine oxidase.
The specificity of the reaction is confirmed by the either omitting the substrate NADPH or including a specific inhibitor for NADPH oxidase, diphenyleneiodonium chloride (DPI) in the reaction medium (Ellis et al., 1998; Ellis and Grant, 2002).
In addition to detecting the cerium perhydroxide precipitate by CLSM or EM, the precipitate can be used for polymerization and cyclization of diaminobenzidine (DAB), resulting in an amplification of the primary reaction product, that can be visualized by conventional light microscopy. Cobalt chloride can be used to enhance the contrast (Gossrau et al., 1989).
Skin Pigmentation and Its Analysis
with a factor of 35, but this factor has been corrected to 160, due to reports of massive under-estimation of the eumelanin content (Alaluf et al., 2001; Wakamatsu and Ito, 2002).
NH
NH
O
NH HOOC
(COOH) (COOH) O
O O H
O H
Eumelanin
KMnO4
NH
HOOC COOH
HOOC
PTCA
Figure 11: Oxidative breakdown of eumelanin. Eumelanin is degraded oxidatively with potassium permanganate (KMnO4), resulting in specific degradation products: pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA). PTCA is produced at a yield of 2.8 %, while the yield of PDCA is negligible. The degradation products are detected and quantified by HPLC, and PTCA is used as a specific indicator for eumelanin. The measured amount of PTCA is multiplied by a conversion factor of 160 to determine the actual eumelanin content of the investigated sample.
S NH
S N
N S
N
N
H2 COOH
OH HOOC
COOH OH
O
N
H2 COOH
OH
I H
NH2 COOH O
H
N H2
Pheomelanin AHP
Figure 12: Reductive hydrolysis of pheomelanin. Pheomelanin is chemically degraded by hydrogen iodide, producing the pheomelanin-specific marker 4-amino-3-hydroxyphenylalanine (4-AHP) at a yield of 11 %.
4-AHP can be quantitatively determined by HPLC. The obtained 4-AHP content of the respective sample is multiplied by a conversion factor of 9 to determine the pheomelanin content.
Skin Pigmentation and Its Analysis
III The Project – Challenging Questions of Skin Pigmentation
The type and amount of melanin and its epidermal distribution pattern determines the actual skin colour. But while the biosynthesis of melanin within the melanocytes is well charac-terized, neither transfer of melanosomes from the melanocytes to the surrounding keratino-cytes, nor the ultimate fate of transferred melanosomes within the keratinocytes are elucidated up to date (Borovanský and Elleder, 2003; Van Den Bossche et al., 2006). This project there-fore aims at finding morphological and biochemical functional clues for the description of melanin processing and its degradation pathway. A further aim is to establish potentially unique criteria for various skin types, including dyspigmentations, and the potential relation to their morphological and functional background.
As understanding the metabolism of melanin requires basic knowledge of its epidermal distri-bution, a first step is to investigate the distribution of melanin and/ or melanosomes in differ-ent epidermal layers in skin of Caucasian, Asian and Negroid origin. A further step covers the morphological description of melanosomes before and after transfer from melanocyte to re-cipient keratinocyte, with a special focus on the number of melanosomal membranes to find proof for one or more of the existing theories regarding melanosome transfer. The mode of transfer is of special interest, as it may provide an explanation for the distribution patterns within the keratinocytes as well as the ensuing processing of the melanosomes. Further em-phasis is put on the distinctions of lentigo senilis compared to normal elderly skin, including the number of melanocytes, the epidermal distribution of melanosomes and the ultrastructure of the skin. Several staining and microscopy techniques are available for the characterization of skin pigmentation, and an appropriate method for each respective task is to be identified.
Supplementary, a chemical analysis of the total eumelanin and pheomelanin content of skin biopsies is performed, and a comparison of the results of this method with microscopic inves-tigation is aspired.
Based on the assumption of Borovanský and Elleder (2003; see also chapter I.5.2), that NADPH oxidase may be involved in the melanin degradation process by providing ROS capable of degrading the extremely stable polymer, a cytochemical assay is established to demonstrate the sites of hydrogen peroxide production in the epidermis and identify a potential colocalisation with melanosomes or cellular structures implied in melanin processing.
C M ATERIALS & M ETHODS
I Standard Buffers
Buffers that are repeatedly used for different applications are listed in this chapter. The preparation of specific solutions and buffers is described in the respective chapters. Chemicals and reagents are usually purchased from Sigma-Aldrich, Munich, Germany. Variant suppliers are mentioned additionally.
Dulbecco's Modified Eagle Medium (DMEM)
The cell culture medium is readily purchased from Invitrogen, Karlsruhe, Germany.
Phosphate-Buffered Saline (PBS) pH 7.2
The Buffer is readily purchased from Invitrogen, Karlsruhe, Germany.
HEPES Buffer 0.05 M, pH 7.4
HEPES (N-2-Hydroxyethyl piperazine-N´-2´-ethane sulphonic acid)
(Roth, Karlsruhe, Germany) 11.91 g
Aqua bidest. ad 1000 ml
Sodium hydroxide solution (Merck, Darmstadt, Germany) ad pH 7.4 Sodium Cacodylate Buffer 0.1 M, pH 7.4
Sodium cacodylate trihydrate 21.4 g
Aqua bidest. ad 1000 ml
Hydrochloric acid ad pH 7.4
Sodium Cacodylate Buffer 0.15 M, pH 7.4
Sodium cacodylate trihydrate 32.1 g
Aqua bidest. ad 1000 ml
Hydrochloric acid ad pH 7.4
Sodium cacodylate contains arsenic and is therefore toxic.
Materials & Methods
Tris Hydrochlorine Buffer (Tris-HCl Buffer) 0.05 M, pH 7.6
Tris (Tris[hydroxymethyl]aminomethane) (USB Corp., Cleveland, Ohio, USA) 6.06 g
Aqua bidest. ad 1000 ml
Hydrochloric acid ad pH 7.6
All buffers can be stored at 4 °C for several weeks.
II Skin Samples
For this project three different sample collectives were investigated, that were obtained during two studies approved by the Hamburg ethics committee (internal Beiersdorf AG study num-bers: 10684 and 18655). And all participants were required to give informed consent before partaking.
During the first study (2000 - 2001) biopsies of the nates (diameter 4-5 mm) were obtained from thirty volunteers with healthy skin of the following ethnic groupings: European (n = 10), Asian (n = 10) and African (n = 11). Further eleven volunteers contributed biopsies from the back of their hands (diameter 2-3 mm), one each covering an age spot (lentigo senilis, le-sional) and one from the periphery of the respective age spot (perilele-sional). Biopsies were taken in the University Medical Centre Hamburg-Eppendorf and placed – epidermis upward – onto cellulose, soaked in cell culture medium (DMEM), to ensure adequate supply of nutri-ents and to prevent desiccation during transport to Beiersdorf AG. The biopsies were proc-essed for high-pressure freezing immediately, yielding five samples per volunteer, and stored in liquid nitrogen until further processing.
During a second study (2005) additional thirty volunteers – five of each of Fitzpatrick’s skin phototypes I-VI – gave two punch biopsies each (diameter 4 mm) from the nates. The samples were transported from the University Medical Centre to Beiersdorf AG as described before.
After removal of the adipose tissue, one biopsy of each volunteer was processed for micro-scopic investigation, while the other biopsy was prepared for chemical analysis of the melanin content by Dr. Wakamatsu of the Fujita Health University in Toyoake, Japan (S. Ito and Jimbow, 1983; Wakamatsu and Ito, 2002).
For microscopy, each biopsy was divided into six smaller parts: three 2 mm-punch biopsies were extracted and high-pressure frozen followed by low-temperature dehydration and
em-Materials & Methods bedding (see chapters III.1.2.2 and III.2.2). One part was fixed in IEM and uranyl acetate for room temperature dehydration and embedding (chapters III.1.1 and III.2.1) and the remaining two parts were plunge-frozen for immunohistochemistry (III.1.2.1). The second biopsy was weighted to determine the moist mass, before plunge-freezing, followed by freeze-drying and shipment to Japan for chemical analysis. Table 2 displays the usage and applied investigative techniques of the different sample collectives.
Additionally, discard material from cosmetic surgery was used for several microscopic inves-tigations, including the assessment of staining techniques and the NADPH oxidase detection assay. Patients gave written consent. The cosmetic surgery biopsies were transported and stored as described earlier.
Sections of paraffin embedded Negroid skin samples were obtained from Dr. Brandner, Labo-ratory for Cell Biology, Department of Dermatology and Venerology, University Medical Centre Hamburg-Eppendorf.
Table 2: Designated use of samples for this project.
Sample collective Investigation of… Microscopy mode
Results described in chapter:
Melanosome distribution
and melanin clearing TEM E.I.1.1
Caucasian, Asian
and Negroid skin Structural organisation of
melanosomes TEM E.IV.2
Melanocyte distribution CLSM E.II.3 Melanosome distribution
and melanin clearing TEM E.I.1.3, E.I.2.2, E.III BDF-Study
Nr. 10684
Lentigo senilis lesional and perilesional
Characteristics
of lentigo senilis LM E.III
Melanocyte distribution CLSM E.II.2 Melanosome distribution
and melanin clearing LM and TEM E.I.1.2 Structural organisation of
melanosomes TEM E.IV.2
BDF-Study Nr. 18655
Fitzpatrick’s Skin Phototypes I-VI
Chemical analysis of
melanin content - E.I.3
Assessment of staining techniques
LM, CLSM
and TEM D
Cosmetic surgery discard material
NADPH oxidase
detection assay LM and TEM E.V.2
Materials & Methods
III Sample Preparation for Microscopy
The procedure of sample preparation for microscopy comprises four major steps: (1) fixation, (2) dehydration and embedding, (3) sectioning and (4) staining. For each step various alternatives and modifications are possible and the suitable variant is chosen depending on the respective experiment and intended mode of microscopy, sometimes also requiring combinations of two methods. The preparative techniques applied during this project are described in the following chapter. Table 3 gives an overview of the employed combinations.
Chemically fixed samples are either processed for resin embedding at room temperature, or plunge-frozen and sectioned in the cryostat. Physically or cryofixed specimen can also be transferred directly to the cryostat for sectioning or freeze substituted, followed by cryostat cutting or low-temperature embedding and subsequent ultramicrotomy. After suitable staining, cryostat sections and semithin resin sections can be investigated using a light microscope (LM) or confocal laser scanning microscopy (CLSM). Ultrathin resin sections are stained with heavy metal salts for transmission electron microscopy (TEM).
Table 3: Sample preparation and resulting microscopic applications.
Fixation Dehydration Embedding Sectioning Staining Microscopy
Histocytochemistry LM Chemical
sample fixation
At room temp. At room temp. Ultramicrotomy
Heavy metal staining TEM Chem. &
phys. fix. - - Cryostat Histocytochemistry
Immunofluorescence LM
- - Cryostat Histocytochemistry
Immunofluorescence LM CLSM
- Cryostat Immunofluorescence CLSM
Histocytochemistry CLSM Physical
sample
fixation Freeze
substitution Low-temp.
embedding Ultramicrotomy
Heavy metal staining TEM