Comparison HPLC and TEM/stereology quantifying eumelanins and pheome-lanins

PUR-POSE

2013.05.04 Eun kyung Noh

INTRODUCTION_concept

l MELANOCYTE lPigment cells that originate in the neural crestSpecialized organelle synthesizing melanin

l MELANIN lTwo types : eumelanin (black-brown, elliptic and lamellar/fibrillar in mammals) pheomelanin (yellow-reddish, spherical with a spotty pattern in mammals)

l MELANOGENESIS lL-tyrosine to L-3,4-dihydroxyphenylalanine(L-DOPA) and oxidation to DOPA-quinone by tyrosinaseAfter two stages, seperate to eumelanogenesis and pheomelanogenesis - eumelanogenesis : DOPA-quinone to DOPAchrome, DOPAchrome to DHICA-eumelanin by TRP-2(DCT) and TRP1 DOPAchrome to DHI-eumelanin - pheomelanogenesis : DOPA-quinone to cysteinylDOPA

PART 1

INTRODUCTION_concept

l QUANTIFICATION OF TWO MELANINS lHigh performance liquid chromatography(HPLC)Stereological image analysis

PART 1

MATERIAL and METHODS_stereological analysis

PART 2

Cell lines : HBL, LND1 DOR, BEU IGR3, ARN

Medium : Ham F10 medium+10% FCS+1% penicillinc-strepto-mycin+1% kanamycin +1% of 200mM glutamine sol.

Melanoma cells

Melanoma cells fixation in sus-pension

The technique to obtain (near) spheri-cal melanocytes and embed them as pellet

Melanoma cell were detached, pel-leted, washed, fixed in suspension → ethanol dehydration → infiltration with ethanol : 1,2-epoxypropane, 1,2-epoxypropane, 1,2-epoxypropane : embeding medium▶ Homogenization and Sampling in pyramidal tipped plastic capsules

Cut into eight pieces(4 summit part, 4 basal part)

Randomly selected, re-embeded in EPON

Fixation and Em-bedding

70 nm ultrathin section were cutwith Ultracut S ultramicrotome equipped with a diamond knife (Diatome)

Submittion to chloroform vapor to correct possible deformation due to compression

Collecting with 150 mesh copper grids

Staining with 2% uranyl acetate & post-staining 0.2% lead citrate

Observation with a Jeol 1200-EX Transmission Electron Micro-scope

Transmission electron mi-

croscopy

MATERIAL and METHODS_stereological analysis

PART 2

MATERIAL and METHODS_stereological analysis

PART 2

Permittion to dissolve pheomelaninon ultrathin sections with an acceptable specificity

Internal structure of the spheroid melanosomes is dissolved by treatment with 0.5 N NaOH solu-tion, whereas the ellipsoid melanosomes are not affected(J Invest Dermatol, 1993, Vol. 100 ,pp. 172S-175S)

Each grid supporting the ultrathin section was deposited on a drop of 300mM NaOH, 45 min

Rinsed with bi-distilled water

Alkali elution of pheome-

laninMicrograph-scannerSemper 6 plus image analysis software

Data : An(nuclear area) Ace(cell area) Ami(melanin area) Nmp(number of melanin particles) Nco(number of connection between melanin parti-cles)

Primary data/2-D measurements were calculated to 3-D melaniza-tion parameters by stereology

Pheomelanin to eumelanin ratio(P/E)

Image analysis and stereology

30 cells systematically sampled

Ellipsoidal shape formula :

(D1 : long axis diameter, D2 : short axis diameter)

Mean cell diameter(MCD) :

(vce : cell volume)

Estimation of mean cell vol-

ume

MATERIAL and METHODS_HPLC analysisPART 2

Concentration of eumelanin and pheomelanin in extraction of 106 cells

• Eumelanin to pyrrole-2,3,5-tricarboxylic acid(PTCA) : permanganate oxidation

• Pheomelanin to aminohydroxyphenylalanine(AHP) : hydriodic acid hydrolysis

▶ total melanin amount formula : TM(μg/106cells) = 50xPTCA(μg/106cells) + 50xAHP(μg/106cells)

HPLC determination of eumelanin and pheomelanin in melanoma cells

RESULTS and DISCUSSION_stereological data

PART 3

Figure 2 Total melanin stereological data ob-tained for the 6 melanoma cell lines used in this studyA : Melanin volume per average melanoma cellsB : Melanosomal maturation or mean melanin area per average melanized melanosome

RESULTS and DISCUSSION_stereological data

PART 3

Figure 3 Pheomelanin stereological data obtained for the 6 melanoma cell lines used in this studyA : Phomelanin volume per average melanoma cellsB : Melanosomal maturation or mean melanin area per average melanized melanosome

Figure 4 Eumelanin stereological data obtained for the 6 melanoma cell lines used in this studyA : Eumelanin volume per average melanoma cellsB : Melanosomal maturation or mean melanin area per average melanized melanosome

RESULTS and DISCUSSION_HPLC dataPART 3

Figure 5 Linear regression analysis of HPLC and stereological data(cytoplasmic volume density of melanin) for total melanin

RESULTS and DISCUSSION_HPLC dataPART 3

Figure 6 Linear regression analysis of HPLC and stereological data for eumelanin and pheomelanin.In both cases the number of melanized melanosomes was uesd as stereological parameter.A : EumelaninB : Pheomelanin

Figure 7 Linear regression analysis of the relation-ship between the HPLC-derived P/E ratio and stere-ology-derived P/E ratios (mean of the P/E ratios ob-tained in S and B sections)

CONCLUSIONPART 4

• Stereological image analysis method could be an interesting approach for the quantitative of melanins

• Advantage of Stereological method is that it requires a low number of pigment cells

• Stereological method needs supplementary confrontations with the HPLC chemical approach to clarify these results

(J Invest Dermatol, 1993, Vol. 100 ,pp. 172S-175S)