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Comparison HPLC and TEM/stereology quantifying eumelanins and pheomelaninsPURPOSE2013.05.04 Eun kyung Noh
INTRODUCTION_conceptl MELANOCYTE lPigment cells that originate in the neural crestSpecialized organelle synthesizing melan...
INTRODUCTION_conceptl QUANTIFICATION OF TWO MELANINS lHigh performance liquid chromatography(HPLC)Stereological image anal...
MATERIAL and METHODS_stereological analysisPART2Cell lines : HBL, LND1DOR, BEUIGR3, ARNMedium : Ham F10 medium+10% FCS+1% ...
MATERIAL and METHODS_stereological analysisPART2
MATERIAL and METHODS_stereological analysisPART2Permittion to dissolve pheomelaninon ultrathin sectionswith an acceptable ...
MATERIAL and METHODS_HPLC analysisPART2Concentration of eumelanin and pheomelanin in extraction of 106 cells• Eumelanin to...
RESULTS and DISCUSSION_stereological dataPART3Figure 2 Total melanin stereological data obtainedfor the 6 melanoma cell li...
RESULTS and DISCUSSION_stereological dataPART3Figure 3 Pheomelanin stereological data obtained for the6 melanoma cell line...
RESULTS and DISCUSSION_HPLC dataPART3Figure 5 Linear regression analysis of HPLC andstereological data(cytoplasmic volume ...
RESULTS and DISCUSSION_HPLC dataPART3Figure 6 Linear regression analysis of HPLC andstereological data for eumelanin and p...
CONCLUSIONPART4• Stereological image analysis method could be an interesting approach for the quantitative of melanins• Ad...
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130504 paper 은경

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130504 paper 은경

  1. 1. Comparison HPLC and TEM/stereology quantifying eumelanins and pheomelaninsPURPOSE2013.05.04 Eun kyung Noh
  2. 2. INTRODUCTION_conceptl MELANOCYTE lPigment cells that originate in the neural crestSpecialized organelle synthesizing melaninl 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 TRP1DOPAchrome to DHI-eumelanin- pheomelanogenesis : DOPA-quinone to cysteinylDOPAPART1
  3. 3. INTRODUCTION_conceptl QUANTIFICATION OF TWO MELANINS lHigh performance liquid chromatography(HPLC)Stereological image analysisPART1
  4. 4. MATERIAL and METHODS_stereological analysisPART2Cell lines : HBL, LND1DOR, BEUIGR3, ARNMedium : Ham F10 medium+10% FCS+1% penicillinc-streptomycin+1% kanamycin+1% of 200mM glutamine sol.Melanoma cellsMelanoma cells fixation in suspensionThe technique to obtain (near) sphericalmelanocytes and embed them as pelletMelanoma cell were detached, pelleted,washed, fixed in suspension → ethanoldehydration → infiltration with ethanol : 1,2-epoxypropane, 1,2-epoxypropane, 1,2-epoxypropane : embeding medium▶ Homogenization and Samplingin pyramidal tipped plastic capsulesCut into eight pieces(4 summit part, 4 basal part)Randomly selected, re-embeded in EPONFixation and Embedding70 nm ultrathin section were cutwith Ultracut S ultramicrotomeequipped with a diamond knife(Diatome)Submittion to chloroform vapor tocorrect possible deformation due tocompressionCollecting with 150 mesh coppergridsStaining with 2% uranyl acetate &post-staining 0.2% lead citrateObservation with a Jeol 1200-EXTransmission Electron MicroscopeTransmissionelectronmicroscopy
  5. 5. MATERIAL and METHODS_stereological analysisPART2
  6. 6. MATERIAL and METHODS_stereological analysisPART2Permittion to dissolve pheomelaninon ultrathin sectionswith an acceptable specificityInternal structure of the spheroidmelanosomes is dissolved bytreatment with 0.5 N NaOH solution,whereas the ellipsoid melanosomesare not affected(J Invest Dermatol, 1993, Vol. 100 ,pp. 172S-175S)Each grid supporting the ultrathinsection was deposited on a drop of300mM NaOH, 45 minRinsed with bi-distilled waterAlkali elution ofpheomelaninMicrograph-scannerSemper 6 plus image analysissoftwareData : An(nuclear area)Ace(cell area)Ami(melanin area)Nmp(number of melaninparticles)Nco(number of connectionbetween melanin particles)Primary data/2-D measurementswere calculated to 3-D melanizationparameters by stereologyPheomelanin to eumelanin ratio(P/E)Image analysisand stereology30 cells systematically sampledEllipsoidal shape formula :(D1 : long axis diameter, D2 : shortaxis diameter)Mean cell diameter(MCD) :(vce : cell volume)Estimation of meancell volume
  7. 7. MATERIAL and METHODS_HPLC analysisPART2Concentration 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
  8. 8. RESULTS and DISCUSSION_stereological dataPART3Figure 2 Total melanin stereological data obtainedfor the 6 melanoma cell lines used in this studyA : Melanin volume per average melanoma cellsB : Melanosomal maturation or mean melanin areaper average melanized melanosome
  9. 9. RESULTS and DISCUSSION_stereological dataPART3Figure 3 Pheomelanin stereological data obtained for the6 melanoma cell lines used in this studyA : Phomelanin volume per average melanoma cellsB : Melanosomal maturation or mean melanin area peraverage melanized melanosomeFigure 4 Eumelanin stereological data obtained for the 6melanoma cell lines used in this studyA : Eumelanin volume per average melanoma cellsB : Melanosomal maturation or mean melanin area peraverage melanized melanosome
  10. 10. RESULTS and DISCUSSION_HPLC dataPART3Figure 5 Linear regression analysis of HPLC andstereological data(cytoplasmic volume density ofmelanin) for total melanin
  11. 11. RESULTS and DISCUSSION_HPLC dataPART3Figure 6 Linear regression analysis of HPLC andstereological data for eumelanin and pheomelanin.In both cases the number of melanized melanosomes wasuesd as stereological parameter.A : EumelaninB : PheomelaninFigure 7 Linear regression analysis of the relationshipbetween the HPLC-derived P/E ratio and stereology-derived P/E ratios (mean of the P/E ratios obtained in Sand B sections)
  12. 12. CONCLUSIONPART4• 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)

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