1. Alejandro Gugliucci , Eriko Kinugasa, Hiroaki Ogata , Russell Caccavello , Satoshi KimuraJorge Ferreira & Sydney Goldfeder molecular biology Medicine Student III Semester UPB

2. INTRODUCTIONMillions of patients around the world depends on replacement therapy, 80% recieve HD, the problem resides on cardiovascular effects. dyslipoproteinemia of ESRD is caused by triglycerides and IDL accumulation. Other fact on vascular Affects is HDL, it has antioxidative and anti inflammatory properties. PON-1 is essential for the function of HDL. show PON -1 activity is an important determinant of the endpoints in renal failure cardiovascular 3. HDL 25% of total cholesterol in blood Formed in the liver and intestine as discoidal, (pre- HDL) .Density: Main component: Protein Apoproteins: AI, AII (C, E) Diameter: 5-12 3 types: HDLn,HDL2,HDL3Reverse cholesterol transport Exchange with other lipoproteins. steroidogenic tissues 4. Esterase activity hydrolyzed derivatives of OP compounds Substrates: triphosphates (paraoxon and diazoxon)1946 Mazurinsecticide metabolites (toxic), parathion Hydrolyze aromatic esters, phenylacetate and naphthylacetate 5. PARAOXONASE Glycoprotein composed of 354 aa. Weight 43 kDa -helix, 6 blades and 4 strands Free sulfhydryl group at position 284 is essential for both activities, blocking, or replacement of this cysteine by Ala or Ser mutants for PON 1 affects recombination activityCa 2+7.4 ACa 2+ 6. PARAOXONASEPON 1PON 2PON3 7. RELATIONHDLVLDL PARAOXONASEHEMODIALYSIS 8. GENERAL OBJECTIVE Demonstrate the mechanism of PON-1 activation during hemodialysis, by identifying PON-1 on HDL subclasses and put forth the HDL remodeling and shift of PON 1 among HDL particle. 9. MATERIALES Y MTODOS SUJETOS A ESTUDIAR ESRD Escuela universitaria de medicina ShowaUniversidad del norte de yokohama- hospital Tsuzuki-kuYokohama, Japn 42 pacientes (18 mujeres-24 hombres 6312 aos) HD 6.4 aos(1-19 aos)- regular 2meses, 3 veces por semana 3-4 horas147.4C 15min.Citrato de sodio o EDTA 5mmol/l86.8009.xg80C52Poblacin saludable de trabajadores del hospital, no diabetes ni enfermedad renal3. 3-4hrs 10. MATERIALES Y MTODOSColesterol + HDL+ TG= mtodos enzimticos.Pruebas en generalAlbumina, urea, creatininaConcentracin de protenas: Mtodo Bradford (BioRad) 465-595nm 11. MATERIALES Y MTODOS20hrs. Lipoprotena con Apo B ULTRACENTRIFUGACIN HDL24hrs. HDL-2HDL-3Ultracentrfuga: 1923,Sueco Svedberg, velocidades 100.000rpm, velocidad dependiente de masa.por gradiente de densidades, en el cual es posible la separacin de las lipoprotenas a partir de sus diferentes densidades, incluso sus fracciones (HDL-2 Y HDL3) 12. MATERIALES Y MTODOSActividad PON 1Lactona natural que deriva de la ciclizacin de un derivado de ac. cinmicoActividad LactonasaDHCanin p-nitrofenolPON-3paraoxn pnitrofenol + dietilfosfato405nm 13. MATERIALES Y MTODOS Deteccin de la actividad de PON1 in situZimogramaPAGE Nativa tris-glicina (rango de pH 8.3 a 9.5)Geles se corrieron clula de electroforesis XCell Sure Lock (Novex , Invitrogen), se apilan durante 30 min a 65 V y a continuacin, se ejecutan durante 16 horas a 100 V a 4 C. 14. MATERIALES Y MTODOS Deteccin HDL Y LDL En el mismo gel, luego del Zimograma HDLDetecta protenas por interaccin antgenoAnticuerpo Western BlotTransfieren protenas de gel a membrana, Bloqueo de unin protenas membrana, deteccin protenas con anticuerpos enzimas.ElectrotransferenciaSEMISECA Rpida, poco calor, varios geles Aparato con superficie de grafito, genera campo uniforme Sndwich: papel filtro: humedece con tris-glicina, gel, Membrana. Membrana PVDF: ms utilizadas, varias coloraciones, estables, hidrofbicas.HMEDAElectroforesisDifusin simple 15. MATERIALES Y MTODOSpr- seLas detectan por:Colorante de PVDF: Azul de croomasieAnticuerpos secundarios conjugados Con enzimas. HRP.Inmunodeteccin: anticuerpos monoclonaes o policlonalesQuimioluminescenica 16. Pacientes en comparacin con los sujetos control. Creatinina y urea de HDL plasmtico Las 3 actividades de la PON1 HDL (4,6%) LDL (13%) VLDL (30%)Liplisis aumentada de la VLDL que es transformada en LDL Ningn cambio de las subclases de HDL fue significante. 17. HDL2b HDL2a HDL3a HDL3b HDL3c 18. HDL2b HDL2a HDL3a HDL3b HDL3c 19. ApoE Cambios en apoE junto con activacin de PON1 y traslado entre partculas.apoE notable en HDL2No hay cambios significativos para apoA-I ni apoA-II 20. 30% dialysis 25% shifts to HDL and VLDLPON1 lact. and aryl. activation in short time (4h)Increase of PON1 in large HDL2, reflects maturation of smaller HDL2 21. WHAT HAPPENS TO VLDL?What happens to apoE? 22. AuthorWhat they saidYes or NoOrsoni(27), et alPrecisely we have recently reported that a Yes similar PON1 activation and transffer can be reproduced ex vivo in a few hours.Ribeiro S, et alPON1 status predicts cardiovascular outcome Yes in CRF. 23. AuthorWhat they saidYes or NoGugliucci A(29), et alThis reinforces the concept that a process Yes of remodeling with shifts in peripheral HDL proteins is enhanced by dialysis, as we have also recently shown ex vivo.Deakin S, Manning PJ(30-32), VLDL has been suggested as a vehicle for Yes et al PON1 secretion from hepatocytes and it may well also be a PON1 reservoir. 24. PON1 activation offers protection against cardiovascular diseases, atherosclerosis and reduces oxidative stress.PON1 can be a potential target for both molecular and pharmacological impacts. 25. Hemodialysis gives a big benefit in a short time frame of 4h by the activation of PON1.The mechanism of PON1 is not totally understood yet; it needs further research to complete this gap in our knowledge.