mohsin al-saleh - porphyrins & porphobilinogen · 2017-03-10 · • c-18 column • gradient...
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Porphyrins&Porphobilinogen
MohsenAl-SalehSeniorBiomedicalScien3st
Porphyrins• Heterocyclicmacrocycleorganiccompounds• Composedoffourmodifiedpyrrolesubunits(tetrapyrrole)bymethenebridges• Ithas26(delocalized)pielectrons,Ithashighlyconjugateddoublebonds
• The4nitrogenatomsatthecenteroftheringareexcellentatconjugaCngmetalsbecauseoftheirlonepairs
• IthasveryintenseabsorpConbandsinthevisibleregionatSoretpeak(band)in
blueregionofvisiblespectrumnear400-410nmwavelength• Itformeddeepviolet/bluecolorduetoanelectrondipolemovement(π-π)trans.• Thename"porphyrin"comesfromtheGreekwordporphyros,meaningpurple• Porphyrinsaretheconjugateacidsofligandsthatbindmetalstoformcomplexes
Physicalproper3es• Color:– ColoraConaround405nm– Usuallyred
• Fluorescence:– around620nm– Reddish-pinkcolor
• Chela3on:– ArrangementofnitrogenatomsallowschelaConofmetalatomssuchasiron,thatparCcipateinoxidaCvemetabolism
Porphyrinstypes• Uroporphyrin:URO
– Watersoluble– Hemeprecursor– Foundinurine
• Coproporphyrin:COPRO– Watersoluble– Hemeprecursor– Foundinurineandfeces
• Protoporphyrin:PROTO– Waterinsoluble– Hemeprecursor– Foundinfeces
• OneofthefirstindicaConofporphyriaisproducConofdarkcoloredurinesample
• PorphyrinshavecharacterisCcUV/VISabsorpConpeak(Soretpeak)intheregion400-410nm
• CoproporphyrinhaspeakabsorpConbetween402-403nm
• UroporphyrinhaspeakabsorpConbetween406-407nm
Totalporphyrinsscreening
Substanceswhichinterfereintest
• Aminosalicylicacid• Birthcontrolpills• Barbiturates• Ethylalcohol• Morphine
Porphyrinssampleprepara3on
• Urinesample:(PrefersampleisDU-Urinesample)1. Samplemustbecoveredwithaluminumfoil2. SampleiscentrifugeandfilteredoutfromsolidparCcles3. Sample(0.5mL)isacidifiedwith2.7MofHCL(2mL)4. IncubaConindarkforanhour5. Sampleisscannedfrom300to500nm->spectrumposiCon6. ConstrucConofbaselinepeaksfromeithersideofmainpeak
(Around390to420nm)
UV/VISspectroscopy(190-700nm)
Total-porphyrinspeakanalysis
Totalporphyrinsinug/L=2A(λmax)–(A390+A420)e;wheree=4740ug/L
HeightofSoretband
Baseline
Repor3ngporphyrin
• Iftheofurineresult<300nmol/L,reportasnega3ve
• Resultsofurine>300nmol/L;its(posi3ve)shouldbeexpressedas(nmolporphyrins/mmolcreaCnine)
• Urineporphyrin/creaCnineraCo:<35nmol/mmolcreaCnine
• ThetotalporphyrinconcentraConpermmolofcreaCnineisthebestindicatoroftotalporphyrinexcreCon.
Samplecollec3on• Itcanalsobemeasuredonblood,urine,biologicalCssues
andfecalsamples• Venousbloodsampleshouldbecollectedandtransport
coveredinice• PaCentshouldbe12-14hoursfasCng• Threetypesofporphyrinscanbemeasured:1. Coproporphyrin2. Protoporphyrin(lieledifficult)3. Uroporphyrin
Othertechniques&methods
1. UV/VISspectrophotometerofferingnarrowslitswithaidofskilledBMS
2. Fluorescencespectroscopy;excitaConandemissionwavelengths=beeerselecCvitye.g.goodforfecalporphyrinsanalysis
3. HPLCforurine,plasma,&Cssueextracts
fecal(falsenega0ve)
Chromsystemkitforporphyrinsinurine
LotsoffracConaCon&extracConmethodsavailable;Urine->Johnsonetal.,&Feces->Lockwoodetal.,&Pudeketal.,&Plasma->Kennedy&
James
Porphyrinsinurine/plasma• ItsfastandreliablemethodfordifferenCaldiagnosis• PreparaConrequiresonlyonemixing&onecentrifugaConstep.(Sample+stabilizaCon+Priming+ISTD)
• C-18column• Gradientsystemwithtwomobilephase• Fluorescencedetectorat405-620nm
Resultedpeaksforurineporphyrins
Inplasma,Uroporphyrincurveissplitinto(I&III)
Porphobilinogen(PBG)
Porphobilinogen(PBG)
• Itspyrrolewithmolarmassof226.229g/L• Itisgeneratedfrom(ALA)bytheenzymeALAdehydratase
PBGTestproceduresinurinesample:
1. UrinepHisadjustedto6–8
2. Syringecontainingwithorganicresinsisfilledwithsample
3. Expeltheurinesamplewash-upwithdisClledwaterX2
4. EluConreagentisfilledinsyringeandmixed
5. ExpelthesoluConintoreacContubecontainingDMAB
Watson-Schwartztest
• PBG+dimethylaminobenzaldehyde(DMAB)+acidsoluConàMagentacolor
Watson-Schwartztest:interferences
• Specificity:UrobilinogeninurinealsoproducesmagentacolorinsimilarreacConcondiCon
• Thus,OrganicextracConiscarriedtoremovetheseinterferingsubstance
• SyringecontainananionexchangeresinwhichbindstoPBGandotherinterferencearewash-out
Watson-Schwartztest:interferences
++
+ +
+ ++
+
+
+
++
UrobilinogenPBG
TheEnd
• QuesCons?