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

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+ +

+ ++

+

+

+

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UrobilinogenPBG

TheEnd

•  QuesCons?