haemoglobin metabolism and.its clinical applications pptx

Hemoglobinmetabolismanditsclinicalapplications

Dr.Rohini CSane

FunctionsofHemoglobinmoleculeinoxygentransport

FunctionsofHemoglobinmoleculeincarbondioxidetransport

DegradationofHemetobilepigments

Macrophagesofreticuloendothelial (RE)inspleen/liver/bonemarrow-Hb

RBCà 12Odays

Nonprotein‘Heme’Globin

6gm /dayheme brokendown/resynthesisà Bilirubin(300mg/day)=250mg/dayHb+50mg/daymyoglobin

Fateglobin(reutilization)

• FormationofHb(resynthesis)• Degradedtoaminoacids–metabolismincludingformationofHb

SourcesofHeme

Heme

20%immatureRBC/Myoglobin/globin/cytochrome/peroxidase/catalaseTrppyrrolase80%RBCHb

(GREEN–excretedbybirds&amphibians)→

Transferrin

←αGlobinchains

←βGlobinchains

RBC(Agederythrocytes)withHbphagocytosisbymacrophages→

specificityforα methylenebridge←

Presenceinmammals←

Yellowwith36H←

→Bilirubin- AlbumincomplexinBlood

+

Hemeoxygenase

1. Microsomalenzymes2. NADPH,O₂,Methylenebridgesbetweentwopyrroleringsà

biliverdin3. Fe²⁺à Fe³⁺

Heme oxygenaseHemeBiliverdin+Fe³⁺+CO• Biliverdinà excretedbybirds,amphibians&mammals

DegradationofHemetobilepigments

BiliverdinreductaseBiliverdin(green) Bilirubin(nonfunctionalthereforeexcreted)1gmhemoglobin*à 35mgBilirubinBiliverdin+ Bilirubinà Hemederivatives• (*sources- 80%RBC+10%ineffectiveerythropoiesis+10%Myoglobin)• 6gm /dayheme brokendown/resynthesisà Bilirubin(300mg/day)=250mg/dayHb+50mg/daymyoglobin

TransportofBilirubintoLiver

• Bilirubin- (lipophilic–insolubleinwater)• Bilirubin+Albuminà Transportedinplasma• Per100mlplasmaà 25mgBilirubinboundtighttoAlbumin• BilirubinhaslowaffinityforAlbuminà easilydetached&entertissue

Drugs :sulphonomides /salicylates/PenicillindisplaceBilirubinfromAlbuminbindingsites

BilirubinentersCNS

Damagetoneuronsà Kernicterus

DegradationofHemetobilepigments

Conjugation0fBilirubinLiverAlbumin–Bilirubincomplex

IUptake carriermediatedactivetransportSinusoidalsurfaceofhepatocytes

Bilirubin+protein(Ligandin )2UDPGlucoronate

IIConjugation UDPEndoplasmic-reticulum(hepaticmicroosomes ) BilirubinGlucuronyl transferase(chromosome2)

Bilirubin+2moleculesofGlucuronate80%BilirubinDiglucuronide+20%Bilirubinmonoglucuronide

(MOAT–multiplespecificorganiciontransport)-presentincanalculi

IIIsecretionBilirubinOligonucleotide

Bilesalts

FateofBilirubininhumanbody

FactorsaffectingconjugationofBilirubininliver

1. DrugslikePrimaquine/Novobiocin/Chloramphenicol,/Androgen/Pregnanediolinterferewithconjugationprocessmaycausejaundice.

2. DecreaseconcentrationofUDP-Glucuronyl Transferase3. PhenobarbitalinducesUDP-Glucuronyl Transferase

Production&excretionofBilirubinRetiiculoendothelialsystem(RES)

Hb↓

Biliverdin(38H)↓

Bilirubin(36H)

LiverBilirubin

Diglucuronide

BilirubinDiglucuronide↓DeconjugationFreeBilirubin(36H)

↓reductionUrobilinogen (44H)byEcoli

(colorless)

Reductionofvinylgroupofstercobilinogen (48H)

↓stercoblin(46H)

DARKBROWN /200MG/DAY

BLOODUROBILINOGEN

KIDNEYUROBILINOGEN

UROBILLINOGEN(44H)↓

UROBILININURINE(42H)(<4GM/DAY)

BILEDUCT→←PORTALCIRCULATION

EHC↓

←SMALLPORTION

EHC–Entero hepaticcirculation

ExcretionofBilirubinintobile

ConjugatedBilirubinActivetransport*(againstconcentrationgradient)Ratelimitingstep

Bile>98%Bilirubinentersbileinconjugatedform

*InducedbyPhenobarbitone

Production&excretionofBilirubin

• ExcretionofBiliverdinà greencolorstool(children&prolongedantibioticstherapy)• Blackcolorstoolduringconstipation• Schlesingertest:Stercobilin (SB)&Urobilin (UB)+Zn²⁺à greenfluroscent

Fouchet’s DiagnostictestforBilirubininurine

Bilirubin–yellow,Biliverdin–green ,Bilinofuschin –Red, Bilicyanin-violet

←URINE→

Gmelin’s Test

←CONCHNO₃→

Bilirubin–yellow,Biliverdin–green ,Bilifuschin–Red, Bilicyanin-violet

FateofBilirubinBilirubinGlucuronides

Hydrolysis↓bacterialenzyme(β Glucuronidase )Bilirubin↓smallportionreabsorbed

Urobilinogen (colorless)à SBG(Erhlich +ve )↙↘

Stercobilin Urobilin(excretedinstool-)(excretedinurinebykidney)↓↓BrowncolorofstoolpaleyellowcolorofurineSerumBilirubinà VanDerBergTest,UrineBilirubinà Fouchet’s Test,Gmelin Test

Conjugatedandunconjugatedbilirubin(Directandindirectbilirubin)

Stercobilin andUrobilin

FormationofconjugatedbilirubininHepatocytes

TypesofBilirubinFreebilirubin Conjugatedbilirubin

1 InH₂O insoluble soluble2 Inalcohol soluble soluble3 Normalplasmalevels 0.2mg/dl 0.2-0.8mg/dl4 Inbile Absent present5 Inurine Alwaysabsent Normallyabsent(presentin

hemolytic&Obstructivejaundice)6 AbsorptionfromGIT Absorbed NotAbsorbed7 Diffusionintissue Diffusestocauseyellow Dosenotdiffuse

8 VanderBerghTest Indirectpositive directpositive

Bilirubin&itsreductionproductsnumberofHatoms color

Bilirubin(BR) 36 RedyellowMeso bilirubin(MB) 40 yellowUrobillinogen (UBG) 44 colorlessStercobilinogen (SBG) 48 colorlessUrobilin (UB) 42 OrangebrownStercobillin (SB) 46 Darkbrown

Jaundice- HyperbilirubinemiaPhysiological– TotalBilirubinconcentrationinserum=(0.2- 0.8mg/dl)

0.2-0.6mg/dl(unconjugated)0- 0.2mg/dl(conjugated)Yellowcolorsclera&skin(depositionofbilirubin)-conc ofserumbilirubin>2mg/dl

q ConjugatedHyperbilirubinemiaq UnconjugatedHyperbilirubinemiavSymptoms1. Nausea2. Vomiting3. AppetiteØ LatentJaundice(1-2mg/dl)

*Diagnosticlaboratorytosetupownqualitycontrolsforreferenceranges

Prehepatic,Hepaticandposthepaticjaundice– aclassificationbasedonsite(causeofofHyperbilirubinemia)

ClassificationofJaundicebasedontypeofBilirubin

Intrahepaticjaundice(Genetic/inheritedcauses)

Cause• HemolyticJaundice

• Causes– HemolysisMalariabloodtransfusionsicklecellanemia

• LiverfailstoconjugateexcessofBilirubin

• Therefore↑unconjugatedbilirubin↑Urobilinogen↑stercobilinogen(browncolorstool)

• SGPT/ALP-Normal

• HepaticJaundice• Causes–dysfunctionofLiverHepatitisalinfectionpoisons/toxinscirrhosis/CCF

• ↑unconjugatedbilirubin↑conjugatedbilirubin↑Urobilinogen↑stercobilinogen(browncolorstool)↑SGPT/ALP

• ObstructiveJaundice• Gallstonestoolcontainsfatunavailabilityofbilesalts

• ↑conjugatedbilirubin↑Urobilinogen↓ stercobilinogen (palecolorstool)↑SGPT/ALP

ComparisonofHemolytic/hepaticandobstructiveJaundice

DifferentialdiagnosisJaundice/Icterus

HemolyticDiseaseofAdultsà unconjugatedHyperbilirubinemia

1. increaseinunconjugatedbilirubininblood2. Absenceofbilirubininurine3. Excretionofurobilinogenà (EhrlichTestpositive)4. Excretionofstercobilinogen infaecesvDiseasesassociatedwitha) congenitalspherocytosisb) G6PDdeficiencyc) Autoimmunehemolyticanemiad) Toxincarbontetrachloride

HemolyticDiseaseofAdultsà unconjugatedHyperbilirubinemiaIncompatibilitybetweenmaternal&fetalbloodgroupsAntiantibodiesABOIgM typecannotbetransferredtoplacentavRh incompatibilityFetusmotherRh(+ve )Rh(-ve )RBCRBC elicitimmuneresponseAnti-D(IgG)

Destruction Anti-D (IgG )

Of RBC←Placenta

Secondpregnancy(beforebirth–destructionofRBC)à CHILDbornwithseverehemolyticdiseaseàErythroblastosis fetalis

Erythroblastosis Fetalis• SerumBilirubinà >20mg/dlà nomoreboundtoAlbumin

• BilirubinBrain(Kernicterus-depositionofbilirubininbrain)• Basalgangliaàmentalretardation• ↓ATPasemitochondriaà fits,spasticity,toxicencephalitis• Treatment:(1)phototherapybeforeage<1yearà

isomerizationZZà ZE(2)Bloodtransfusion

HepatocellularJaundice

1. ViralHepatitis(virusesA,B,C,DorG)à purehepatocellulardiseases

2. ↓conjugatedbilirubintherefore↑freeBilirubin3. Inflammatoryodemaofcellsà intracellularcanalculicompressed

à obstruction(atsiteofbileformation)4. Increaseobstructionà↑obstructionà↑conjugatedBilirubin

thereforeBiphasic5. Bilirubinuria6. UrobilinogenàNORMAL ordecreasedinhepatocellularJaundice

ObstructiveJaundiceà conjugatedHyperbilirubinemia1. ↑conjugatedbilirubin2. Bilirubininurine3. ↓urobilinogen (nilifobstructiveiscomplete)4. Faeces claycolor(↓stercobilinogen )5. Absenceofbilesaltsà Stetorrhoeamayresult6. CausesIIntrahepaticCholestasisa) Activehepatitisb) Biliarycirrhosisc) Lymphomasd) Hepatomae) Viralhepatitis

ObstructiveJaundiceà conjugatedHyperbilirubinemia

• CausesIIExtrahepaticCholestasis1. Stonesinbiliarytract2. Stonesingallbladder3. Biliaryatresia4. Carcinomaheadofpancreasà lymphglandsenlarged5. Porta hepatitis

Acquiredhyperbilinogen(unconjugatedhyperbilinogen)

vPhysiologicalJaundiceofnewborn(neonates)1. Afterseconddayoflife,transienthyperbilinogen –Jaundice2. ↑rateofdestructionofRBC(transient)3. à immaturehepaticsystemofconjugation4. à Bilirubindonot exceed5mg/dl5. à 2weeksphototherapy+barbitonetoinduceconjugation(ZZ

bilirubinà ZE+EEà excretedwithoutconjugationinurine)6. Undueprolongationà crenism7. Breastmilkjaundice(estrogenà↓enzymeGlucuronyl transferase)

Bilepigment DiagnosticTestBilirubin VanDerBergh(serum),Fouchet’s Test&Gmelin ‘stest

inUrineUrobilinogen (UBG) Ehrlich’sTestUrobilin Schlesinger’stest

DIRECTBILIRUBIIN- Azo pigment(pH5)àPURPLECOLOR-↓

INDIRECTREACTION–FREEBILIRUBIN(H2Oinsoluble,alcoholsoluble)

*Diagnosticlaboratorytosetupownqualitycontrolsforreferenceranges

EnzymeestimationshelpindifferentialdiagnosisofJaundice

EnzymeestimationshelpindifferentialdiagnosisofJaundice

*Diagnosticlaboratorytosetupownqualitycontrolsforreferenceranges

EnzymesindicatingHepatocellulardamage

v↑ALT↑ASTViralHepatitis

v↑IHDHepatocellularnecrosis

v ALT/ALT>1AlcoholliverdiseaseObstructiveliverdisease:cholestasis/hepaticcarcinoma/parenchymalcell

damage

Parameter HemolyticJaundice HepaticJaundice

ObstructiveJaundice

1 Bloodfreebilirubin ↑ ↑ normal2 Conjugatedbilirubin normal ↑ ↑

3 ALP normal ↑ ↑↑

4 Bilesalts Nil Nil PRESENT5 UrineBilirubin Nil Nil PRESENT6 Urineurobilinogen ↑ Nil Nil7 Stool Darkbrown Claycolor

DifferentialDiagnosisofJaundice

FateofBilirubin

CongenitalHyperbilirubinemiavAbnormaluptake,conjugationorexcretionofbilirubinduetoinheriteddefects• 1.GilbertDisease–defectinuptake• 2.CriglerNajjar syndrome–defectinconjugation

CongenitalHyperbilirubinemiav(1) Gilbert’sDiseasea) Autosomaldominanttraitb) DefectuptakeofBilirubinc) Asymptomaticd) Presenceofjaundice(Bilirubinà 3mg/dl)

CongenitalHyperbilirubinemia(2)CriglerNajjar syndrome:DefectinconjugationTypeIa) CongenitalnonhemolyticJaundiceb) DeficiencyofUDP–Glucuronyl transferasec) Fatal(deathbeforetwoyearsofage)d) Jaundiceappearswithin24hrsoflifee) Unconjugatedbilirubin(>20mg/dl)f) Kernicterusg) Barbituratesnoeffect(enzymenodefect)

CongenitalHyperbilirubinemia(2)CriglerNajjar syndrome:DefectinconjugationTypeIIa) CongenitalnonhemolyticJaundiceb) DeficiencyofUDP–Glucuronyl transferasec) Diseaserunsmorebenigncoursed) Bilirubin–mononucleotidepresente) Totalbilirubinseldome rises:Unconjugatedbilirubin(>15mg/dl)f) NoKernicterusg) Barbituratesuseintreatmentà Jaundiceimproves

ManagementofCrigler Najjar Syndrome

CongenitalHyperbilirubinemiavDubin Johnson’ssyndromea) Autosomalrecessivetraitb) ↑conjugatedbilirubininbloodc) DefectinexcretionofconjugatedBilirubin(ATPase dependent

organicanionTransporterprotein-MOATmutationd) DefectATPdependentorganicaniontransportinbilecanalculie) BilirubingetsdepositedinliverthereforeBilirubingetsdepositedin

liver(BlackLiverJaundice)f) DiagnosticTest:BromosulphthaleinTest

CongenitalHyperbilirubinemiavDubin Johnson’ssyndromeDiagnosticTest:BromosulphthaleinTest250mgBromosulphthalein(intravenous)

Normal(dyeremainingplasma) Dubin Johnson’ssyndrome(dyeremainingplasma)

45min <5% >2hrslevels(<2%)2hrs <2% <45minlevels(>5%)

(2hrlevelmorethan45min)

At45minBSPtakenupbyhepatocytes/thereforedecreaseinblood levels

EXCRETORYDEFECT:BSPregurgates inblood

CongenitalHyperbilirubinemiavRotorsyndromea) Causenotknown(autosomalrecessive)b) Bilirubinexcretiondefectivec) Nodepositionofpigmentinliver

?

ComparisonofDubin Johnson’sandRotor‘ssyndrome

ComparisonofGilbert‘sandCrigler- Najjar syndrome

BiosynthesisofHeme(Porphyrinring)• Siteà Liver/RBCproducingcellsofbonemarrow(erythroid cells)/othertissue• ExcretiontoruleàmatureRBClackingmitochondria• IFormationofδ aminoLevulinate (mitochondria)Glycine+succinylCoA

* Pyridoxalphosphatedependentδ aminoLevulinate synthtase

δ aminoLevulinate (ALA)*ratecontrollingstep

BiosynthesisofHeme(Porphyrinring)IISynthesisofPorphobilinogen2. δ aminoLevulinate (ALA)

ALADehydratase *

Porphobilinogen(PBG)• ActivitydecreasesbyPb /Hg&Zinccontainingenzyme

BiosynthesisofHeme(Porphyrinring)1. SuccinylCoA+Glycine→ALA2. ALA+ALAà Porphobilinogen(PBG)+2H₂O3. 4X(PBG)4. Uroporbilinogen III(UBG)5. Coporphyrinogen III(CPGIII)+CO₂6. Protoporphyrinogen III(PPGIII)7. HEME

BiosynthesisofHeme(Porphyrinring)

SuccinylCoA+Glycine→ALA

ALA+ALAà Porphobilinogen(PBG)+2H₂O4

4X(PBG)Uroporphyrinogen

Coporphyrinogen III(CPGIII))

Protoporphyrinogen III(PPGIII)

HEME

HEMGLOBIN1. ALA SYNTHTASE 2.ALA DEHYDRATASE 3.PBG DEAMINASE&UPGIIICOSYNTHTASE 4.UROPORPHYRIN

DECARBOXYLASE 5COPORPHYRINOGENOXIDASE 6.Hemesynthtase

→4CO₂

NADP+O₂→ →NADPH+H+2CO₂

ACETYLàMETHYL

PROPINYLàVINYL

ACETYLàMETHYL,4CO₂

PROPINYLà VINYL+2CO₂

BiosynthesisofHeme

Metallo- porphyrin

1. UroporphyrinI&III2. Coporphyrin I&III3. PROTOPORPHYRINIX&HEMEvHemecontainingproteins- hemoglobin,cytochromes,catalase,tryptophanpyrrolase

Hemesynthesis

• Site :mitochondriaofallmammalian tissueexceptRBCpredominantlyinLiver&bonemarrow

vStepI :*ALAsynthasePLP CO₂CoASH

SuccinylCoA+Glycineδ ALA• δ ALA:DeltaaminoLevullinic acid• *Regulatoryenzyme=ratelimitingstepqINHtreatmentà PLP↓à ALASYNTHESIS↓

Hemesynthesis

vStepII :Site–MITOCHONDRIA

*ALADEHYDRATASEPLP 2H₂O2MoleculesofALAPorphobilinogen(PBG )*activitydecreasedbyheavymetals

Hemesynthesis

vStepIII : Site–MITOCHONDRIA

*PBGDEAMINASE4NH₃4MoleculesofPBGUroporphyrinogen(UBG )

• *UroporphyrinogenIsynthtase&UroporphyrinogenIIIsynthtase• *deficiencyofUroporphyrinogenIIIsynthtaseleadstoformationofTypeIUBGà Porphyria

Hemesynthesis

vStepIV :Site–MITOCHONDRIA

*Uroporphyrinogendecarboxylase4CO₂• Uroporphyrinogen(UBG )Type III CoproporphyrinogenIII

• *AcetylàMethyl• *deficiencyofà Porphyria

Hemesynthesis

vStepV : Site-CYTOSOLIC

*Coproporphyrinogenoxidase2CO₂Coproporphyrinogen IIIProtoporphyrinogenIII

O₂• *Methylà Vinyl• *deficiencyofCoproporphyrinogenoxidaseà Porphyria

Hemesynthesis

vStepVI :Site-CYTOSOLIC

*Protoporphyrinogenoxidase4HProtoporphyrinogenIIIProtoporphyrin III (IX)

Methylene(CH2)àMethenyl (CH- )• *deficiencyofProtoporphyrinogen oxidaseà Porphyria

Hemesynthesis

vStepVII :Site-CYTOSOLIC

*HemesynthtaseProtoporphyrinIII (IX)Heme

Fe²⁺• Ferrochelatase• Heme+ Protein= HEMOGLOBIN

1.SuccinylCoA+Glycineà

ALA

ALA

2.ALAà PBG

3.PBGà UBGIII 4.UBGà CPGIII

CPGIII

CPGIII

5.CPGIIIàPPGIII 6.PPGà PP 7.PPà HEME

CYTOSOLIC-2,3,4

MITOCHONDRIAL

HEMESYNTHESIS

RegulationofHemesynthesis1.↑Glucoseà↑catabolismofCRP(repressorprotein)à decreaseALA(inductionprevented,Glucoseadministeredfortreatmentofporphyria

2.↓ALAsynthasebyHematin,ExcessoffreeHeme(Fe²⁺à Fe³⁺)

3.Compartmentizationofenzymesà easierforregulation,ratelimitingstepsinmitochondria

4. Hemesynthesiscontrolledbya)globinsynthesisb)Druglikebarbiturates(requirecytochrome450)

5.lead/Mercury↓ALASynthase↓Ferrochelatase

RegulationofHemesynthesis

ThreemechanismscontrollingactivityofALAsynthasebyHeme/HematinFe³⁺

a.Feedbackinhibition(Heme/HematinFe³⁺)

b.RepressionofALAsynthase

C.InhibitionofALAsynthasetransportfromcytosoltomitochondria(thesiteofaction)

Hemesynthesissite:liver(ALAsynthaseà regulatoryenzyme)

EffectofdrugsonALAsynthaseactivity1. *Phenobarbital2. *Insecticides ↑activityofALAsynthase3. *Carcinogen* MetabolizedbyHEMEcontainingproteincytochrome450

↓IncreasedincorporationofHemeincytochrome450

↓↓cellularlevelofHeme

↓↑ALAsynthase(derepression)tomeetcellulardemands

RegulationintheErythroidcells

vALAsynthasedosenotregulateHemesynthesisinErythroidcellsvUroporphyrinogensynthase&Ferrochelatase regulateHemesynthesis

vCellularuptakeofironregulateHemesynthesisvHemestimulateglobinsynthesis

RegulationofHemesynthesis

Porphyrin&BilirubinMetabolism

• Porphyrin :Groupofcompoundsof4substitutedpyrroleringslinkedbyMethanebridges•à formationofcomplexeswithmetalionsboundtoN₂atomofpyrrolering

IRON

Heme

Hb

MAGNASIUM

Chlorophyll

Photosynthetic pigment

Porphyrin&BilirubinMetabolism

vIronPorphyrins :1. Hemoglobin2. Myooglobin3. Cytochrome4. Catalase&peroxidase5. Tryptophanpyrrolase

PorphyrinMetabolism• Porphyria's:GroupofinbornerrorsofmetabolismassociatedwiththebiosynthesisofHeme• Characteristics of Porphyrias :increaseinproduction&excretionofporphyrin&theirporphyrinprecursorsALA&PBG• AutosomaldominantorAutosomalrecessive• Diagnosis ofPorphyria's:• 1.urine(exoposure toUVà red fluorescence )• 2.urine+CHCl₃à extraction• Aqueouslayer(PBG)+Ehrlichreagentà pink color

Porphyria's:

• TypesPorphyria's:1.inheriteda) Erythropoietin :enzymesdeficiencyoccursinerythrocytesb) Hepatic : enzymesdeficiencyliesintheliver2.Acquired💟

Autosomalrecessive

Autosomaldominant

Erythrodontia

RBC-FLUORESCENCE

Liver,skinfluorescence

AcuteintermittentPorphyrias• DeficiencyofenzymesUroporphyrinogensynthaseI

SuccinylCoA+Glycine

δ aminolevilinate(ALA)↑

Porphobilinogen ↑

UroporphyrinogenIII

δ aminolevilinatesynthase

ALADehydratase

Uroporphyrinogen synthase

Uroporphyrinogen I

↑ALA↑PORPHYRINOGEN

1.AcuteintermittentPorphyria• Age :afterpuberty(Artist–Vincet ,KingGeorgeIII–MAD)• Deficient enzyme :UroporphyrinogensynthaseI(↑ALA–nofeedbackmechanism)• Noporphyria,nophotosensitivity• Excretion inurine:δ aminolevilinate(ALA)&porphobilinogen (PBG)• Exposureofporphobilinogen (PBG)toairdarkens(formationofPorphobilin )• Diagnosis:urinaryconc ofPBG(darkenson exposuretoair)• Symptoms:Abdominalpain,Vomiting,cardiovascularabnormalities,neuropsychiatricdisturbances(therefore↓Trppyrrolase ,↑Trp,5ʹOHTyramine• Treatment:Hematinà↓ALA, ↓PBG• Adverse effects :Barbiturate(ppt ofattack)à↑ALAsynthase(ascytochrome450)àPBG↑,increasewithcarbohydratediet&menopause

2.Congenital erythropoietin PorphyriavCongenitalvDeficient enzyme :↓UroporphyrinogencosynthtaseIII• Excretioninurine:UroporphyrinogenI&Coporphyrinogen Ià

↓oxidation↓UroporphyrinI&Coporphyrin I

Symptoms :1.↑Hemolysis2.Erythrodontia(teeth),portwinecolorurine3.ExposureOFSKINà UVsensitizationofporphyrinà absorbed&emitredfluorescentlight4.Dermatitis,scarringburning&itchingofskin- ear&nose(Leprosylike),(asROS↑à accumulationofPorphyrin)5.portwinecolorurine(UroporphyrinI&Coporphyrin I)

Congenital erythropoietin Porphyria

PhotosensitivityErythrodontia (teeth )

Congenital erythropoietin Porphyria

Dermatitis,scarringburning&itchingofskin- ear&nose(Leprosylike,(asROS↑à accumulationofPorphyrins )

Dermatitis,scarringburning&itchingofskin- ear&nose(Leprosylike)(asROS↑à accumulationofPorphyrins

Congenital erythropoietin Porphyria

3.Porphyria Cutanea Tarda• Cutaneoushepaticporphyria• Deficient enzyme :↓Uroporphyrinogendecarboxylase• Excretioninurine:Porphobilinogen&Uroporphyrin(I&III)rarely• Symptoms :1. Cutaneous2. Photosensitivity3. LiverexhibitsfluorescenceTreatment :Hematin(↓ALAsynthase,↓accumulationvariousintermediate)

4.Hereditary Coporphyria• Deficient enzyme :↓Coporphyrinogenoxidase• Excretioninurine:UroporphyrinogenI&Coporphyrinogen Ià

↓oxidation↓UroporphyrinI&Coporphyrin I

• Urinedarkcolor:presenceof UroporphyrinI ,Coporphyrin I,ALA,PBG• Treatment :Hematin(↓ALAsynthase,↓accumulationvariousintermediate)•

5.Variegate porphyria• Deficient enzyme :Protoporphyrinogen oxidase• Accumulationinplasma&excretioninurineof:PorphobilinogenUroporphyrinogen,Uroporphyrin,Coproporphyrinogen,Coproporphyrin,Protoporphyrin(↓Hemesynthesis)• Photosensitivity exhibited• Plasmaexhibitsredfluorescence(duepresenceofCoproporphyrinogen• Neuropsychiatric manifestation

6.Herediatory Protophyria (Erythropoietic protoporphyria )

• Deficient enzyme:Ferrochelatase• Accumulationinplasma(increaseinconcentration) ,excretioninurine (increaseinconcentration)& faeces (increaseinconcentration)of:PROTOPORPHYRINIX• RBC,skinexhibitredfluorescence•

7.Acquired (toxic )Porphyrias• Exposureofbodytotoxiccompounds(toys/Xeroxink)Examples:Heavymetals(Lead)↓ALADehydratase

Hexchlorobenzene ↓UroporphyrinsynthaseIDrugs(Griseoflavin )↓Ferrochelatase

Therefore↓Heme↑ALAsynthaseAquried porphyrias associatedwithanamia

ReactionsofHeme synthesis

PrenataldiagnosisofPorphyriasEnzymeestimations/PCR

Anemia• 75%Indianpopulationsufferfromanemia• Hbconcentrationdecreasesinanemia<10gm%(normalconcentrationofbloodHb– 14-16mg%-male,13-15mg%-female)vIIronDeficiencyanemia–mostcommona) Nutritionalb) LackofabsorptionofIron(Gastrectomy&Achlorhydria )c) Hookworminfection(0.3ml/day/hookworm)d) Repeatedpregnancy(↓hemoglobin-1gmperdelivery)e) Nephrosisà Glomerularfiltration→proteinuriaf) LossofHaptoglobin/Hemopexin /Transferring) Heavymetalpoisoningh) Lossofblood(menustrual cycle,piles,pepticulcers,Uterinehemorrhages)

AnemiaII-ImpairedproductionofRBC

DefectinHemesynthesis

• Nutritional• Deficiencyofiron,Copper,VitaminB12,VitaminC

• Leadpoisoning

DeficiencyofERYTHROPOITIN

• Physiological–kidneycellssynthesizeERYTHROPOIETIN

↓• ↑RBCsynthesis• Chronicrenalfailureàno ERYTHROPOIETIN

Decreaseinstemcells

• APLASTICANEMIA

• MALIGNANTINFITRATION

• INFLECTION

Hemin crystals

• Fe²⁺↔Fe³⁺à Fe³⁺+Cl⁻à HematinchlorideorHEMINvMedicallegalcases• Blood+Bloodstains+Nippe ’sfluid(1%KCL+KBr +KI+Glacialaceticacidà darkbrownRHOMBICcrystalàmicroscopicinspectionHemepartofBloodà Testpositive

Hemin crystals

HemoglobinestimationvDrabkin’s TestforHemoglobinestimation:Cyanmeth HemoglobinMethod• Blood(Hb)+Drabkin’s reagentàCyanmeth Hemoglobin(Absorbance-540nm)à colorimetricestimation• Hb+potassiumferricyanideàMeth- Hb

Sulph Hemoglobinemia• Oxy–Hb+H₂Sà Sulph –Hb(absorptionpeakat620nm)

vFormationofSulph –Hbbysulphonamide ,Phenacetin ,Dapsone ,AcetonevBasophilicstripingofRBC

←Irreversible

AbnormalHborHbvariants1.Sicklesyndromea) 1. sicklecelltrait(AS)b) 2.sicklecelldiseasewithSS,SC,SD,SO,Sβ Thalassemia2.UnstableHaemoglobinsCongenitalHeinzbodyanemia–Hb Zurich3.HbwithabnormaloxygenaffinityA.Highaffinityà Polycythemia(familial)à HbChesapeake,OlympiaB.Low affinityà Cyanosis(familial)à HbM,Hb–Kansas,Hb- Hoppe4. StructuralvariationleadingtoThalassemia'sphenotypeA. AlphaThalassemiaà HbConstantspring,DeltabetaThalassemia,Hb–

LeporeB. BetaThalassemia:HbQuongC. 5.Hemoglobinthatdonot produceanyclinicalsymptoms:HbP HbQ ,HbJ

SICKLE CELL DISEASEvCharacteristics of Sickle cell disease a) Glutamicacidà Valine (6th positiononbetachain)b) Hydrophilicà Hydrophobic(stickinessonsurfaceofmolecule)c) PolymerizationofHb inRBCàdistortionofRBCintosickleshapedd) DeoxyHbS hasprotrusionononeside&cavityonothersideà

manymoleculesadheretogether

SICKLE CELL DISEASE

SignsandsymptomsofThalassemiamajor

InheritanceofThalassemia

AbnormalHborHbvariantsIIIHemolyticanaemias duetointra-corpusculardefecta) Hemoglobinopathies :HbS ,Hbc ,HbMb) Thalessemias :major&minorc) Abnormalshapespherocytosis&elliptocytosisd) Enzymedeficiencyà Glucose6PhosphodehydrogenaseIVHemolyticanemiadueextracorpuscularcause/defectsa) Infection–malarialparasites,streptococcusb) AutoimmuneHemolysis–RBCmembranecomponent,Syphilis,

Lympholenticular neoplasiac) Isoimmune hemolysisà RhincompatibilityinNewbornd) Hemolysisduetodrugsensitization–Dopa quinone (fixedonRBC)à

Abnormalantibodies againstalteredmembrane

AbnormalHborHbvariantsVhemorrhages

a) Hematuriab) Hematomesisc) Hemoptysisd) Pepticulcerse) Hemorrhoidesf) Thrombocytopeniag) Menorrhagiah) Bleedingtendencies