MEDICAL BIOCHEMISTRY THIS PAGE ISBLANKCopyright 2006, 2002 New Age International (P) Ltd., PublishersPublished by New Age International (P) Ltd., PublishersAll rights reserved.No part of this ebook may be reproduced in any form, by photostat, microfilm,xerography, or any other means, or incorporated into any information retrievalsystem, electronic or mechanical, without the written permission of the publisher.All inquiries should be emailed to rights@newagepublishers.comISBN: 978-81-224-2300-6PUBLISHING FOR ONE WORLDNEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS4835/24, Ansari Road, Daryaganj, New Delhi - 110002Visit us at www.newagepublishers.comT TT TTom om om om omy yy yyElderdaughter Elderdaughter Elderdaughter Elderdaughter ElderdaughterLateNallur LateNallur LateNallur LateNallur LateNalluriKiranmayiChowdary iKiranmayiChowdary iKiranmayiChowdary iKiranmayiChowdary iKiranmayiChowdaryTHIS PAGE ISBLANK(vii)PREFACE TO THE SECOND EDITIONIattemptedtoprovideessentialinformationonmolecularbasisofhealthanddiseasethat is mainly related to life of surviving cell(s) in the first edition of the book. However lifecycle of cell(s) includes cell(s) birth and cell(s) death apart from survival. For the last coupleofyearsthesefrontierareasareadvancingrapidlywhichisviewedbymanyasgoodsignfordevelopmentof:(a)newtherapyortherapeuticsforcancer(b)immortalizedcells.Thelatterfuelsgrowthofbiotechnologyandpharmaceuticalindustriesalso.Hence,inthesecondeditiontwochapters1.Biochemistryofcellcycle(cellbirth)2.Biochemistryofapoptosis(celldeath)areadded.Aslivingorganismsevolvedfromsimpleunicellulartohighlycomplexmulticellularmammals,severalnewsystemsandorgansweredeveloped.Forexample,bloodwhichactsasvehicleorcommunicationbetweenvariouslocationsofbody,immunesystemwhichprotectsbodyfromintrudersorforeignorganisms.Parkinsonism,psychosis,depression,Schizophrenia,lossoftasteandolfactionareduetodisturbancesinnervous,tasteandolfactorysystems.Variousorganspresentinbodyperformseveralorganspecificfunctionswhichareessentialforlife.Iffunctionsoftheseorgans are disturbed, diseases in which may culminate in death. So, in this edition biochemistryofbloodincludingimmuneresponseinChapter-32;molecularandcellularmechanismoflearning,memory,behaviour,tasteandolfactoryinbiochemicalcommunicationsChapter;tests,proceduresthataredoneinhospitalbiochemistrylaboratorytoassessfunctionsofliver,kidneyinChapter-33andthyroidinChapter-29aredetailed.Dependingondiseaseaparticularconstituentofbloodiseitherelevatedorlowered.Diagnosisandprognosisofdiseaseusuallyinvolvesdetectionandmeasurementofvariousbloodconstituentsinhospitalbiochemistrylaboratory.Thereforeadvancedtechniqueslikehighperformanceliquidchromatography(HPLC),affinitychromatography,andgeneraltechniqueslikecentrifugationanddialysis,instrumentsfromspectrophotometertoautoanalyzerandmethodsusedfordetectionandquantitationofbloodconstituentslikecarbohydrates, proteins, lipids, nucleic acids, enzymes, electrolytes etc., in health and diseasearedetailedinChapter-34.Humansandothermammalsareabletoremovewasteproducts,toxins,foreigncompoundsfrombloodandorgansintheformofurine.Indisease,compositionofurinevaries from that of healthy state. So, detection, quantitation of various constituents of urineis carried out in hospital biochemistry laboratory to confirm diagnosis of diseases. In Chapter-34,methodsfordetectionandestimationofurineconstituentundernormalanddiseasedconditionsarealsodetailed.Moststrikingfeatureinthiseditionofthebookisinclusionofbiochemicalaspectsofdiseases or disease causing organisms common to tropical countries like malaria, tuberculosis,pepticulcerorgastritis,pneumonia,leishmaniasis,giardiasis,trypanosomiasisetc.Sincemostoftheorganismsaredevelopingresistancetotheexistingdrugs,thereisneedfordevelopment of new drugs which requires thorough biochemical knowledge of these diseasesas well as disease causing organisms. Apart from adding new chapters, all existing chaptershavebeenupdatedbyaddingnewsubjectmatter.Referencesofeachchapterupdatedbyincludingreviews,books,researcharticlesetc.Further,numberofunsolvedproblemshavebeenincreasedinmostofthechapters.Ihopethiseditionwillbewellreceivedbyteachersandstudentsofvariousmedical,dental,pharmacy,biotechnology,physiotherapy,medicallaboratorytechnology,biomedicalengineering, life sciences under graduate and post graduate courses, Suggestions or commentsfromteachersandstudentsarewelcome.IamgratefultoSri.R.K.Gupta,Chairman;SriSaumyaGupta,ManagingDirectorofNewAgeInternational,NewDelhi,forpublishingsecondedition.N.MALLIKARJUNARAO(viii)PREFACE TO THE FIRST EDITIONThisbookexplainsthefundamentalsofbiochemical(molecular)basesofhealthanddisease.Henceitmeetsmedicalandalliedhealthsciencesstudentsneeds.Asateacherofmedical,dental,pharmacy,biomedicalengineeringandsciencestudentsforthelasttwodecades,Iknowtheproblemsfacedbystudentsinmastering(conceptualizing)thesubjectwithin a limited time. Most of these students need a book for their routine day-to-day studywhichcontainsonlythenecessaryinformationinasimpleandconciseway.Therefore,thisbookiswritteninsimplelanguageinsuchawaythatastudentwithverylittlechemistryor biology background can easily follow the various aspects of biochemistry that are presented.Thisbookisalsousefulforthosewhoarespecializinginbiochemistry(M.Sc.orM.D.)becauseadvancesinfrontierareasofbiochemistryarepresentedinasystemicway.Ofcourseadvancesinotherareasthatarerelevanttomedicalstudentsarealsoincludedtoalimitedextent.Aninterestingfeatureofthisbookisthatthemedicalandbiologicalimportance of each chapter is highlighted in simple numbered statements. Further, in somechapters,diseases,drugs(treatments)ortoxinsofparticularsubjectmatteraredescribedundermedicalimportanceheads.Further,eachchapterstextisdesignedtofacilitateeasyflowofinformationinaninteresting,thoughtprovokingandlogicalmanner.Exercises(cases)givenattheendofeachchapterhelpinmasteringofthesubjectbystudentandutilization of biochemical principles by the student in solving health problems. To enthusiasticstudents,referencesgivenattheendofeachchapterprovideadditionalinformation.There are 29 chapters in the book. First six chapters deal with the composition, structure,function and life cycle of cells and the goal of biochemistry; occurrence, chemistry, structureandfunctionsofbiomoleculeslikeaminoacids,peptides,proteins,enzymes,carbohydratesandlipids.Thisisthenfollowedbychapters7and8thatdealwithmembranestructure,various transporters that move biomolecules across membrane and disintegration of complexmoleculesoffoodandabsorptionofresultingproducts,respectively.Chapters 9-12 deal with the production and utilization of energy in various pathways ofcarbohydrate,lipidandaminoacidmetabolisms.Regulatorymechanismsofsomeoftheimportant pathways are also outlined. Further synthesis of biologically (medically) importantcompoundsincluding non-essential amino acids is detailed. In chapter 11 the ultimate wayof producing energy from all energy yielding compounds in the respiratory chain is described.Changes in the flow of metabolites into various pathways of carbohydrate, lipid and proteinmetabolism that occur among tissues in well fed state, diabetes and starvation are describedinchapter13.Fundamentalsofmolecularbiologyi.e.,occurrence,chemistry,structure,functions,metabolismofnucleotides,nucleicacidsandcontrolofgeneexpressionaswellasappliedmolecular biology i.e., recombinant DNA technology are detailed in chapters 14-20. Biomedical(ix)(chemical)aspectsoftwomajorhealthproblemsofthe20thcenturycancerandAIDSarebriefed in chapter 21. In chapter 22 occurrence, chemistry, structure, functions and metabolismofporphyrinsandhemoglobinaredescribed.Clinically related topics like vitamins, minerals, macro nutrients, energy, nutraceuticalsoffood,electrolytes,acid-basebalanceanddetoxificationaredescribedinchapters23-27.Chemistry,production,detectionandusesofisotopesinbiochemistryandmedicinearedetailedinchapter28.Chapter29dealswithmechanismsofcommunicationbetweencells.IhopebothteachersandstudentsofBiochemistryatundergraduateandpostgraduatelevelsusethisbookextensivelyandtheirsuggestionstoimprovethebookfurtheraremostwelcome.IexpressmysincerethankstoNewAgeInternational,Publishersforpublishingthebook.N.MALLIKARJUNARAO(x)CONTENTS I'"fffffto1MSt.md&Ii/ion>ii) I'"ff{octtofluFi,..,Edition(u) ..Coli , ,. AminoAcids andPeptides " 3.Proteins ,. ElUymesalogy... ". Cancer and Aids.90 22.Porphyrin andHaemOjlobi n Metabolism "" 23.Vitamirul '" ". Minerala ,n (:ai) " Energy,Nutrients. Medie;nft and 'Ibxins of Food .. 26.Wate_r.Electrolytes and AcidBue Balanoo 629 " Dernxification ofXenobiotiCI ,' ...... _-... _Or"' --- j-"........ ... _.... - "" Too .'- -y-...... .._--- , 486 MedicalBiochemistry11. PCR and DNA polymorphism Now several PCR based assays are developed to detectDNAvariationsorDNApolymorphism.Someexamplesare(a)RandomlyamplifiedpolymorphicDNA(RAPD)(b)Amplifiedfragmentlengthpolymorphism(AFLP)(c)Sequencerelatedamplifiedpolymorphism(SRAP)etc.RAPDThegenerationofRAPDsinvolvesuseofsingleshortrandomoligoneucliotides.WhentheserandomprimersaremixedwithsampleDNAandsubjectedtoPCRamplificationofseveralfragmentsoccurs.TheDNAamplificationwithrandomprimersexposepolymorphismsdistributedthroughoutthegenome.RAPDisalsousedingenomemappingandgenetagging.AFLPThisPCR-basedtechniquepermitsinspectionofpolymorphismatlargenumberoflociwithinshortperiodoftimeandrequiresverysmallamountofDNA.AFLPispotentiallyusedingenomefingerprintingandmapping.Restrictionfragmentlengthpolymorphism(RFLP)It is another technique based on hybridization principle. DNA is a polymorphic molecule, i.e.,existinseveralforms.DNAofanindividualvariesfromothers.SequenceofDNAofanindividualisunique.Further,mutationsinDNAgeneratespolymorphicDNAinsameindividual, which occurs in diseases. So, DNA polymorphism is due to variations in sequence.WhenDNAofanindividualissubjectedtodigestionwithrestrictionenzymefragmentsofvaryingsizesorlengthsthatareuniquetoinvidualssequenceorcellareproduced.RFLP may also result from presence of variable numbers of tandem repeates (VNTR) inDNA. These are short sequences of DNA that are scattered locations in genome and repeatedintandem.Thenumberoftheserepeatsareuniquetoindividual.WhenDNAoftwoindividualsissubjectedtodigestionwithrestrictionenzymesfragmentsthatvaryinlengthandnumberaregenerated.Therefore,RFLPoftwoindividualsresultsfromthedifferencesinthelocationandnumberofcleavagesites.DifferencesinDNAoftwoindividualmaybeduetoevolutionarychanges.RFLPissimilartosouthernblottinginmanyaspects.InitialstepofRFLPinvolvesdigestion of more DNA samples with restriction enzymes where as in southern blotting onlyoneDNAsampleisdigested.RestofthestepsofRFLParethoseofsouthernblotting.Hence, in RFLP next step probes are used for hybridization. Probes hybridizes with fragmentcontainingcomplementarysequences.Thenpolymorphismsaredetectedbypresenceorabsenceofbandsafterhybridization.Applications1. RFLPisusedasadiagnostictestofinheriteddisease.Forexample,HbS:InHbSgenethereislossofonerestrictionsiteforrestrictionenzymeduetomutationwhereasnormalHbAgenehastwocleavagesites.So,RFLPofsickle-cellanemiapatientshowstwobandswhereasinRFLPofnormalindividualthreebandsappear.2. RFLPisalsousedtoidentifychromosomaldifference.RecombinantDNATechnology 4873. RFLPisusedforisolationandsequencingofcloselyrelatedgenes.4. RFLPiscombinationwithPCRisusedtodetectDNAvariations.Bioinformatics1. It is the combination of IT (Information Technology) and Life Sciences like Biochemistry,MolecularBiology,Biotechnologyetc.2. Itisdefinedasapplicationofinformationtechnologyandsciencefororganisationmanagement,mininganduseoflifesciences.3. Mainapplicationareasofbioinformaticsaregenomics,proteomics,pharmacogenomics,chemiinformaticsetc.4. Oneoftheearliestapplicationareaofbioinformaticsisindrugdesignprocess.Bioinformatics revolutionized traditional approach of drug discovery from target discoveryand screening to discovery and development of therapeutic agents whose role in preventionofcureofadiseaseiswellvalidated.Further,drugssodesignedhavelessfailures.5. Followingstepsofbioinformaticsbasedmethodofdesigningdrugthatisanenzymeinhibitor.(a) Selectionofchemicalfragmentsfrommolecularlibrary.(b) Assemblyofchemicalfragmentsinapiece-wisemannerintopossibleinhibitormolecule.(c) Usingdockingalgorithmallthepossibleinhibitormoleculesarescreenedtoselecthighlypotentinhibitorwhichpreciselyfitsinthebindingcavityofenzyme.6. Knowledge of genome sequence allows structure activity based drug designing. Followingarestepsofdrugdesigningprocess,whichinvolvesgenomesequenceknowledge.(a) DeterminationofproteinsequenceusingDNAsequence.(b) Prediction algorithms are used to visualise structure adopted by the protein molecule.(c) Using docking algorithm a molecule that binds and alters protein function is identifiedasadrug.REFERENCES1. Wu, K., Grossman, L. and Moldave, K. (Eds.). Recombinant DNA methodology. AcademicPress,NewYork,1989.2. Berger,S.L.andKimmal,A.R.MethodsinEnzymology,Vol.152,AcademicPress,California, 1987.3. Kantoff,P.W.Prospectsforgenetherapyforimmunodeficiencydisease.Ann.Rev.Immunol,6,5894,1988.4. Agarwal,S.andJang,J.GEM91.Ananti-senseoligonucleotidephosphorothioateasatherapeuticagentforAIDS.Anti-senseRes.Dev.2,261266,1992.5. Rangarajan,P.N.andPadmanabhan,G.Genetherapy:principles,practice,problemsandprospects.Curr.Sci.71(5),360367,1996.6. Marx,J.L.DNAfingerprintingtakeswitnessstand.Science240,16161618,1988.488 MedicalBiochemistry7. Mullus,K.B.Theunusualoriginofpolymerasechainreaction.Sci.Am.P.56,April1990.8. Meada,M.H.DairyGene.TheSciences.NewYorkAcademyofSciences,NewYork,P.21,October1997.9. Michel, K. and Schmidtke. J. DNA finger printing. BIOS Scientific Pub. USA, Canada, 1994.10. Schena, Mark, (Ed.) DNA micro arrays: A practical appraoch, Oxford University Press, 1999.11. Siebert,P.(Ed.).ThePCRtechnique.RT-PCR.EatonPublishing,MA,USA,1998.12. Mcpherson,M-JandMoller,S.G.PCR,Springer-VerlagTalos,2000.13. Dieffenbach, Carl. W. and Gabriel, S.D. PCR primer: A Laboratory manual. Cold SpringHarborLaboratoryPress,NY,2003.14. K.Shahetal.Molecularimagingofgenetherapyforcancer.GeneTherapy.11,11751187, 2004.15. Li,G.andQuiors.Sequencerelatedamplifiedpolymorphism(SRAP)anewmarkersystembasedonsimplePCRreaction:itsapplicationtomappingandgenetagginginBrassica.Theor.Appl.Genet.103,455461,2001.16. vander wurff. A.W.G. Chan, Y.L. vanstraalan, N.M. and Schouten. J. TE-AFLP; Combiningrapidity and robustness in DNA finger printing. Nucleic acids Research. 28, 105109, 2000.17. Song,H.etal.NeuralstemcellsfromadulthippocampusdevelopessentialpropertiesoffunctionalCNSneurons.Nature417,3944,2002.18. Gage,F.H.Mammalianneuralstemcells.Science.287,14331438,2000.19. Lesk, A.M. (Ed.). Introduction to Bioinformatics. Oxford University Press, New York, 2002.20. Isner,J.M.Myocardialgenetherapy.Nature.415,234239,2002.21. Austin,C.P.etal.Theknockoutmouseproject.NatureGenetics.36,921924,2004.22. Kubota,C.etal.Serialbullcloningbysomaticcellnucleartransfer.NatureBiotechnology.22,693694,2004.23. Susan M. Rhind et al. Human cloning: can it be made safe. Nat. Rev. Geneti. 4, 855864, 2003.24. RebecaJ.Morrisetal.Capturingandprofilingadulthairfolliclestemcells.NatureBiotechnology.22,411417,2004.25. Arekawa,T.etal.EfficacyoffoodplantbasedoralcholeratoxinBsubunitvaccine.Nature.Biotechnol.16,292297,1998.26. Karatzas, C.N. Designer milk from transgenic clones. Nature. Biotechnol. 21, 138139, 2003.27. Nishimura,E.K.etal.Mechanismofhairgraying:IncompletemelanocytestemcellmaintenanceintheNiche.Science.307,720-724,2005.EXERCISESESSAY QUESTIONS1. DefinerecombinantDNA.ExplainstepsofrecombinantDNAtechnologyusedforproductionofhumangeneproductsinabiotechnologycompany.RecombinantDNATechnology 4892. Giveanaccountofgenetherapy.3. Describeproductionandapplicationofhybridomas.4. Describehybridizationtechniques.5. Describepolymerasechainreaction(PCR).Shortquestions1. Writestepsinvolvedinproductionoftransgenicanimals.2. Definecloning.Writestepsnecessaryforproductionofclonedsheep.3. WriteanoteonDNAvaccines.4. Defineediblevaccines.Writestepsofediblevaccination.5. Write a biosensor working principle. Name components of a biosensor and write their applications.6. Explainwesternblottechnique.Writeitssignificance.7. WriterecombinantDNAtechnologyapplications.Fillintheblanks1. RecombinantDNAtechnologymayleadtocreationof.................species.2. cDNA probe is ................. molecule prepared from 32P labelled nucleoside triphosphates.3. Cloningisa.................reproductionofmammals.4. .................cellsareimmortal.5. ACAAACTisrepetitivesequenceoffruitfly..................490 MedicalBiochemistryMEDICAL AND BIOLOGICAL IMPORTANCE1. Cancer is a major health problem affecting humans throughout the world. Several typesof cancers affecting major organs like lung, brain, kidney, colon, breast, oesophagus andstomachhavebeenidentified.2. Rateofincidenceofcancerofparticularorganinparticularpopulationdependsonseveral factors like age, sex, dietary habits, environment, geographical location, geneticmakeup,culture,physicalexerciseetc.ForexampleinIndiaoralcanceriscommoninbetal nut chewing regions and in reverse smokers. Stomach cancer is more in JapaneseandChinesepeople.Coloncanceriscommoninadvancedcountriesandlungcanceriscommon in smokers. Old people are more prone to any type of cancer. Brain cancer andbloodcancerarecommoninchildren.Menabove50arepronetoprostatecancer.Womenabove45arepronetobreast,ovarianandcervicalcancers.3. Rateofincidenceofcancerofparticularorganvariesfromdevelopedcountriestodevelopingcountries.(Table21.1).Lungandcolorectalcancersarehighindevelopedcountrieswhilestomachandcervicalcanceraremoreindevelopingcountries.FurtherinIndiapharyngealcancersarehighinWesternIndiawhereasstomachcancersaremorecommoninSouthernIndia.Nearly 10 million new cases of cancer are diagnosed globally every year. It is estimatedthatby2020tenmillionpersonswoulddieofcancereveryyearWorldwide.Table21.1CancerincidencerateindevelopedanddevelopingCountriesCancersite Developedcountries DevelopingcountriesLung 62 24Colonandrectum 20-45 2-8Stomach 10 60Cervix 14 30Prostate 30 10Mouthandpharynx 13 2521CHAPTERCANCER AND AIDS490CancerandAIDS 4914. Nucleararchitectureisalteredincancercells.Newanticancerdrugsthatrevertthesechangesmaybedeveloped.Likewisenewtumourmarkerbasedonnuclearstructuralchangesmaybeusedincancerdiagnosis.5. Extensiveresearchcarriedoutforthelasttwodecadesonvarioustypesofcancerledtodevelopmentofpropertreatmentforatleastsometypesofcancers.However,itgreatlyexpandedourknowledgeonmolecularmechanismofcancer.6. AIDSisanothermajorhealthproblemthatsurfacedaroundsecondhalf20thcentury.AccordingtoWHO(WorldHealthOrganization)estimationabout20millionpeopleareaffected by AIDS. At least about 5-10000 people get infected for every 24 hours. Spreadofthisinfectiousdiseasealsodependsonseveralfactors.Indevelopingcountriesitisspreadingfasterduetoprevalentsocioeconomicconditions.7. ThoughthevariousfacetsofcancerandAIDSarebeingprobedthoroughlyforthelasttwodecadespropercureisnotinsightparticularlyforAIDS.CANCERGrowthofalltypesofcellsiscontrolledinthebody.Ifthegrowthofcellisnotcontrolledtheycontinuetoproliferatewhichleadstomalignancy.Socancerismalignantgrowth(uncontrolledgrowth)ofcells.Malignantgrowthofcellisalsocalledastumour.Cancerofaparticularorganortissuedevelopswhenthecellsofthatorganhavelostgrowthcontrol.In addition cancer cells has other abilities a) Invasion b) Metastasis. Cancer cells are carriedto other parts of the body by circulation where they develop further. So, Cancer of one organifnotdetectedcanspreadtootherorgans(Figure21.1).Fig.21.1Cancerdevelopmentfromnormalcell.NomenclatureandclassificationofCancersGenerallycancersarenamedaccordingtotheorganaffected.Howevertheyareclassifiedbasedonthethreeembryonicgermlayersfromwhichtissueororganisderived.1.CarcinomasArethecancerofcellsororgansderivedfromeitherectodermorendoderm.Cancersofepithelialtissues,nervoustissues,glandsetc.arenamedascarcinomas.492 MedicalBiochemistryExample:(a) Adenocarcinoma:Cancerofgland.(b) Squamouscellcarcinoma:Cancerofsquamouscellsofepithelialtissues.(c) Gliomas:Cancerofbrainnervoustissues.2.SarcomasAre cancers of tissues of mesodermal origin. Generally cancers of bone, cartilage, connectivetissue,muscleetcarecalledassarcomas.Examples:(a) Osteosarcoma:Bonecancer.(b) Fibrosarcoma:Connectivetissuecancer.Cancer is primarily due to DNA damage or damage of genes. DNA damage may result fromtheactionofbiological,chemical,physicalandenvironmentalagentsonDNA.Incidenceofcanceralsodependsonthegeneticmakeupofanindividual.CancergenesOncogenes.Aregenesresponsiblefordevelopmentofcancer.Protooncogenes.Theyareprecursorsofoncogenes.Theyareconvertedtooncogenesbyactivation.Tumoursuppressorgenes.Theyarepresentinnormalhealthypeople.Productsofthempreventcancerdevelopment.Theproductofoncogenesdisturbsthenormalcellgrowthcontrolmechanismleadingtocancer. Usually products of oncogenes are protein kinases that phosphorylate tyrosine residuesofproteins.(Tyrosinekinase.)Bothcellularandviraloncogenesarefound.Examplesforoncogenesandprotooncogenesaregivenbelow.1. Cellularoncogenethatcausesratsarcomaisdesignatedasc-rasoncogene.Likewisec-rasprotooncogene.2. Viraloncogenethatcausesratsarcomaisdesignatedasv-rasoncogene.Likewisev-rasprotooncogene.3. Oncogeneofrousesarcomaisdesignatedassrc-oncogene.4. Oncogeneofsimiansarcomaisdesignatedsis-oncogene.5. Oncogeneofchickenmyelocytomaisdesignatedmyc-oncogene.CarcinogenesisByseveralwayscarcinogenesisoccursinhumansandotheranimals.Usuallytheyarenamed according causative agent or factor. Different types of carcinogenesis are given below:1. Biologicalagentsthatcausecancerorbiological(viral)carcinogenesis.SomeDNA and RNA viruses are carcinogenic and hence they are called as oncogenic viruses.Whennormalcellsareculturedwithoncogenicviruses,thenormalcellsaretrans-formedintocancer(tumour)cells.Oncogenesofthevirusesareresponsibleforthedevelopmentofcancer.CancerandAIDS 493Examples1.HepatitisBviruscauselivercancerinhumans.2.Retrovirusesalsocausecancerinhumans.2. Chemicalcarcinogensormutagensorchemicalcarcinogenesis.ManychemicalsubstancescausemutationsinDNA.Theyarecalledmutagens.Sometimesthismuta-tioninDNAmayconvertnormalcelltocancercell.Thentheyarecalledascarcino-gens.Examples1.Cigarettesmokecauseslungcancerinhumans.2.Aflatoxinsarecarcinogens.3.Nitrosamine,Benzapyrinsandasbestosalsocausecancer.3. Physical agents that cause cancer or physical carcinogenesis. Exposure to radia-tion may damage DNA. UV light exposure causes mutation in DNA of skin cells. MutantDNAmediatescarcinogenesisbyactivationofoncogeneswhichleadstodevelopmentofcancerofskinormultipletumoursofskin.Cancerduetogeneticfactors:SomegenesinDNAareassociatedwithdevelopmentofcancerinsusceptibleindividu-als.Examples:1. Retinoblastoma,cancerofeyedevelopsinpeoplecarryingRBIgene.2. Wilmstumour,kidneycancerdevelopsinchildrenhavinggeneWTI.ActivationofprotooncogenetooncogeneByseveralwaysactivationofprotooncogenetooncogenecanoccur.Someofthemaregivenbelow.(a) Pointmutation.Pointmutationconvertsprotooncogenetooncogene.Humanbladdercarcinomaisduetopointmutation.Mutationmaybeduetoerrorduringreplication.(b) Geneamplification.Amplificationofoncogenesresultsintheformationofproductsofthesegenesbyseveralfolds.Thisinturnconvertsnormalcellstocancercells.(c) Chromosomaltranslocation,promoter/enhancerinsertionalsoleadstoactivationofprotooncogenetooncogene.Mechanismofactionofoncogenesor MechanismofcarcinogenesisTheproductofoncogeneconvertsnormalcelltocancercellbyseveralways.1. The product ofc-ras oncogene is a less active GTPase. This leads to prolonged activationofadenylatecyclaseandhenceactivitiesofcAMPdependentproteinkinase.Asaresultcellularmetabolismisalteredandnormalcellistransformedintocancercell.2. Myc-oncogeneproductisDNAbindingproteinortranscriptionfactor(TF).Itregulatesexpressionofcellcyclegenes.Asaresultcellcycleisaltered.3. Src-oncogeneproductistyrosinekinase.Itphosphorylatescyclinsandcyclindependentkinasesofcellcycle.Thisresultsincellcyclealteration.4. Someoncogeneproductsarepolypeptidegrowthfactorsthataffectcellcycleandmitosis.494 MedicalBiochemistryMechanismofvirusmediatedcarcinogenesis1. Severalhumantumourvirusesinduceimmortalizationofhumantissuecells.Itisfollowedbymalignantconversionwhichinvolvesseveralsteps.2. Humanpapillomaviruses(HPV),humanT-lymphocytevirus(HTLV)possessdefinedoncogenesthatstimulateproliferationofhumancells.3. Humanpapillomaviruscausescervicalcancer.Cancerofcervixisthenumberonecancer in Indian women. In India about 100,000 women develop this cancer every year.4. HumanT-lymphocyteviruscausesT-lymphocyteleukemia.5. The oncogenes of HPV are E6 and E7. They are able to immortalize keratinocytes. Theycontainallnecessaryinformationforimmortalization.6. The E7 protein releases transcription factor which activates genes engaged in cell cycleprogression.7. TheE6proteinbindsP53andabolishesitstumoursuppressiveandTransactivationalproperties.ItalsopromotesubiquitinisationofP53anditssubsequentproteolysis.8. ThusE6andE7areabletoimmortalizecellsindependentlyandbothgenescooperateeffectivelyinimmortalizationofcells.MetabolismofcarcinogenMetabolismofcarcinogenafterenteringthebodyismainlydirectedtowardsproducingmetaboliteswhichcanbeexcreted.Theenzymesystemsofphase-Iandphase-IIreactionsof metabolism of xenobiotics are mainly responsible for the formation of excretory metabolites.Sometimes these compounds lead to tumour formation. If it leads to formation of malignanttumour then it is known as Cancer. Cytochrome P450 enzymes of phase-I are involved in theformationofacarcinogen.Mechanismofacarcinogenmediatedcarcinogenesis1. Carcinogenesisbyacarcinogeninvolvesseveralsteps.2. Firststepistheinductionofmolecularlesion.3. SecondstepisthefixationofmolecularlesionbyDNAreplication.4. TheultimatecarcinogenicformsofcarcinogensarehighlyreactiveelectrophileswhicharereactivetowardsDNA.5. TheybindcovalentlywithDNAtoproduceDNAadduct.6. ThistypeofDNAmodificationismajordrivingforceforcancerdevelopment.NuclearstructureofCancercells1. Incancercellsnucleararchitectureisaltered.Thesealterationsarecharacteristicsoftumourtype.2. Componentsofnuclearmatrixplayaroleinorganizationofchromosomesandnuclearcomponents.Proteincompositionofnuclearmatrixisalteredincancercells.3. Oncogenesinducetumor-specificnuclearchangesandtheseinturnchangesgeneregu-lation.4. Incancercellschromosomalterritoriesandgenelociarechanged.CancerandAIDS 4955. Structural changes in tumour cells lead to changes in nucleoli and perinuclear compart-ment.6. Thesechangescanbeusedaspotentialtumourmarkersandtargetsforanti-cancerdrugs.TreatmentofcancerSeveraltypesoftreatmentsareavailableforcancermanagement.Somearegivenbelow.Cancergenetherapyisexplainedinearlierchapter.1. ChemotherapyCompoundsthatblockreplicationofcellsandantimetabolitesthatblocknucleotidebiosynthesisareusedasanticanceragentsorinchemotherapyofcancer.(a) Mercaptopurine.Itisapurineanalogusedinthetreatmentofluekaemia.Itisconverted into nucleotidein vivo and incorporated into nucleic acids and interferes withreplication.(b) Fluoro uracil.It is a pyrimidine analog and used in the treatment of colorectal cancer.Invivoitisconvertedfluorodeoxyuridinephosphateandinhibitsreplication.(c) Methotrexate.Itisafolicacidanalogandusedinthetreatmentofchoriocarcinoma.(d) Azaserine.Itisaglutamineanalogusedincancertreatment.Itblocksnucleicacidbiosynthesis(replication)byinhibitingglutaminedependentmetabolicreactions.(e) Acivicin. Another glutamine analog used as anticancer agent. It is a competitive inhibi-torofglutamineutilizingenzyme.Methotrexate,azaserineandacivicinareantimetabolitesusedincancertreatment.They are called as anti metabolites because they block nucleic acid synthesis by anatgonizingmetabolicroleofglutamine.2. RadiotherapyRadiation can break phosphodiester linkages of DNA and interferes with replication process.Asaresultgrowthofcancercellscancomedown.Basedonthisprincipleradiationisusedtotreattumours.3. SurgeryItisthetreatmentofchoiceintheadvancedstagesofcancer.Cancer(tumour)tissueisremovedbysurgery.Usuallysurgeryisperformedwithoperataingmicroscope.4. Photo chemotherapyItisanewlyintroducedtreatmentforcancer.Itusesaphotosensitivedrugandlaserlighttodestroycancercells.5. Suicide Gene Therapy (Molecular surgery)Itisakindofgenetherapyusedinthetreatmentofsolidtumourswheretherapeuticgeneistargetedattumourcellskillingcellswhichexpressingit.Itisalsoknownasmolecularsurgery.Thesuicidegenesareenzymeswhichactivateslowtoxicprodrugtotoxicpotentdrug.Herpessimplexthymidinekinase(HSTK)andcytosinedeaminase(CD)aretwosuchenzymes. HSTK converts non-toxic anti-viral drug ganciclovir to toxic form by phosphorylation.496 MedicalBiochemistryCDconvertsnon-toxicfluorocytosineintotoxicfluorouracil.Vectorscarryinggenesoftheseenzymesareinjecteddirectlyintotumour.Itisfollowedbyintratumouralinjectionofprodrug.TumourMarkersCancer(tumourormalignant)cellsproduceabnormalsubstances.Usuallythesesubstancesare not produced by normal cells. The abnormal substances produced by the cancer cells areenzymes,hormonesandproteins.Thesesubstancesarereleasedintobloodbycancercells.Asaresulttheirlevelinbloodrises.Measurementofthesesubstancesinbloodorserumprovides useful information about cancer. Hence, they are called as tumour markers, Nowa-daysmeasurementoftumourmarkersinbloodisanintegralpartofoncology.Tumourmarkermeasurementisusedin(a) Detectionofcancer.(b) Diagnosisofcancer.(c) Prognosisofcancer.(d) Determinationofcancerstage.(e) Determinationoflocationofcancerinthebody.(f) Determinationoforganinvolvedincancer.(g) Cancertherapy.Someclinicallyimportanttumourmarkersare1. -Fetoprotein(AFP).Itisaplasmaproteinandusuallyabsentinnormalpeopleplasma.Itistumourmarkerforlivercancerandgermcellcancer.2. Calcitonin.Itisahormone.Itistumourmarkerforthyroidcancer.3. Carcinoembryonicantigen(CEA).Itisaproteinanditistumourmarkerforlungcancer,breastcancer,coloncancerandpancreascancer.4. Humanchorionicgonodotropin(HCG).Itistropichormone.Itistumourmarkerforgermcellcancerandtrophoblastcancer.5. Acidphosphatase.Itistumourmarkerforprostatecancer.6. Highmobilitygroupchromosomalproteins(HMGCP).Theyarefamilyofnon-histonechromosomalproteinsthatserveasarchitecturalelementsinchromatin.Innormaltissuestheseproteinsareexpressedatverylowlevels.Theirleveliselevatedinmanyhumancancers.Thissmallmolecularweightproteins'expressionisincreasedinneoplastictransformationofcellsandmetastaticoftumourprogression.Theycanserveasnoveldiagnostictumourmarkers.Disadvantagesoftumourmarkers1. Thesetumourmarkersusuallydetectcanceratadvancedstage.Sotheyareoflittlehelpinsavinglives.2. Agivenmarkerisusefulinthedetectionofonlyonetypeofcancer.3. Sometimesmeasurementofmorethanonetypeoftumourmarkermaybehelpfulorrequired.CancerandAIDS 497AIDSItistheabbreviatedformofAcquiredImmunoDeficiencySyndrome.Itisanacquireddisease. It is an infectious disease. In this disease body immune or defense system weakens.Itisnamedassyndromebecausefullblowndiseasemakesupmanydiseases.AIDSiscausedbyaviruscalledasHumanImmunoDeficiencyVirustype-I(HIV-I).Itisaretrovirus,ItconsistsofRNAwhichissurroundedbytwotypesofproteins.TheRNAcoreisenvelopedinmembranelipidbilayercontainingglycoproteins(Figure21.2).WhenHIVinfectshumansitinfectsT-cellsoflymphocyteswhichformanimportantpartofimmunesystem.Thelymphocytes(T-cells)fightdiseasesbykillingdiseasecausingagents.ThecellsurfaceofT-cellscontainsaglycoproteinreceptorknownasCD-4receptor.TheT-cellsarealsocalledasCD-4cellsbecauseofthis.AIDSvirusattacksCD-4cellsandkillsthem.SowhenapersonisinfectedwithHIVforprolongedperiodhisCD-4cellcountdecreasesandheissusceptibletoinfections.Fig.21.2StructureofAIDSvirusHIVLifecycle1. HIVgeneticmaterialissinglestrandedRNA.2. WhenHIVentersintobody,itgetsattachedtoT-cellthroughCD-4receptor.3. Then HIV internalizes in the cell after fusing with membrane of CD-4 cell. Its contentsarereleasedintotheCD-4cell.4. ThegeneticmaterialofHIVistransformedintoDNAbyreversetranscriptase.5. TheHIVDNAisintegratedintohostDNA.6. ExpressionofHIVRNAandtranslationofRNAproducesproproteinsintheCD-4cell.7. Proproteinsareprocessedbyprotease(HIV)toperfectproteinsofHIV.8. AssemblingofRNAandHIVproteinsintonewHIVparticles.9. NewlyformedHIVcomesoutofCD-4cellbykillingitorwhenCD-4celldies.VariouseventsinvolvedinHIVlifecycleareshowninFigure21.3.498 MedicalBiochemistryFig.21.3HIVLifecycleSymptoms of AIDSIn the early phase HIV infected people develop flu associated symptoms like fever, headache,swollen lymph glands, stomach ache and swollen joints. These initial symptoms subside afterfew days and infected people remain normal for a period ranging from 6 months to 10 yearsorevenlonger.DuringthisperiodHIVmultipliesinthebodyandkillsmanyT-cells.AsaresultCD-4cellscountdecrease.InnormalpeopletheT-cellcountisbetween500to1500/mlofblood.AstheT-cell(CD-4)countdecreasesinthebloodHIVinfectionsymptomslikenightsweats,diarrhoeaandfeversurfacesandremainforfewdaystofewweeks.HIVinfectionbecomesAIDSwhenT-cellcountgoesdownto200/mlofblood.Atthisstage,HIVinfectedpeoplecontactopportunisticinfectionsliketuberculosis,pneumonia,weightloss,tumoursandfungalinfections.Recoveryfromtheseconditionsisslowandrequiresextensivetreatment.LaboratoryDiagnosisWhenapersonisinfectedwithHIVantibodiestoHIVareproducedinthebodylikeinanyotherinfection.SothepresenceofHIVantibodiesinthebloodindicatesinfection.Mostofthe AIDS detection tests are based on identification of HIV antibodies in the blood. Enzymelinkedimmunosorptiveassay(ELISA)andwesternblottechniqueareusedtodetectHIVantibodiesinblood.SomeAIDSdetectiontestsarebasedongeneticmaterialofHIV.AIDSTherapyCurrentlyAIDStreatmentinvolvesuseoftwoclassesofdrugs.Theyare(a) Inhibitors of reverse transcriptaseSince reverse transcriptase is important for replication of HIV blocking reverse transcriptaseactioncancontrolHIVproliferation.SomeofthereversetranscriptaseinhibitorsusedasdrugsareAZT,ddI,ddc,d4Tand3TC.CancerandAIDS 499(b) Protease inhibitorsSinceHIVproteaseisessentialforprocessingofproteins,blockingofthisenzymealsocanarrestHIVproliferation.SomeofHIVproteaseinhibitorsusedasdrugareindinavir,saquinavir,ritonavirandnelfinavir.HIV AND CANCERInIndia,HIV-1ismostlyresponsibleforAIDS.HIV-2iscommoninWestAfricaandfoundinIndiaalso.BothHIV-1andHIV-2arenotdirectlyoncogenic.However,KaposissarcomaofAIDSpatientsislargelyanattributeofHIV.Kaposi'ssarcoma(KS)isararetumourfoundonlyinmenoversixtyyearsincertainEastern European and Mediterranean population. However, risk of KS in HIV infected adultmalehomosexualslessthansixtyyearsoldissometenthousandfoldhigherthanthatoftheir counterparts in general population. With AIDS, endemic KS has become most commonofalltumoursinSubSaharanAfrica.TheepidemologyofKSbeforeandafterAIDSsug-gestedatransmissibleagentmayunderlietumour.Humanherpesvirus-8(HHV-8)orKS-associatedherpesvirus(KSHV)isdiscoveredasresponsibleforKSinhumans.KSHVDNAisfoundinKSbiopsiesaloneandinHIVpositivepatients.BothKSHVandHIVinfectionsare independent and highly risk factors in the development of KS in AIDS patients. The riskofKSinKSHVpositivepatients'sincreaseswithdecreasingCD-4TlymphocytesasoccursinAIDS.ItisbelievedthatTatproteinofHIV-1hasroleinKSpathogenesis.Itactssynergisticallywithcellulargrowthfactors.However,KScommonlyoccursinKSHVpa-tients with HIV-1 than with HIV-2 infection. In HIV positive patient KSHV is also associatedwithlymphoproliferativedisease.Burkittslymphoma(BL)andNon-Hodgkinlymphoma(NHL)aretwolymphomasfre-quentlyseeninAIDScases.NHLinAIDSoccursinbrainandBLingut.IncidenceofanycancerincreasesinAIDSpatientsduetoimmunesuppression.TheimmunesuppressioninducedbyHIVacceleratesprogressionofmalignancy.Livercancer,skincancer,testicularcancer and treatocarcinoma are more in AIDS patients. Hence, cancers associated with AIDSareprobablyopportunisticneoplasmslikeopportunisticinfections.REFERENCES1. Weinberg,R.A.Amolecularbasisofcancer.Sci.Am.249(5),126-142,1983.2. Boyle,P.Nutritionalfactorsandcancer.InHumanNutritionandDietetics.Garrow,J.S.andJames,W.P.T.(Eds.)9thed.ChurchillLivingstone,Edinburgh,1993.3. Lavecchia, C. Bidoli, E. and Barra, L. Types of Cigarettes and cancer of upper digestiveandrespiratorytract.Cancercausescontrol.1,69-74,1990.4. Dwyer,M.J.BiomedicalaspectofHIVandAIDS.Curr.Sci.69(10),823-827,1995.5. Wlodawer,A.andEricikson,J.W.StructurebasedinhibitorsofHIV-1proteases.Ann.Rev.Biochem.62,543-585,1993.6. Roberts, N.A. Drug resistance patterns of saquinavir and other HIV protease inhibitors.AIDS,9, 527-532, 1995.7. J.Cohen,HIV/AIDSinAsia,Science,June,2004.500 MedicalBiochemistry8. HIV/AIDSinIndia,Science,April2004.9. Ryan, K.M. and Vousden, K. Cancer: Pinning a change on P53. Nature 419, 795-797,2002.10. Mikail,V.Blagosklonny.CellimmortalityandHallmarksofcancer.Cellcycle.2,296-299, 2003.11. Kristi, G. Bache.et al. Defective down regulation of receptor tyrosine kinases in cancer.The EMBO Journal.23,2707-2714,2004.12. Donald,W.K.etal.(Eds.)6thed.Cancermedicine,BCDecker,2003.13. Veldwij,K.M.R.etal.SuicidegenetherapyofSarcomacelllinesusingrecombinantadenoassociatedvirusvectors.Cancergenetherapy.11,577-580,2004.14. Bandura,J.L.andCalvi,B.R.Duplicationofgenomeinnormalandcancercellcycle.CancerBiol.Ther.1,8-13,2002.15. Zink,D.etal.Nuclearstructureincancercells.Nat.Rev.Cancer.4,677-687,2004.16. Parkin,D.M.etal.Estimatingworldcancerburden.Int.J.,Cancer.4,153-156,2001.17. Mathew, A. Cancer registration with emphasis on Indian Scenario. In Basic informationfor cancer registry documentation (Ed. Mathew. A.), Regional Cancer Centre, Trivendrum,pp11-17, 2003.18. Davis,M.I.etal.Crystalstructureofprostatespecificantigenatumormarkerandpeptidase.Proc.Natl.Acad.Sci.USA.102,5881-5986,2005.EXERCISESSHORT QUESTIONS1. Defineoncogenes,protooncogenesandtumoursuppressorgenes.Giveexamplesforchemicalcarcinogens.Explainhowtheycausecancer.2. Whataretumourmarkers?Giveanexampleanditsclinicalimportance.3. ExpandHIV.WriteanoteonHIVlifecycle.4. WritesymptomsandtreatmentsavailableforAIDS.MEDICAL AND BIOLOGICAL IMPORTANCE1. Porphyrinsarepresentinbiologicalfluidslikeblood,bile,urineandfecesofanimalsandinvertebrates.Theyarealsofoundinplantsandbacteria.2. Porphyrinsarecomponentsofhemeproteinsofanimalsandinvertebrates.Hemeismetalloporphyrin.Itcontainsmetalironinthecentreofporphyrinring.Hencehemeproteinsarereferredasmetalloporphyrinoproteins.3. HemeproteinslikehemoglobinandmyoglobinareinvolvedinO2transportinanimalsandvertebrates.4. In invertebrates erythrocruorins which are also hemeproteins are responsible for the O2transport.5. Hemeproteinslikecytochromesandcytochromeoxidasearecomponentsofrespiratorychainandinvolvedinelectrontransport.6. CytochromeP450whichisinvolvedindetoxificationofdrugsisahemeprotein.7. Somehemeproteinsareinvolvedinmetabolism.Forexampletryptophandioxygenaseanenzymeoftryptophancatabolismisametelloprophyrinoproteinandcyclooxygenaseanenzymeofprostaglandinsynthesisisahemeprotein.8. HemeproteinslikecatalaseandperoxidaseareinvolvedintheremovalofH2O2.9. Inplantsporphyrinsarecomponentsofchlorophyllandphycobilins.10. Inbacteriaporphyrinsarecomponentsofcyanocobalamin.11. Agroupofinheriteddiseasesknownasporphyriasareduetoabnormalitiesinheme(porphyrin)biosynthesis.Leadpoisoningalsoblocksporphyrinbiosynthesis.12. Acommondiseasejaundiceisduetoexcessivecatabolismofporphyrinsorhemecon-tainingcompounds.Hepatitisandcancerofpancreasalsocancausejaundice.13. A group of inherited diseases known as hemoglobinopathies are due to abnormalities inproductionofhemoglobin.14. Carbonmonoxideapoisonousgaspresentinautomobileexhaustworksbycombiningwithhemoglobin.22CHAPTERPORPHYRIN ANDHAEMOGLOBIN METABOLISM501502 MedicalBiochemistry15. Photosensitivepropertyofporphyrinsisusedincancerphotochemotherapy.16. Bilirubinendproductofhemecatabolismactasantioxidant.17. Hemoglobinisasourceofproteinformalarialparasiteduringmalaria.Enzymesofhemoglobindegradationpathwayareexploitedfornewdrugdesign.PorphyrinsChemistryPorphyrinsarederivedfromaparentcompoundporphin.Porphinisatetrapyrroleanditisacycliccompound.Inporphin,4pyrrolesarelinkedthroughmethenyl(CH=)bridges.FourpyrroleringsofporphinareshownwithRomannumbersI,II,IIIandIV.MethenylbridgesareindicatedbyGreeknumbers,, and.SubstituentpositionsofI,II,IIIandIVringsareindicatedwithIndo-Arabicnumbers1,2,3,4,5,6and7,8respectively(Fig.22.1).Theeightnumberedsubstituentpositionscorrespondstoeighthydrogenatomsofpyrrolerings.ShorthandrepresentationofPorphyrinsNaturallyoccurringporphyrinscontainvarioussidechainsinplaceof8hydrogenatoms.Theydifferonlyinsidechainsattachedtofourpyrrolerings.HenceFischerproposedashorthandformforporphyrinsinwhichonlysubstitutionsareparticularlyshown.Inthisshort hand form as shown in Fig. 22.1 each pyrrole ring with numbered substituent positionsisshownasbracket.ThefourbracketsareindicatedwithRomannumbersandtheyarejoinedbyeliminatingthemethenylbridgestoformcrossshape.Fig.22.1Structuresofpyrrole,porphinandporphyrinPorphyrinandHaemoglobinMetabolism 503PorphyrinsIsomersPorphyrinsareintermediatesofhemebiosynthesis.Eachporphyrincanexistinmanyisomericformswhichdependsonkindsofsidechainsandarrangementofsidechains.Forexampleaporphyrinlikeuroporphyrinwithtwotypeofsidechainsacetate(A),propionate(P) can exist in four isomeric forms or four types. They are type I, type II, type III and typeIV.InuroporphyrinIsidechainsarearrangedsymmetrically.InothertypessidechainsA,Parearrangedasymmetrically(Fig.22.2).InnatureIandIIItypeporphyrinsaremore.HowevertypeIIIispredominantandmoreimportantforhemebiosynthesis.Protoporphyrinwiththreetypesofsidechaincanexistinfifteenisomericforms.Inprotoporphyrins two pyrrole rings contain methyl (M) and propionate (P) side chains. Othertwo pyrrole rings contain methyl (M) and Vinyl (V) side chains. Heme of hemoglobin containsprotoporphyrinIX(Fig.22.2).Fig.22.2(a)Shorthandformsofuroporphyrinandprotoporphyrin.(b)ConversionofuroporphyrinogenIIItouroporphyrinIII.PropertiesofPorphyrins1. Solubility.Ifaprophyrinhaspolarsidechainsthenitismoresolubleinaqueous504 MedicalBiochemistryenvironmentofblood.Ifaporphyrinhasnonpolarsidechainsthenitislesssolubleinblood.2. Lightabsorption.Allporphyrinsabsorblightmaximallyat400nm.Porphyrinsalsocanabsorblightinthevisibleregion.Whenporphyrincombineswithmetalitsabsorp-tionchanges.ForexampleprotoporphyrinIXabsorbslightat645nmwhereashemewhichisthecombinationofprotoporphyrinIXandironabsorblightat545nm.Lightabsorptionpropertiesofporphyrinsareusedfortheiridentification.3. Fluoroescence.Porphyrinsshowfluoroescenceinorganicsolventswhentheyareex-posedtoUV-light.Thispropertyisusedtodetectporphyrinsinbiologicalfluids.4. Allporphyrinsarecoloredcompounds.MedicalImportance1. Photosensitivepropertyofporphyrinsisusedincancerphotochemotherapy.Itisacom-binationtherapyandinvolvesuseoflightsensitiveporphyrincontainingdrugandlaserlight.Tumorcellconcentratemoreofporphyrincontainingdrugthannormalcells.Onexposuretolaserlightporphyrinsintumorcellsgetexcitedanddestroystumorcells.2. Photosensitivitywhichisoneofclinicalsymptomseeninsomeporphyriasisduetolightabsorptionpropertyofporphyrins.Porphyrinswhichaccumulateinareassur-rounding skin get excited on exposure to sunlight. They react with O2 and may generatefreeradicalsofoxygenwhichdamageslysosomesandothercellstructures.Thedegradativeenzymesreleasedfromdamagedlysosomesmaycauseskindamage.3. Sinceporphyrinsarecoloredmoleculestheiraccumulationintissuesandexcretioninurineasseeninsomeporphyriasgivesrisetocharacteristiccolortourineandtissues.PorphyrinogensTheyareformedduringhemebiosynthesis.Inporphyrinogensthepyrroleringsarelinkedthroughmethylene(CH2)bridgesandallthenitrogensofpyrroleringsarehydrogen-ated. Hence they contain six extra hydrogen atoms than corresponding porphyrins. They canbe auto oxidized to their corresponding porphyrins (Fig. 22.2). Moreover porphyrinogens arecolorlesswhereasporphyrinsarecoloredcompounds.HemebiosynthesisHemeissynthesizedbymostofthecellsexceptmatureerythrocytes.Howeverbonemarrowandliverarechieforgansinvolvedinhemeproduction.Bonemarrowproducesabout80%andrestisproducedbyliver.ReactionSequenceInhemesynthesisfirstandlastthreereactionsoccursinmitochondriaandintermediatereactionstakesplaceincytosol.Aminoacidglycineandsuccinyl-CoAofcitricacidcyclearestartingmaterialsforthehemeformation(Fig.22.3).1. Formationof-aminolevulinicacid(ALA)fromsuccinyl-CoAandglycineisthefirstreactionofhemebiosynthesisandinvolvesCCbondformation.ThereactioniscatalyzedbyaminolevulinicacidsynthaseorALAsynthasewhichtakesplaceintwosteps. The first step of reaction is mediated by pyridoxal phosphate the prosthetic groupof ALA synthase. It forms a Schiff base with glycine which then condenses with succinyl-PorphyrinandHaemoglobinMetabolism 505Fig. 22.3 Reaction sequence of Heme biosynthesis. Arrow mark indicates newly formed CCand CN linkages.HCNH2H COOH +COOHCH2CH2C=OSCoAGlycineSuccinyl-CoACoASHP-PO41COOHCH2CH2C=OHCNH2amino--ketoadipic acid- COOHCO21COOHCH2CH2C=OCH2NH2aminolevulinic acid(ALA)-COOHCH2CH2O=CHNCH22 22HO2APAPCH2NH2Porphobilinogen(PBG)ENH4N H3 aCH2EN HCH2NH2APN HPBGNH43 b APAPCH2EN HCH2N HDipyrryl EnzymeAPN HPBGCH2NH2NH43 cAPN HAPN HAPN HAPN H PBGAPN HAPN HAPN HAPN HCH2ECH2CH2CH2NH2CH2CH2CH2CH2E3 dNH4Methylbilane enzyme3 eHydroxy methyl bilaneTripyrryl EnzymeALAAdductEHO2

PM

I CHz CHz "I Mr{?MCHM MrQ=rMCH,M PCH:::: P PMPM4C02 COOH < " A J ~ ~ . A/ " V , " '-"". --C_ Coo7 --" , < " " " .. .....,..-.""""- QO