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 TTo m o m o m o m o my yy yyElder daughter Elder daughter Elder daughter Elder daughter Elder daughterLate Nallur Late Nallur Late Nallur Late Nallur Late Nalluri Kiranmayi Chowdary i Kiranmayi Chowdary i Kiranmayi Chowdary i Kiranmayi Chowdary i Kiranmayi ChowdaryTHIS PAGE ISBLANK(vii)PREFACE TO THE SECOND EDITIONI attempted to provide essential information on molecular basis of health and diseasethat 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 coupleof years these frontier areas are advancing rapidly which is viewed by many as good signfor development of :(a) new therapy or therapeutics for cancer(b) immortalized cells.The latter fuels growth of biotechnology and pharmaceutical industries also. Hence, inthe second edition two chapters1. Biochemistry of cell cycle (cell birth) 2. Biochemistry ofapoptosis (cell death) are added.As living organisms evolved from simple unicellular to highly complex multicellularmammals, several new systems and organs were developed. For example, blood which actsas vehicle or communication between various locations of body, immune system whichprotects body from intruders or foreign organisms.Parkinsonism, psychosis, depression, Schizophrenia, loss of taste and olfaction are dueto disturbances in nervous, taste and olfactory systems. Various organs present in bodyperform several organ specific functions which are essential for life. If functions of theseorgans are disturbed, diseases in which may culminate in death. So, in this edition biochemistryof blood including immune response in Chapter-32; molecular and cellular mechanism oflearning, memory, behaviour, taste and olfactory in biochemical communications Chapter;tests, procedures that are done in hospital biochemistry laboratory to assess functions ofliver, kidney in Chapter-33 and thyroid in Chapter-29 are detailed.Depending on disease a particular constituent of blood is either elevated or lowered.Diagnosis and prognosis of disease usually involves detection and measurement of variousblood constituents in hospital biochemistry laboratory. Therefore advanced techniques likehigh performance liquid chromatography (HPLC), affinity chromatography, and generaltechniques like centrifugation and dialysis, instruments from spectrophotometer to autoanalyzer and methods used for detection and quantitation of blood constituents likecarbohydrates, proteins, lipids, nucleic acids, enzymes, electrolytes etc., in health and diseaseare detailed in Chapter-34.Humans and other mammals are able to remove waste products, toxins, foreigncompounds from blood and organs in the form of urine. In disease, composition of urinevaries 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, methods for detection and estimation of urine constituent under normal and diseasedconditions are also detailed.Most striking feature in this edition of the book is inclusion of biochemical aspects ofdiseases or disease causing organisms common to tropical countries like malaria, tuberculosis,peptic ulcer or gastritis, pneumonia, leishmaniasis, giardiasis, trypanosomiasis etc. Sincemost of the organisms are developing resistance to the existing drugs, there is need fordevelopment of new drugs which requires thorough biochemical knowledge of these diseasesas well as disease causing organisms. Apart from adding new chapters, all existing chaptershave been updated by adding new subject matter. References of each chapter updated byincluding reviews, books, research articles etc. Further, number of unsolved problems havebeen increased in most of the chapters.I hope this edition will be well received by teachers and students of various medical,dental, pharmacy, biotechnology, physiotherapy, medical laboratory technology, biomedicalengineering, life sciences under graduate and post graduate courses, Suggestions or commentsfrom teachers and students are welcome. I am grateful to Sri. R.K. Gupta, Chairman; SriSaumya Gupta, Managing Director of New Age International, New Delhi, for publishingsecond edition.N. MALLIKARJUNA RAO(viii)PREFACE TO THE FIRST EDITIONThis book explains the fundamentals of biochemical (molecular) bases of health anddisease. Hence it meets medical and allied health sciences students needs. As a teacher ofmedical, dental, pharmacy, biomedical engineering and science students for the last twodecades, I know the problems faced by students in mastering (conceptualizing) the subjectwithin a limited time. Most of these students need a book for their routine day-to-day studywhich contains only the necessary information in a simple and concise way. Therefore, thisbook is written in simple language in such a way that a student with very little chemistryor biology background can easily follow the various aspects of biochemistry that are presented.This book is also useful for those who are specializing in biochemistry (M.Sc. or M.D.)because advances in frontier areas of biochemistry are presented in a systemic way. Ofcourse advances in other areas that are relevant to medical students are also included toa limited extent. An interesting feature of this book is that the medical and biologicalimportance of each chapter is highlighted in simple numbered statements. Further, in somechapters, diseases, drugs (treatments) or toxins of particular subject matter are describedunder medical importance heads. Further, each chapters text is designed to facilitate easyflow of information in an interesting, thought provoking and logical manner. Exercises(cases) given at the end of each chapter help in mastering of the subject by student andutilization of biochemical principles by the student in solving health problems. To enthusiasticstudents, references given at the end of each chapter provide additional information.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, structureand functions of biomolecules like amino acids, peptides, proteins, enzymes, carbohydratesand lipids. This is then followed by chapters 7 and 8 that deal with membrane structure,various transporters that move biomolecules across membrane and disintegration of complexmolecules of food and absorption of resulting products, respectively.Chapters 9-12 deal with the production and utilization of energy in various pathways ofcarbohydrate, lipid and aminoacid metabolisms. Regulatory mechanisms of some of theimportant pathways are also outlined. Further synthesis of biologically (medically) importantcompounds including 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 describedin chapter 13.Fundamentals of molecular biology i.e., occurrence, chemistry, structure, functions,metabolism of nucleotides, nucleic acids and control of gene expression as well as appliedmolecular biology i.e., recombinant DNA technology are detailed in chapters 14-20. Biomedical(ix)(chemical) aspects of two major health problems of the 20th centurycancer and AIDS arebriefed in chapter 21. In chapter 22 occurrence, chemistry, structure, functions and metabolismof porphyrins and hemoglobin are described.Clinically related topics like vitamins, minerals, macro nutrients, energy, nutraceuticalsof food, electrolytes, acid-base balance and detoxification are described in chapters 23-27.Chemistry, production, detection and uses of isotopes in biochemistry and medicine aredetailed in chapter 28. Chapter 29 deals with mechanisms of communication between cells.I hope both teachers and students of Biochemistry at undergraduate and postgraduatelevels use this book extensively and their suggestions to improve the book further are mostwelcome. I express my sincere thanks to New Age International, Publishers for publishingthe book.N. MALLIKARJUNA RAO(x)CONTENTS I'"fffff to 1M St.md &Ii/ion >ii) I'"ff{oct to flu Fi,.., Edition (u) .. Coli , ,. AminoAcids and Peptides " 3. Proteins ,. ElUymes alogy ... ". Cancer and Aids .90 22. Porphyrin and HaemOjlobi n Metabolism "" 23. Vitamirul '" ". Minerala ,n (:ai) " Energy, Nutrients. Medie;nft and 'Ibxins of Food .. 26. Wate_r. Electrolytes and Acid Bue Balanoo 629 " Dernxification ofXenobiotiCI ,' ...... _-... _Or" ' --- j-" ........ ... _ .... - "" Too .'- -y -.... .. .. _--- , 486 Medical Biochemistry11. PCR and DNA polymorphism Now several PCR based assays are developed to detectDNA variations or DNA polymorphism. Some examples are(a) Randomly amplified polymorphic DNA (RAPD)(b) Amplified fragment length polymorphism (AFLP)(c) Sequence related amplified polymorphism (SRAP) etc.RAPDThe generation of RAPDs involves use of single short random oligoneucliotides. When theserandom primers are mixed with sample DNA and subjected to PCR amplification of severalfragments occurs. The DNA amplification with random primers expose polymorphismsdistributed through out the genome. RAPD is also used in genome mapping and genetagging.AFLPThis PCR-based technique permits inspection of polymorphism at large number of loci within short period of time and requires very small amount of DNA. AFLP is potentially usedin genome finger printing and mapping.Restriction fragment length polymorphism (RFLP)It is another technique based on hybridization principle. DNA is a polymorphic molecule, i.e.,exist in several forms. DNA of an individual varies from others. Sequence of DNA of anindividual is unique. Further, mutations in DNA generates polymorphic DNA in sameindividual, which occurs in diseases. So, DNA polymorphism is due to variations in sequence.When DNA of an individual is subjected to digestion with restriction enzyme fragments ofvarying sizes or lengths that are unique to inviduals sequence or cell are produced.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 repeatedin tandem. The number of these repeats are unique to individual. When DNA of twoindividuals is subjected to digestion with restriction enzymes fragments that vary in lengthand number are generated. Therefore, RFLP of two individuals results from the differencesin the location and number of cleavage sites. Differences in DNA of two individual may bedue to evolutionary changes.RFLP is similar to southern blotting in many aspects. Initial step of RFLP involvesdigestion of more DNA samples with restriction enzymes where as in southern blotting onlyone DNA sample is digested. Rest of the steps of RFLP are those of southern blotting.Hence, in RFLP next step probes are used for hybridization. Probes hybridizes with fragmentcontaining complementary sequences. Then polymorphisms are detected by presence orabsence of bands after hybridization.Applications1. RFLP is used as a diagnostic test of inherited disease. For example, HbS: In HbS genethere is loss of one restriction site for restriction enzyme due to mutation where asnormal HbA gene has two cleavage sites. So, RFLP of sickle-cell anemia patient showstwo bands where as in RFLP of normal individual three bands appear.2. RFLP is also used to identify chromosomal difference.Recombinant DNA Technology 4873. RFLP is used for isolation and sequencing of closely related genes.4. RFLP is combination with PCR is used to detect DNA variations.Bioinformatics1. It is the combination of IT (Information Technology) and Life Sciences like Biochemistry,Molecular Biology, Biotechnology etc.2. It is defined as application of information technology and science for organisationmanagement, mining and use of life sciences.3. Main application areas of bioinformatics are genomics, proteomics, pharmacogenomics,chemiinformatics etc.4. One of the earliest application are a of bioinformatics is in drug design process.Bioinformatics revolutionized traditional approach of drug discovery from target discoveryand screening to discovery and development of therapeutic agents whose role in preventionof cure of a disease is well validated. Further, drugs so designed have less failures.5. Following steps of bioinformatics based method of designing drug that is an enzymeinhibitor.(a) Selection of chemical fragments from molecular library.(b) Assembly of chemical fragments in a piece-wise manner into possible inhibitormolecule.(c) Using docking algorithm all the possible inhibitor molecules are screened to selecthighly potent inhibitor which precisely fits in the binding cavity of enzyme.6. Knowledge of genome sequence allows structure activity based drug designing. Followingare steps of drug designing process, which involves genome sequence knowledge.(a) Determination of protein sequence using DNA sequence.(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 identifiedas a drug.REFERENCES1. Wu, K., Grossman, L. and Moldave, K. (Eds.). Recombinant DNA methodology. AcademicPress, New York, 1989.2. Berger, S.L. and Kimmal, A.R. Methods in Enzymology, Vol. 152, Academic Press,California, 1987.3. Kantoff, P.W. Prospects for gene therapy for immuno deficiency disease. Ann. Rev.Immunol, 6, 5894, 1988.4. Agarwal, S. and Jang, J. GEM 91. An anti-sense oligo nucleotide phosphorothioate asa therapeutic agent for AIDS. Anti-sense Res. Dev. 2, 261266, 1992.5. Rangarajan, P.N. and Padmanabhan, G. Gene therapy: principles, practice, problemsand prospects. Curr. Sci. 71 (5), 360367, 1996.6. Marx, J.L. DNA finger printing takes witness stand. Science 240, 16161618, 1988.488 Medical Biochemistry7. Mullus, K.B. The unusual origin of polymerase chain reaction. Sci. Am. P. 56, April1990.8. Meada, M.H. Dairy Gene. The Sciences. New York Academy of Sciences, New York, P.21, October 1997.9. 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.). The PCR technique. RT-PCR. Eaton Publishing, MA, USA, 1998.12. Mcpherson, M-J and Moller, S.G. PCR, Springer-Verlag Talos, 2000.13. Dieffenbach, Carl. W. and Gabriel, S.D. PCR primer: A Laboratory manual. Cold SpringHarbor Laboratory Press, NY, 2003.14. K. Shah et al. Molecular imaging of gene therapy for cancer. Gene Therapy. 11, 11751187, 2004.15. Li, G. and Quiors. Sequence related amplified polymorphism (SRAP) a new markersystem based on simple PCR reaction: its application to mapping and gene tagging inBrassica. 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. et al. Neural stem cells from adult hippo campus develop essential propertiesof functional CNS neurons. Nature 417, 3944, 2002.18. Gage, F.H. Mammalian neural stem cells. Science. 287, 14331438, 2000.19. Lesk, A.M. (Ed.). Introduction to Bioinformatics. Oxford University Press, New York, 2002.20. Isner, J.M. Myocardial gene therapy. Nature. 415, 234239, 2002.21. Austin, C.P. et al. The knockout mouse project. Nature Genetics. 36, 921924, 2004.22. Kubota, C. et al. Serial bull cloning by somatic cell nuclear transfer. NatureBiotechnology. 22, 693694, 2004.23. Susan M. Rhind et al. Human cloning: can it be made safe. Nat. Rev. Geneti. 4, 855864, 2003.24. Rebeca J. Morris et al. Capturing and profiling adult hair follicle stem cells. NatureBiotechnology. 22, 411417, 2004.25. Arekawa, T. et al. Efficacy of food plant based oral cholera toxin B sub unit vaccine.Nature. Biotechnol. 16, 292297, 1998.26. Karatzas, C.N. Designer milk from transgenic clones. Nature. Biotechnol. 21, 138139, 2003.27. Nishimura, E. K. et al. Mechanism of hair graying: Incomplete melanocyte stem cellmaintenance in the Niche. Science. 307, 720-724, 2005.EXERCISESESSAY QUESTIONS1. Define recombinant DNA. Explain steps of recombinant DNA technology used for production ofhuman gene products in a biotechnology company.Recombinant DNA Technology 4892. Give an account of gene therapy.3. Describe production and application of hybridomas.4. Describe hybridization techniques.5. Describe polymerase chain reaction (PCR).Short questions1. Write steps involved in production of transgenic animals.2. Define cloning. Write steps necessary for production of cloned sheep.3. Write a note on DNA vaccines.4. Define edible vaccines. Write steps of edible vaccination.5. Write a biosensor working principle. Name components of a biosensor and write their applications.6. Explain western blot technique. Write its significance.7. Write recombinant DNA technology applications.Fill in the blanks1. Recombinant DNA technology may lead to creation of ................. species.2. cDNA probe is ................. molecule prepared from 32P labelled nucleoside triphosphates.3. Cloning is a ................. reproduction of mammals.4. ................. cells are immortal.5. ACAAACT is repetitive sequence of fruit fly ................. .490 Medical BiochemistryMEDICAL 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 andstomach have been identified.2. Rate of incidence of cancer of particular organ in particular population depends onseveral factors like age, sex, dietary habits, environment, geographical location, geneticmake up, culture, physical exercise etc. For example in India oral cancer is common inbetal nut chewing regions and in reverse smokers. Stomach cancer is more in Japaneseand Chinese people. Colon cancer is common in advanced countries and lung cancer iscommon in smokers. Old people are more prone to any type of cancer. Brain cancer andblood cancer are common in children. Men above 50 are prone to prostate cancer.Women above 45 are prone to breast, ovarian and cervical cancers.3. Rate of incidence of cancer of particular organ varies from developed countries todeveloping countries. (Table 21.1). Lung and colorectal cancers are high in developedcountries while stomach and cervical cancer are more in developing countries. Furtherin India pharyngeal cancers are high in Western India where as stomach cancers aremore common in Southern India.Nearly 10 million new cases of cancer are diagnosed globally every year. It is estimatedthat by 2020 ten million persons would die of cancer every year World wide.Table 21.1 Cancer incidence rate in developed and developing CountriesCancer site Developed countries Developing countriesLung 62 24Colon and rectum 20-45 2-8Stomach 10 60Cervix 14 30Prostate 30 10Mouth and pharynx 13 2521CHAPTERCANCER AND AIDS490Cancer and AIDS 4914. Nuclear architecture is altered in cancer cells. New anticancer drugs that revert thesechanges may be developed. Like wise new tumour marker based on nuclear structuralchanges may be used in cancer diagnosis.5. Extensive research carried out for the last two decades on various types of cancer ledto development of proper treatment for at least some types of cancers. However, itgreatly expanded our knowledge on molecular mechanism of cancer.6. AIDS is another major health problem that surfaced around second half 20th century.According to WHO (World Health Organization) estimation about 20 million people areaffected by AIDS. At least about 5-10000 people get infected for every 24 hours. Spreadof this infectious disease also depends on several factors. In developing countries it isspreading faster due to prevalent socioeconomic conditions.7. Though the various facets of cancer and AIDS are being probed thoroughly for the lasttwo decades proper cure is not in sight particularly for AIDS.CANCERGrowth of all types of cells is controlled in the body. If the growth of cell is not controlledthey continue to proliferate which leads to malignancy. So cancer is malignant growth (uncontrolled growth) of cells. Malignant growth of cell is also called as tumour. Cancer of aparticular organ or tissue develops when the cells of that organ have lost growth control.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 organif not detected can spread to other organs (Figure 21.1).Fig. 21.1 Cancer development from normal cell.Nomenclature and classification of CancersGenerally cancers are named according to the organ affected. However they are classifiedbased on the three embryonic germ layers from which tissue or organ is derived.1. CarcinomasAre the cancer of cells or organs derived from either ectoderm or endoderm. Cancers ofepithelial tissues, nervous tissues, glands etc. are named as carcinomas.492 Medical BiochemistryExample:(a) Adenocarcinoma: Cancer of gland.(b) Squamous cell carcinoma: Cancer of squamous cells of epithelial tissues.(c) Gliomas: Cancer of brain nervous tissues.2. SarcomasAre cancers of tissues of mesodermal origin. Generally cancers of bone, cartilage, connectivetissue, muscle etc are called as sarcomas.Examples:(a) Osteosarcoma: Bone cancer.(b) Fibrosarcoma: Connective tissue cancer.Cancer is primarily due to DNA damage or damage of genes. DNA damage may result fromthe action of biological, chemical, physical and environmental agents on DNA. Incidence ofcancer also depends on the genetic make up of an individual.Cancer genesOncogenes. Are genes responsible for development of cancer.Proto oncogenes. They are precursors of oncogenes. They are converted to oncogenes byactivation.Tumour suppressor genes. They are present in normal healthy people. Products of themprevent cancer development.The product of oncogenes disturbs the normal cell growth control mechanism leading tocancer. Usually products of oncogenes are protein kinases that phosphorylate tyrosine residuesof proteins. (Tyrosine kinase.) Both cellular and viral oncogenes are found. Examples foroncogenes and protooncogenes are given below.1. Cellular oncogene that causes rat sarcoma is designated as c-ras oncogene. Likewisec-ras protooncogene.2. Viral oncogene that causes rat sarcoma is designated as v-ras oncogene. Likewisev-ras protooncogene.3. Oncogene of rouse sarcoma is designated as src-oncogene.4. Oncogene of simian sarcoma is designated sis-oncogene.5. Oncogene of chicken myelocytoma is designated myc-oncogene.CarcinogenesisBy several ways carcinogenesis occurs in humans and other animals. Usually they arenamed according causative agent or factor. Different types of carcinogenesis are given below:1. Biological agents that cause cancer or biological (viral) carcinogenesis. SomeDNA and RNA viruses are carcinogenic and hence they are called as oncogenic viruses.When normal cells are cultured with oncogenic viruses, the normal cells are trans-formed into cancer (tumour) cells. Oncogenes of the viruses are responsible for thedevelopment of cancer.Cancer and AIDS 493Examples1. Hepatitis B virus cause liver cancer in humans.2. Retro viruses also cause cancer in humans.2. Chemical carcinogens or mutagens or chemical carcinogenesis. Many chemicalsubstances cause mutations in DNA. They are called mutagens. Sometimes this muta-tion in DNA may convert normal cell to cancer cell. Then they are called as carcino-gens.Examples1. Cigarette smoke causes lung cancer in humans.2. Aflatoxins are carcinogens.3. Nitrosamine, Benzapyrins and asbestos also cause cancer.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. MutantDNA mediates carcinogenesis by activation of oncogenes which leads to development ofcancer of skin or multiple tumours of skin.Cancer due to genetic factors:Some genes in DNA are associated with development of cancer in susceptible individu-als. Examples:1. Retinoblastoma, cancer of eye develops in people carrying RBI gene.2. Wilms tumour, kidney cancer develops in children having gene WTI.Activation of protooncogene to oncogeneBy several ways activation of protooncogene to oncogene can occur. Some of them are givenbelow.(a) Point mutation. Point mutation converts protooncogene to oncogene. Human bladdercarcinoma is due to point mutation. Mutation may be due to error during replication.(b) Gene amplification. Amplification of oncogenes results in the formation of productsof these genes by several folds. This in turn converts normal cells to cancer cells.(c) Chromosomal translocation, promoter/enhancer insertion also leads to activation ofprotooncogene to oncogene.Mechanism of action of oncogenes or Mechanism of carcinogenesisThe product of oncogene converts normal cell to cancer cell by several ways.1. The product of c-ras oncogene is a less active GTPase. This leads to prolonged activationof adenylate cyclase and hence activities of cAMP dependent proteinkinase. As a resultcellular metabolism is altered and normal cell is transformed into cancer cell.2. Myc-oncogene product is DNA binding protein or transcription factor (TF). It regulatesexpression of cell cycle genes. As a result cell cycle is altered.3. Src-oncogene product is tyrosinekinase. It phosphorylates cyclins and cyclin dependentkinases of cell cycle. This results in cell cycle alteration.4. Some oncogene products are polypeptide growth factors that affect cell cycle andmitosis.494 Medical BiochemistryMechanism of virus mediated carcinogenesis1. Several human tumour viruses induce immortalization of human tissue cells. It isfollowed by malignant conversion which involves several steps.2. Human papilloma viruses (HPV), human T-lymphocyte virus (HTLV) possess definedoncogenes that stimulate proliferation of human cells.3. Human papilloma virus causes cervical cancer. Cancer of cervix is the number onecancer in Indian women. In India about 100,000 women develop this cancer every year.4. Human T-lymphocyte virus causes T-lymphocyte leukemia.5. The oncogenes of HPV are E6 and E7. They are able to immortalize keratinocytes. Theycontain all necessary information for immortalization.6. The E7 protein releases transcription factor which activates genes engaged in cell cycleprogression.7. The E6 protein binds P53 and abolishes its tumour suppressive and Trans activationalproperties. It also promotes ubiquitinisation of P53 and its subsequent proteolysis.8. Thus E6 and E7 are able to immortalize cells independently and both genes cooperateeffectively in immortalization of cells.Metabolism of carcinogenMetabolism of carcinogen after entering the body is mainly directed towards producingmetabolites which can be excreted. The enzyme systems of phase-I and phase-II reactionsof 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 theformation of a carcinogen.Mechanism of a carcinogen mediated carcinogenesis1. Carcinogenesis by a carcinogen involves several steps.2. First step is the induction of molecular lesion.3. Second step is the fixation of molecular lesion by DNA replication.4. The ultimate carcinogenic forms of carcinogens are highly reactive electrophiles whichare reactive towards DNA.5. They bind covalently with DNA to produce DNA adduct.6. This type of DNA modification is major driving force for cancer development.Nuclear structure of Cancer cells1. In cancer cells nuclear architecture is altered. These alterations are characteristics oftumour type.2. Components of nuclear matrix play a role in organization of chromosomes and nuclearcomponents. Protein composition of nuclear matrix is altered in cancer cells.3. Oncogenes induce tumor-specific nuclear changes and these in turn changes gene regu-lation.4. In cancer cells chromosomal territories and gene loci are changed.Cancer and AIDS 4955. Structural changes in tumour cells lead to changes in nucleoli and perinuclear compart-ment.6. These changes can be used as potential tumour markers and targets for anti-cancerdrugs.Treatment of cancerSeveral types of treatments are available for cancer management. Some are given below.Cancer gene therapy is explained in earlier chapter.1. ChemotherapyCompounds that block replication of cells and anti metabolites that block nucleotidebiosynthesis are used as anticancer agents or in chemotherapy of cancer.(a) Mercapto purine. It is a purine analog used in the treatment of luekaemia. It isconverted into nucleotide in 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.In vivo it is converted fluorodeoxy uridine phosphate and inhibits replication.(c) Methotrexate. It is a folic acid analog and used in the treatment of chorio carcinoma.(d) Azaserine. It is a glutamine analog used in cancer treatment. It blocks nucleic acidbiosynthesis (replication) by inhibiting glutamine dependent metabolic reactions.(e) Acivicin. Another glutamine analog used as anticancer agent. It is a competitive inhibi-tor of glutamine utilizing enzyme.Methotrexate, azaserine and acivicin are anti metabolites used in cancer treatment.They are called as anti metabolites because they block nucleic acid synthesis by anatgonizingmetabolic role of glutamine.2. RadiotherapyRadiation can break phosphodiester linkages of DNA and interferes with replication process.As a result growth of cancer cells can come down. Based on this principle radiation is usedto treat tumours.3. SurgeryIt is the treatment of choice in the advanced stages of cancer. Cancer (tumour) tissue isremoved by surgery. Usually surgery is performed with operataing microscope.4. Photo chemotherapyIt is a newly introduced treatment for cancer. It uses a photosensitive drug and laser lightto destroy cancer cells.5. Suicide Gene Therapy (Molecular surgery)It is a kind of gene therapy used in the treatment of solid tumours where therapeutic geneis targeted at tumour cells killing cells which expressing it. It is also known as molecularsurgery. The suicide genes are enzymes which activates low toxic prodrug to toxic potentdrug. Herpes simplex thymidine kinase (HSTK) and cytosine deaminase (CD) are two suchenzymes. HSTK converts non-toxic anti-viral drug ganciclovir to toxic form by phosphorylation.496 Medical BiochemistryCD converts non-toxic fluorocytosine into toxic fluorouracil. Vectors carrying genes of theseenzymes are injected directly into tumour. It is followed by intratumoural injection ofprodrug.Tumour MarkersCancer (tumour or malignant) cells produce abnormal substances. Usually these substancesare not produced by normal cells. The abnormal substances produced by the cancer cells areenzymes, hormones and proteins. These substances are released into blood by cancer cells.As a result their level in blood rises. Measurement of these substances in blood or serumprovides useful information about cancer. Hence, they are called as tumour markers, Nowa-days measurement of tumour markers in blood is an integral part of oncology. Tumourmarker measurement is used in(a) Detection of cancer.(b) Diagnosis of cancer.(c) Prognosis of cancer.(d) Determination of cancer stage.(e) Determination of location of cancer in the body.(f) Determination of organ involved in cancer.(g) Cancer therapy.Some clinically important tumour markers are1. -Feto protein(AFP). It is a plasma protein and usually absent in normal peopleplasma. It is tumour marker for liver cancer and germ cell cancer.2. Calcitonin. It is a hormone. It is tumour marker for thyroid cancer.3. Carcino embryonic antigen (CEA). It is a protein and it is tumour marker for lungcancer, breast cancer, colon cancer and pancreas cancer.4. Human chorionic gonodotropin (HCG). It is tropic hormone. It is tumour markerfor germ cell cancer and trophoblast cancer.5. Acid phosphatase. It is tumour marker for prostate cancer.6. High mobility group chromosomal proteins (HMGCP). They are family of non-histone chromosomal proteins that serve as architectural elements in chromatin . Innormal tissues these proteins are expressed at very low levels. Their level is elevatedin many human cancers. This small molecular weight proteins' expression is increasedin neoplastic transformation of cells and metastatic of tumour progression. They canserve as novel diagnostic tumour markers.Disadvantages of tumour markers1. These tumour markers usually detect cancer at advanced stage. So they are of littlehelp in saving lives.2. A given marker is useful in the detection of only one type of cancer.3. Sometimes measurement of more than one type of tumour marker may be helpful orrequired.Cancer and AIDS 497AIDSIt is the abbreviated form of Acquired Immuno Deficiency Syndrome. It is an acquireddisease. It is an infectious disease. In this disease body immune or defense system weakens.It is named as syndrome because full blown disease makes up many diseases.AIDS is caused by a virus called as Human Immuno Deficiency Virus type-I (HIV-I). Itis a retrovirus, It consists of RNA which is surrounded by two types of proteins. The RNAcore is enveloped in membrane lipid bilayer containing glycoproteins (Figure 21.2). WhenHIV infects humans it infects T-cells of lymphocytes which form an important part ofimmune system. The lymphocytes (T-cells) fight diseases by killing disease causing agents.The cell surface of T-cells contains a glycoprotein receptor known as CD-4 receptor. The T-cells are also called as CD-4 cells because of this. AIDS virus attacks CD-4 cells and killsthem. So when a person is infected with HIV for prolonged period his CD-4 cell countdecreases and he is susceptible to infections.Fig. 21.2 Structure of AIDS virusHIV Life cycle1. HIV genetic material is single stranded RNA.2. When HIV enters into body, it gets attached to T-cell through CD-4 receptor.3. Then HIV internalizes in the cell after fusing with membrane of CD-4 cell. Its contentsare released into the CD-4 cell.4. The genetic material of HIV is transformed into DNA by reverse transcriptase.5. The HIV DNA is integrated into host DNA.6. Expression of HIV RNA and translation of RNA produces proproteins in the CD-4 cell.7. Pro proteins are processed by protease (HIV) to perfect proteins of HIV.8. Assembling of RNA and HIV proteins into new HIV particles.9. Newly formed HIV comes out of CD-4 cell by killing it or when CD-4 cell dies.Various events involved in HIV life cycle are shown in Figure 21.3.498 Medical BiochemistryFig. 21.3 HIV Life cycleSymptoms 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 yearsor even longer. During this period HIV multiplies in the body and kills many T-cells. As aresult CD-4 cells count decrease. In normal people the T-cell count is between 500 to 1500/ml of blood. As the T-cell (CD-4) count decreases in the blood HIV infection symptoms likenight sweats, diarrhoea and fever surfaces and remain for few days to few weeks.HIV infection becomes AIDS when T-cell count goes down to 200/ml of blood. At this stage,HIV infected people contact opportunistic infections like tuberculosis, pneumonia, weightloss, tumours and fungal infections. Recovery from these conditions is slow and requiresextensive treatment.Laboratory DiagnosisWhen a person is infected with HIV antibodies to HIV are produced in the body like in anyother infection. So the presence of HIV antibodies in the blood indicates infection. Most ofthe AIDS detection tests are based on identification of HIV antibodies in the blood. Enzymelinked immunosorptive assay (ELISA) and western blot technique are used to detect HIVantibodies in blood. Some AIDS detection tests are based on genetic material of HIV.AIDS TherapyCurrently AIDS treatment involves use of two classes of drugs. They are(a) Inhibitors of reverse transcriptaseSince reverse transcriptase is important for replication of HIV blocking reverse transcriptaseaction can control HIV proliferation. Some of the reverse transcriptase inhibitors used asdrugs are AZT, ddI, ddc, d4T and 3TC.Cancer and AIDS 499(b) Protease inhibitorsSince HIV protease is essential for processing of proteins, blocking of this enzyme also canarrest HIV proliferation. Some of HIV protease inhibitors used as drug are indinavir,saquinavir, ritonavir and nelfinavir.HIV AND CANCERIn India, HIV-1 is mostly responsible for AIDS. HIV-2 is common in West Africa and foundin India also. Both HIV-1 and HIV-2 are not directly oncogenic. However, Kaposis sarcomaof AIDS patients is largely an attribute of HIV.Kaposi's sarcoma (KS) is a rare tumour found only in men over sixty years in certainEastern European and Mediterranean population. However, risk of KS in HIV infected adultmale homosexuals less than sixty years old is some ten thousand fold higher than that oftheir counterparts in general population. With AIDS, endemic KS has become most commonof all tumours in Sub Saharan Africa. The epidemology of KS before and after AIDS sug-gested a transmissible agent may underlie tumour. Human herpes virus-8 (HHV-8) or KS-associated herpes virus (KSHV) is discovered as responsible for KS in humans. KSHV DNAis found in KS biopsies alone and in HIV positive patients. Both KSHV and HIV infectionsare independent and highly risk factors in the development of KS in AIDS patients. The riskof KS in KSHV positive patients's increases with decreasing CD-4 T lymphocytes as occursin AIDS. It is believed that Tat protein of HIV-1 has role in KS pathogenesis. It actssynergistically with cellular growth factors. However, KS commonly occurs in KSHV pa-tients with HIV-1 than with HIV-2 infection. In HIV positive patient KSHV is also associatedwith lympho proliferative disease.Burkitts lymphoma (BL) and Non-Hodgkin lymphoma (NHL) are two lymphomas fre-quently seen in AIDS cases. NHL in AIDS occurs in brain and BL in gut. Incidence of anycancer increases in AIDS patients due to immune suppression. The immune suppressioninduced by HIV accelerates progression of malignancy. Liver cancer, skin cancer, testicularcancer and treatocarcinoma are more in AIDS patients. Hence, cancers associated with AIDSare probably opportunistic neoplasms like opportunistic infections.REFERENCES1. Weinberg, R.A. A molecular basis of cancer. Sci. Am. 249(5), 126-142, 1983.2. Boyle, P. Nutritional factors and cancer. In Human Nutrition and Dietetics. Garrow,J.S. and James, W.P.T. (Eds.) 9th ed. Churchill Livingstone, Edinburgh, 1993.3. Lavecchia, C. Bidoli, E. and Barra, L. Types of Cigarettes and cancer of upper digestiveand respiratory tract. Cancer causes control. 1, 69-74, 1990.4. Dwyer, M.J. Biomedical aspect of HIV and AIDS. Curr. Sci. 69(10), 823-827, 1995.5. Wlodawer, A. and Ericikson, J.W. Structure based inhibitors of HIV-1 proteases. 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/AIDS in Asia, Science, June, 2004.500 Medical Biochemistry8. HIV/AIDS in India, Science, April 2004.9. Ryan, K.M. and Vousden, K. Cancer: Pinning a change on P53. Nature 419, 795-797,2002.10. Mikail, V. Blagosklonny. Cell immortality and Hall marks of cancer. Cell cycle. 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. et al. (Eds.) 6th ed. Cancer medicine, BC Decker, 2003.13. Veldwij, K.M.R. et al. Suicide gene therapy of Sarcoma cell lines using recombinantadeno associated virus vectors. Cancer gene therapy. 11, 577-580, 2004.14. Bandura, J.L. and Calvi, B.R. Duplication of genome in normal and cancer cell cycle.Cancer Biol. Ther. 1, 8-13, 2002.15. Zink, D. et al. Nuclear structure in cancer cells. Nat. Rev. Cancer. 4, 677-687, 2004.16. Parkin, D.M. et al. Estimating world cancer burden. 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. et al. Crystal structure of prostate specific antigen a tumor marker andpeptidase. Proc. Natl. Acad. Sci. USA. 102, 5881-5986, 2005.EXERCISESSHORT QUESTIONS1. Define oncogenes, protooncogenes and tumour suppressor genes. Give examples for chemicalcarcinogens. Explain how they cause cancer.2. What are tumour markers ? Give an example and its clinical importance.3. Expand HIV. Write a note on HIV life cycle.4. Write symptoms and treatments available for AIDS.MEDICAL AND BIOLOGICAL IMPORTANCE1. Porphyrins are present in biological fluids like blood, bile, urine and feces of animalsand invertebrates. They are also found in plants and bacteria.2. Porphyrins are components of hemeproteins of animals and invertebrates. Heme ismetalloporphyrin. It contains metal iron in the centre of porphyrin ring. Hence hemeproteins are referred as metalloporphyrinoproteins.3. Hemeproteins like hemoglobin and myoglobin are involved in O2 transport in animalsand vertebrates.4. In invertebrates erythrocruorins which are also hemeproteins are responsible for the O2transport.5. Hemeproteins like cytochromes and cytochrome oxidase are components of respiratorychain and involved in electron transport.6. Cytochrome P450 which is involved in detoxification of drugs is a hemeprotein.7. Some hemeproteins are involved in metabolism. For example tryptophan dioxygenasean enzyme of tryptophan catabolism is a metelloprophyrinoprotein and cyclooxygenasean enzyme of prostaglandin synthesis is a hemeprotein.8. Hemeproteins like catalase and peroxidase are involved in the removal of H2O2.9. In plants porphyrins are components of chlorophyll and phycobilins.10. In bacteria porphyrins are components of cyanocobalamin.11. A group of inherited diseases known as porphyrias are due to abnormalities in heme(porphyrin) biosynthesis. Lead poisoning also blocks porphyrin biosynthesis.12. A common disease jaundice is due to excessive catabolism of porphyrins or heme con-taining compounds. Hepatitis and cancer of pancreas also can cause jaundice.13. A group of inherited diseases known as hemoglobinopathies are due to abnormalities inproduction of hemoglobin.14. Carbon monoxide a poisonous gas present in automobile exhaust works by combiningwith hemoglobin.22CHAPTERPORPHYRIN ANDHAEMOGLOBIN METABOLISM501502 Medical Biochemistry15. Photosensitive property of porphyrins is used in cancer photochemotherapy.16. Bilirubin end product of heme catabolism act as antioxidant.17. Hemoglobin is a source of protein for malarial parasite during malaria. Enzymes ofhemoglobin degradation pathway are exploited for new drug design.Porphyrins ChemistryPorphyrins are derived from a parent compound porphin. Porphin is a tetrapyrrole and itis a cyclic compound. In porphin, 4 pyrroles are linked through methenyl (CH=) bridges.Four pyrrole rings of porphin are shown with Roman numbers I, II, III and IV. Methenylbridges are indicated by Greek numbers , , and . Substituent positions of I, II, III andIV rings are indicated with Indo-Arabic numbers 1, 2, 3, 4, 5, 6 and 7, 8 respectively(Fig. 22.1). The eight numbered substituent positions corresponds to eight hydrogen atomsof pyrrole rings.Short hand representation of PorphyrinsNaturally occurring porphyrins contain various side chains in place of 8 hydrogen atoms.They differ only in side chains attached to four pyrrole rings. Hence Fischer proposed ashort hand form for porphyrins in which only substitutions are particularly shown. In thisshort hand form as shown in Fig. 22.1 each pyrrole ring with numbered substituent positionsis shown as bracket. The four brackets are indicated with Roman numbers and they arejoined by eliminating the methenyl bridges to form cross shape.Fig. 22.1 Structures of pyrrole, porphin and porphyrinPorphyrin and Haemoglobin Metabolism 503Porphyrins IsomersPorphyrins are intermediates of heme biosynthesis. Each porphyrin can exist in manyisomeric forms which depends on kinds of side chains and arrangement of side chains. Forexample a porphyrin like uroporphyrin with two type of side chains acetate (A), propionate(P) can exist in four isomeric forms or four types. They are type I, type II, type III and typeIV. In uroporphyrin I side chains are arranged symmetrically. In other types side chains A,P are arranged asymmetrically (Fig. 22.2). In nature I and III type porphyrins are more.However type III is predominant and more important for heme biosynthesis.Protoporphyrin with three types of side chain can exist in fifteen isomeric forms. 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 containsprotoporphyrin IX (Fig. 22.2).Fig. 22.2 (a) Short hand forms of uroporphyrin and protoporphyrin.(b) Conversion of uroporphyrinogen III to uroporphyrin III.Properties of Porphyrins1. Solubility. If a prophyrin has polar side chains then it is more soluble in aqueous504 Medical Biochemistryenvironment of blood. If a porphyrin has nonpolar side chains then it is less soluble inblood.2. Light absorption. All porphyrins absorb light maximally at 400 nm. Porphyrins alsocan absorb light in the visible region. When porphyrin combines with metal its absorp-tion changes. For example protoporphyrin IX absorbs light at 645 nm whereas hemewhich is the combination of protoporphyrin IX and iron absorb light at 545 nm. Lightabsorption properties of porphyrins are used for their identification.3. Fluoroescence. Porphyrins show fluoroescence in organic solvents when they are ex-posed to UV-light. This property is used to detect porphyrins in biological fluids.4. All porphyrins are colored compounds.Medical Importance1. Photosensitive property of porphyrins is used in cancer photochemotherapy. It is a com-bination therapy and involves use of light sensitive porphyrin containing drug and laserlight. Tumor cell concentrate more of porphyrin containing drug than normal cells. Onexposure to laser light porphyrins in tumor cells get excited and destroys tumor cells.2. Photosensitivity which is one of clinical symptom seen in some porphyrias is due tolight absorption property of porphyrins. Porphyrins which accumulate in areas sur-rounding skin get excited on exposure to sunlight. They react with O2 and may generatefree radicals of oxygen which damages lysosomes and other cell structures. Thedegradative enzymes released from damaged lysosomes may cause skin damage.3. Since porphyrins are colored molecules their accumulation in tissues and excretion inurine as seen in some porphyrias gives rise to characteristic color to urine and tissues.PorphyrinogensThey are formed during heme biosynthesis. In porphyrinogens the pyrrole rings are linkedthrough methylene (CH2) bridges and all the nitrogens of pyrrole rings are hydrogen-ated. Hence they contain six extra hydrogen atoms than corresponding porphyrins. They canbe auto oxidized to their corresponding porphyrins (Fig. 22.2). Moreover porphyrinogens arecolorless whereas porphyrins are colored compounds.Heme biosynthesisHeme is synthesized by most of the cells except mature erythrocytes. However bonemarrowand liver are chief organs involved in heme production. Bonemarrow produces about 80%and rest is produced by liver.Reaction SequenceIn heme synthesis first and last three reactions occurs in mitochondria and intermediatereactions takes place in cytosol. Amino acid glycine and succinyl-CoA of citric acid cycle arestarting materials for the heme formation (Fig. 22.3).1. Formation of -aminolevulinic acid (ALA) from succinyl-CoA and glycine is the firstreaction of heme biosynthesis and involves CC bond formation. The reaction iscatalyzed by aminolevulinic acid synthase or ALA synthase which takes place in twosteps. 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-Porphyrin and Haemoglobin Metabolism 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 P M I CHz CHz " I Mr{?M CH M MrQ=rM CH, M P CH:::: P PM PM 4C02 COOH < " A J ~ ~ . A / " V , " '-"". --C_ Coo 7 --" , < " " " .. .....,..-."""" - QO