1. Dr, lJ, Satyanarayana M.Sc,.Ph.D.,F.l.C.,F.A.C.B. Professor of Biochemistry Siddhartha Medical Colle g e (NTR University of Health Sciences) Vijayawada, 4.P., India Dr, lJ, Chakrapani M.B.B,S.,M.S. BCDCDT(SAn|D ALLTED lPf Ltd. No.1-E(1) "SHUBHAMPLAzA" (lst Floor) 83/I, BBLrRcrnrn MarN Roeo, Korrere 700010 (Ixora) k:i : (+9| -33)6535-3844,2241-8573 oFax : (033)2358-2127 e-mail : books@cal.vsnl.net.in

2. Eiochemistrg First Published : March 1999 Reprinted: 1999 RevisedReprint: August 2000 Reprinted: 2OQO,2001, 2QO2 Second Revised Edition : June 2002 Reprinted: 2003 RevisedReprint: 2004 RevisedReprint: 2005 Third Revised Edition (multicolour) : 2006 Revised Reprint : 2007 @Copyright reserued by Dn U. Satyanarayana. Publishing rights and Printing rights reserved by the Publisher. Marketing rights, Distributing rights & Selling rights reserued by the Publisher. All rights reserved. No part of this publication may be reproduced or transmifted in any form or by any means, electronic, mechanical, photo-copying, recording or any informatign storage and retrieval system, without the prior wiitten permission of the Publisher. Exclusive rights reserued by the Publisher for publishing, printing, sale, marketing, distribution, expoft and translation of this book for all editions and reprints thereof. Cover Design Depicts the universal energy currency of the living world-ATP, predominantly synthesized by the mitochondria ol the cell (the functional unit of life), in comparison with the intemational currencies--$, t, , Rs, Y. Publisher Typesetter Printer ArunabhaSen BOOKS AND ALLIED (P) Lro. 8/1 ChintamoniDas Lane, Kolkata700009 BOOKS AND ALLIED (P) Lro. 8/1 ChintamoniDas Lane, Kolkata700009 SWAPNA PRINTINGWORKS (P) Lro. 52 Raja RammohanRoy Sarani,Kolkata700009 ShyamalBhattacharyaProject Supervisor : tsBN Bt-8?!1q-80-t Price: Rs.575.00lRupeesFivehundredandSeventy{ive)only US$12.00only AuthorsSponsored& Supportedby : UIBFALAAUTFIOR-PUBTISHERINTERTINKS D.No.: 48-16-10,NagarjunaNagar,MahanaduRoad,Vrjayawada-520008(A"Pl 3. Prefaceto the Third Edition Theresponseto the first andthe secondeditionsof my book'Biochemistry'(reprintedseveraltimesin just 6 years)from the studentsandteachersis simplyoverwhelming.I wasfloodedwith highlyappreciative lettersfrom all cornersof Indiaandabroad!Thisgivesme immensesatisfactionandencouragemLntin this academicventure. I havecorrespondedwith manybiochernistryteachers,invitingtheir commentsandopinionsfor further improvingthe book.Mostof them havebeenkind enoughto offerconstructivesuggestions.I alsovisited severalcollegesandhadpersonalinteractionwith facultymembersandstudents.Theseexercises,spreadover the past 6 years,have helpedme to get direct feedbackon my book, besidesrealisingthe additional requirementsof students. I havegreatpleasurein presentingthe third editionof my bookwith severalunique/novelfeatures,some high-lightsof which are listedbelow. . A thoroughrevisionandupdatingof eachchapterwith latestadvances- . Multicolouredillustrationsfor a betterunderstandingof chemicalstructuresandbiochemicalreactions. . Increasein the font sizeof the text for morepleasantandcomfortablereading. o Incorporationof a newSectionon MolecularBiologyandBiotechnology. . Additionof ten new chapters-humangenomeproject,genetherapy,bioinformatics,free radicalsand antioxidants,tissueproteinsandbodyfluids,environmentalbiochemistry,genetics,immunologyetc. . An improvedorientationand treatmentof humanbiochemistryin healthanddisease. . Additionof practicalbiochemistryandclinicalbiochemistrylaboratoryin the appendix. It is true that I representa selectedgroupof individualsauthoringbooks,havingsometime at disposal, besideshardwork,determinationanddedication.I considermyselfan eternallearneranda regularstudent of biochemistry.However,it is beyondmy capabilityto keeptrackof theevergrowingadvancesin biochemistry dueto the exponentialSrowthof the subject.And this makesme nervous,wheneverI think of revisingthe book.I honestlyadmitthat I haveto dependon maturereadersfor subsequenteditionsof this book. AN INVITATION TO READERS It is not all the time possiblefor me to meetthe readersindividuallyandgettheir feedback,despitemy ferventwish.Of course,I dowrite to somepeoplepersonaliyseekingtheir opinions.However,I wishto have the commentsandsuggestionsof eachoneof the readersof my book.I sincerelyinvitethe readersto feelfree andwrite to me expressingtheir frank opinions,criticalcommentsandconstructivesuggestions. DT.U. SATYANARAYANA trl 4. I owea deepdebtof gratitudeto my parents,the lateSri U.VenkataSubbaiah,andSmt. Vajramma,for cultivatingin me the habitof earlyrising.Thewriting of this bookwouldneverhavebeenpossiblewithout this healthyhabit.I am gratefulto Dr. B. S.NarasingaRao(formerDirector,NationalInstituteof Nutrition, Hyderabad)for discipliningmy professionallife, andto my eldestbrother Dr. U. Gudaru(formerProfessorof PowerSystems,WalchandCollegeof Engineering,Sangli)for discipliningmy personallife. My elder son, U. Chakrapani(MBBS)deservesa specialplacein this book. He madea significant contributionat everystageof its preparation-writing, verification,proof-readingandwhat not. I hadthe rare privilegeof teachingmy sonashehappenedto bea studentof our college.Anda majorpartof this bookwas writtenwhilehewaslearningbiochemistry.Thus,hewasthe firstpersonto learnthesubjectof biochemistry from my handwrittenmanuscript.Thestudent-teacherrelation(ratherthan the father-son)hashelpedme in receivinSlconstantfeedbackfrom him and restructurethe book in a way an undergraduatestudentwould expecta biochemistrytextbookto be. Next,I thankDr. G.PitcheswaraRao(formerProfessorof Anatomy,SMC,Vijayawada)for his constructive criticism and advice,and Dr. B. Sivakumar(Director,NationalInstituteof Nutrition, Hyderabad)for his helpful sugi5lestionson the microfigures.I am gratefulto my nephew,Mr. U. SrinivasaRao,for helping me in drawingsomefigures. Last but not least,I thank my wife Krishna Kumari and my youngerson,Amrutpani,without whose cooperationand encouragementthis book could never have beenwritten. The manuscriptwas carefully nurturedlike a newborn babyandthe bookhasnow becomea full-pledgedmemberof our family. ACKNOWLEDGEMENTSTO THE THIRD EDITION I amindebtedto a largenumberof friends,pen-friendsandstudentswhohelpedmeto reviseandimprove the qualityof this book.I haveindividuallyandpersonallythankedall of them (whonumbera fewhundreds!). I onceagainexpressmy gratitudeto them. I thank my friend and colleague,Mr. M.S.T.JaganMohan,who has helpedme with his frequent interactionsto improvethe book,andmakeit morestudent-friendly.I wouldlike to placeon recordmy deep senseof appreciationto my post-graduate(M.D.)students,Dr. (Mrs.)U.B.VijayaLakshmiandDr. (Mrs.)Vidya DesaiSripad,whoseperiodicalacademicinteractionandfeedbackhavecontributedto the improvementof the biomedicaVclinicalaspectsin somechapters.I acknowledgethe helpof my friend,Dr. P.Ramanujam(Reader in English,AndhraLoyolaCollege,Vijayawada)for his helpandencouragementin revisingthe book. I expressmy gratitudeto Mr. ArunabhaSen, Director,Books & Allied (P) Ltd. Kolkata,for his wholeheartedsupportand constantencouragementin revisingthe bookin multicolour,and takingall the painsto bring it out to my satisfaction.I thank Mr. ShyamalBhattacharyafor his excellentpage-makingand graphics-workin the book.I am indebtedto Mr. PrasenjitHalderfor the coverdesignof this book. I thank my wife, Krishna Kumari, and my younger son, Amrutpani, for their constantsupport and encouragement.I am grateful to UppalaAuthor-PublisherInterlinks, Vijayawada,for sponsoringand supportingme to bring out this edition. Iiii] DT.U. SAIYANARAYANA 5. Biochemistry The term Biochemistrywas introducedby Carl Neubergin 1903.Biochemistrybroadlydealswith the chemistrvof life and living processes.Thereis no exaggerationin the statement,'Thescopeof biochemistrg is asuastaslilb itself!' Everyaspectof life-birth,growth,reproduction,aginganddeath,involvesbiochemistry. For that matter,everymovementof life is packedwith hundredsof biochemicalreactions.Biochemistryis the mostrapidlydevelopingandmostinnovativesubjectin medicine.Thisbecomesevidentfromthe factthat over the years,the major shareof NobelPrizesearmarkedfor Medicineand Physiologyhasgoneto researchers engagedir: biochemistry. The disciplineof biochemistryservesas a torch light to trace the intricate complexicitiesof biology, besidesunravellingthe chemicalmysteriesof life.Biochemicalresearchhasamplydemonstratedthat all living thingsarecloselyrelatedat the molecularlevel.Thusbiochemistryis the subjectof unity in the diversified living kingdom. Advancesin biochemistryhavetremendousimpacton humanwelfare,andhavelargelybenefitedmankind and their living styles.Theseincludethe applicationof biochernistryin the laboratoryfor the diagnosisof diseases.the products(insulin,interferon,rowthhormoneetc.)obtainedfrom geneticengiineering,andthe possibleuseof genetherapyin the nearfuture. 0rganizationof the Book This texthook,comprising43 chapters,is orgianizedinto serrensecl:ionsin the heirarchicalorder of learninSbiochemistry. . SectionI dealswith the chemicalconstituentsof life-carbohydrates,lipids,proteinsandaminoacids, nucleicacidsandenzymes. . SectionII physiologicalchemistryincludesdigestionandahsorption,plasmaproteins,hemoglobinand prophyrins,andbiologicaloxidation. . SectionIII incorporatesall the metabolisms(carbohydrates,lipids,aminoacids,nucleotides,minerals) . Section[V covershormones,organfunctiontests,water,electrolyteandacid-basebalance,tissueproteins andtrodi'fluids,andnutrition. . SectionV is exclusivelydevotedto molecularbiologyandbiotechnology(DNA-replication,recombination, ar"lnrepair,transcriptionandtranslation,regulationof geneexpression,recombinantDNAandbiotechnology) . SectionVI givesrelevantinformation on current topics such a^shuman genomeproject,genetherapy, bioirrtormatics,prostaglandins,diabetes,cancer,AIDSetc. . Section VII dealswith the basic aspectsfor learning and understandingbiochemistry (bioorganic chenristry',hiophysicalchemistrytoolsof biochemistry,genetics,immunology). Each chapterin this book is carefully craftedwith colour illustrations, headingsand subheadingsto facilitatequickunderstanding.Theimportantapplicationsof biochemistryto humanhealthanddiseaseareput togetherasbiomedical/clinicalconcepts.Iconsare usedat appropriateplacesto serveas 'landmarks'. The origins of biochemicalwords, confusablesin biochemistry,practicalbiochemistryand clinical biochemistrylaboratory,givenin the appendixare novelfeatures. Thebriokis so organizedasto equipthe readerswith a comprehensiveknowledgeof biochemistry. Iiu] 6. Gontents SECTION ONE ChemicalConstituentsof Life 1 > Biomoleculesandthecell 2 > Carbohydrates 3 > Lioids 4 > Proteinsandaminoacids 5 > Nucleicacidsandnucleotides 6 > Enzymes 7 > Vitamins SECTION TWO PhysiologicalBiochemistry B > Digestionandabsorption 9 > Plasmaoroteins 10 > Hemoglobinandporphyrins 11 > Biologicaloxidation SECTION THBEE q3 > Metabolismofcarbohydrates *4 > Metabolismoflioids F-, Metabolismofaminoacids 16 > Int6grationofmetabolism 17 > Metabolismofnucleotides 1B > Mineralmetabolism SECTION FOUR ClinicalBiochemistrvand Nutrition 19 > Hormones 20 > Organfunctiontests 21 > Water,electrolyteand acid-basebqlance 22 > Tissueproteinsandbodyfluids 23 > Nutrition- SECTION FIVE MolecularBiologyand Biotechnology 24 > DNA-replication,recombinationandrepair523 25 > Transcriotionandtranslation 542 26 > Regulationofgeneexpression 566 27 b RecombinantDNAandbiotechnology578 sEcTtcN stx Current Topics 28 > Humangenomeproject 619 29 > Genetherapy 625 30 F Bioinformatics 634 31 p 'lvletabolismofxenobiotics(detoxification)638 32 >' Prostaglandinsandrelatedcompounds644 33 > Biologicalmembranesandtransport 650 34 b Freeradicalsandantioxidants 655 35 > Environmentalbiochemistry 662 36 l" Insulin,glucosehomeostasis, 3 9 28 43 69 85 176 165 182 196 221 anddiabetesmellitus Cancer 669 58s37> 38> Acquiredimmunodeficiency syndrome(AIDS) 695 241 244 285 330. 380 387 403 427 453 SECTION SEVEN Basicsto LearnBiochemistrv 39 > Introductiontobioorganicchemistry 40 > Overviewofbiophysicalchemistry 41 > Toolsofbiochemistrv 42 > lmmunology 43 > Genetics APPENDICES AnswerstoSelf-assessmenlExercises I Abbreviationsusedinthisbook' ll Greekalphabets lll Originsolimportantbiochemicalwords lV Commonconfusablesinbiochemistry V Practicalbiochemistry-principles Vl Clinicalbiochemistrylaboratory INEEX 703 708 719 732 737 745 751 756 tJt 760 764 770 773 468 487 502 7. fi Protuinsand Amino acids 4: Nucleicacidsand Nucleotides 69 8. BflomnoXeeutrssaildthsCelll -l- hu living matter is composedof mainly six I elements-carbon, hydrogen, oxygenl nitrogen, phosphorus and sulfur. Theseelements togetherconstituteabout 90% of the dry weight of the human body. Severalother functionally importantelementsare also found in the cells. Theseinclude Ca, K, Na, Cl, Mg, Fe,Cu, Co, l, Zn, F, Mo and Se. earbon-a unique element of life Carbonis the most predominantand versatile elementof life. lt possessesa unique propertyto form infinite number of compounds. This is attributedto the ability of carbon to form stable covalentbonds and C-C chains of unlimited length. lt is estimated that about 90% of compounds found in living system invariably contain carbon. Ghemical molecules of li#e Life is composed of lifeless chemical molecules. A single cell of the bacterium, Escherichiacoli containsabout 6.000 different organiccompounds.lt is believedthat man may contain about 100,000 different types of moleculesalthough only a few of them have been characterized. Sornpiex *riomoleeules The organiccompoundssuchasamino acids, nucleotidesand monosaccharidesserve as the monomeric unitsor building blocksof complex biomolecules-proteins,nucleicacids(DNA and RNA) and polysaccharides,respectively.The important biomolecules(macromolecules)with their respective building blocks and major functions are given in Table 1.1. As regards lipids, it may be noted that they are not biopolymers in a strict sense,but majority of them contain fatty acids. Structural heirarehy off asn organisnl The macromolecules(proteins,Iipids,nucleic acidsand polysaccharides)form supramolecular assemblies(e.g. membranes)which in turn organize into organelles,cells, tissues,organs and finally the whole organism. 3 9. BIOCHEMISTFIY Biomolecule Buildingblock (repeatingunit) Major functions 1. Protein Aminoacids 2. Deoxyribonucleicacid(DNA) Deoxyribonucleotides Ribonucleotides3. Ribonucleicacid(RNA) 4. Polysaccharide(glycogen)Monosaccharides(glucose) Fundamentalbasisofstructureand functionofcell(staticanddynamicfunctions). fl_eq_o_sitoryo.l.!9199iFryi{9l1llgt Essentiallyrequiredlorproteinbiosynthesis. Storageformofenergytomeetshortterm demands. 5. Lipid Fattyacids,glycerol Storagetormofenergytomeetlongterm demands;structuralcomponentsofmembranes. Chem*ca! composition of man The chemicalcompositionof a normal man, weighing 65 kg, is given in Table 1.2.Water is the solventof life and contributesto more than 60"h of the weight. This is followed by protein (mostlyin muscle)and lipid (mostlyin adipose tissue).The carbohydratecontent is rather low which is in the form of glycogen. The cell is the structuraland functional unit of life. ft may be also regardedas the basic unit of hiological activity. The concept of cell originated from the contributionsof Schleidenand Schwann(1838). However, it was only after 1940, the complexitiesof cell structurewere exposed. Constituent Percent(7") Weight (kg) Prokaryotic and eukaryotic cells The cells of the living kingdom may be divided into two categories 1. Prokaryotes(Creek: pro - before;karyon- nucleus)lacka well definednucleusand possess relatively simple structure.These include the variousbacteria. 2. Eukaryotes(Greek: eu-true; karyon- nucleus)possessa well definednucleusand are more complex in their structureand function. The higher organisms(animalsand plants)are composedof eukaryoticcells. A comparisonof the characteristicsbetween prokaryotesand eukaryotesis listedin Table 1.3. The human body is composedof about 1014 cells.There are about 250 typesof specialized cel{s in- the human body'G.g. erythrocytes, nerve-cells, muscle cells, B cells of pancreas. An eukaryoticcell is generally10 to 100 pm in diameter. A diagrammatic representation of a typical rat liver cell is depicted in Fig.I.t. The plant cell differsfrom an animalcell by possessinga rigid cell wall (mostlycomposedof cellulose)and chloroplasts.The latter are the sitesof photosynthesis. Water Protein Lipid Carbohydrate Minerals 61.6 17.0 13.8 6.1 40 11 I '| 4 10. Chapter 1 : BIOMOLECULESAND THE CELL Characteristic Prokaryotic cell Eukaryoticcell 1. Size Small(generally1-10pm) Large(generally10-100pm) 2. Cellmembrane Cellisenvelopedbyaflexibleplasmamembrane Distinctorganellesarefound (e.9.mitochondria,nucleus,lysosomes) 3. Sub-cellular organelles 4, Nucleus Notwelldefined;DNAisfound asnucleoid,histonesareabsent Nucleusiswelldefined,surroundedbya membrane:DNAisassociatedwithhistones 5. Energymetabolism Mitochondriaabsent,enzymesof energymetabolismboundto Enzymesolenergymetabolismarelocated inmitochondria membrane 6. Celldivision 7. Cytoplasm Usuallyfissionandnomitosis Mitosis 0rganellesandcytoskeleton absent Containsorganellesandcytoskeleton (anetworkoftubulesandfilaments) The cell consistsof well definedsubcellular organelles,envelopedby a plasma membrane. By differential centrifugation of tissue homogenate, it is possible to isolate each cellular organelle in a relatively pure form (Refer Chapter 41). The distribution of major enzymes and metabolic pathways in different cellular organelles is given in the chapter on enzymes (Refer Fig.6.6). The subcellular organellesare briefly describedin the following pages. Nucleus Nucleus is the largest cellular organelle, surroundedbv a double membrane nuclear envelope.The outer membraneis continuous with the membranesof endoplasmicreticulum. At certainintervals,the two nuclearmembranes have nuclearporeswith a diameterof about 90 nm. Theseporespermit the free passageof the products synthesizedin the nucleus into the surrounding cytoplasm. Roughendoplasmicreticulum Golgiapparatus Lysosome Mitochondrion Plasmamembrane Vacuole Ribosomes Peroxisome Cytoskeleton Cytosol Coatedpits Ftg. 1.1: Diagrammaticrepresentationof a nt liverell. 11. BIOCHEMISTF|Y Nucleus contains DNA, the repository of genetic information. Eukaryotic DNA is associatedwith basic protein (histones)in the ratio of 1 : 1, to form nucleosomes.An assembly of nucleosomesconstituteschromatin fibres of chromosomes(Creek'.chroma - colour; soma- body). Thus, a single human chromosomeis comoosedof abouta million nucleosomes.The number of chromosomes is a characteristic feature of the species. Humans have 46 chromosomes,compactlypackedin the nucleus. The nucleusof the eukaryoticcell containsa dense bodv known as nucleolus.lt is rich in RNA, particularlythe ribosomal RNA which entersthe cytosolthrough nuclearpores. The ground materialof the nucleus is often referredto as nucleoplasm.lt is rich in enzymes such as DNA polymerases and RNA polymerases.To the surpriseof biochemists,the enzymes of glycolysis,citric acid cycle and hexose monophosphateshunt have also been detectedin the nucleoplasm. Mitochondria The mitochondria (Creek'. mitos- thread; chondros- granule) are the centres for the cellularrespirationand energymetabolism.They are regarded as the power housesof the cell with variablesize and shape.Mitochondriaare rod-like or filamentousbodies, usuallv with dimensions of 1.0 x 3 pm. About 2,0O0 mitochondria,occupyingabout 1/5thof the total cell volume,are presentin a typicalcell. The mitochondriaare comoosedof a double membrane system. The outer membrane is smooth and completelyenvelopsthe organelle. The inner membrane is folded to form cristae (Latin- crests)which occupy a larger surface area. The internal chamber of mitochondriais referred to as matrix or mitosol. The componentsof electron transportchain and oxidative phosphorylation (flavoprotein, cytochromesb, c1, C, a and a3 and coupling factors)are buried in the inner mitochondrial membrane.The matrixcontainsseveralenzvmes concerned with the energy metabolism of carbohydrates,lipidsandaminoacids(e.g.,citric acid cycle, p-oxidation).The matrix enzymes also parlicipate in the synthesisof heme and urea. Mitochondria are the principal producers of ATP in the aerobic cells. ATP, the energy currency,generatedin mitochondriais exported to all partsof the cell to provideenergyfor the cellularwork. The mitochondrialmatrixcontainsa circular double stranded DNA (mtDNA), RNA and ribosomes.Thus,the mitochondriaareequipped with an independent protein synthesizing machinery.It is estimatedthat about 10% of the mitochondrial oroteins are produced in the mitochondria. The structureand functionsof mitochondria closely resemble prokaryotic cells. lt is hypothesizedthat mitochondria have evolved from aerobicbacteria.Further,it is believedthat duringevolution,the aerobicbacteriadeveloped a symbiotic relationship with primordial anaerobiceukaryoticcellsthat ultimatelyled to the arrival of aerobiceukaryotes. Endoplasmic reticulum The network of membraneenclosedspaces that extends throughout the cytoplasm constitutesendoplasmicreticulum(ER).Someof these thread-like structuresextend from the nuclearporesto the plasmamembrane. A large portion of the ER is studded with ribosomesto give a granularappearancewhich is referred ro as rough endoplasmic reticulum. Ribosomes are the factories of protein biosynthesis. During the process of cell fractionation,roughERisdisruptedto form small vesiclesknown as microsomes.It may be noted that microsomesas such do not occur in the cell. The smoothendoplasmicreticulumdoes not containribosomes.lt is involvedin the synthesis of lipids (triacylglycerols,phospholipids,sterols) and metabolismof drugs,besidessupplyingCa'?. for the cellularfunctions. Golgi apparats,r$ Eukaryoticcells contain a unique clusterof membrane vesicles known as dictyosomes 12. Chapter 1 : BIOMOLECULESAND THE CELL which, in turn, constituteColgi apparatus(or Colgi complex).The newly synthesizedproteins are handed over to the Colgi apparatuswhich catalysethe addition of carbohydrates,lipids or sulfatemoietiesto the proteins.Thesechemical modificationsare necessaryfor the transportof proteinsacrossthe plasmamembrane. Certainproteinsand enzymesareenclosedin membrane vesicles of Colgi apparatusand secreted from the cell after the appropriate signals.The digestiveenzymesof pancreasare oroducedin this fashion. Colgi apparatusare also involved in the membrane synthesis, particularly for the formation of intracellular organelles (e.g. peroxisomes,lysosomes). Lysosornes Lysosomesare sphericalvesiclesenveloped by a singlemembrane.Lysosomesare regarded as the digestivetract of the cell, sincethey are actively involved in digestion of cellular substances-namely proteins, lipids, carbo- hydratesand nucleic acids.Lysosomalenzymes are categorizedas hydrolases.Theseinclude the following enzymes(with substratein brackets) a-C lucosidase(glycogen) Cathepsins(proteins) Lipases(lipids) Ribonucleases(RNA) The pH of the lysosomalmatrixis moreacidic (pH< 5) than the cytosol (pH-7) and this facilitatesthe degradationof differentcompounds. The lysosomal enzymes are responsiblefor maintaining the cellular compounds in a dynamic stafe, by their degradationand recycling.The degradedproductsleavethe lysosomes,usually by diffusion, for reutilization by the cell. Sometimes,however,certain residualproducts, rich in lipidsand proteins,collectivelyknown as Iipofuscinaccumulatein the cell. Lipofuscinis the agepigmentor wear and tearpigmentwhich has been implicatedin ageingprocess. The digestiveenzymesof cellularcompounds are confinedto the lvsosomesin the bestinterest of the cell. Escapeof theseenzymesinto cytosol will destroythe functionalmacromoleculesof tne cell and result in many complications.The occurrence of several diseases(e.g. arthritis, musclediseases,allergicdisorders)hasbeenpartly attributedto the releaseof lysosomalenzymes. Feroxisomes Peroxisomes,also known as microbodies, are single membranecellularorganelles.They are spherical or oval in shape and contain the enzyme catalase.Catalaseprotectsthe cell from the toxic effectsof HrO, by converting it to HrO and Or. Peroxisomesare also involved in tne oxidation of long chain fatty acids (> C,s),and synthesisof plasmalogensand glycolipids.Plants contain glyoxysomes, a specialized type of BTOMED|eAL/ CLINICAL COIUCEPTS A liuing cell is a true representotiueof life with its own organizotionand specialized lunctions. Accumulotion oJ lipofuscin,a pigment rich in lipids and proteins, in the cell hasbeen implicated in ogeing process. Leokageof lysosomalenzymesinto the cell degrodesseuerolfunctional macromolecules and this may leod to certain disorders (e.9. arthritis). rq Zellweger syndrome is a rare diseose characterized by the absence of functional peroxisomes. 13. E}IOCHEMISTF|Y peroxisomes, which are involved in the glyoxylate pathway. Peroxisome biogenesisdisorders (PBDs), are a Broup of rare diseasesinvolving the enzyme activities of peroxisomes. The biochemical abnormalitiesassociatedwith PBDs incluoe increasedlevelsof very long chain fatty acids (C2aand C26)and decreasedconcentrationsof plasmalogens.The most severeform of PBDsis Zellweger syndrome, a condition characterized by the absenceof functional peroxisomes.The victimsof this diseasemav die within one vear after birth. {iytosol and cytoskeleton The cellular matrix is collectively referredto as cytosol. Cytosol is basicallya compartment containing several enzymes/ metabolites and saltsin an aqueousgel like medium.More recent studies however, indicate that the cytoplasm actuallycontainsa complex network of protein filaments, spread throughout, that constitutes cytoskeleton.The cytoplasmicfilamentsare of three types- microtubules, actin filaments and intermediatefilaments.The filamentswhich are polymers of proteins are responsiblefor the structure,shapeand organizationof the cell. INTEGRATIOI{ OF CELLULAR FUNCTIONS The eukaryoticcells performa wide rangeof complex reactionsfunctionsto maintaintissues, and for the ultimatewell-beingof the whole organism. For this purpose, the various intracellularprocessesand biochemicalreactions are tightly controlledand integrated.Divisionof a cell intotwo daughtercellsis goodexampleof the orderlyoccurrenceof an integratedseriesof cellularreactions. Apoptosisis the programmedcell death or cell suicide. This occurs when the cell has fulfilled its biologicalfunctions.Apoptosismay be regardedas a natural cell deathand it differs from the cell death caused by injury due to radiation,anoxiaetc. Programmedcell death is a highly regulatedprocess. 1. 2. 3. Life is composed ol lifeless chemical molecules. The complex biomolecules, proteins, nucleic ocids (DNA and RNA), polysaccharidesand lipids are formed by the monomeric units amino acids,nucleotides,monosaccharidesand fotty acids,respectluely. The cell is the structuroland functional unit of life. The eukoryoticcell consisfsof well det'inedsubcellulororganelles,enuelopedin a plasma membrane. The nucleus contoinsDNA, the repositoryol genetic int'ormation.DNA, in association with proteins (histones),forms nucleosomeswhich,in turn, make up the chromosomes. The mitochondria qre the centresfor energymetobolism. Theyare the principalproducers of ATP which is exported to all parts of the cell to ptouide energylor cellular work. Endoplosmic reticulum (ER) ts the network of membrane enclosed spocesthat extends throughout the cytoplosm. ER studded with ribosomes, the factorles of protein biosynfhesis, ts relerred to as rough ER. Golgi opparatus sre a cluster of membrane uesiclesto uthich the newlg synthesizedproteins are handed ouer for t'urther processing ond export. Lysosomesare the digestiue bodiesol the cell, actiuely involued in the degradotion of cellular compounds. Peroxisomescontoln the enzymecatalosethat protects the cell lrom the toxic elfects of HrOr. The cellular ground motrix is referred to as cytosol which, in fact, is composed of a network ot' protein t'ilaments, the cytoskeleton. Theeukaryoticcellsperform a widerangeof complex lunctionsin a well coordinatedand integrated fashion. Apoptosis is the processol programmed cell death or cell suicide. 5. 6. 7. 14. 1^ arbohydratesare the most abundantorganic - molecules in nature. They are primarily composedof the elementscarbon, hydrogen and oxygen.The name carbohydrateliterallymeans 'hydratesof carbon'.Someof the carbohydrates possessthe empiricalformula (C.H2O)nwhere n 3 3, satisfyingthat thesecarbohydratesare in fact carbonhydrates.However,thereare several non-carbohydratecompounds(e.g. acetic acid, C2HaO2;lacticacid,C3H6O3)which alsoappear as hydratesof carbon. Further, some of the genuine carbohydrates (e.g. rhamnohexose, C6H12O5ideoxyribose,C5H16Oa)do not satisfy the generalformula.Hencecarbohydratescannot be alwaysconsideredas hydratesof carbon. Carbohydrates may be defined as polyhydroxyaldehydes or ketones or compounds which produce them on hydrolysis. The term 'sugar' is applied to carbohydratessoluble in water and sweet to taste. #-ur*c;tEerEsof earbohydrates Carbohydratesparticipatein a wide rangeof functions 1. Theyarethe mostabundantdietarysource of energy (a Cal/S)for all organisms. 2. Carbohydratesare precursorsfor many organic compounds(fats,amino acids). 3. Carbohydrates(asglycoproteinsand glyco- lipids) participate in the structure of cell membraneand cellular functionssuch as cell growth, adhesionand fertilization. 4. They are structuralcomponentsof many organisms.Theseincludethe fiber (cellulose)of plants,exoskeletonof some insectsand the cell wall of microorganisms. 5. Carbohydratesalso serve as the storage form of energy(glycogen)to meetthe immediate energydemandsof the body. CLASSIFICATION OF GARBOHYDRATES Carbohydrates are often referred to as saccharides (Greek: sakcharon-sugar).They are broadlyclassifiedinto three major groups- monosaccharides, oligosaccharides and polysaccharides.This categorizationis basedon 15. t0 BIOCHEMISTRY Monosaccharides(empiricalformula) AIdose Ketose Trioses(CgHoOg) Telroses(C+HoO+) Pentoses(CsHroOs) Hexoses(CoHrzOo) Heptoses(CzHr+Oz) Glyceraldehyde Erythrose Ribose Glucose Glucoheptose Dihydroxyacetone Erythrulose Ribulose Fructose Sedoheptulose the number of sugar units. Mono- and oligo- saccharidesare sweet to taste, crystalline in characterand soluble in water, hence thev are commonly known as sugars. FJtonosaccharides Monosaccharides(Greek: mono-one)are the simplestgroup of carbohydratesand are often referred to as simple sugars.They have the generalformula Cn(H20)n,and they cannot be further hydrolysed.The monosaccharidesare divided into differentcategories,based on the functionalgroupandthe numberof carbonatoms Aldoses : When the functional group in IHmonosaccharidesis an aldehydel-C:oi, ,h"u are known as aldoses e.g. glyceraldehyde, glucose. Ketoses: When the functionalgroup is a keto lt-C:O.l group, they are referredto as ketoses e.g. dihydroxyacetone,fructose. Basedon the number of carbon atoms,the monosaccharidesare regarded as trioses (3C), tetroses (4C), pentoses (5C), hexoses (6C) and heptoses(7C).Thesetermsalongwith functional groupsare usedwhile namingmonosaccharides. For instance, glucose is an aldohexose while fructose is a ketohexose(Table 2,1). Thecommonmonosaccharidesand disaccha- rides of biological importanceare given in the Table 2.2. SSlgosaccharides Oligosaccharides(Creek: oligo-few) contain 2-1O monosaccharidemolecules which are liberatedon hydrolysis.Basedon the numberof monosaccharide units present, the oligo- saccharides are further subdivided to disaccharides,trisaccharidesetc. Polysace harides Polysacchari6ls(Creek:poly-many)are poly- mers of mondficcharide units with high mole- cular weight (up to a million).They are usually tasteless(non-sugars)and form colloids with water. The polysaccharidesare of two types- homopolysaccharidesand heteropolysaccharides. Stereoisomerismis an importantcharacterof monosaccharides. Stereoisomers are the compounds that have the same structural formulaebut differ in their spatialconfiguration. A carbon is said to be asymmetric when it is attached to four different atoms or groups. Ihe number of asymmetric carbon atoms (n) determines the possible isomers of a given compound which is equal to 2n. Clucose contains4 asymmetriccarbons,and thus has 16 tsomers. Glyeeraldehyde -tfu e ref erqlrt*e cff rb$hyd$'er'&3 Clyceraldehyde(triose)is the simplestmono- saccharidewith one asymmetriccarbonatom. lt existsastwo stereoisomersand hasbeenchosen as the referencecarbohydrateio representthe structureof all other carbohvdrates. 16. Ghapter 2 : CARBOHYDRATES 11 Trioses Glyceraldehyde Dihydroxyacetone Tetroses D-Erythrose Foundincellsasphosphate Foundincellsasphosphate i Widespread I Widespreadasaconstituentof I RNAandnucleotides i AsaconstituentofDNA : Producedduringmetabolism i Asaconstituentofglycoproteins i anogums i ls anintermediateinuronicacidpathway i Heartmuscle i --. --. -- --.. ---.. -.. -. --. Asaconstituentolpolysaccharides (starch,glycogen,cellulose)and disaccharides(maltose,lactose, sucrose).Alsofoundinfruits Asaconstituentoflactose (milksugar) Foundinplantpolysaccharides andanimalglycoproteins Fruitsandhoney,asaconstituent ofsucroseandinulin Foundinolants i Glyceraldehyde3-phosphateisanintermediate i inglycolysis i ttst-pnosphateisanintermediateinglycolysis ----t------..-..-.--...--- ForthestructureofRNAandnucleotide coenzymes(ATP,NAD+,NADP+) ForthestructureolDNA Itisanimportantmetaboliteinhexose monophosphateshunt Involvedinthefunctionofglycoproteins Excretedinurineinessenlialpentosuria Asaconstituentollvxollavinofheartmuscle The'sugarfuel'oflife;excretedinurinein diabetes.Structuralunitofcelluloseinplants Convertedtoglucose,failureleadsto galactosemia Forthestructureofpolysaccharides Itsphosphatesareintermediatesofglycolysis Its7-phosphateisanintermediateinhexose monophosphateshunt,andinphotosynthesis Pentoses D-Ribose D-Deoxyribose D-Ribulose D-Xylose L-Xylulose D-Lyxose Hexoses D-Glucose D-Galactose D-Mannose D-Fructose Heptoses D-Sedoheptulose Disaccharides Occurrence Biochemical importance Sucrose Lactose Asaconstituentofcanesugarand beetsugar,pineapple Milksugar Productofstarchhydrolysis, occursingerminatingseeds Mostcommonlyusedtablesugarsupplying calories Exclusivecarbohydratesourcetobreastfed infants.Lactasedeficiency(lactoseintolerance) leadstodianheaandflatulence Animportantintermediateinlhedigestionof starch Maltose 17. 12 E}IOCHEMISTFIY H-C:O I H-C-OH cH2oH D-Glyceraldehyde H-C:O HO-C-H cH2oH L-Glyceraldehyde H-C:O I HO-C-H H-C-OH I HO-C-H HO-C-H cH2oH L-Glucose Fig.2.1 : DandL- formsof glucosecomparedwith D and L- glyceraldehydes (the reference carbohydrate). D" and L-isomers The D and L isomersare mirror imagesof each other. The spatialorientationof -H and -OH groups on the carbon atom (Cs for glucose)that is adjacentto the terminal primary alcohol carbon determineswhetherthe sugaris D- or L-isomer.lf the -OH group is on the right side,the sugaris of D-series,and if on the left side, it belongs to L-series.The structuresof D- and L-glucosebasedon the referencemono- saccharide, D- and L-glyceraldehyde (glycerose) are depicted in Fig.2.1. It may be noted that the naturallyoccurring monosaccharidesin the mammaliantissuesare mostlyof D-configuration.Theenzymemachinery of cells is specific to metaboliseD-seriesof monosaccharides. fn the medical practice, the term dextroseis used for glucosein solution. This is becauseof the dextrorotatorynature of glucose. Optlcal activity of sugars Optical activity is a characteristicfeature of compounds with asymmetric carbon atom. When a beam of polarized light is passed througha solutionof an optical isomer,it will be rotated either to the right or left. The term dextrorotatory (+) and levorotatory (-) are used to compoundsthat respectivelyrotatethe plane of polarizedlight to the right or to the left. An optical isomer may be designatedas D(+), D(-), L(+)and L(-) basedon its structural relation with glyceraldehyde.lt may be noted that the D- and L-configurationsof sugarsare primarily based on the structure of glyceraldehyde,the optical activitieshowever, may be different. Racemicmixture : lf D- and L-isomersare presentin equal concentration,it is known as racemicmixtureor DL mixture.Racemicmixture does not exhibit any optical activity, since the dextro- and levorotatorv activities cancel each other. Configuration of D-aldoses The configuration of possible D-aldoses startingfrom D-glyceraldehydeis depicted in Fig.2.2. This is a representation of Killiani- Fischersynthesis,by increasingthe chain length of an aldose,by one carbon at a time. Thus, startingwith an aldotriose(3C),aldotetroses(4C), aldopentoses(5C) and aldohexoses(6C) are formed. Of the 8 aldohexoses,glucose,mannose and galactoseare the most familiar. Among these, D-glucose is the only aldose mono- saccharidethat predominantlyoccurs in nature. Gonfiguration of D-ketoses Startingfrom dihydroxyacetone(triose),there are five keto-sugarswhich are physiologicallr important.Their structuresare given in Fig,2.3 Epimers ff two monosaccharides differ from eac- other in their configuration around a singk specificcarbon (otherthan anomeric)atom. L*ei are referred to as epimersto each orher '.Fig,21 For instance, glucoseand galactose are efilwl with regardto carbon 4 (Ca-epimers- -^:i 's they differ in the arrangementof -OH g.'ELcr Ca. Clucose and mannose are epi-'e--' q drl regardto carbon 2 (C2-epimers). The interconversionof epimers e - I r::r'e to galactose and vice versai s i - -^,'- a* H-C:O I H-C-OH I HO-C-H I H-C-OH I H-Q-OH I cHzoH D-Glucose 18. Ghapter 2 : CABBOHYDFATES 13 Aldotriose (3c) Aldotetroses (4c) cHo I HOCH I Aldo toses HOCH ) I HCOH I cH2oH D-Lyxoee cHo HOCH I I Aldo- HOCH HOCH hexoses HoCH noCH (6c) tl HCOH HCOH tt cHzoH cHzoH D-Galactose D-Talose 'l t- cHo HOCH I HCOH HCOH cH2oH D-Arabinose / JT cHo cHo HCOH HOCH rl HOCH HOCH tl HCOH HCOH tl HCOH HCOH ll cH2oH cH2oH D-Glucose D-Mannose HCOH I HCOH cH2oH D-Ribose / JT cHo cHo tl HCOH HOCH HCOH HCOH tl HCOH HCOH tl HCOH HCOH ll cH2oH cH2oH D-Allose D-Altrose cHo HCOH cHo I HCOH I HCOH I HOCH I HCOH cH2oH D-Gulose cHo I HCOH cHo I HOCH I HCOH I HOCH HCOH I cH2oH D-ldose cHo I HCOH I cH2oH D-Erythrose D.Threooe cHo I HCOH I HOCH I HCOH cH2oH D-Xylose I / / *+ Fig.2.2 : ThestructuralrelationshipbetweenD-aldosesshownin Fischerprojection. (TheconfigurationaroundC2(ed) distinguishesthemembersof eachpair). epimerization, and a group of enzymes- namely-epimerases catalysethis reaction. Enantiomers Enantiomers are a special type of stereoisomers that are mirror images of eachother. The two membersare designatedas D- and L-sugars.Enantiomersof glucose are depicted in Fig.2.5. Majority of the sugarsin the higher animals (includingman) are of D-type (Fig.2.5'1. The term diastereomersis used to represent the sfereoisomers that are not mirror imagesof one another. For a better understanding structure, let us consider the hemiacetals and hemiketals, producedwhen an aldehydeor a with alcohol. of glucose formation of respectively ketone reacts 19. 14 E}IOCHEMISTRY ?H2oH C:O I cH2oH Dlhydroxyacetone cH2oH I C:O I HOCH HCOH I cH2oH D-Xylulose cH20H I C:O HCOH HCOH I cH2oH D-Ribulose cH2oH I C:O HOCH I HCOH I HCOH I cH2oH D-Fructose cH2oH I C:O I HOCH I HCOH I HCOH I HCOH I cH2oH D-Sedoheptulose Fig.2.3 : Structuresof ketosesof physiologicalimportance. ,H nt-C.1^ + R2-oH l- Rr- LJ Aldefry COOH) resultsin the formationof gluconicacid. 2. Oxidation of terminal alcohol group (CH2OH------+COOH) leadsto the production of glucuronicacid. Reduetion When treatedwith reducing agentssuch as sodiumamalgam,the aldehydeor keto groupof monosaccharideis reduced to corresponding alcohol, as indicatedby the generalformula : H H-C:O H-C-Ol-t I RR The important monosaccharidesand their correspondingalcoholsare given below. D-Glucose D-Galactose------+D-Dulcitol D-Mannose ------+D-Mannitol D-Fructose --) D-Mannitol+ D-Sorbitol D-Ribose -+ D-Ribitol Sorbitol and dulcitol when accumulate in tissuesin large amounts cause strong osmotic effectsfeadingto swelling of cells,and certain pathologicalconditions.e.g.cataract,peripheral neuropathy,nephropathy.Mannitol is usefulto reduce intracranialtensionbv forced diuresis. 23. Ghapter 2 : CAFIBOHYDRATES 17 H-C--O I H-C-OH I HO-C-H I H-C-OH I H-C-OH I cH2oH D-Glucose H-C:O I H-C:O I cH20H Hydrorymethylfurfural H-C:O I Formation of esters The alcoholic groups of monosaccharides may be esterified by non-enzymatic or enzymatic reactions. Esterificationof carbo- hydrate with phosphoric acid is a common reaction in metabolism.Glucose 6-phosphate and glucose 1-phosphateare good examples. ATP donates the phosphate moiety in ester formation. lClycoside bond formation (see below) and mutarotation(discussedalready) may also be referred to, as these are also the characteristic propertiesof monosaccharides.l GLYCOSIDES Glycosidesare formed when the hemiacetal or hemiketal hydroxyl group (of anomeric carbon)of a carbohydratereactswith a hydroxyl group of another carbohydrate or a non- carbohydrate (e.g. methyl alcohol, phenol, glycerol). The bond so formed is known as glycosidic bond and the non-carbohydrate moiety (when present)is referredto as aglycone. The monosaccharidesare held together by glycosidic bonds to result in di-, oligo- or polysaccharides(seelaterfor structures). H-C=O I _ + HrN-NH-CuHu H-C-OH R Glucose Phenylhydrazine H-C:N-NH-CoHs I H-C-OH I R Glucohydrazone l7-H2N-NH-C6H' I H-C:N-NH-CoHs I C:N-NH-CoHs I R Glucosazone Fig.2.10: A summatyof osazonefomation H-C-OH C----r tlll H-C-OH Conc.HeSoo H-Q L I rH I U H-C-OH '1 H-C I CHrou 3H2o H-d---l D-Ribose Furfural Fig.2.9 : Dehydration of monosaccharides with concentrated H "SO o. Dehydration When treatedwith concentratedsulfuricacid, monosaccharidesundergodehydrationwith an eliminationof 3 water molecules.Thus hexoses give hydroxymethylfurfuralwhile pentosesgive furfural on dehydration (Fi9.2.9).Thesefurfurals can condense with phenolic compounds (a-naphthol)to form coloured products.This is the chemical basisof the popular Molisch test. In case of oligo- and polysaccharides,they are firsthydrolysedto monosaccharidesby acid,and this is followed by dehydration. Osazone formation Phenylhydrazinein acetic acid, when boiled with reducing sugars, forms osazones in a reactionsummarizedin Fig,2,10. As is evident from the reaction, the first two carbons (Cr and C2) are involved in osazone formation. The sugars that differ in their configuration on these two carbons give the same type of osazones,since the differenceis maskedby bindingwith phenylhydrazine.Thus glucose,fructoseand mannosegive the same type (needle-shaped)osazones. Reducingdisaccharidesalso give osazones- maltose sunflower-shaped,and lactose powder- puff shaped. (RrcprcsentsCrto Crofglucose). 24. t8 BIOCHEMISTRY Naming of glycosidic bond : The nomenclatureof glycosidic bonds is based on the Iinkagesbetweenthe carbon atomsand the status of the anomeric carbon (o or p). For instance,lactose-which is formed by a bond between C1 of p-galactoseand Ca of glucose- is namedas 0(.1-+ 4) glycosidicbond. The other glycosidicbonds are describedin the structure of di- and polysaccharides. Physiologieally important glycosides 1. Glucovanillin (vanillin-D-glucoside)is a naturalsubstancethat impartsvanilla flavour. 2. Cardiac glycosides(steroidalglycosides): Digoxin and digitoxin contain the aglycone steroidand they stimulatemusclecontraction. 3. Streptomycin, an antibiotic used in the treatmentof tuberculosisis a glycoside. 4. Ouabain inhibits Na+- K+ ATPase and blocksthe activetransportof Na+. DERIVATIVESOF MONOSACCHARIDES Thereare severalderivativesof monosaccha- rides, some of which are physiologically important 1. Sugar acids : Oxidation of aldehyde or primaryalcoholgroupin monosaccharideresults in sugaracids.Cluconic acid is producedfrom glucose by oxidation of aldehyde (C1 group) whereasglucuronicacid is formedwhen primary alcoholgroup (C6)is oxidized. 2. Sugar alcohols (polyols) : They are producedby reductionof aldosesor ketoses.For instance,sorbitol is formed from glucose and mannitol from mannose. 3. Alditols : The monosaccharides, on reduction,yield polyhydroxyalcohols,known as alditols. Ribitol is a constituent of flavin coenzymes; glycerol and myo-inositol are componentsof lipids.Xylitol is a sweetenerused in sugarlessgumsand candies. 4. Amino sugars : When one or more hydroxyl groups of the monosaccharidesare replaced by amino groups, the products formed are amino sugarse.g. D-glucosamine, D-galactosamine.They are present as consti- tuentsof heteropolysaccharides. The amino groups of amino sugars are sometimes acetylated e.g. N-acetyl D-gluco- samrne. N-Acetylneuraminic acid (NANA) is a derivativeof N-acetylmannoseand pyruvicacid. It is an important constituentof glycoproteins and glycolipids.The term sialicacid is usedto include NANA and its other derivatives. Certain antibiotics contain amino sugars which may be involvedin the antibioticactivity e.g. erythromycin. 5. Deoxysugars: These are the sugarsthat contain one oxygen lessthan that presentin the parent molecule. The groups -CHOH and -CH2OH become-CH2 and -CH3 due to the absenceof oxygen.D-2-Deoxyriboseis the most important deoxysugarsince it is a structural constituentof DNA (in contrastto D-ribose in RNA). 6. L-Ascorbic acid (vitamin C) : This is a water-solublevitamin, the structureof which closelyresemblesthat of a monosaccharide. The structuresof selected monosaccharide derivativesare depictedin Fig.2.l1. Among the oligosaccharides,disaccharides are the most common (Fig.2,l2).As is evident from the name, a disaccharideconsistsof two monosaccharideunits(similaror dissimilar)held together by a glycosidic hond. They are crystalline,water-solubleand sweetto taste.The disaccharidesare of two types '1. Reducingdisaccharideswith free aldehyde or keto group e.g. maltose, lactose. 2. Non-reducingdisaccharideswith no free aldehyde or keto group e.g. sucrose,trehalose. Maltose Maltose is composed of two a-D-glucose unitsheldtogetherby cl (1 -+ 4) glycosidicbond. Thefreealdehydegrouppresenton C1of second glucoseanswersthe reducingreactions,besides 25. Ghapter & : CAFIBOHYDRATES 19 H-C:O I H-C-OH I HO-C-H I H-C-OH I H-C-OH I COOH D-Glucuronicacid OHH D-2-Deoxyribose cH2oH I H-C-OH I cH2oH Glycerol H NHz D-Glucosamine HOH myo-lnositol H3C-C--HN HOH N-Acetylneuraminicacid Fiq.2.11 : Structuresol monosaccharidederivatives(selectedexamples). the osazone formations (sunflower-shaped). Maltosecan be hydrolysedby dilute acid or the enzyme maltaseto liberate two moleculesof cr-D-glucose. ln isomaltose,the glucose units are held togetherby o (1 --+6) glycosidiclinkage. Cellobioseis another disaccharide,identical in structurewith maltose,exceptthat the former has p (1 -r 4) glycosidiclinkage.Cellobioseis formedduringthe hydrolysisof cellulose. Suoroee Sucrose(canesugar)isthe sugarof commerce, mostlyproducedby sugarcane and sugarbeets. Sucrose is made up of a-D-glucose and p- D-fructose.The two monosaccharidesare held togetherby a glycosidicbond (a1-+ B2),between Cj of c-glucose and C2 of B-fructose.The reducing groups of glucose and fructose are involvedin glycosidicbond, hencesucroseis a non-reducing sugar,and it cannot form osazones. Sucroseis the major carbohydrateproduced in photosynthesis. lt is transported into the storageorgansof plants (such as roots, tubers and seeds).Sucroseis the mostabundantamong the naturallyoccurring sugars.lt has distinct advantagesover other sugarsas a storageand transoortform. This is due to the fact that in sucrose,both the functional groups (aldehyde and keto)are held togetherand protectedfrom oxidativeattacks. Sucrose is an important source of dietary carbohydrate.lt is sweeter than most other commonsugars(exceptfructose)namelyglucose, lactoseand maltose.Sucroseis employed as a sweeteningagentin food industry.The intestinal enzyme-sucrase-hydrolysessucroseto glucose and fructosewhich are absorbed. F-aetsse Lactoseis more commonlv known as milk sugarsinceit is the disaccharidefound in milk. Lactoseis composed ol p-D-galactoseand B-D- glucoseheld togetherby 0 (1 -r a) glycosidic bond.The anomericcarbonof C1glucoseis free, hence lactoseexhibits reducing propertiesand formsosazones(powder-puffor hedgehogshape). Lactose of milk is the most important carbohydratein the nutritionof young mammals. It is hydrolysedby the intestinalenzyme lactase to glucoseand galactose. lnversion ef suerose Sucrose,as such is dextrorotatory(+66.5o). But, r,r,hen hydrolysed, sucrose becomes levorotatory(-28.2"). The processof change in optical rotation from dextrorotatory (+) to levorotatory(-) is referredto as inversion.The 26. BIOCHEMISTF|Y HOH Glucose Fructose Sucrose (a-D-glucosyl(1 --+2)p-D-fructose) Galactose Lactose (p-D-galactosyl(1 -+ a)p-D-glucose) Fig. 2.12 : Structures of disaccharides -maltose, sucrose and lactose. hydrolysed mixture of sucrose, containing gfucoseand fructose, is known as invert sugar. The processof inversionis explainedbelow. Hydrolysisof sucroseby the enzyme sucrase (invertasdor dilute acid liberatesone molecure each of glucoseand fructose.ft is postulatedthat sucrose (dextro) is first split into a-D- glucopyranose(+52.5") and p-D-fructofuranose, both being dextrorotatory. However, p-D- fructofuranoseis lessstableand immediatelygets converted to p-D-fructopyranose which is stronglylevorotatory(-92"). The overalleffectis that dextro sucrose (+66.5") on inversion is converted to levo form (28.2'. Polysaccharides(or simply glycans)consistof repeat units of monosaccharides or their derivatives,held togetherby glycosidicbonds. Theyareprimarilyconcernedwith two important functions-structural,and storageof energy. Polysaccharides are linear as well as branched polymers. This is in contrast to structureof proteinsand nucleicacidswhich are only linear polymers. The occurrence of branchesin polysaccharidesis due to the fact that glycosidic linkagescan be formed at any one of the hydroxylBroupsof a monosaccharide. Polysaccharidesare of two types 1. Homopolysaccharideswhich on hydrolysis yield only a singletype of monosaccharide.They are named based on the nature of the monosaccharideunit. Thus,glucans arepolymers of glucose whereas fructosans are polymers of fructose. 2. Heteropofysaccharideson hydrolysisyield a mixture of a few monosaccharidesor their derivatives. $tarch Starch is the carbohydrate reserveof plants which is the most importantdietary sourcefor higheranimals,includingman. High contentof starchis found in cereals,roots,tubers,vegetables etc. Starch is a homopolymer composed of D-glucoseunits held by a-glycosidicbonds. lt is known as glucosan or glucan. Starch consists of two polysaccharide components-water soluble amylose (15-20o/ol and a water insoluble amylopectin (80-85%). Chemically, amylose is a long unbranched chain with 200-1,00OD-glucoseunitsheld by c (1 + 4) glycosidiclinkages.Amylopectin,on the other hand, is a branchedchain with a (1 --r 6t glycosidicbondsat the branchingpointsand c (1 -; 4) linkages everywhere else (Fig.2.13). Amylopectin molecule containing a few 27. ChapteF 2 : CARBOHYDFATES 21 D-Glucose D-Glucose Amylopectin o-Amylose +- (1-* 6) Branch MainchainLg 6nu vt t2 thousandglucoseunits looks like a branched tree (20-30 glucoseunits per branch). Starches are hydrolysed by amylase (pancreaticor salivary)to liberatedextrins,and finally maltoseand glucoseunits.Amylaseacts specificallyon a (1 -+ 4) glycosidicbonds. Dextrins Dextrins are the breakdown products of starchby the enzyme amylaseor dilute acids. Starch is sequentially hydrolysed through different dextrins and, finally, to maltose and glucose.The variousintermediates(identifiedby iodine colouration) are soluble starch (blue), amylodextrin (violet), erythrodextrin (red) and achrodextrin (no colour). Inulin fnulin is a polymerof fructosei.e., fructosan. It occursin dahlia bulbs,garlic,onion etc. lt is a low molecularweight (around5,000) poly- saccharideeasilysoluble in water. Inulin is not utilized by the body. lt is used for assessing kidney function through measurement of glomerular filtration rate (GFR). Glycogen Clycogen is the carbohydrate reserve in animals,henceoften referredro asanimal starch. It is present in high concentration in liver, followed by muscle,brainetc.Clycogenis also found in plantsthat do not possesschlorophyll (e.9.yeast,fungi). The structureof glycogenis similarto that of amylopectin with more number of branches. Glucoseis the repeatingunit in glycogenjoined togetherby u (1 + 4) glycosidicbonds,and a (1 + 6) glycosidic bonds at branching points (Fi9.2.1Q.The molecularweight (up to 1 x 108) and the numberof glucoseunits (up to 25,000) vary in glycogendependingon the sourcefrom which glycogenis obtained. 28. 22 BIOCHEMISTRY Fiq.2.14: Structureofglycogen(A)Generalstructure (B)Enlargedat a branchpoint. Cellulose Celluloseoccursexclusivelyin plantsand it is the most abundant organic substancein plant kingdom. lt is a predominantconstituentof plant cell wall. Celluloseis totally absent in animal body. Cellulose is composed of p-D-glucose units linked by 9 0 -+ 4) glycosidic bonds(Fi9.2.1fl. Cellulosecannot be digestedby mammals- includingman-due to lack of the enzymethat cleavesB-glycosidicbonds(a amylasebreakscr bondsonly). Certainruminantsand herbivorous animalscontainmicroorganismsin thegutwhich produce enzymesthat can cleave p-glycosidic bonds. Hydrolysis of cellulose yields a disaccharide cellobiose, followed by P-D- glucose. Cellulose, though not digested, has great importancein human nutrition. lt is a major constituentol fiber, the non-digestablecarbo- hydrate.The functions of dietary fiber include decreasing the absorption of glucose and cholesterolfrom the intestine,besidesincreasing the bulk of feces. (For details,Chapter 23) Ghitin Chitin is composed of N-acetyl D- glucosamineunits held togetherby F (1 -+ a) glycosidicbonds.lt isa structuralpolysaccharide found in the exoskeletonof some invertebrates e.g. insects,crustaceans. When the polysaccharidesare composedof differenttypesof sugarsor their derivatives,they are referred to as heteropolvsaccharidesor heteroglycans. MUCOPOLYSACCHARIDES Mucopolysaccharidesare heteroglycansmade up of repeatingunitsof sugarderivatives,namely amino sugarsand uronic acids.Theseare more commonly known as glycosaminoglycans (GAG).Acetylatedamino groups,besidessulfate and carboxyl groups are generally present in CAC structure.The presenceof sulfate and carboxyl groups contributesto acidity of the molecules, making them acid mucopoly,- saccharides. Someof the mucopolysaccharidesare found in combination with proteins to forrn mucoproteins or mucoids or proteoglycans (Fig.2.l6l.Mucoproteinsmay containup to 95o, carbohydrate and 5o/"protein. S-D-Glucose T N T Ot (B) 9H2OH uqt, CH2oH y'-O., , F--o. ,r4-Or - (+ i) - r+ ,L^_K. X^_-oJ, ,./'o-'- J - L-/ " --l Fig. 2.15 : Structureof cellulose(The repeat:r; -- ' may be several thousands). 29. CARBOHYDRATES 23 Fig. 2.16 : Diagrammaticrepresentationof a prateoglycan complex. Mucopolysaccharidesareessentialcomponents of tissue structure.The extracellularspacesof tissue (particularlyconnective tissue-cartilage, skin, blood vessels,tendons)consistof collagen and elastinfibersembeddedin a matrixor ground substance.Thegroundsubstanceis predominantly composedof CAC. The importantmucopolysaccharidesincluoe hyaluronicacid, chondroitin4-sulfate,heparin, dermatansulfateand keratansulfate(Fig.Z.'[1. j'i' ,ir:r:, | '.i. :,{,tiiiEl'l Hyaluronicacid is an importantGAC found in the groundsubstanceof synovialfluid of joints and vitreoushumor of eyes.it is also presentas a ground substancein connectivetissues,and forms a gel aroundthe ovum. Hyaluronicacid servesas a lubricantand shock absorbantin joints. BToMEDtCAt/ CLtft|ICALCO$CEpTS Hyaluronicacid rlr Glucose is the most important energy sourceol carbohgdratesto the mammals (except ruminants).The bulk of dietary carbohydrote(starch)is dlgestedond finally obsorbedas glucose into the body. Ea Dextrose (glucosein solution in dextrorotatory form) is frequently used in medical Rq'- CF practice. Fructoseis obundantly found in the semen which is utilized by the spermsfor energy. Seueral diseoses are associated with carbohydrate.se.g., diabetes mellitus, glycogen storage diseoses,galactosemia. trs Accumulation of sorbitol and dulcitol in the fissuesmoy cause certoin pathological conditionse.g. cotaract, nephropothy. t-s' Inulin, a polymer of t'ructose,is used fo ossessrenal function by meosuringglomerular filtration rate (GFR). ue The non-digestiblecarbohydratecellulose playsa signilicant role in human nutriticsn. These include decreasing the intestinal absorption ol glucose and cholesterol, qnd increasingbulk of feces to ouoid eonstipation. rt The mucopolysaccharidehyaluronic acid seruesas a lubricant and shock absorbantin ioints. The enzgmehgaluronidaseof semendegradesthe gel (contains hyaluronic acid)around the ouum. This qllows eft'ectiuepenetration of sperm into the ouum. The mucopolysaccharideheparin is an onticoagulant(preuentsblood clotting). The suruiual of Antarctic lish below -2"C is attributed to the antit'reeze glycoproteins. streptomycin is a glycosideemployed in the treatment oJ tuberculosis. !3:. [j- IF s: -;-- s' -sS't/:- -'- 30. 24 BIOCHEMISTFIY Hyaluronic acid is composed of alternate units of D-glucuronic acid and N-acetyl D-glucosamine.These two molecules form disaccharideunits held togetherby 0 (t -+ S) glycosidic bond (Fi9,2,15).Hyaluronic acid containsabout 250-25,000 disaccharideunits (heldby p 1 -+ 4 bonds)with a molecularweight uo to 4 million. Hyaluronidase is an enzyme that breaks (B1 -+ 4 linkages)hyaluronic acid and other CAC. This enzyme is present in high concentrationin testes,seminalfluid, and in certainsnakeand insectvenoms.Hyaluronidase of semen is assignedan important role in fertilization as this enzyme clears the gel (hyaluronicacid) around the ovum allowing a better penetration of sperm into the ovum. Hyaluronidaseof bacteriahelps their invasion into the animaltissues. Ghondroitin sulfates Chondroitin 4-sulfate (Greek: chondros- cartilage) is a major constituent of various mammalian tissues(bone, cartilage,tendons, heart,valves,skin,corneaetc.).Structurally,it is comparablewith hyaluronicacid. Chondroitin 4-sulfateconsistsof repeatingdisaccharideunits composedof D-glucuronicacid and N-acetyl D-galactosamine4-sulfate(Fig.2.lV. Chondroitin5-sulfateis alsopresentin many tissues.As evident from the name, the sulfate group is found on C6 insteadof Ca. Heparin Heparin is an anticoagulant(preventsblood clotting)thatoccursin blood,lung,liver,kidney, spleenetc. Heparin helps in the releaseof the enzyme lipoprotein lipase which helps in clearingthe turbidityof lipemic plasma. Heparin is composedof alternatingunits of N-sulfoD-glucosamine6-sulfateand glucuronate 2-sulfate(Fi9.2.17). Dermatan sulfate The name dermatansulfateis derived from the fact that this compoundmostlyoccursin the skin. lt is structurallyrelated to chondroitin D-Glucuronicacid N-Acetylglucosamine Hyaluronic acid H NH-CO-CH3 N-Acetylgalactosamine 4-sulfate Chondroitin 4-sulfate o- D-Glucuronate-2-sulfateN-Sulfoglucosamine 6-sulfate Heparin t -o'r H NH_CO_CH. N-Acetylgalactosamine 4-sulfate Dermatansulfate H NH_CO :- N-Acetylglucosamine 6-sulfate Keratansulfate Fiq.2.17 : Structuresof commonglycosaminogi',-;-: - D-Glucuronicacid H O-SO; -o-so3 H NH-SOa qH2oH o the disaccharidesas repeatingunits. 31. Ghapter 2 : CAFIBOHYDHATES 25 Glycosaminoglycan Composition Tissuedistribution Function(s) Hyaluronicacid D-Glucuronicacid, N-acetylglucosamine Connectivetissue,synovialfluid, vitroushumor Servesasalubricant.and shockabsorber.Promotes woundhealing Chondroitinsulfate D-Glucuronicacid, N-acetylgalactosamine 4-sulfate Cartilage,bone,skin,bloodvessel walls Helpstomaintainthestructure andshapesoftissues Heparin D-Glucuronate2-sulfate,Blood,lung,liver,kidney,spleen N-sulfoglucosamine 6-sulfate Actsasananticoagulant Dermatansulfate L-lduronicacid,N-acetyl- galactosamine4-sulfate Bloodvesselvalves,heartvalves, Maintainstheshapesoftissues skin Keratansulfate D-Galactose,N-acetyl- glucosamine6-sulfate Cartilage,cornea,connective tissues Keepscorneatransparent 4-sulfate.The only differenceis that there is an inversion in the configuration around C5 of D-glucuronic acid to form L-iduronic acid (Fi9.2.1V. Keratan sulfate It is a heterogeneousCAG with a variable sulfate content, besides small amounts of mannose, fructose, sialic acid etc. Keratan sulfateessentiallyconsistsof alternatingunitsof D-galactosamine and N-acetylglucosamine 6-sulfate. A summaryof the glycosaminoglycanswith regardto composition,distributionand functions is given in Table 2.3. Several proteins are covalently bound to carbohydrateswhich are referredto as glyco- proteins. The carbohydrate content of glycoproteinvariesfrom 1o/oto 90o/oby weight, Sometimes the term mucoprotein is used for glycoprotein with carbohydrateconcentration more than 4"/o. Clycoproteins are very widely distributedin the cells and perform variety of functions.Theseincludetheir role as enzymes, hormones,transportproteins,structuralproteins and receptors.A selectedlist of glycoproteins and their major functionsis given in Table2.4. The carbohydratesfound in glycoproteins include mannose, galactose, N-acetyl- glucosamine, N-acetylgalactosamine,xylose, L-fucoseand N-acetylneuraminicacid (NANA). NANA is an importantsialicacid (SeeFig.2,l1). Antifreeze glycoproteins : The Antarctic fish live below -2oC, a temperatureat which the Glycoprotein(s) Major function(s) Collagen Hydrolases,proteases, glycosidases Ceruloplasmin lmmunoglobulins Synovialglycoproteins Thyrotropin,erylhropoietin Bloodgroupsubstances Fibronectin,laminin Intrinsicfactor Fibrinogen Structure Enzymes Transport Defenseagainstinfection Lubrication Hormones Antigens Cell-cellrecognitionand adhesion Absorptionofvitamin8,, Bloodclotting 32. 26 ElIOCHEMISTF|Y blood would {reeze.lt is now known that ihese fish contain antifreezeglycogtrateinwhich lower the freezingpoint of waterand interferewith tne crystalformationof ice. Antifreezegiycoproteins consistof 50 repeatingunits of the tripeptide, alanine-alawine-threonine. Each threonine residue is bound to B-galactosyl(1 + 3) o( N-acetylgalactosamine. ri#i .f*iCA# '? r,.4F.!"r.Ii $:F"r1.*fi { "'.3 i4 t: * :il The blood group antigens (of erythrocyte membrane) contain carbohydratesas glyco- proteinsor glycolipids.N-,A.cetylgaiactosamine, galactose,fucose,sialic acid etc. are found in the blood group substances.The carbohydrate contentalso playsa determinantrole in blood Eroup!n8. X. Carbohydrs,tesare the polyhydroxyaldehydesor ketones,or campounds whichproduce them on hydrolysis.The term sugor is applied to carbohydratessoluble in water and stDeetto taste. Carbahgdratesqre the major dietary energy sources,besidestheir inualuementin cell structure and uariousother t'unctions. 2. Carbohydrqtesare broadly c/ossiJiedinta 3 groups-ffionasqccharides,oligosoccharides and ytoiysaccharides.The monosacchsridesare further diuided into dit't'erentcategories bqsedan the presenceaf t'wnctionalgroups {oldosesar ketoses)and the number of carbon atoms (trioses,tetroses,pentases,hexosesand heptcses). 3. Glyceraldehyde{triose) is the simplestcarbohydrateand is chosen as a reJerenceto write the cont'iguratian of all other rnonasaccharides(D- anc L- forms). It' two rnonosaccharidesdiffer in their structurearound o singlecarbonatom, they ore known as eplmers.Glucoseand galactoseare C4-epimers. 4. D'Glucose is the most predominant naturally occurring aldosdmonosaccharide. Giucoseexisfscs a and p anemerswith dit'Jerentopticalrotations. The interconuersion of a and B anomericforms with changein theopticalrotatianis knoun asmutsratation. 5. Manosaccharidespariicipate in seuercl recctions"Theseinclude oxidation, reduction. dehydration, asazone formetion etc. Formatian ol esters and glycosides by manosacchqridesis af specialsignificanceln biochemical reactions. 6. Among the oligosacchqrides,disoccharidesare the most common. Theseinclude the reducing disaccharidesnamely lactose(rnilk sugar)and maltase(malt sugar)and the non-reducingsucrose(canesugar). 7. Palysacclwridesare the poiymersot' monosaccharidesor their deriuatiues,held together by glycosidic bonds.Homopalysaccharidessre compasedot' a single manosaccharicle (e.g., starch,glycogen,cellulose, inulin). Heteropolysaccharidescontain a mixture af Jew monasaceharidesor thetr derluatiues(e.g., rnucapolysacaharides). 8. Slorch and glgcogensre the carbohydratereseruesot' plants and animalsrespectiuelg. Cellulose,exclusiuelyt'ound in plants, is the structural constituent.Inulin is utilized to ossesskidney tunction bg measuringglomerular t'iltration rate (GFR). 9. Mucopoiysaccharides(glycosominoglycans)are the essential companents o/ tlssue structure. Theyprouide the mstrix or grownd substanceof extracellular tissuespacesin whtchcollagenand elastinfibers are embedded.Hyaluranic ocid,chondroitin 4'sult'ote, heporin, are amang the important glycosaminaglgcdns. 70. Glycoproteins are a group of biochernically important compaunds with a uariable composition of carbohyd.rate(7-900/o),caualently bound to protein. Seueralenzyrnes, hormanes,structura! proteinsand cellular receptorsare in fact glycoproteins. 33. Ghapter 2 : CAFIBOHYDHATES I. Essayquestions 1. Define and classifycarbohydrateswith suitableexamples.Add a note on the functionsof carbohydrates. 2. Describethe structureand functionsof mucopolysaccharides. 3. Cive an accountof the structuralconfigurationof monosaccharides,with specialreferenceto glucose. 4. Discussthe structureand functionsof 3 biochemicallyimportantdisaccharides. 5. Definepolysaccharidesand describethe structureof 3 homopolysaccharides. Short notes (a)Epimers,(b)Mutarotation,(c)Osazoneformation,(d)Clycosidicbond,(e)Sugarderivatives,(fl Anomers,(g)Enediol,(h)Amino su8ars,(i) Inversionof sucrose,(j) Deoxysugars. Fill in the blanks 1. Namea non-reducingdisaccharide 2. The carbohydratethat is taken as a referencefor writing the configurationof others 3. lf two monosaccharidesdifferin configurationarounda singlecarbonatom,they are known as 27 II. III. 4. 5. 6. 7. B. 9. 10. The s and B cyclicformsof D-glucoseare referredto as The non-carbohydratemoietyfound in glycosidesis known as Cive an exampleof a glycosideantibiotic Theglycosidicbondsat the branchingpointsin the structureof starchare The polysaccharideemployedfor the assessmentof kidneyfunction The glycosaminoglycanthat servesas a lubricantand shockabsorbantof joints Namethe sialicacid,mostlyfound in the structureof glycoproteinsand glycolipids IV. Multiple choice questions 11. Riboseand deoxyribosedifferin structurearounda singlecarbon,namely (a)Cr (b)Cz (c)C: (d)Cq. 12. One of the followingis not an aldose (a)Clucose(b)Calactose(c) Mannose(d) Fructose. 13. The glycosaminoglycanthat servesas an anticoagulant (a) Heparin(b) Hyaluronicacid (c)Chondroitinsulfate(d) Dermatansulfate. 14. The followingpolysaccharideis composedof B-glycosidicbonds (a)Starch(b)Clycogen(c) Dextrin(d)Cellulose. 15. The carbonatomsinvolvedin the osazoneformation (a)'l and 2 (b) 2 and 3 (c) 3 and 4 (d) 5 and 6. 34. Lirpirdls fl ?"'-o- i R--c-o1H frCH2-H R3 The Jat speaks : "ffith uater, I say, 'Touch menot': T'otlte tongue,I am tasteful; IY'ithin limits, I am datiful; fn excess,I am dangerous!" I ipids (Creek: lipos-fat) are of Breat L importance to the body as the chief concentratedstorageform of energy, besides their role in cellularstructureand variousother biochemicalfunctions.As such. lioids are a heterogeneous group of compounds ano, therefore,it is rather difficult to define them preciselv. Lipidsmay be regarded as organic substances relatively insoluble in water, soluble in organic solvents (alcohol, ether etc.), actually or potentially related to fatty acidsand utilized by the living cells. Unlike the polysaccharides,proteins and nucleic acids,lipids are not polymers.Further, lipidsare mostlysmall molecules. Lipids are broadlyclassified(modifiedfrom Bloor) into simple, complex, derived and miscellaneouslipids,whicharefurthersubdivided into differentgroups 1. Simple lipids: Estersof fatty acids with alcohols.Theseare mainly of two types (a) Fatsand oils (triacylglycerols): Theseare estersof fatty acids with glycerol. The difference between fat and oil is only physical.Thus,oil is a liquid while fat is a solid at room temperature. (b) Waxes: Estersof fattyacids(usuallylong chain)with alcoholsotherthan glycerol. These alcohols may be aliphatic or alicyclic.Cetylalcoholis mostcommonly found in waxes. 2. Complex(or compound)lipids: Theseare estersof fatty acids with alcohols containing additional groups such as phosphate, nitrogenousbase, carbohydrate,protein etc They are furtherdividedas follows (a) Phospholipids:They containphosphor,c acid and frequentlya nitrogenousbase This is in addition to alcohol and fai:. acids. 28 35. Chapter 3 : LIPIDS 29 (i) Glycerophospholipids: Thesephospho- lipids containglycerolas the alcohol e.9.,lecithin,cephalin. (ii) Sphingophospholipids: Sphingosineis the alcohol in this group of phospho- lipidse.g.,sphingomyelin. (b) Glycolipids: Theselipids contain a fatty acid, carbohydrateand nitrogenousbase. The alcohol is sphingosine,hence they are also called as glycosphingolipids. Clycerol and phosphateare absente.g., cerebrosides,gangliosides. (c) Lipoproteins: Macromolecularcomplexes of lipids with proteins. (d) Other complexlipids: Sulfolipids,amino- lipidsand lipopolysaccharidesareamong the othercomplex lipids. 3. Derived lipids: Theseare the derivatives obtainedon the hydrolysisof group 1 and group 2lipids which possessthe characteristicsof lipids.Theseincludeglycerolandotheralcohols, fatty acids,mono- and diacylglycerols,lipid (fat) soluble vitamins, steroid hormones, hydro- carbonsand ketonebodies. 4. Miscellaneouslipids: These include a large number of compounds possessingthe characteristics of lipids .g., carotenoids, squalene,hydrocarbonssuch as pentacosane(in bees wax), terpenesetc. NEUTRAT LIPIDS: The lipids which are unchargedare referredto asneutrallipids.These are mono-, di-, and triacylglycerols,cholesterol and cholesterylesters. Functions of lipids Lipids performseveralimportantfunctions 1. They are the concentratedfuel reserveof the body (triacylglycerols). 2. Lipids are the constituentsof membrane structure and regulate the membrane permeability(phospholipidsand cholesterol). 3. They serve as a source of fat soluble vitamins(4, D, E and K). 4. Lipidsare importantas cellularmetabolic regulators(steroidhormonesand prostaglandins). 5. Lipidsprotectthe internalorgans,serveas insulatingmaterialsand give shapeand smooth appearanceto the body. Fatty acids are carboxylic acids with hydrocarbonside chain. They are the simplest form of lipids. Occurrence Fattyacidsmainly occur in the esterifiedform as major constituentsof variouslipids.They are also present as free (unesterified)fatty acids. Fattyacidsof animalorgin are much simplerin structure in contrast to those of plant origin which oftencontaingroupssuch asepoxy,keto, hydroxy and cyclopentanerings. Even and odd carbon fatty acids Most of the fatty acids that occur in natural lipids are of even carbons(usually 14C-2OC). This is due to the fact that biosynthesisof fatty acidsmainly occurswith the sequentialaddition of 2 carbon units. Palmitic acid (l6C) and stearicacid (l$C) are the most common. Among the odd chain fatty acids, propionic acid (3C) and valericacid (5C)are well known. Saturated and unsaturated fatty acids Saturatedfatty acids do not contain double bonds,while unsaturatedfattyacidscontainone or more double bonds. Both saturated and unsaturatedfatty acids almost equally occur in the natural lipids. Fatty acids with one double bond are monounsaturated,and thosewith 2 or more double bonds are collectivelv known as polyunsaturated fafty acids (PIJFA). Nomenclature of fatty acids The namingof a fatty acid (systematicname) is basedon the hydrocarbonfrom which it is derived. The saturatedfatty acids end with a suffix -anoic (e.g., octanoic acid) while the unsaturatedfatty acids end with a suffix -enoic 36. 30 BIOCHEMISTF|Y (e.9., octadecanoic acid). In addition to systematicnames/ fatty acids have common nameswhich are more widely used (Iable J. l). Numbering of carbon atoms : lt startsfrom the carboxylcarbonwhich is takenas number1. The carbonsadjacentto this (carboxylC) are2, 3, 4 and so on or alternatelya, F, T and so on. The terminalcarbon containingmethyl group is known omega (or) carbon. Starting from the methylend, the carbonatomsin a fattyacid are numberedas omega 1, 2, 3 etc. The numbering of carbon atoms in two different ways is given below 7654321 cH3 - cH2 - cH2- cH2-cH2 - cH2 - COOH 01 a2 o)3 ()4 ol5 (t)6 Length of hydrocarbon cha:n of fatty acids Dependingon the length of carbon chains, fatty acids are categorizedinto 3 groups-short chain with less than 6 carbons; medium chain with 8 to 14 carbons and long cfiain with 16 to 24 carbons. Shorthand representation of latty aclds lnstead of writing the full structures, biochemists employ shorthand notations (by numbers)to representfatty acids. The general rule is that the total numberof carbonatomsare written first,followed by the nunrberof double bonds and finally the (firstcarbon) position of Common Name Systematicname Abbreviationx Structure l.Saturatedfattyaclds Aceticacid Propionicacid Butyricacid Valericacid Caproicacid Caprylicacid Capricacid Lauricacid Myristicacid Palmiticacid Stearicacid Arachidicacid Behenicacid Lignocericacid Ethanoicacid n-Propanoicacid n-Butanoicacid n-Pentanoicacid n-Hexanoicacid n-Octanoicacid n-Decanoicacid n-Dodecanoicacid n-Tetradecanoicacid n-Hexadecanoicacid n-Octadecanoicacid n-Eicosanoicacid n-Docosanoicacid n-Tetracosanoicacid CHsCO0H CHgCHzCOOH CHs(CHz)z0O0H CHo(CHz)gCOOH CHs(CHe)+COOH CHe(CHz)oCOOH CHs(CHz)eC0OH CHs(CHz)roCOOH CHs(CHzhzCOOH CHg(CHz)t+CO0H CHs(CHz)roC0OH CHg(CHz)reCOOH CHs(CHz)zo00OH CH3(CHz)zzCOOH 2:0 3:0 4:0 F.n 6:0 8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0 ll. Unsaturatedfattyacids Palmitoleicacid Oleicacid Linoleicacid** Linolenicacid*x Arachidonicacid cr1s9-Hexadecenoicacid cls-9-Octadecenoicacid cls,cls-9,12-Octadeca- dienoicacid Allce9,12,15-0cta- decatrienoicacid Allcls-5,8,11,14- 16:1;9 18:1;9 18:2;9,12 18:3;9,12,'15 20:4;5,8,11,14 CHg(CHz)sCH=CH(CHz)zCOOH CHs(CHz)zCH=CH(CHz)zCOOH CHg(CHz)+CH=CHCHzCH=CH(CHz)zCOOH CHoCHzCH=CHCHzCH=CHCHzCH =CH(CHz)zCO0H CHg(CHz)+CH=CHCHzCH=CHCHzCH Elc0:a!tr3e!o!1ci1___ __=9H9'tcl=_cl9F!)49oli * Totalnunberofcarbonatons,followedbythenumberotdoublebondsandthefirctcarbonposrtionotthedoublebond(s). ** Essentialfawacids. 37. Ghapten 3 : LIPIDS 31 double bonds, startingfrom the carboxyl end. Thus,saturatedfattyacid, palmiticacid iswritten as.l 6:0, oleic acid as 18:1;9, arachidonic acid as 20 : 4; 5, 8, 11, 14. There are other conventionsof representing the double bonds.Ae indicatesthat the double bond is between9 and 10 of the fatty acid. o 9 representsthe double bond position(9 and 10) from the CH3-(CH2)12-CH:CH-CH-?H-NH-C-R ' , *.CHg r_-CHz-CHz-Nf9,Tt Choline 'n3 (8)Sphlngomyelln Fig. 3.3 : Sttucturesof phospholipids. 42. 36 BIOCHEMISTF|Y (a) Dipalmitoyl lecithin is an important phosphatidylcholinefoundin lungs,lt isa surface active agent and prevents the adherence of inner surface of the lungsdue to surfacetension.Respiratory distresssyndromein infantsis a disorder characterizedby the absenceof dipalmitoyl lecithin. (b) Lysolecithinis formed by removalof the fatty acid either at C, or C, of lecithin. 3. Cephafins (phosphatidylethanolamine): Ethanolamineis the nitrogenousbasepresentin cephalins,Thus,lecithinandcephalindifferwith regardto the base. 4. Phosphatidylinositol: The steroisomer myo-inositolis attachedto phosphatidicacid to givephosphatidylinositol(Pl).Thisisan important comDonentof cell membranes.The action of certain hormones(e.9.oxytocin, vasopressin)is mediatedthroughPl. 5. Phosphatidylserine:The amino acid serineis presentin this group of glycerophos- pholipids.Phosphatidylthreonineis alsofound in certaintissues. 6. Plasmalogens: When a fatty acid is attachedby an etherlinkageat C1 of glycerolin the glycerophospholipids, the resultant compound is plasmalogen. Phosphatidal- ethanolamineis the most imoortantwhich is similarin structureto phosphatidylethanolamine but for the ether linkage(in place of ester).An unsaturatedfatty acid occurs at C1. Choline, inositoland serinemay substituteethanolamine to give other plasmalogens. Z. Cardiolipin: lt is so named as it was first isolated from heart muscle. Structurally, a cardiolipin consists of two molecules of phosphatidicacid held by an additionalglycerol through phosphategroups. lt is an important componentof inner mitochondrialmembrane. Cardiolipin is the only phosphoglyceridethat possessesantigenic properties. Sphingomyelins Sphingosineis an amino alcohol presentin sphingomyelins(sphingophospholipids).They do notcontainglycerolat all. Sphingosineisattached by an amide linkageto a fatty acid to produce ceramide.The alcohol group of sphingosineis bound to phosphorylcholinein sphingomyelin structure(Fig.3.3).Sphingomyelinsare important constituentsof myelin and are found in good quantityin brain and nervoustissues. Action of phospholipases Phospholipasesare a group of enzymesthat hydrolysephospholipids.Thereare four distinct phospholipases(Ar, 42, C and D), eachone of them specificallyactson a particularbond. For details,refer lipid metabolism(Chapter l4). Functions of phospholipids Phospholipidsconstitutean importantgroup of compound lipids that performa wide variety of functions 1. In associationwith proteins,phospholipids form the structural components of membranes and regulatemembranepermeability. 2. Phospholipids (lecithin, cephalin and cardiolipin)in the mitochondriaare responsible for maintaining the conformation of electron transportchain components,and thus cellular respiration. 3. Phospholipidsparticipatein the absorption of fat from the intestine. 4. Phospholipids are essential for the synthesisof different lipoproteins,and thus participate in the transport of lipids. 5. Accumulationof fat in liver(fattyliver)can be preventedby phospholipids,hence they are regarded as lipotropic factors. 6. Arachidonicacid,an unsaturatedfattyacid liberated from phospholipids, serves as a precursorfor the synthesisof eicosanoids(prosta- glandins,prostacyclins,thromboxanesetc.). 7. Phospholipidsparticipatein the reverse cholesteroltransport and thus help in the removalof cholesterolfrom the body. 8. Phospholipidsact as surfactants(agenL. lowering surface tension). For instance dipalmitoylphosphatidylcholineis an importar: fung surfactant. Respiratory distresssyndrome ^ infantsis associatedwith insufficientproductio^ of this surfactant. 43. Chapter 3 r LIPIDS 37 Sphingosine loHloYIY] o-cH2 Fig. 3.4 : Structure ot galactosylceramide(R = H). Fot sulfagalactosylceramideR is a sulfatide (R = SOi-). 9. Cephalins,an importantgroupof phospho- lipids participatein blood clotting. 10. Phospholipids(phosphatidylinositol)are involvedin signaltransmissionacrossmembranes. ClycoIipids (glycosphingol ipids)are important constituentsof cell membrane and nervous tissues(particularlythe brain). Cerebrosidesare the simplestform of glycolipids.Theycontaina ceramide(sphingosineattachgdto a fatty acid) and one or more sugars.Galactocerebroside (galactosylceramide)and glucocerebrosideare the most importantglycolipids.The structureof galactosylceramideis givenin Fig3.a. lt contains the fatty acid cerebronic acid. Sulfagalactosylceramideis the sulfatide derived from galactosylceramide. Gangliosides: Theseare predominantlyfound in ganglionsand are the most complex form of glycosphingolipids.They are the derivativesof cerebrosidesand containone or moremolecules of N-acetylneuraminicacid (NANA), the most imoortantsialicacid. The structureof NANA is givenin carbohydratechemistry(ReferFig.2.l1. The most important gangliosidespresentin the brain are CM1, CM2, CD, and CT, (G representsgangliosidewhile M, D and T indicate rnono-, di- or tri- sialic acid residues, and the number denotes the carbohydrate sequence attached to the ceramide). The ganglioside,CM2 that accumulatesin Tay-Sachs diseaseis reoresentednext (outlinestructure). Ceramide IGlucose f Galactos tl N-Acetyl- N-Acetyl- galactosamine neuraminicacid Lipoproteinsare molecular complexes of lipids with proteins. They are the transport vehiclesfor lipids in the circulation.Thereare five types of lipoproteins,namely chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density Iipoproteins (HDL) and free fatty acid-albumin complexes. Their structure, separation, metabolismand diseasesare discussedtogether (Chapter l4). Steroids are the compounds containing a cyclic steroid nucleus (or ring) namely cyclopentanoperhydrophenanthrene (CPPP). lr consistsof a phenanthrenenucleus(ringsA, B and C) to which a cyclopentanering (D) is attached. The structure and numbering of CPPP are shown in Fi9.3.5.The steroidnucleusrepresents saturatedcarbons,unlessspecificallyshown as double bonds.The methyl side chains(19 and 44. 38 BIOCHEMISTF|Y Fig. 3.5 : Sttucturcs of steroids (A, B, C-Perhydro- phenanthrene; D-Cyclopentane). 18) attachedto carbons10 and 13 are shownas single bonds. At carbon 17, steroidsusually containa sidechain. There are severalsteroidsin the biological system.Theseinclude cholesterol,bile acids, vitamin D, sex hormones, adrenocortical hormones,sitosterols,cardiac glycosidesand alkaloids.lf the steroid,containsone or more hydroxyl groups it is commonly known as sterol (meanssolid alcohol). CI{OLESTEROL Chofesterol, exclusively found in animals, is the most abundantanimal sterol.lt is widely distributedin all cellsand is a major component of cell membranesand lipoproteins.Cholesterol (Creek: chole-bile)was first isolatedfrom bile. Cholesterolliterally means 'solid alcohol from bile.' Structure and occurrence The structure of cholesterol (C27Ha6O)is depictedin Fig.3.5.lt hasone hydroxylgroup at C3 and a double bond between C5 and C6. An 8 carbon aliphaticside chain is attachedto C17. Cholesterolcontains a total of 5 methyl Sroups. Due to the presence of an -OH group, cholesterolisweaklyamphiphilic.As a structural component of plasma membranes,cholesterol is an important determinant of membrane permeabilityr,properties. The occurrence of cholesterolis much higher in the membranesof sub-celIular organelles. Cholesterolis founi in associationwith fany acids to- form cholestervlesters(esterification occursat the OH group of C3). Properties and reactions: Cholesterol is an yellowish crystallinesolid. The crystals,under the microscope, show a notched (E) appearance.Cholesterolis insoluble in water and soluble in organic solvents such as chloroform,benzene,ether etc. Several reactions given by cholesterolare useful for its qualitative identification and quantitativeestimation.TheseincludeSalkowski's test, Liebermann-Burchardreaction and Zak's test. Functions of cholesterol: Cholesterolis a poor conductorof heat and electricity,since it has a high dielectric constant.lt is presentin abundance in nervous tissues.lt appearsthat cholesterolfunctionsas an insulatingcover for the transmissionof electricalimpulsesin the nervous tissue. Cholesterol performs several other biochemicalfunctionswhich include its role in membranestructureand function, in the synthesisof bile acids, hormones (sex and cortical) and vitamin D (for details, Refer Chapters 7 and l9). ERGOSTEROL Ergosteroloccursin plants.lt is alsofound as a structuralconstituentof membranesin yeast and fungi. Ergosterol(Fig.3.5)is an important precursorfor vitamin D. When exposedto light, 45. fTi Cfrraptee3 : LIPIDS 39 li I cH3(cHdn-coo- Hydrophobic Hydrophiic hydrocarbonchain carboxylgroup (tail) (head) (A) Fatty acid the ring B of ergosterolopensand it is converted to ergocalciferol, a compound containing vitamin D activity. Theothersterolspresentin plantcellsinclude stigmasterol and ftsitosterol. (C)Amphipathic lipid otl Hydrophobic HYdroPhilic tail head (B) Phospholipid As per definition,lipidsare insoluble(hydro- phobic) in water. This is primarilydue to the predominantpresenceof hydrocarbongroups. However, some of the lipids possesspolar or hydrophilicgroupswhich tend to be solublein water. Molecules which contain both hydrophobicand hydrophilicgroupsare known as amphipathic (Creek : amphi-both, pathos- passion). Examplesof amphipathiclipids: Among the lipids,fatty acids,phospholipids,sphingolipids, bile salts and cholesterol(to some extent)are amphipathicin nature. Phospholipidshavea hydrophilichead(phos- phategroup attachedto choline, ethanolamine, inositol etc.) and a long hydrophobictail. The generalstructureof an amphipathicIipid may be representedas a polar or hydrophilicheadwith a non-polaror hydrophobictail (Fig.3.6). Fattyacidscontaina hydrocarbonchain with a carboxyl (COO-) group at physiologicalpH. The carboxyl group is polar in nature with affinityto water (hydrophilic)while hydrocarbon chain of fatty acid is hydrophobic. Orientation amphipathic lipids: When the amphipathiclipids are mixed in water (aqueous phase), the polar groups (heads) orient themselvestowards aqueousphase while the non-polar (tails) orient towards the opposite directions.This leadsto the formation of mr'celles (Fi9.3.6).Micelle formation, facilitatedby bile salts is very important for lipid digestion and absorption (Chapter 8). Me*nhrane bilayers In caseof biologicalmembranes,a bilayerof lipidsis formedorientingthe polar headsto the Aqueous pnase OOOOC Aqueousphase ttttlltttl Nonpolarphase ooAqueousphase (E)Lipidbilayer Fig. 3.6 : Summary of amphipathic lipids in the formation of micelle and lipid bilayer. (D)Micelle 46. 40 BIOCHEMISTRY BIOMEDICAL/ CLIITIICALCOilCEPTS !ei- g The phospholipid4ipalmitoyl lecithin-preuents the adherence of inner surface of the lungs, the absenceof which is ossociofed with respiratory disfress syndrome in infants. Cepholinsparticipate in blood clotting. The action of certain hormones is mediated through phosphatidylinositol. Phospholipidsare important for the synthesisand transport of lipoproteins ond reuerse tronsport ol cholesterol. Cholesterolis essentialfor the synfhesisol bile ocids,hormones(sexand cortical)and uitaminD. Lipoproteinsoccur in the membronestructure, besidesseruingos o meansol transport uehiclesfor lipids. Lipids are associatedwith certain disorders----obesityond atherosclerosis. outer aqueous phase on either side and the nonpolar tails into the interior (Fig.3.6.fhe formation of a lipid bilayer is the basis of membranestructure. Liposomes: They are producedwhen amphi- pathic lipids in aqueousmedium are subjected to sonification. They have intermittent aqueous phases in the lipid bilayer. Liposomes,in combinationwith tissuespecificantiBens,are used as carriersof drugsto tarBettissues. Emulsions: These are produced when non- polar lipids (e.g. triacylglycerols)are mixed with water. The particlesare largerin size and stabilized by emulsifying agents (usuallr amphipathic lipids), such as bile salts and phospholipids. 47. LIPIDS substancesrelatiuely inso,lublein water, soluble in organicactually or potentially related to totti-o"iJl'ord or" utilized 2 Lipids are crassifiedinto simpre(fats ,and.,oirs), c,omprex(phosphoripids,grgcolipids),deriued (fatty acids, steriodn"r_"n"rl and miscelloneous(carotenoids). 3' Fatty acidsare the maior constituentsof.uariousripids.saturatedand unsaturotedfattyacidsarmostequarsoccurin "tt"*i'1io11i, ri'['i":ur"rrirrated fattyacids(pr'FA) ii[i:"0"i:':;::i:i and tinoteni,o,id o," the.s'se,'tiaiioitv o,ia, thatneedto be 4' Triacylglycerols(simply fats)are the esters 7f glycerol with fatty acids.They are foundin adipose tissueand pr:imoriry ,*nrtior.o, ',u"li.r"rr"- oj oli^otr. seuerar tests(iodinenumber,RMnumber)areemprogedin tn" ioiorrJo"/iio"i"i'r"inepurityof fotsand oirs,5' Phosphotipidsare complex lipids cctntainin.g phosphoricacid. Glycerophospholipids ;;:I:,:Ji:'::;',:i;,:i:;,:ot andtheseinct-ude'r"'"it"'i-"intin, phosphatravrinoiitot, 6' sphingophospholipids(sp.hingornyelins)contain sphingosineas the alcohol in place oftJi:;fi,!:,ttvcerophosphoti;idsi.-ih-osphoripid;";,;;i;;';";:, constituentsof ptasma 7 cerebrosidesare the .simprestform of grycoripidswhich occur in the membranesofneruoustissue. Gangriosidesare predominontrg round in ti" gorstions. Theycontainone or more moteculesof N-acetylneuraminic.";,; ifi;:;r;:,)i." t 8. Steroidscontain th,eringcyclopentanoperhydrophenanthrene. Timportanceincrude. "nlutt"ii,""i,,tJ'ir,ar,r,rr-iir'n,";;;.'I::#r::t i!^i,"Jlfr,,iZ,,hormones-A steroid containing'or."or"*or. hydroxyr groupsis known as steror.9' cholesteraris the most abundqnt animarsterar.It containsone hydroxyrgroup (at cz),a double bond (c51) and an "igl,t co,rao.nside choin,ti"rn.a b cp. cholesterol-is 2."?;:::::'t:.":"T"#[::;:::;,:x:_i:ao,, ,,,",riiJ"ii,'"ioJ",,,,nesiso/bireacids, r0 The lipids that oor:::r both hvdrophobic(non -porar)and hydrophiric(porar) groupsareknown as amphipathic. rhese in,i"a" iori, o,,;;.;:;;;h;i;,:;:, sphinsoripidsand bite ';:;r:#::.''athic ripidson i^poioi constituentsin the biiaeersof the bioroqical 48. 42 BIOCHEMISTRY Essayquestions 1. Writean accountof classificationof lipidswith suitabl