Biochemistry

Dr, lJ, Satyan arayana M . S c,.P h.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 .

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Eiochemistrg First Published : March 1999 Reprinted: 1999 Revised Reprint : August 2000 Reprinted: 2OQO,2001, 2QO2 Second Revised Edition : June 2002 Reprinted: 2003 Revised Reprint : 2004 Revised Reprint : 2005 Third Revised Edition (multicolour) : 2006 Revised Reprint : 2007

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Prefaceto the Third Edition

The responseto the first and the secondeditionsof my book 'Biochemistry'(reprintedseveraltimes in just 6 years)from the studentsand teachersis simply overwhelming.I was flooded with highly appreciative lettersfrom all cornersof India and abroad!This givesme immensesatisfactionand encouragemLnt in this academicventure. I havecorresponded with many biochernistry teachers,inviting their commentsand opinionsfor further improvingthe book. Most of them havebeenkind enoughto offer constructivesuggestions. I also visited severalcollegesand had personalinteractionwith facultymembersand students.Theseexercises, spreadover the past 6 years,have helped me to get direct feedbackon my book, besidesrealisingthe additional requirementsof students. I havegreat pleasurein presentingthe third edition of my book with severalunique/novelfeatures,some high-lightsof which are listedbelow. . A thoroughrevisionand updatingof eachchapterwith latestadvances. Multicolouredillustrationsfor a better understanding of chemicalstructuresand biochemicalreactions. . Increasein the font size of the text for more pleasantand comfortablereading. o Incorporationof a new Sectionon MolecularBiologyand Biotechnology. . Addition of ten new chapters-human genomeproject,gene therapy,bioinformatics,free radicals and antioxidants,tissueproteinsand body fluids,environmentalbiochemistry,genetics,immunologyetc. . An improvedorientationand treatmentof human biochemistryin healthand disease. . Additionof practicalbiochemistryand clinical biochemistrylaboratoryin the appendix. It is true that I representa selectedgroup of individualsauthoringbooks,havingsometime at disposal, besideshard work, determinationand dedication.I considermyselfan eternallearnerand a regularstudent of biochemistry. However,it is beyondmy capabilityto keeptrack of the evergrowing advances in biochemistry due to the exponentialSrowth of the subject.And this makesme nervous,wheneverI think of revising the book. I honestlyadmit that I haveto dependon mature readersfor subsequent editionsof this book. AN INVITATION TO READERS It is not all the time possiblefor me to meetthe readersindividuallyand get their feedback, despitemy ferventwish. Of course,I do write to somepeoplepersonaliyseekingtheir opinions.However,I wish to have the commentsand suggestions of eachoneof the readersof my book.I sincerelyinvite the readersto feelfree and write to me expressing their frank opinions,critical commentsand constructivesuggestions. DT. U. SATYANARAYANA

tr l

I owe a deepdebt of gratitude to my parents,the late Sri U. VenkataSubbaiah,and Smt. Vajramma,for cultivatingin me the habit of earlyrising.The writing of this bookwould neverhavebeenpossiblewithout this healthyhabit. I am gratefulto Dr. B. S. NarasingaRao(formerDirector,NationalInstituteof Nutrition, Hyderabad)for disciplining my professionallife, and to my eldestbrother Dr. U. Gudaru(former Professorof PowerSystems,WalchandCollegeof Engineering,Sangli)for discipliningmy personallife. My elder son, U. Chakrapani(MBBS)deservesa specialplace in this book. He made a significant contribution at everystageof its preparation-writing, verification,proof-readingand what not. I had the rare privilegeof teachingmy son as he happened to be a studentof our college.And a major part of this bookwas written while he waslearningbiochemistry. Thus,he wasthe first personto learnthe subjectof biochemistry from my handwrittenmanuscript.The student-teacherrelation (rather than the father-son)has helpedme in receivinSlconstant feedbackfrom him and restructure the book in a way an undergraduatestudent would expecta biochemistrytextbookto be. Next,I thank Dr. G. PitcheswaraRao(former Professorof Anatomy,SMC,Vijayawada) for his constructive criticism and advice,and Dr. B. Sivakumar(Director,NationalInstitute of Nutrition, Hyderabad)for his helpful sugi5lestions on the microfigures.I am grateful to my nephew,Mr. U. SrinivasaRao,for helping me in drawingsomefigures. Last but not least, I thank my wife Krishna Kumari and my younger son, Amrutpani, without whose cooperationand encouragementthis book could never have been written. The manuscript was carefully nurtured like a new born babyand the book has now becomea full-pledgedmemberof our family. ACKNOWLEDGEMENTS TO THE THIRD EDITION I am indebtedto a largenumberof friends,pen-friends andstudentswho helpedme to reviseand improve the qualityof this book.I haveindividuallyand personallythankedall of them (whonumbera fewhundreds!). I onceagainexpressmy gratitudeto them. I thank my friend and colleague,Mr. M.S.T.JaganMohan, who has helped me with his frequent interactionsto improvethe book,and makeit more student-friendly. I would like to placeon recordmy deep (M.D.)students,Dr. (Mrs.)U.B.VijayaLakshmiand Dr. (Mrs.)Vidya senseof appreciation to my post-graduate DesaiSripad,whoseperiodicalacademicinteractionand feedback havecontributedto the improvementof the biomedicaVclinical aspectsin somechapters.I acknowledge the help of my friend,Dr. P. Ramanujam(Reader in English,AndhraLoyolaCollege,Vijayawada) for his help and encouragement in revisingthe book. I expressmy gratitude to Mr. ArunabhaSen, Director, Books & Allied (P) Ltd. Kolkata, for his wholehearted supportand constantencouragement in revisingthe book in multicolour,and taking all the pains to 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 constant support and encouragement.I am grateful to UppalaAuthor-PublisherInterlinks, Vijayawada,for sponsoringand supportingme to bring out this edition.

DT. U. SAIYANARAYANA Ii i i ]

Biochemistry

The term Biochemistry was introduced by Carl Neuberg in 1903. Biochemistrybroadly dealswith the chemistrv of life and living processes. There is no exaggerationin the statement,'Thescopeof biochemistrg is as uastas lilb itself !' Everyaspectof life-birth, growth, reproduction,agingand death,involvesbiochemistry. For that matter, everymovementof life is packedwith hundredsof biochemicalreactions.Biochemistryis the mostrapidlydeveloping and mostinnovativesubjectin medicine.This becomesevidentfrom the factthat over the years,the major share of Nobel Prizesearmarkedfor Medicineand Physiologyhas gone to researchers engagedir: biochemistry. The discipline of biochemistryservesas a torch light to trace the intricate complexicitiesof biology, that all living besidesunravellingthe chemicalmysteriesof life. Biochemicalresearchhasamplydemonstrated things are closelyrelatedat the molecularlevel.Thus biochemistryis the subjectof unity in the diversified living kingdom. Advancesin biochemistryhavetremendousimpact on human welfare,and havelargelybenefitedmankind and their living styles.Theseincludethe applicationof biochernistryin the laboratoryfor the diagnosisof and the diseases. the products(insulin,interferon,€rowth hormoneetc.)obtainedfrom geneticengiineering, possibleuse of genetherapy in the near future. 0rganization of the Book This texthook,comprising43 chapters,is orgianizedinto serrensecl:ionsin the heirarchicalorder of learninSbiochemistry. . SectionI dealswith the chemicalconstituentsof life-carbohydrates,lipids,proteinsand amino acids, nucleicacidsand enzymes. . SectionII physiologicalchemistryincludesdigestionand ahsorption,plasmaproteins,hemoglobinand prophyrins,and biologicaloxidation. . SectionIII incorporatesall the metabolisms(carbohydrates, lipids,amino acids,nucleotides,minerals) . Section [V covershormones,organ function tests,water,electrolyteand acid-basebalance,tissueproteins and trodi'fluids,and nutrition. . Section V is exclusivelydevotedto molecularbiologyand biotechnology(DNA-replication, recombination, DNAandbiotechnology) ar"lnrepair,transcriptionandtranslation, recombinant regulationof geneexpression, . Section VI gives relevant information on current topics such a^shuman genomeproject, gene therapy, prostaglandins, bioirrtormatics, diabetes,cancer,AIDS etc. . Section VII deals with the basic aspectsfor learning and understandingbiochemistry (bioorganic chenristry', hiophysicalchemistrytools of biochemistry,genetics,immunology). Each chapter in this book is carefully crafted with colour illustrations, headingsand subheadingsto areput facilitatequick understanding. The importantapplications of biochemistryto humanhealthand disease places 'landmarks'. together as biomedical/clinicalconcepts.Icons are used at appropriate to serveas The origins of biochemicalwords, confusablesin biochemistry,practical biochemistryand clinical biochemistrylaboratory,given in the appendixare novel features. The briok is so organizedas to equip the readerswith a comprehensive knowledgeof biochemistry. Ii u]

Gontents

SECTION ONE ChemicalConstituentsof Life 1 > Biomolecules andthecell

3

2 > Carbohydrates

9

3 > Lioids

28

4 > Proteins andamino acids

43

acidsandnucleotides 5 > Nucleic

69

6 > Enzymes

85

7 > Vitamins

176

TWO SECTION PhysiologicalBiochemistry B > Digestion andabsorption

165

9 > Plasma oroteins

182

10 > Hemoglobin andporphyrins

196

11 > Biologicaloxidation

221

SECTION

SECTION FIVE Molecular Biologyand Biotechnology 24 > DNA-replication, recombination andrepair 523 25 > Transcriotion andtranslation 542 26 > Regulation of geneexpression 566 27 b Recombinant DNAandbiotechnology 578

sEcTtcN Current 28 > 29 > 30 F 31 p 32 33 34 35 36

>' > b > l"

37> 38>

THBEE

stx

Topics project Humangenome

619 Genetherapy 625 Bioinformatics 634 'lvletabolism (detoxification) of xenobiotics 638 Prostaglandins andrelated compounds644 Biological membranes andtransport 650 Freeradicals andantioxidants 655 Environmental biochemistry 662 glucose Insulin, homeostasis, mellitus anddiabetes 669 Cancer 58s

Acquired immunodeficiency (AIDS) syndrome

695

241

q3 > Metabolism of carbohydrates

244

*4 > Metabolism of lioids

285

Metabolism of aminoacids

F-, 16 > Int6gration of metabolism

330. 380

17 > Metabolism of nucleotides

387

metabolism 1B > Mineral

403

FOUR SECTION Clinical Biochemistrvand Nutrition 19 > Hormones function tests 20 > Organ 21 > Water, electrolyte and bqlance acid-base > proteins Tissue andbodyfluids 22 23 > Nutrition-

SECTION SEVEN Basicsto Learn Biochemistrv 39 > Introduction to bioorganic chemistry 40 > Overview of biophysical chemistry 41 > Toolsof biochemistrv 42 > lmmunology 43 > Genetics

487

APPENDICES Answers to Self-assessmenl Exercises I Abbreviations usedin thisbook' ll Greek alphabets lll Origins words ol important biochemical lV Common confusables in biochemistry V Practical biochemistry-principles Vl Clinical laboratory biochemistry

502

INEEX

427 453 468

703 708 71 9 732 73 7 745 751 756 tJt

760 764 77 0 77 3

fi

Protuinsand Amino acids

4:

Nucleic acidsand Nucleotides 69

BflomnoXeeutrss aildthsCelll

-l- hu living matter is composed of mainly six hydrogen, oxygenl I elements-carbon, nitrogen, phosphorus and sulfur. These elements togetherconstituteabout 90% of the dry weight of the human body. Severalother functionally important elementsare also found in the cells. These include Ca, K, Na, Cl, Mg, Fe, Cu, Co, l, Zn, F, Mo and Se.

organic compounds.lt is believedthat man may contain about 100,000 different types of molecules although only a few of them have been characterized. Sornpiex

*riomoleeules

The organic compoundssuch as amino acids, nucleotidesand monosaccharidesserve as the monomeric units or building blocks of complex biomolecules-proteins,nucleic acids (DNA and earbon-a unique element of life RNA) and polysaccharides,respectively.The Carbon is the most predominantand versatile important biomolecules(macromolecules) with elementof life. lt possesses a unique propertyto their respective building blocks and major form infinite number of compounds. This is functions are given in Table 1.1. As regards attributed to the ability of carbon to form stable lipids, it may be noted that they are not c ov ale n t b o n d s a n d C -C c h a i n s of unl i mi ted biopolymers in a strict sense, but majority of length. lt is estimated that about 90% of them contain fatty acids. compounds found in living system invariably contain carbon. Structural heirarehy off asn organisnl The macromolecules(proteins,Iipids, nucleic acids and polysaccharides) form supramolecular Life is composed of lifeless chemical assembl i es(e.g. membranes)w hi ch i n tu r n molecules. A single cell of the bacterium, organize into organelles,cells, tissues,organs Escherichiacoli contains about 6.000 different and fi nal l y the w hol e organi sm.

Ghemical

molecules

of li#e

3

B IOC H E MIS TFIY

Major functions

Building block (repeatingunit)

Biomolecule 1. Protein

Amino acids

and Fundamental basisofstructure functions). function anddynamic ofcell(static

acid(DNA) 2. Deoxyribonucleic

Deoxyribonucleotides Ribonucleotides

o.l.! 9199 iFryi{9l1llgt fl_eq_o_sitory

acid(RNA) 3. Ribonucleic

(glucose) 4. Polysaccharide(glycogen) Monosaccharides Fattyacids,glycerol

5. Lipid

Chem *c a !

c o m p o s i ti o n

biosynthesis. required lorprotein Essentially tomeetshortterm formofenergy Storage demands. tomeetlongterm tormofenergy Storage of membranes. components demands; structural

Prokaryotic

o f ma n

The chemical compositionof a normal man, weighing 65 kg, is given in Table 1.2. Water is the solvent of life and contributesto more than 60"h of the weight. This is followed by protein ( m os t lyin mu s c l e )a n d l i p i d (mo s tl yi n adi pose 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.

and eukaryotic

cells

The cel l s of the l i vi ng ki ngdom may be divided into two categories 1. Prokaryotes(Creek : pro - before; karyon nucl eus)l ack a w el l defi nednucl eusand possess relatively simple structure. These include the various bacteria. 2. E ukaryotes(Greek: eu-true; karyonnucleus)possessa well defined nucleusand are more complex in their structure and function. The hi gher organi sms(ani mal sand pl ants)are composedof eukaryoticcells. A comparisonof the characteristicsbetween prokaryotesand eukaryotesis listed in Table 1.3.

The concept of cell originated from the c ont r ibut i o n so f S c h l e i d e na n d S c h w a n n(1838). However, it was only after 1940, the complexitiesof cell structurewere exposed.

Percent(7")

Weight (kg)

Water

6 1 .6

40

Protein

17.0

11

Lipid

1 3 .8

I '|

6 .1

4

Constituent

Carbohydrate Minerals

The human body is composedof about 1014 cells. There are about 250 types of specialized cel{s in- the human body'G.g. erythrocytes, nerve-cells, muscle cells, B cells of pancreas. A n eukaryoti ccel l i s general l y10 to 100 pm in diameter. A diagrammatic representation of a typical rat liver cell is depicted in Fi g.I.t. The pl ant cel l di ffersfrom an ani mal cel l by possessing a rigid cell wall (mostlycomposedof cellulose) and chloroplasts.The latter are the sitesof photosynthesis.

AND THE CELL Chapter 1 : BIOMOLECULES

Eukaryotic cell

Prokaryotic cell

Characteristic

1-10pm) Small(generally

1. Size

2. Cellmembrane 3. Sub-cellular organelles 4, Nucleus metabolism 5. Energy

6. Celldivision 7. Cytoplasm

DNAisfound Notwelldefined; histones areabsent asnucleoid,

of Mitochondria absent, enzymes metabolism bound to energy membrane fission Usually andnomitosis andcytoskeleton 0rganelles absent

T he c e l l c o n s i s tso f w e l l d e fi n e d subcel l ul ar organelles,enveloped by a plasma membrane. tissue By differential centrifugation of homogenate, it is possible to isolate each c ellular o rg a n e l l e i n a re l a ti v e l y p ure form (Refer Chapter 41). The distribution of major enzymes and metabolic pathways in different c ellular o rg a n e l l e s i s g i v e n i n th e chapter on enzymes (Refer Fig.6.6). The subcellular organellesare briefly describedin the following pages.

pm) Large(generally 10-100 plasma flexible Cellisenveloped bya membrane Distinct organelles arefound (e.9.mitochondria, nucleus, lysosomes) Nucleus iswelldefined, surrounded bya membrane: DNAisassociated withhistones Enzymes metabolism ol energy arelocated inmitochondria Mitosis

Contains organelles andcytoskeleton (anetwork oftubules andfilaments)

N ucl eus N ucl eus i s the l argest cel l ul ar organel l e , surrounded bv a doubl e membrane nucl ear envel ope. The outer membrane i s conti nuous w i th the membranesof endopl asmi creti cul um . At certain intervals,the two nuclear membranes have nuclear pores with a diameterof about 90 nm. These pores permit the free passageof the products synthesizedin the nucleus into the surroundi ng cytopl asm.

Mitochondrion

Plasmamembrane Vacuole

Roughendoplasmicreticulum

Ribosomes

Golgi apparatus

Lysosome

Peroxisome Cytoskeleton Cytosol Coatedpits

Ftg. 1.1: Diagrammaticrepresentationof a nt liverell.

B IOC H E MIS TF|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 nucleosomesconstitutes chromatin fibres of chromosomes(Creek'.chroma - colour; soma bod y ). T h u s , a s i n g l e h u ma n c h romosome i s c om o o s e do f a b o u t a m i l l i o n n u cl eosomes. The number of chromosomes is a characteristic feature of the species. Humans have 46 chromosomes,compactlypacked in the nucleus. The nucleusof the eukaryoticcell containsa dense bodv known as nucleolus.lt is rich in RNA, p a rti c u l a rl y th e ri b o s o mal R N A w hi ch entersthe cytosol through nuclear pores.

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 parts of the cell to provide energy for the cel l ul arw ork. The mi tochondri almatri x contai nsa ci rcular double stranded DNA (mtDNA), RNA and ribosomes.Thus, the mitochondriaare equipped with an independent protein synthesizing machinery.It is estimatedthat about 10% of the mitochondrial oroteins are produced in the mi tochondri a.

The ground material of the nucleus is often referredto as nucleoplasm. lt is rich in enzymes p o l y me ra s e s and R N A s uc h a s D N A polymerases.To the surpriseof biochemists,the enzy m e s o f g l y c o l y s i s ,c i tri c a ci d cycl e and hexose monophosphateshunt have also been detected in the nucleoplasm.

The structureand functions of mitochondria closely resemble prokaryotic cells. lt is hypothesizedthat mitochondria have evolved from aerobic bacteria.Further,it is believedthat during evolution,the aerobicbacteriadeveloped a symbi oti c rel ati onshi p w i th pri mor dial anaerobiceukaryoticcells that ultimately led to the arrival of aerobic eukaryotes.

Mitochondria

Endoplasmic

The mitochondria (Creek'. mitos - thread; chondros- granule) are the centres for the c ell u l a rre s p i ra ti o n a n d e n e rg ymetabol i sm.They are regarded as the power houses of the cell wit h v a ri a b l es i z e a n d s h a p e .Mi t ochondri aare r od -l i k e o r fi l a me n to u s b o d i e s , usual l v w i th dim e n s i o n s o f 1 .0 x 3 p m. A bout 2,0O0 mitochondria,occupying about 1/5thof the total c ell v o l u me , a re p re s e n ti n a ty p ical cel l .

The network of membrane enclosed spaces that extends throughout the cytoplasm constitutesendoplasmicreticulum (ER).Some of these thread-like structures extend from the nuclear pores to the plasma membrane.

reticulum

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 During the process of cell biosynthesis. The mitochondriaare comoosedof a double fractionation, rough ER is disruptedto form small membrane system. The outer membrane is It may be noted vesicles known as microsomes. smooth and completely envelopsthe organelle. not occur in the microsomes as such do that The inner membrane is folded to form cristae (Latin- crests) which occupy a larger surface cel l . area. The internal chamber of mitochondria is The smooth endoplasmicreticulum does not referred to as matrix or mitosol. contain ribosomes.lt is involved in the synthesis phospholipids,sterols) The componentsof electron transportchain of lipids (triacylglycerols, of drugs, besidessupplyingCa'?. and metabolism and oxidative phosphorylation (flavoprotein, functi ons. for the cel l ul ar c y t o c h ro m e sb , c 1 , C , a a n d a 3 and coupl i ng factors) are buried in the inner mitochondrial Golgi apparats,r$ membrane.The matrix containsseveralenzvmes concerned with the energy metabolism of E ukaryoti ccel l s contai n a uni que cl ust erof c ar b o h y d ra te sl i,p i d sa n d a m i n o a c i ds(e.g.,ci tri c membrane vesicles known as dictyosomes

AND THE CELL Chapter 1 : BIOMOLECULES whic h, in tu rn , c o n s ti tu teC o l g i a p p a ratus(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 plasma membrane.

The pH of the lysosomalmatrix is more acidic (pH < 5) than the cytosol (pH-7) and this facilitatesthe degradationof differentcompounds. The lysosomal enzymes are responsible for maintaining the cellular compounds in a dynamic stafe, by their degradationand recycling. The degradedproducts leave the lysosomes,usually by diffusion, for reutilization by the cell. Certainproteinsand enzymesare enclosedin Sometimes,however, certain residual products, m em br ane v e s i c l e s o f C o l g i a p p a ra t us and rich in lipids and proteins,collectivelyknown as secreted from the cell after the appropriate Iipofuscinaccumulatein the cell. Lipofuscinis signals.The digestiveenzymes of pancreasare the age pigment or wear and tear pigmentwhich or oduc edi n th i s fa s h i o n . has been implicatedin ageingprocess. Colgi a p p a ra tu sa re a l s o i n v o l v e d i n the The digestiveenzymesof cellular compounds membrane synthesis, particularly for the are confinedto the lvsosomesin the best interest f or m at ion o f i n tra c e l l u l a r o rg a n e l l e s (e.g. of the cell. Escapeof theseenzymesinto cytosol peroxisomes,lysosomes). will destroythe functionalmacromolecules of tne cel l and resul t i n many compl i cati ons.The Lysosornes occurrence of several diseases(e.g. arthritis, allergicdisorders) hasbeenpartly Lysosomesare spherical vesicles enveloped musclediseases, by a single membrane.Lysosomesare regarded attributedto the releaseof lysosomalenzymes. as the digestivetract of the cell, since they are ac t iv ely in v o l v e d i n d i g e s ti o n o f cel l ul ar Feroxi somes s ubs t anc e s -n a m e l y p ro te i n s , l i p i d s , carboPeroxisomes,also known as microbodies, are hydratesand nucleic acids. Lysosomalenzymes si ngl e membranecel l ul ar organel l es.They are are categorizedas hydrolases.These include the spherical or oval in shape and contain the following enzymes(with substratein brackets) enzyme catalase.Catalaseprotects the cell from a-C lucosidase(glycogen) the toxic effectsof HrO, by converting it to HrO and Or. Peroxisomesare also involved in tne Cathepsins(proteins) oxidation of long chain fatty acids (> C,s),and L i p a s e s(l i p i d s ) synthesisof plasmalogens and glycolipids.Plants (R N A ) glyoxysomes, contain Ribonucleases a specialized type of

BTOMED|eAL/ CLINICAL COIUCEPTS

A liuing cell is a true representotiueof life with its own organizotion and specialized lunctions. Accumulotion oJ lipofuscin, a pigment rich in lipids and proteins, in the cell has been 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.

E } IOC H E MIS TF| Y per ox is o m e s , w h i c h glyoxylate pathway.

a re i n v o l v e d i n

the

Peroxisome biogenesis disorders (PBDs), are a Broup of rare diseasesinvolving the enzyme activities of peroxisomes. The biochemical abnor m a l i ti e sa s s o c i a te dw i th P BD s i ncl uoe increasedlevels of very long chain fatty acids (C2aand C26) and decreasedconcentrationsof plasmalogens. The most severeform of PBDs is Zellweger syndrome, a condition characterized by the absenceof functional peroxisomes.The v ic t im s o f th i s d i s e a s em a v d i e w i th i n one vear after birth. {iytosol

and cytoskeleton

The cellular matrix is collectively referred to as cytosol. Cytosol is basically a compartment containing several enzymes/ metabolites and saltsin an aqueousgel like medium. More recent studies however, indicate that the cytoplasm actually contains a complex network of protein filaments, spread throughout, that constitutes cytoskeleton.The cytoplasmic filaments are of

three types- microtubules, actin filaments and intermediatefilaments.The filamentswhich are polymers of proteins are responsiblefor the structure,shape and organizationof the cell. IN TE GR A TIOI{ OF C E LLU LA R FU N C TION S The eukaryoticcells perform a wide rangeof complex reactionsfunctionsto maintain tissues, and for the ul ti mate w el l -bei ng of the w hol e organi sm. For thi s purpose, the vari ous intracellularprocesses and biochemicalreactions are tightly controlled and integrated.Division of a cel l i nto tw o daughtercel l s i s good exampl eof the orderly occurrenceof an integratedseriesof cel l ul ar reacti ons. Apoptosisis the programmed cell death or cel l sui ci de. Thi s occurs w hen the cel l has ful fi l l ed i ts bi ol ogi calfuncti ons.A poptosi smay be regardedas a natural cell death and it differs from the cell death caused by injury due to radiation,anoxia etc. Programmedcell death is a highly regulatedprocess.

1. 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, monosaccharides and fotty acids, respectluely. 2 . The cell is the structurol and functional unit of life. The eukoryotic cell consisfsof well det'inedsubcellulor organelles,enueloped in a plasma membrane. 3. The nucleus contoins DNA, the repository ol genetic int'ormation.DNA, in association with proteins (histones),forms nucleosomeswhich, in turn, make up the chromosomes. The mitochondria qre the centresfor energy metobolism. They are the principal producers of ATP which is exported to all parts of the cell to ptouide energy lor cellular work. 5. Endoplosmic reticulum (ER) ts the network of membrane enclosed spoces that 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 synthesized proteins are handed ouer for t'urther processing ond export. 6. Lysosomes are the digestiue bodies ol the cell, actiuely involued in the degradotion of cellular compounds. Peroxisomescontoln the enzyme catalose that 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. 7. The eukaryoticcellsperform a wide rangeof complex lunctionsin a well coordinatedand integrated fashion. Apoptosis is the processol programmed cell death or cell suicide.

1^ arbohydratesare the most abundant organic \ - m o l e c u l e s i n n a tu re . T h e y a re pri mari l y composedof the elementscarbon, hydrogen and oxygen.The name carbohydrateliterally means 'hydratesof carbon'. Some of the carbohydrates pos s es sth e e mp i ri c a l fo rmu l a (C .H 2O)nw here n 3 3, satisfyingthat these carbohydratesare in fact carbon hydrates.However,there are several non-carbohydratecompounds (e.g. acetic acid, C2HaO2;lactic acid, C3H6O3)which also appear as hydrates of carbon. Further, some of the genuine carbohydrates (e.g. rhamnohexose, C6H12O5ideoxyribose,C5H16Oa)do not satisfy the generalformula.Hence carbohydrates cannot be always consideredas hydratesof carbon. Carbohydrates may be defined as polyhydroxyaldehydes or ketones or compounds which produce them on hydrolysis. The term ' s ugar ' i s a p p l i e d to c a rb o h y d ra te ssol ubl e i n water and sweet to taste. #-ur*c;tEerEsof earbohydrates Carbohydratesparticipatein a wide range of f unc t io n s

1. They are the most abundantdietarysource of energy (a Cal/S)for all organisms. 2. Carbohydratesare precursors for many organic compounds (fats,amino acids). (asglycoproteinsand glyco3. Carbohydrates lipids) participate in the structure of cell membraneand cel l ul ar functi onssuch as cell growth, adhesionand fertilization. 4. They are structuralcomponentsof many organi sms.Thesei ncl udethe fi ber (cel l ul ose)o f plants,exoskeletonof some insectsand the cell w al l of mi croorgani sms. 5. Carbohydratesalso serve as the storage form of energy(glycogen)to meet the immediate energy demandsof the body. C LA S S IFIC A TION

OF GARBOHYDRATES Carbohydrates are often referred to as saccharides (Greek: sakcharon-sugar). They are broadly classifiedinto three major groupsmonosaccharides, oligosaccharides and polysaccharides.This categorization is based on

t0

B IOC H E MIS TR Y

Monosaccharides(empirical formula)

AIdose

(CgHoOg) Trioses (C+HoO+) Telroses (CsHroOs) Pentoses (CoHrzOo) Hexoses (CzHr+Oz) Heptoses

Glyceraldehyde Erythrose Ribose Glucose Glucoheptose

Ketose

Dihydroxyacetone Erythrulose Ribulose Fructose Sedoheptulose

the number of sugar units. Mono- and oligosaccharides are sweet to taste, crystalline in characterand soluble in water, hence thev are commonly known as sugars.

liberatedon hydrolysis.Basedon the number of monosaccharide units present, the oligosaccharides are further subdivided to disaccharides,trisaccharides etc.

FJtonosaccharides

P ol ysace hari des

(Greek : mono-one)are the Monosaccharides group simplest 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 different categories,based on the functionalgroup and the numberof carbonatoms

Polysacchari6ls(Creek:poly-many)are polymers of mondficcharide units with high molecul ar w ei ght (up to a mi l l i on).They are usual l y tasteless(non-sugars)and form colloids with water. The polysaccharidesare of two typeshomopolysaccharides and heteropolysaccharides.

Aldoses : When the functional group in IH monosaccharidesis an aldehyde l-C:oi,

\ ,h"u

are known as aldoses e.g. glyceraldehyde, gluc os e.

is an importantcharacterof Stereoisomerism the monosaccharides. Stereoisomers are Ketoses: When the functionalgroup is a keto compounds that have the same structural \ lt formulae but differ in their spatialconfiguration. \-C:O.l group, they are referred to as ketoses e.g. dihydroxyacetone,fructose.

A carbon is said to be asymmetric when it is to four different atoms or groups. Ihe attached Based on the number of carbon atoms, the of asymmetric carbon atoms (n) number monosaccharides are regarded as trioses (3C), the possible isomers of a given determines tetroses (4C), pentoses (5C), hexoses (6C) and w hi ch i s equal to 2n. C l ucose compound heptoses(7C).Theseterms along with functional 4 contains asymmetric carbons,and thus has 16 groupsare usedwhile naming monosaccharides. tsomers. For instance, glucose is an aldohexose while fructose is a ketohexose(Table 2,1). Glyeeraldehyde T he c om mo n m o n o s a c c h a ri d easn d d i saccha-tfu e ref erqlrt*e cff rb$hyd$'er'&€3 rides of biological importanceare given in the Clyceraldehyde(triose)is the simplestmonoTable 2.2. saccharidewith one asymmetriccarbon atom. lt S S lgos ac c h a ri d e s and has been chosen existsas two stereoisomers (Creek: io representthe the reference carbohydrate Oligosaccharides oligo-few) contain as 2- 1O m ono s a c c h a ri d em o l e c u l e s w h i ch are structureof all other carbohvdrates.

Ghapter 2 : CARBOHYDRATES

11

Trioses incellsasphosphate Glyceraldehyde Found DihydroxyacetoneFound incellsasphosphate Tetroses D-Erythrose

i i i

Glyceraldehyde 3-phosphate isanintermediate inglycolysis ttst -pnosphate isanintermediate inglycolysis

----t - -- - --.. -.. -. --... -- -

i Widespread

Pentoses D-Ribose

asa constituent of I Widespread I RNAandnucleotides

D-Deoxyribose i Asa constituent ofDNA D-Ribulose

during metabolism : Produced

D-Xylose

i i i i

L-Xylulose D-Lyxose

Asa constituent ofglycoproteins anogums ls anintermediate inuronic acidpathway Heart muscle

i --. --. -- --.. ---.. -.. -. --.

Hexoses D-Glucose

D-Galactose D-Mannose D-Fructose

Asa constituent ol polysaccharides (starch, glycogen, cellulose) and (maltose, disaccharides lactose, Alsofoundinfruits sucrose). Asa constituent oflactose (milk sugar) Found inplantpolysaccharides glycoproteins andanimal Fruits andhoney, asa constituent andinulin ofsucrose

Heptoses inolants D-SedoheptuloseFound Disaccharides

Sucrose Lactose

Maltose

Occurrence

Asa constituent ofcanesugarand pineapple beetsugar, Milksugar

Product ofstarch hydrolysis, ingerminating seeds occurs

Forthestructure ofRNAandnucleotide (ATP, coenzymes NAD+, NADP+) Forthestructure ol DNA Itisanimportant metabolite inhexose monophosphate shunt Involved inthefunction ofglycoproteins pentosuria Excreted inurineinessenlial Asa constituent ol lvxollavin ofheartmuscle The'sugar fuel'oflife;excreted inurine in diabetes. Structural unitofcellulose inplants

Converted toglucose, failure leads to galactosemia Forthestructure ofpolysaccharides Itsphosphates areintermediates ofglycolysis

Its7-phosphate isan intermediate in hexose monophosphate shunt,andin photosynthesis Biochemical importance

Most commonly used table sugar supplying calories Exclusive carbohydrate source tobreast fed (lactose infants. Lactase deficiency intolerance) leads todianhea andflatulence Animportant intermediate inlhedigestion of starch

E}IOCHEMISTFIY

12 H-C:O I H-C-OH cH2oH D-Glyceraldehyde H-C:O I H-C-OH I HO-C-H I H-C-OH I H-Q-OH I

cHzoH D-Glucose

H-C:O HO-C-H

to compoundsthat respectivelyrotatethe plane of polarized light to the right or to the left.

cH2oH

An optical isomer may be designated as D(+), D(-), L(+) and L(-) based on its structural L-Glyceraldehyde relation with glyceraldehyde.lt may be noted that the D- and L-configurationsof sugarsare H-C:O I primarily based on structure of the HO-C-H glyceraldehyde,the optical activities however, H-C-OH may be different.

I HO-C-H HO-C-H

cH2oH L-Glucose

Fig.2.1 : DandL- forms of glucose compared with D and L- glyceraldehydes (the reference carbohydrate).

Racemic mixture : lf D- and L-isomersare present in equal concentration,it is known as racemi cmi xtureor D L mi xture.R acemi cmi xture does not exhibit any optical activity, since the dextro- and levorotatorv activities cancel each other. Configuration

D" and L-isomers The D and L isomers are mirror images of each other. The spatial orientation of -H and -OH groups on the carbon atom (Cs for glucose)that is adjacentto the terminal primary alcohol carbon determineswhether the sugar is D- or L-isomer.lf the -OH group is on the right side, the sugar is of D-series,and if on the left side, it belongs to L-series.The structuresof D- and L-glucosebased on the referencemonosaccharide, D- and L-glyceraldehyde (glycerose) are depicted in Fig.2.1.

of D-aldoses

The configuration of possible D-aldoses starting from D-glyceraldehydeis depicted in Fig.2.2. This is a representation of KillianiFischersynthesis,by increasingthe chain length of an aldose, by one carbon at a time. Thus, (4C), startingwith an aldotriose(3C),aldotetroses aldopentoses (5C) and aldohexoses(6C) are formed. Of the 8 aldohexoses,glucose, mannose and galactose are the most familiar. Among these, D -gl ucose i s the onl y al dose monosaccharidethat predominantlyoccurs in nature.

Gonfiguration of D-ketoses It may be noted that the naturallyoccurring Startingfrom dihydroxyacetone(triose),there monosaccharides in the mammalian tissuesare are five keto-sugarswhich are physiologicallr mostlyof D-configuration. The enzymemachinery important.Their structuresare given in Fig,2.3 of cells is specific to metabolise D-series of monosaccharides.

Epimers

fn the medical practice, the term dextrose is used for glucose in solution. This is because of the dextrorotatory nature of glucose.

ff two monosaccharides differ from eacother in their configuration around a singk specificcarbon (other than anomeric) atom. L*ei are referred to as epimers to each orher '.Fig,21 Optlcal activity of sugars For instance, glucose and galactose are efilwl with regard to carbon 4 (Ca-epimers - -^:i 's Optical activity is a characteristicfeature of they differ in the arrangementof -OH g.'ELcr compounds with asymmetric carbon atom. Clucose and mannose are epi-'e--' q drl When a beam of polarized light is passed Ca. regardto carbon 2 (C2-epimers). througha solutionof an optical isomer,it will be rotated either to the right or left. The term The interconversionof epimers e I r::r'e dextrorotatory (+) and levorotatory (-) are used to galactose and vice versai s i- - -^,'- a*

Ghapter 2 : CABBOHYDFATES

13

cHo

I HCOH I cH2oH

'l

Aldotriose

(3c)

t-

Aldotetroses (4c) D-Erythrose

D.Threooe

cHo

cHo

H C OH

H OC H I HCOH

HCOH I HCOH cH2oH D-Ribose

cHo

HCOH

I H C OH I HOCH I HCOH

cH2oH D-Arabinose

cH2oH D-Xylose

cHo

I HOCH Aldo I HOCH I

toses )

HCOH I cH2oH D-Lyxoee

I /\

JT

cHo

tl HCOH HCOH tl HCOH tl HCOH ll cH2oH D-Allose

/\

/\

cHo HOCH HCOH

JT

cHo HCOH

rl

cHo HOCH

HOCH HOCH tl HCOH HCOH HCOH tl HCOH HCOH HCOH ll cH2oH cH2oH cH2oH D-Altrose D-Glucose D-Mannose

*/\+

cHo

cHo

I H C OH I HCOH I HOCH I HCOH

cH2oH D-Gulose

cHo

I HCOH

I

HOCH I HCOH I HOCH

I HOCH

HoCH

noCH

HCOH I

cH2oH D-ldose

cHo H OC H I AldoHOCH hexoses

tl HCOH tt

cHzoH

(6c)

HCOH

cHzoH

D-Galactose D-Talose

Fig.2.2 : ThestructuralrelationshipbetweenD-aldosesshownin Fischerprojection. (TheconfigurationaroundC2(ed) distinguishesthe membersof eachpair).

epimerization, and a group of enzymesnamely-epimerases catalyse this reaction.

The term diastereomersis used to represent the sfereoisomers that are not mirror images of one another.

E nanti o m e rs Enantiomers are a special type of stereoisomers that are mirror images of each other. The two members are designatedas D- and L-sugars.Enantiomersof glucose are depicted in Fig.2.5.

For a better understanding of glucose structure, let us consider the formation of hemiacetals and hemiketals, respectively Majority of the sugarsin the higher animals producedwhen an aldehydeor a ketone reacts (including man) are of D-type (Fig.2.5'1. w i th al cohol .

E } IOC H E MIS TR Y

14

cH2oH cH2oH I

cH2oH

?H2oH

C :O

cH20H I

I

C:O HCOH I I cH2oH cH2oH Dlhydroxyacetone D-Xylulose

HOCH I HCOH

C :O

C :O I HOCH

HCOH

I

HCOH I cH2oH

HCOH I

cH2oH D-Ribulose

D-Fructose

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-

Anomers-nrutarotation

Rr-

The a and p cyclic forms of D-glucose are known as anomers. Thev differ from each other Alcohol AldefryB

sp

,9 € 9a )C rD rE

A is the initial substrate,B, C, and D are the intermediatesand E is the end product, in a pathway catalysed by four different enzymes (e1, e-2,e3, e4).The very first step (A -+ B by the enzyme e1) is the most effective for regulating the pathway, by the final end product E. This type of control is often called negative feedback regulation since increasedlevels of end product will result in its (er) decreasedsynthesis.This is a real cellular economy to save the cell from the wasteful expenditure of synthesizing a compound which is alreadyavailablewithin the c ell. Feedbackinhibition or end product inhibition is a specialised type of allosteric inhibition necessaryto control metabolic pathways for efficient cellular function.

ADP AMP, NADADP, AcetylCoA

AMP

Palmitale

N- Acetylglutamate L- Tryptophan lsocitrale

Carbamoylphosphate+ Aspartate

Feedback control

Carbamoyl aspartate + Pi I Y Cytidinetriphosphate(CTP)

Carbamoyl phosphateundergoesa sequence of reactionsfor synthesisof the end product, CTP. When CTP accumulates,it allosterically inhibitsthe enzyme aspartatetranscarbamoylase by a feedback mechanism. Feedback regulation or feedback inhibition? Sometimesa distinction is made between these two usages.Feedback regulation representsa phenomenonwhile feedbackinhibition involves the mechanism of regulation. Thus, in a true sense,they are not synonymous.For instance, dietary cholesteroldecreaseshepaticcholesterol biosynthesisthrough feedback regulation.This does not i nvol ve feedback i nhi bi ti on, si nce dietary cholesteroldoes not directly inhibit the regulatory enzyme HMG CoA reductase. However, the activity of gene encoding this enzyme is reduced (repression)by cholesterol.

Aspartate transcarbamoylase (ATCase) is of latent enzymes a good example of an allosteric enzyme 2. Activation Latent enzymes,as such, are inactive.Some inhibited by a feedback mechanism. ATCase catalysesthe very first reaction in pyrimidine enzymes are synthesized as Proenzymes or zymogens which undergo irreversiblecovalent biosynthesis.

103

Ghapter 6 : ENZYMES

There are some enzymeswhich are active in activation by the breakdown of one or more peptidebonds.For instance,proenzymes -namely dephosphorylatedstate and become inactive pepsinogenand plasminogen, when phosphorylatede.g. glycogen synthase, chymotrypsinogen, are respectively convertedto the active enzymes acetyl CoA carboxylase. chymotrypsin,pepsin and plasmin. A few enzymesare activeonly with sulfhydryl Certain enzvmes exist in the active and (-SH) groups, €.8. succinate dehydrogenase, inactive forms which are interconvertible, urease.Substanceslike glutathionebring about depending on the needs of the body. The the stabilityof these enzymes. interconversion is brought about by the E -S H + E -S H reversible covalent modifications, namely E-S-S-E Reduced phosphorylation and dephosphorylation, and Oxidised GS-SG inactive 2G-SH active oxidation and reduction of disulfide bonds. Clycogen phosphorylaseis a muscle enzyme that breaks dow'n glycogen to provide energy. This enzyme is a homodimer (two identical subunits)and existsin two interconvertible forms. Phosphorylase b (dephosphoenzyme)is inactive which is convertedby phosphorylationof serine residuesto active form phosphorylasea. The inactiveenzyme phosphorylase b is producedon dephosphorylationas illustratedbelow.

E

Phosphorylaseb (inactive). .,-_-\ '

P

.-....-.

phosphatase

/'

E

2Pi

Organelle

______----l

P Phosphorylasea z (active) _./

3. Gompartnnentation Thereare certainsubstancesin the body (e.g., fatty acids,glycogen)which are synthesizedand also degraded.Thereis no point for simultaneous occurrenceof both the pathways.Cenerally,the synthetic (anabolic) and hreakdown (catabolic) pathways are operative in different cellular organellesto achieve maximum economy. For instance,enzymes for fatty acid synthesisare found in the cytosol whereas enzymes for fatty acid oxidation are presentin the mitochondria. Dependingon the needsof the body - through the mediationof hormonal and other controlsfatty acids are either synthesizedor oxidized. The intracellularlocation of certain enzymes and metabolic pathways is given in Table 6.6.

Enzym e/metaboIi c pathway

Cytoplasm

peptidases; glycolysis; Aminotransferases; hexose monophosphate shunt; fattyacid purine andpyrimidine catabolism. synthesis;

Mitochondria

Fattyacidoxidation; amino acidoxidation; Krebs cycle; ureasynthesis; electron phosphorylation. transport chainandoxidative

Nucleus

Biosynthesis ofDNAandRNA.

(microsomes) phospholipid Endoplasmic reticulum Protein biosynthesis;triacylglyceroland synthesis; synthesis steroid and P4Eo; esterase. reduction; cytochrome Lysosomes

phosphatases; phospholipases; proteases; Lysozyme; hydrolases; lipases; nucleases.

Golgiapparatus

glucosyF 5'-nucleotidase; Glucose 6-phosphatase; andgalactosyl-transferases.

Peroxisomes

D-amino urateoxidase; acidoxidase; longchainfattyacidoxidation. Catalase;

I

l04 4" Control I I

B IOC H E MIS TR Y

of enzyme

synthesis

Most of the enzymes, particularly the rate limiting ones, are present in very low the amount of the concentration.Nevertheless, enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate Iimiting enzymes have short half-lives. This helps in the efficient regulationof the enzyme levels. There are two types of enzymei-(a) Constitutive enzymes (house-keeping enzymes)-the levels of which are not controlled and remain fairly constant. (b) Adaptive enzymes-their concentrationsincreaseor decreaseas per body needs and are well-regulated.The synthesisof enzymes (proteinsl is regulated by the genes (Refer Chapter 25).

!

{

I

ln general,the key and regulatoryenzymes are most rapidly degraded.lf not needed, they i mmedi atel y di sappear and, as and w hen Though required,they are quickly sysnthesized. not always true, an enzyme with long half-life is usually sluggishin its catalytic activity. 6. lsoenzymes Multiple forms of the same enzyme will also help in the regulationof enzymeactivity, Many of the isoenzymes are tissue-specific.Although isoenzymesof a given enzyme catalysethe same reaction, they differ in K' V.nu* or both. e.g. isoenzvmesof LDH and CPK.

Induction and repression: The term induction is used to represent increased synthesis of enzyme while repressionindicates its decreased Enzymesare never expressedin termsof their synthesis. Induction or repression which (as mg or pg etc.), but are ultimatelydeterminesthe enzyme concentration concentration at the gene level through the mediation of expressedonly as activities. Various methods have been introduced for the estimation of hormonesor other substances. enzyme activities (particularly for the plasma Examplesof enzyme induction : The hormone enzymes). In fact, the activities have been insulin induces the synthesis of glycogen expressedin many ways, like King-Armstrong synthetase, glucokinase, phosphofructokinase units, Somogyi units, Reitman-Frankelunits, and pyruvate kinase. All these enzymes are spectrophotometricunits etc. inv olv e d i n th e u ti l i z a ti o n o f g l ucose. The hormone cortisol inducesthe synthesisof many Katal enzymes e.B. pyruvate carboxylase, tryptophan oxygenaseand tyrosine aminotransferase. In order to mai ntai n uni formi ty i n th e (as units) Examplesof repression: In many instances, expression of enzyme activities substrate can repress the synthesis of enzyme. worldover, the EnzymeCommissionof IUB has Pyruvate carboxylaseis a key enzyme in the suggestedradical changes.A new unit- namely (abbreviatedas kat)-was introduced.One synthesis of glucose from non-carbohydrate katal sourceslike pyruvateand amino acids.lf there is kat denotes the conversion of one mole (mol/sec).Activity may also sufficientglucoseavailable,there is no necessity substrateper second (mkat), microkatals for its synthesis. This is achieved through be expressedas millikatals (pkat) and so on. repression of pyruvate carboxylase hy glucose. 5. Enzyme

degradation

Enzymesare not immortal,since it will create a seriesof problems.There is a lot of variability in the half-livesof individualenzymes.For some, it is in days while for others in hours or in m inut e s ,e .g . L D H a - 5 to 6 d a y s ; L D H I - 8 to 12 hou rs ;a m v l a s e-3 to 5 h o u rs .

International Units (lUf Some workers preferto use standardunits or Sl units (SystemInternational).One Sl unit or InternationalUnit (lU) is defined as the amount of enzyme activity that catalysesthe conversion of one micromol of suhstrate per minute. Sl units and katal are interconvertible.

Chapter 6 : ENZYMES

= 60 pkatal (or) 1 nkatal = 1 .6 7 l U 1lU

Enzyme Laboratory

use of enzyme

units

In the clinical laboratories, however, the units- namelv katal or Sl units-are vet to find a place. Many investigators still use the old units like King-Armstrongunits, Somogyi units etc. while expressingthe enzyme activities. lt is therefore, essential that the units of enzyme activity, along with the normal values, be invariably stated while expressingthe enzymes for comparison.

Ribozymes Ribozymes are a group ol ribonucleic acids that function as biological catalysts,and they are regardedas non-proteinenzymes.

Application

Therapeutic applications Streptokinase/urokinaseToremove blood clots Asparaginase Incancer therapy Paoain Anti-inf lammatory o,-Antitrypsin Totreatemphysema (breathing ditficulty due todistension oflungs) Analytical application reagents(for estimation) Glucose oxidase andoeroxidase Glucose

Urease Cholesterol oxidase Uricase Lipase Luciferase

Urea Cholesterol Uricacid Triacylglycerols Todetect bacterial contamination offoods Alkalinephosphatase/ Intheanalyticaltechnique

lgpp-n9irl eereriq?ee _qL!94

Applications in geneticengineering A lt ma n a n d h i s c o w o rk e rs ,i n 1 983, found Restriction endonucleases Gene lransfer, DNAtinger that ribonucleaseP- an enzyme till then known pnnrng to cleave precursorsof tRNAs to give tRNAsIag DNApolymerase Polymerase chain was functional due to RNA component present reaction in the enzyme and not the protein part of the enzyme. The RNA part isolated from ribonucleaseP exhibiteda true enzyme activityand also obeyed Michaelis-Menten kinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis. RNA moleculesare known to adapt a tertiary structure just as in the case of proteins (i.e. enzymes). The specific conformation of RNA may be responsiblefor its function as biocatalyst. It is believed that ribozymes IRNAs) were functioningas catalystsbeforethe occurrenceof protein enzymesduring evolution.

Industrialapplications

preparation Cheese Production ofhigh fructose syrup Infoodindustry to convert starch toglucose powder Washing

Rennin Glucose isomerase u,-Amylase Proteases

Enzymes as therapeqtic

agents

preparedfrom streptococcus 1. Streptokinase is useful for clearing the blood clots. Streptokinase activatesplasmaplasminogen to plasminwhich, in turn, attacksfibrin to convert into solubleproducts. Plasminogen I

Certain enzymes are useful as therapeutic genetic agents, analytical reagents, in m anip u l a ti o n sa n d fo r i n d u s tri a l a ppl i cati ons (Table 6.V.

Streptokinase J Plasmin

I

Fibrin-l-* (clot)

Solubleproducts

B IOC H E MIS TR Y

106

2. The enzyme asparaginaseis used in the Tumorcellsare dependent treatmentof leukemias. on asparagineof the host's plasma for their the multiplication.By administeringasparaginase, Estimationof enzyme activities in biological host'splasmalevelsof asparagineare drastically (particularly plasma/serum)is of great fluids reduced.This leadsto depressionin the viability cl i ni cal i mportance.E nzymesi n the ci rcul ati on of t um o r c e l l s . are divided into two Eroups- plasma functional and pl asmanon-functi onal . reagents Enzyrmes as analytica! Some enzymes are useful in the clinical laboratory for the measurementof substrates, drugs, and even the activitiesof other enzymes. The biochemicalcompounds(e.g.glucose,urea, uric acid, cholesterol)can be more accurately and specifically estimated by enzymatic procedures compared to the conventional chemical methods. A good example is the estimationof plasmaglucoseby glucoseoxidase and peroxidasemethod. lmmobilized

enzymes

Enzymescan be used as catalytic agents in industrial and medical applications. Some of theseenzymesare immobilized by binding-.them t o a s o l i d , i n s o l u b l e ma tri x w h i c h w i l l not affect the enzyme stability or its catalytic activity. Beaded gels and cyanogen bromide activated sepharose are commonly used for . h e b o u nd enzymes im m ob i l i z a ti o no f e n z y m e s T can be preservedfor long periodswithout lossof activity.

l.

Fl asma

speei fi c

or P l astna

functional enzYrnes Certain enzymesare normally presentin the plasma and they have specific functions to perform. Cenerally, these enzyme activitiesare higher in plasma than in the tissues.They are mostly synthesizedin the liver and enter the ci rcul ati on e.g. l i poprotei n l i pase, pl asmi n , thrombin, choline esterase,ceruloplasminetc. l mpai rment i n l i ver functi on or genet ic disordersoften leadsto a fall in the activitiesof plasma functional enzymes e.g' deficiency of cerul opl asmi ni n W i l son' s di sease. 2, i{on-piasrma specific or plasrna enzymes non-functional

These enzymes are either totally absent or oresent at a low concentration in plasma compared to their levels found in the tissues. The digestive enzymes of the gastrointestinal tract (e.g. amylase, pepsin, trypsin, lipase etc.) present in the plasma are known as secretory Clucoseoxidaseand peroxidase,immobilized enzymes. All the other plasma enzymes and coated on a strip of paper, are used in the associated with metabolism of the cell are clinical laboratoryfor the detectionof glucosein collectivefy referred to as consfitutive enzymes u r ine. acid transaminases, (e.g. lactatedehydrogenase, phosphocreatine phosphatases, and alkaline xidgg9 t Gluconicacid+ Hro, kinase). Glucose Hzoz o-Toluidine (colourless)

Hzo Oxidizedtoluidine (bluecolour)

Estimation of the activities of non-plasma specific enzymes is very important for the diagnosisand prognosisof severaldiseases.

The normal serum level of an enzyme indicatesthe balance between its synthesisand The intensityof the blue colour dependson releasein the routine cell turnover. The raised the concentrationof glucose. Hence, the strip enzyme l evel scoul d be due to cel l ul ardamag e, estimation increasedrate of cell turnover, proliferationof method is usefulfor semi-quantitative cells, increasedsynthesisof enzymesetc. Serum of gluc o s ei n u ri n e .

Chapten 6 : ENZYMES

Serum enzyme (elevated)

l',

107

Disease(most important)

Amylase

Aculepancreatitis

glutamate pyruvate (SGPT) Serum transaminase

(hepatitis) Liverdiseases

glutamate (SGOT) Serum oxaloacetale transaminase

(myocardial Heartattacks infarction)

phosphatase Alkaline

jaundice Rickets, obstructive

Acidphosphatase

gland Cancer ofprostate

(LDH) Lactate dehydrogenase

Heart attacks, liverdiseases

phosphokinase (CPK) Creatine

Myocardial (earlymarker) infarction

Aldolase

Muscular dystrophy

5'-Nucleolidase

Hepatitis

yGlutamyl (GGT) transpeptidase

Alcoholism

enzymes are conveniently used as markers to It may be noted that SCPTis more specificfor det ect th e c e l l u l a r d a m a g e w h i c h ul ti matel y the di agnosi sof l i ver di seases w hi l e S C OT i s for helps in the diagnosis of diseases. heart diseases.This is mainly becauseof their cel l ul ar di stri buti on- S C P T i s a cytosom al A summary of the important enzymes useful enzyme while SCOT is found in cytosol and f or t he d i a g n o s i so f s p e c i fi cd i s e a sesi s gi ven i n mi tochondri a. Table6.8. Detailedinformationon the diagnostic enzymes including referencevalues is provided Alkaline phosphatase (ALP) : lt is elevated in in Table 5.9. A brief account of selected certain bone and liver diseases(normal 3-13 KA diagnosticenzymes is discussed units/dl). ALP is useful for the diagnosis of rickets, hyperparathyroidism, carcinoma of Amylase : The activity of serum amylase is bone, and obstructive jaundice. increasedin acute pancreatitis (normal 80-180 Somogyi units/dl).The peak value is observed Acid phosphatase(ACP) : lt is increased in \\,ithin 8-12 hours after the onset of disease the cancer of prostate gland (normal 0.5-4 KA rvhich returns to normal by 3rd or 4th day. units/dl).The tartaratelabile ACP (normal' Certain enzymesare utilized os therapeutic agents. Streptokinosein used to dissolue blood clots in circulation while asparaginoseis emploged in the treatment of leukemias. r':' Determination of serum enzyme actiuities is of great importance t'or the diagnosisof seueral diseoses(refer Table 6.8). rt'' Lowered body temperature (hypothermia) is accompained by o decrease in enzyme actiuities' This principle is exploited to reduce metobolic demand. during open heart

surgery or transportotion of organs lor transplantation surgery.

lt2

BIOCHEMISTF|Y Creatine phosphokinase (precisefy isoenzyme MB) is the first enzyme to be released into circulationwithin 6-18 hoursafterthe infarction. Therefore,CPKestimationis highly usefulfor the early diagnosisof Ml. This enzyme reachesa peak value within 24-30 hours, and returnsto normal level by the 2nd or 3rd day.

i o E N

ul 0

6 12f8243036

424A*6066724

Hours

5

6

7

I

9 10 11

Days

Fig. 6.16 : Enzyme paftern in myocadial infarction (CPK-Creatine phosphokinase; SGOT-Serum

Fo;t;nritar}iiries sf alcohof, r$e",fraydrogenr.*se Alcohol dehydrogenase (ADH) has two heterodimer isoenzymes. Among the white Americans and Europeans,cx,p1isoenzyme is predominantwhereas in Japaneseand Chinese (Orientals)oB2 is mostly present.The isomerop2 more rapidly convertsalcohol to acetaldehyde.

Aspartate transaminase(AST or SCOT) rises sharplyafter CPK, and reachesa peak within 48 hours of the myocardial infarction. AST takes 4-5 days to return to normal level. Lactatedehydrogenase(LDHl) generally rises from the second day after infarction, attains a peak by the 3rd or 4th day and takes about 10-15 days to reach normal level. Thus, LDH is the fast enzyme to rise and also the last enzyme to return to normal level in Ml.

Cardiac troponins (CT) : Although not enzymes/ the proteins cardiac troponins are hi ghl y useful for the earl y di agnosi sof M l. Among these, troponin I (inhibitory element of actomysin ATPase) and troponin f (fropomysin binding element)are important.Cardiactroponin Accumulation of acetaldehvdeis associated (CTl) | is releasedinto circulation within four with tachycardia (increase in heart rate) and hours after the onset of Ml, reachesa peak value facial flushing among Orientals which is not by 12-24 hours,and remainselevatedfor about commonlv seen in whites. lt is believed that a week. Japaneseand Chinesehave increasedsensitivity The protein myoglobin is also an early marker to alcohol due to the presenceof ap2-isoenzyme for the diagnosisof Ml. Myoglobin is however, of A D H . not commonly used as it is not specific to cardiac diseases.ln the Table 6.12, a summary of the diagnosticmarkersused in Ml is given.

Hnzymes in liver diseases

For the right diagnosisof a particular disease, The following enzymes-when elevated in it is always better to estimate a few (three or more) serum enzymes, instead of a single serum-are useful for the diagnosis of liver enzyme. Examples of enzyme patterns in dysfunction due to viral hepatitis (jaundice), toxic hepatitis,cirrhosisand hepatic necrosis important diseasesare given here. Hnrymes

in nnyoeardial

infarction

1 . A l ani ne transami nase;

2. Aspartatetransaminase; The enzymes- namelycreatinephosphokinase 3. Lactatedehydrogenase; (CPK), aspartatetransaminase(AST) and lactate dehydrogenase(LDH)-are important for the The enzymes that markedly increase in diagnosis of myocardial infarction (Ml). The intrahepaticand extrahepaticcholestasisare : elevationof theseenzvmesin serum in relation to '1. Alkaline phosphatase, 2. 5'-Nucleotidase hours/daysof Ml is given in the Fig.6.l6.

113

Chapter 6 : ENZYMES

Diagnostic marker

Time of peak elevation

Time of return to normal level

Diagnostic importance

4-Ohrs

20-25hrs

Earliest marker, however notcardiac specific.

I Cardiac trooonin

12-24hrs

5-9days

Earlymarker andcardiac specific.

troponin T Cardiac

18-36hrs

5-14days

Relatively earlymarker andcardiac specific. However, inotherdegenerative elevated diseases.

phosphokinase (MB) Creatine

20-30hrs

24-48hrs

Cardiac specific andearlymarker.

(LDHl) Laclate dehydrogenase

48-72 hrs

10-15 days

Relatively latemarker andcardiac specific.

Asparlate transaminase

3048hrs

4-6days

Notcardiac specific,

Serum-y-glutamyltranspeptidaseis useful in the diagnosisof alcoholic liver diseases.

(tartaratelabile) is specific for the detection of prostaticcarcinoma.

lNote : Prostate specific antigen (PSA; mol wL 32 KD), though not an enzyme, is a more probably In themuscular dystrophies, dueto reliable marker for the detection of prostate the increased leakage of enzymes from the cancer. Normal serum concentrationof PSA is damaged cells, serum levels of certain muscle 1-4 n{mll. enzymes are increased.These include creatine A non-specific increase in certain enzymes phosphokinase, aldolase and aspartate like LDH, alkalinephosphatase and transaminase transaminase. Of these,CPK is the most reliable indicator of muscular diseases,followed by may be associatedwith malignancy in any part of the body. aldolas e .

Enzymes in muscle diseases

p-C l ucuroni dase esti mati oni n uri ne i s usefu l for detecting the cancers of urinary bladder, Increase in the serum acid phosphatase pancreasetc.

Enzymes

in cancers

114 B IOC H E MIS TFI Y

r' Enzymesare the protein biocatarystssynthesizedby the

'!ii,iir

cers.They are classified c/osse's---'ox idoreductaies,transferi s"r, nvi 'iuing rZ nu, r, tyases, isomerases r!ri'"r

2' An enzgm" o to::,r!r-:in its action,possessingactiue site, where the substrate binds form enzgme-substrqtecomplex, i"f;;. to the product is t'ormed. 3. Factorslike cont

xtJ,.!J{j,i{T,zT tr;ti,,:";":i",:! riii:{ii%Ti;'i!"Ji,i;:::{:;:::::;:i, n reuersibte (competitiue, and non-competitiue), ,1,i":,I'f*f."iX'J'"i,,J,i2,,:"Y:::!,bv

S Many enzymesrequire prerenceot' non-protein substances .the called cofactors (coenzyntes) Most of tt''' for i*r"v-o-Lre"iriwtiues of B-comptex- uitamins(e.g. NAD+, 'i;;:::7" FAD, 6

The mechanism

"I,:rrr\",a.ctjo2 is e.xproinedbv tock and keymoder (oJFischer),mare recentty induced fit madet(of Koshtand) ,"i ,"triiiJililn ,n.orr. rateof reaction throughacid-base catatysis, ' [l"o;:':ff:r7:;;::,'n" couarentcatarysis

t ,;,!l;i;:,if"i1n]lf,*, thereis a constant regutation of enzymeteuersbroughtaboutbv sm,actiuation ot'proenzgmes, synthesis anddegrad"ri"i.t etc. "*i^Z 9. Estimation of sert

serum o*,u;*-;;,:f i,;:::T:: ,:,,'J",::;i: !:':,,,;"!,:*i;i,f:::;:,?1.'f:",:1j,,:::::;

hepatitis; aspartate,. trarraminas., lorror" dihgdrogenase"(tDH) phosphokinase(CpK) and creatine i,i.rorrrror,, alkaline phosphatase ^ in ^uoroi,"ot rickets and o,,ia

i,n:::i::i::;::;:f

inolin'io''.-"'nprostatic,o,,,no-[;

vstutamv!transpep-

L0' Isoenzymesare the,,murtip,re fotrys of an enzyme catarysingthe same howeue4 dift'er in their phvsicai'rri reoctian which ii"^,car propertie".iD; has five isoenzgmes hasthree'roui""a ciiz;;;'r",u

i:i::ri::

importantin thedragnosis ot'myocardiar

115

Chapter 6 : ENZYMES

I.

Essayquestiosrs and nomenclature. 1. What are enzymes?Describetheir classification 2. Write an accountof the variousfactorsaffectingenzymeactiviti. 3. Describethe mechanismof enzymeaction. Write brieflyon the role of coenzymesin enzymeaction. 4. What are coenzymes? 5. Write an accountof the importanceof serumenzymesin the diagnosisof diseases.

lL Short notes (a) Enzyme specificity,(b) Competitive inhibition, (c) Coenzymes,(d) Allosteric enzymes/ (f) K, value,(g)Serumenzymesin myocardialinfarction,(h) Lactatedehydrogenase, (e)lsoenzymes, (i) Role of metalsin enzymeaction,(j) Active site. lI L F ill i n th e b l a n k s 1. T h e l i te ra lme a n i n go f e n z y m ei s 2. The classof enzymesinvolvedin syntheticreactionsare 3. The non-proteinpart of holoenzyme 4. Enzymeslose the catalytic activity at temperatureabove 70oCdue to 5. Examplesof two enzymescontainingzinc are

and

6. The place at which substratebinds with the enzyme requiresthe coenzyme dehydrogenase 7. The enzymeglucose6-phosphate rs 8. The E.C.numberfor alcohol dehydrogenase activatedby is allosterically 9. Phsophofructokinase 10. The very first enzymeelevatedin serumin myocardialinfarction I V . M u l ti p l e c h o i c e q u e s ti o n s 11. Pepsinis an examplefor the classof enzymesnamely (c) Hydrolases(d) LiSases. (a) Oxidoreductases(b) Transferases 12. The coenzymenot involvedin hydrogentransfer (a) FMN (b) FAD (c) NADP+(d) FH4. 13. In the feedbackregulation,the end productbinds at (a) Active site (b) Allostericsite (c) E-Scomplex(d) None of these. activityin serumis elevatedin 14. y-Clutamyltranspeptidase (a) Pancreatitis (b) Musculardystrophy(c) Myocardialinfarction(d) Alcoholism. 15. In recent years/a non-proteincompound has been identifiedto bring about catalysisin biologicalsystem.The name of the compoundis (a) DNA (b) RNA (c) Lipids(d) Carbohydrates.

Vitamins t, ++ Fat Water soluble soluble

I t i s d i ffi c u l t to d e fi n e v i ta m i ns preci sel y. I vitamins may be regarded as organic compounds required in the diet in small amounts to perform specific biological functions for normal maintenance of optimum growth and health of the organism. The bacterium E.coli does not requireany vitamin, as it can synthesize all of them. lt is believedthat during the course of e v o l u ti o n ,th e a b i l i ty to s y n th esi zevi tami ns was lost. Hence, the higher organismshave to obt a i nth e m fro m d i e t. T h e v i ta m i nsare requi red in s ma l l a m o u n ts , s i n c e th e i r degradati oni s relativelyslow.

the naturalfoods.Funk (1913)i sol atedan act ive pri nci pl e (an ami ne) from ri ce pol i shi ngsand, l ater i n veast, w hi ch coul d cure beri -be r i in pigeons. He coined the term vitamine (Creek : vita-life) to the accessoryfactors with a belief that all of them were amines.lt was later realised that only few of them are amines. The term vitamin, however, is continued without the final letter 'e'.

The usage of A, B and C to vitamins was i ntroduced i n 1915 by McC ol l um and Davis. They first felt there were only two vitaminsfat soluble A and water soluble I (anti-beriberi factor). Soon another water soluble anti-scurvy His to ry a n d n o m e n c l a tu re factor named vitamin C was described.Vitamin I n th e b e g i n n i n g o f 2 0 th c e ntury, i t w as A was later found to possesstwo componentsc lea rl y u n d e rs to o d th a t th e d i ets contai ni ng one that preventsnight blindness(vitaminA) and purified carbohydrate,protein, fat and minerals anotheranti-ricketfactor named as vitamin D. A were not adequateto maintain the growth and fat sol ubl efactorcal l edvi tami nE , i n the abs ence health of experimentalrats, which the natural of which rats failed to reproduceproperly, was foods (such as milk) could do. discovered. Yet another fat soluble vitamin Hopkins coined the term accessoryfactors to concerned with coagulationwas discovered in the unknown and essentialnutrientspresent in mi d 1930s. l t w as named as vi tami n K . In t he

Chapter 7 : VITAMINS

777

sequenceof alphabets it should have been F, but K was preferred to reflect its function (koagulation). As regardsthe water soluble factors,vitamin C was identifiedas a pure substanceand named as ascorbic acid. Vitamin B was found to be a complex mixtureand nomenclaturealso became complex. B1 was.clearly identified as anti-beriberi factor. Many investigators carried out intensiveresearchbetween 192O and 1930 and went on n a m i n g th e m a s th e w a te r sol ubl e v it am ins82 , 8 3 , 8 4 ,8 5 ,8 6 , 8 7 , 8 6 , B g ,8 19, 811 and 812. Some of them were found to be mixturesof alreadyknown vitamins.And for this reason, a few members (numbers!)of the Bcomplex series disappeared from the scene. Exceptfor 81, Bz, Bo and 812, names are more commonly used for other B-complexvitamins.

hematopoietic (folic acid and 812). Most of the water soluble vitamins exert the functions through their respectivecoenzymeswhile only one fat solublevitamin (K) has been identifiedto function as a coenzyme. $ynthesis of vitannims by intestina! bacteria Vitamins, as per the definition, are not synthesizedin the body. However, the bacteria of the gut can produce some of the vitamins, required by man and animals. The bacteria mainly live and synthesizevitamins in the colon region, where the absorptionis relativelypoor. Some of the animals (e.g. rat, deer etc.) eat their own feces, a phenomenon known as coprophagy.

As far as humansare concerned,it is believed that the normal intestinal bacterialsyntfiesig and of vitamins absorption of vitamin K and biotin may be There are about 15 vitamins, essentialfor sufficient to meet the body requirements. For humans. They are classifiedas fat soluble (A, D, other B-complex vitamins, the synthesis and E and K) and water soluble (C and B-group) absorptionare relatively less.Administrationof vitamins as shown in the Table 7.1. The anitibiotics often kills the vitamin synthesizing B - c om plex v i ta m i n s ma y b e s u b -d i v i d ed i nto bacteria present in the gut, hence additional energy-releasing (81, 82, 86, biotin etc.) and consumptionof vitamins is recommended. Glassification

Vitamin A Vitamin D V1amin E Vitamin K

I Vitamin C I

acid(Bn) l-Folic L-Vitamin 8', (cyanocobalamin)

--.._

118 Fa{ soluble

B IOC H E MI STRY vitamins-general

The four vitamins, namely vitamin A, D, E, and K are known as fat or lipid soluble. Their availabilityin the die! absorptionand transport are associatedwith fat. Thev are soluble in fats and oils and also the fat solvents (alcohol, acetoneetc.). Fat soluble vitaminscan be stored in liver and adiposetissue.They are not readily excreted in urine. Excessconsumptionof these vitamins (particularlyA and D) leads to their accumulationand toxic effects. '

Vitamers The term vitamers representsthe chemically similar substances that possess qualitatively similar vitamin activity. Some good examples of vitamersare given below . Retinol,retinal and retinoic acid are vitamers of vitamin A. . Pyridoxine, pyridoxal and pyridoxamine are vitamersof vitamin B..

Alf the fat soluble vitamins are isoprenoid compounds, since they are made up of one or more of five carbon units namely isoprene units ( -C H = C .C H 3 -C H = C H -). F a r sol ubl evi tami ns In the fol l ow i ng pages, the i n dividual perform diverse functions. Vitamin K has a members of the fat soluble and water soluble specific coenzymefunction. vitamins are discussed with regard to the chemistry,biochemicalfunctions,recommended Water sCIluble vitamlels*seneral dietary/dailyallowances(RDA), dietary sources, The water soluble vitamins are a deficiency manifestationsetc.

heterogenousgroup of compounds since they differ chemically from each other. The only common character shared by them is their solubility in water. Most of these vitamins are readily excreted in urine and they are not toxic to the body. Water soluble vitamins are not stored in the body in large quantities (except 812).For this reason,they must be continuously supplied in the diet. Generally, vitamin deficiencies are multiple rather than individual with overlapping symptoms. lt is often difficult to pinpoint the exact biochemical basis for the symptoms. The water soluble vitamins form coenzymes (Refer Table 5.3) that participate in a variety of biochemical reactions,related to either energy generationor hematopoiesis.lt may be due to this reasonthat the deficiencyof vitaminsresults in a number of overlapping symptoms. The common symptomsof the deficiency of one or more vitamins involved in energy metabolism in c l u d e d e rma ti ti s ,g l o s s i ti s(r ed and sw ol l en tongue),cheilitis (ruptureat the cornersof lips), di a rrh e a , m e n ta l c o n fu s i o n , depressi on and malaise.

The fat soluble vitamin A, as such is present only in foods of animal origin. However, its provitamins carotenes are found in plants. It is recorded in the history that Hippocrates (about 500 B .C .) cured ni ght bl i ndness. He prescribedto the patients ox liver (in honey), which is now known to contain high quantity of vi tami n A . Chennistry In the recent years, the term vitamin A is collectively used to representmany structurally related and biologically active molecules (Fig.7.1).The term retinoids is often used to include the natural and synthetic forms of vitamin A. Retinol, retinal and retinoic acirl arc regardedas vitamersof vitamin A.

1. Retinol (vitamin A alcohol) : lt is a primary alcohol containingp-ionone ring. The side chain has two isoprenoid units, four double bonds and D e fi c i e n c yo f v i ta m i n s8 1 , 8 6 and B 12i s more one hydroxylgroup. R eti noli s presenti n anim al closelyassociated with neurologicalmanifestations. tissuesas retinylesterwith long chain fatty acids.

119

Ghapter 7 : VITAMINS

Retinal -C=O I H Retinolc acid

-C=O I OH

p-lonone

Fig. 7.1 : Structuresof vitaminA and relatedcompounds(Redcolour reptesents the substituentgroupsin the respectivecompounds).

2. Retinal (vitamin A aldehyde) : This is an aldehyde form obtained by the oxidation of retinol. Retinal and retinol are interconvertible. Previously, the name retinine was used for r et inal.

As and when needed, vitamin A is released from the liver as free retinol. lt is believed that zinc plays an important role in retinol mobilization. Retinol is transported in the circulation by the plasma retinol binding protein (RBP; mol. wt. 21,000) in association with 3. Retinoic acid (vitamin A acid) : This is pre-albumin.The retinol-RBPcomplex binds to produced by the oxidation of retinal. However, specific receptors on the cell membrane of retinoic acid cannot give rise to the formationof peripheral tissue and enters the cells. Many retinal or retinol. cells of target tissues contain a cellular retinol4. p-Carotene (provitamin A) : This is found binding protein that carries retinol to the in plant foods. lt is cleaved in the intestineto nucl eus and bi nds to the chromati n (D N A ). produce two moles of retinal. ln humans, this It is here that retinol exerts its function in conversion is inefficient, hence p-carotene a manner analogous to that of a steroid possessesabout one-sixth vitamin A activity hormone. compared to that of retinol.

Absorption, transport and mobilization

BIOCHEMICAL FUNCTIONS

Vitamin A is necessary for a variety of functions such as vision, proper growth and Dietary retinyl esters are hydrolysed by differentiatioryreprbduction and maintenanceof pancreaticor intestinalbrush border hydrolases epithelial cells. In recent years, each form of in the intestine,releasingretinol and free fatty vitamin A has been assignedspecific functions acids. Carotenes are hydrolysed by p-carotene (Fig.7.3). l5-1S'-dioxygenaseof intestinal cells to release Vitamin A and vision : The biochemicalfunc2 moles of retinalwhich is reducedto retinol. In tion of vitamin A in the processof vision was first elucidated by Ceorge Wald (Nobel Prize 1968). The events occur in a cyclic process known as Rhodopsin cycle or Wald's visual cycle (Fig.7.4).

BIOCHEMISTFIY

120

lntestinalcell p-Carotene

j Retinal

J

Retina All-fransretinol

II

+ All-transretinal

II

J

Visual cycle (SeeFig.7.a)

Chylomicrons

RBP-t Retinol-RBP

Nuclear receptor

i j

v Specificproteins I I

+ Cell difierentiation

functions transportandbiochemical : summaryof vitaminA absorption, Fig.7.2 (FFA-Freefaw acid; RBP-Retinolbindingprotein)'

121

Chapter 7 : VITAMINS

Dark adaptationtime : When a person shifts from a bri ght l i ght to a di m l i ght (e.9.entry i nto a dim cine theatre), rhodopsin stores are + depleted and vision is impaired. However, Retinol (steroid hormone--{roMh and difterentiation) within a few minutes,known as dark adaptation time, rhodopsin is resynthesizedand vision is improved. Dark adaptationtime is increasedin RetinylPhosphate Retinal (visualcycle) (glycoproteinsynthesis; vitamin A deficient individuals. B-Carotene (antioxidant)

I

I

+ Retinoicacid (steroidhormone-growth and differentiation)

Flg.7.3 : Summaryof thefunctions of vitaminA compounds.

Rods a n d c o n e s

Bleaching of rhodopsin: When exposed to light, the colour of rhodopsinchangesfrom red to yellow, by a process known as bleaching. B l eachi ng occurs i n a few mi l l i secondsand many unstable intermediatesare formed during this orocess. Rhodopsin

-----) Lumirhodopsin Prelumirhodopsin

The retina of the eye possessestwo types of All-trans-retinal+ ll +-- Metartdopsin Opsin +- Metarhodopsin I cells-rods and cones.The human eve has about 10 m illi o n ro d s a n d 5 m i l l i o n c o n e s.The rods Visual cascadeand cGMP : When light strikes are in the peripherywhile conesare at the centre the reti na, a number of bi ochemi cal change s of retina. Rods are involved in dim light vision l eadi ng to membrane hyperpol ari zati onoccur whereas cones are responsiblefor bright light resul ti ngi n the genesi sof nerve i mpul se.The and c ol o u r v i s i o n . An i m a l s -s u c h a s ow l s and hyperpolarizationof the membrane is brought cats for which night vision is more importantabout by a vi sualcascadei nvol vi ngcycl i c C MP . possessmostly rods. When a photon (from ligh$ is absorbed by rhodopsin, metarhodopsinll is produced. The W ald' s v i s u a l c y c l e protein transducin is activated by metarhodopsin Rhodopsin(mol. wt. 35,000) is a conjugated l l . Thi s i nvol vesan exchangeof C TP for C D P on proteinpresentin rods.lt contains11-crsretinaland inactive transducin. The activated transducin This the proteinopsin.The aldehydegroup (of retinal)is activates cyclic GMP phosphodiesterase. linkedto e-aminogroupof lysine(of opsin). T he p ri ma ry e v e n t i n v i s u a l cycl e, on exposureto light, is the isomerizationof 11-cis: retinal to all-trans retinal. This leads to a c onf or ma ti o n a l c h a n g e i n o p s i n whi ch i s responsiblefor the generationof nerve impulse. The all-trans-retinalis immediately isomerized by r et in a l i s o me ra s e(o f re ti n a l e p i thel i um) .to 1 1- c is - re ti n a l .T h i s c o mb i n e s w i th opsi n.:to regeneraterhodopsin and complete the visual cycle (Fig,7.4).However, the conversionof all frans-retinalto 1 1-crs retinal is incomplete. Therefore, most of the all-frans-retinal is transportedto the liver and convertedto all-frans The all-transretinol by alcbhol dehydrogenase. retinol undergoesisomerizationto 1 1-crsretinol whic h i s th e n o x i d i z e d to 1 1 -c i s reti nal to par t ic ip a tei n th e v i s u a l c y c l e .

Light (photon) Nerve

impulse

lsomerase

I

--" ' -.--- | All- i,'ansretinal 11-cl s-reti('nal -------------+

11-cts-retinol +-

l1ferf+All-trane.retinol

Flg.7.4: Wald'sulsualcycle.

122

B IOC H E MIS TRY synthesisand thus are involved in the cell growth and differentiation.

Rhodopsin

lu'no'o"

2. V i tami n A i s essenti alto mai ntai nhe alt hv epi thel i al ti ssue. Thi s i s due to the fact t hat retinol and retinoic acid are requiredto prevent keratin synthesis(responsiblefor horny surface).

Metarhodopsinll

3. Retinylphosphatesynthesizedfrom retinol is necessary for the synthesis of certain glycoproteinq which are required for growth and mucus secretion.

Phosphodiesterase (inactive)

3',5'-cGMP

51GMP

Fig.7.5 : The visual cascade involving cyclic guanosine monophosphate(3" 5' -cGMP).

enzyme degradescyclic CMP ir\;he rod cells (Fi9.7.5).A rapid decreasein cyclic GMP closes the Na+ channels in the membranesof the rod c el l s . T h i s re s u l tsi n h y p e rp o l a r i zati on w hi ch i s an excitatory responsetransmittedthrough the neuron network to the visual cortex of the brain.

4. R eti noland reti noi c aci d are i nvol v ed in the synthesisof transferrin,the iron transport protein. 5. Vitamin A is consideredto be essentialfor the maintenanceof proper immune system to fight againstvarious infections. 6. Cholesterolsynthesisrequiresvitamin A. Mevalonate,an intermediatein the cholesterol biosynthesis,is diverted for the synthesis of coenzyme Q i n vi tami n A defi ci ency. lt is pertinentto note that the discoveryof coenzyme Q w as ori gi nal l y made i n vi tami n A deficient ani mal s.

7. Carotenor'ds(most important p-carotene) function as antioxidants and reduce the risk of C o n e s a re s p e c i a l i z e di n b ri ght and col our cancers initiated by free radicals and strong vision. Visual cycle comparableto that present oxidants.p-Caroteneis found to be beneficialto in ro d s i s a l s o s e e n i n c b n e s .T h e col our vi si on prevent heart attacks.This is also attributedto is governed by colour sensitive pigmentsthe antioxidantproperty. (red), iodopsin (green) and porphyropsin cyanopsin (hlue). All these pigments are retinaldietary Recommended opsin complexes.When bright light strikesthe (RDA) allowance retina, one or more of these pigments are The dai l y requi rement of vi tami n A is ble a c h e d ,d e p e n d i n go n th e p a rt i cul arcol our of as retinol equivalents(RE)rather than expressed light. The pigmentsdissociateto all-trans-retinal Internati onal U ni ts (l U ). and o p s i n ,a s i n th e c a s eo f rh o d opsi n.A nd thi s reactionpasseson a nerve impulseto brain as a 1 retinol equivalent=1 lrg retinol specific colour-red when porphyropsinsplits, =6 Pg P-carotene greenwhen iodopsinsplitsor blue for cyanopsin. = 12 pg othercarote noids Splitting of these three pigments in different = 3.33 l U of vi tamin A proportionsresultsin the perceptionof different activity from retinol colours by the brain.

Golour vision

Other biochemical functions of vitamin

= ' 10 l U of vi tamin A activityfrom p-carotene A

'1. Retinol and retinoic acid function almost like steroid hormones.They regulatethe protein

The RDA of vitamin A for adults is arouno !"Q0Oretiytolequivalents(3,500 lU) for man and 800 retinol equivalents(2,500 lU) for woman.

Chapter 7 : VITAMINS

123

Effect on reproduction : The reproA)tiu" O ne I nt e rn a ti o n aUl n i t (l U ) e q u a l sto 0 .3 mg of r et inol. T h e re q u i re me n ti n c re a s e si n grow i ng system is adversely affected in vitamin A children,pregnantwomen and lactatingmothers. deficiency.Degenerationof germinalepithelium leads to sterility in males.

Dietary sources Animal sourcescontain (preformed)vitamin A. The best sourcesare liver, kidney, egg yolk, milk, cheese,butter.Fish (cod or shark)liver oils are very rich in vitamin A.

Effect on skin and epithelial cells : The skin becomes rough and dry. Keratinization of epithelial cells of gastrointestinal tract, urinary tract and respiratorytract is noticed. This leadsto increasedbacterialinfection.VitaminA deficiencv is associatedwith formationof urinary stones.

Vegetable sources contain the provitamin The plasma level of retinol binding protein is A-carotenes.Yellow and dark green vegetables decreased in vitamin A deficiency. and fruits are good sources of carotenese.g. carrots,spinach,amaranthus,pumpkins,mango, Hypervitaminosis A papaya etc. Excessiveconsumptionof vitamin A leadsto toxicity. The symptoms of hypervitaminosisA Vitamin A deficiency include dermatitis(drying and rednessof skin), The deficiencysymptomsof vitamin A are not enlargement of liver, skeletal decalcification, immediate,sincethe hepaticstorescan meet the tenderness of long bones, loss of weight, body requirements for quite sometime (2-4 irritability,loss of hair, joint pains etc. months). The deficiency manifestations are Total serum vitamin A level (normal 20-50 relatedto the eyes, skin and growth. pgldl) is elevated in hypervitaminosisA. Free Deficiency manifestationsof the eyes : Nrghf retinol or retinol bound to plasmalipoproteinsis blindness (nyctalopia) is one of the earliest actually harmful to the body. lt is now believed symptoms of vitamin A deficiency. The that the vitamin A toxicosis symptoms appear indiv idu a l sh a v e d i ffi c u l ty to s e e i n d i m l i ght only after retinol binding capacity of retinol since the dark adaptation time is increased. binding protein exceeds. Prolonged deficiency irreversibly damages a Higher concentrationof retinol increasesthe num ber o f v i s u a l c e l l s . , synthesisof lysosomalhydrolases.The manifestations of hypervitaminosisA are attributed to Severe deficiency of vitamin A leads to the destructiveaction of hydrolases,particularly xerophthalmia. This is characterized by dryness on the cell membranes. in conjunctivaand cornea,and keratinizationof epithelial cells. In certain areasof conjunctiva, Beneficial effects of 0.carotene white triangularplaquesknown as Bitot's spots Increased consumption of p-carotene is are seen. associatedwith decreased incidence of heart lf xerophthalmiapersisitsfor a long time, attacks,skin and lung cancers.This is attributed corneal ulcerationand degenerationoccur. This to the antioxidant role of p-carotenewhich is resultsin the destructionof cornea,a condition independentof its role as a precursorof vitamin referred to as keratomalacia, causing total A. Ingestionof high doses of p-carotenefor long VitaminA deficiencyblindnessis mostly periods are not toxic like vitamin A. blindness. common in childrenof the developingcountries. Other

deficiency

manifestations

V i tami n D i s a fat sol ubl e vi tami n. l t Effect on growth : Vitamin A deficiency resultsin growth retardationdue to impairment resemblessterolsin structureand functions like a hormone. in skeletalformation.

124

B IOC H E MIS TRY

Absorption,

tlansport

and storage

Vitamin D is absorbedin the small intestine for w hi ch bi l e i s essenti al .Through l ym ph, vi tami n D entersthe ci rcul ati onbound to pl asm a a2-gl obul i n and i s di stri butedthroughout t he bodv. Liverand other tissuesstoresmall amounts of vi tami n D . ME TA B OLIS M A N D FU N C TION S B IOC H E MIC A L V i tami ns D 2 and D 3, as such, are not biologically active. They are metabolized identically in the body and convefted to active forms of vi tami n D . The metabol i sm a nd bi ochemi calfuncti onsof vi tami n D are depi ct ed in Fig.7.8. (Dr) Ergocalclferol Ftg.7.6 : Formationol ergocalciferolfromergosterol. The symptoms of rickets and the benefical effects of sunlight to prevent rickets have been known for centuries.Hess (1924) reportedthat ir r ad i a ti o nw i th u l tra v i o l e t l i g h t i nduced anti rachitic activity in some foods. Vitamin D was is ola te db y An g u s(1 9 3 1 )w h o n a m e di t cal ci ferol . Ghemistry

Synthesis

of 1,25-DHCC

Cholecalciferolis first hydroxylatedat 25th position to 25-hydroxycholecalciferol(25-OH o3) bv a specific hydroxylasepresent in liver. 25-OH D3 is the major storageand circulatory a specific form of vitamin D. Kidney possesses (calciol) enzyme, 25-hydroxycholecalciferol l -hydroxylase which hydroxylates 25-hydroxycholecalciferolat position 1 to produce 1,25(1,2|-DHCC). 1,25 dihydroxycholecalciferol (1,3 groups and 25 D H C C contai ns3 hydroxyl referred Both to as calcitriol. the carbon) hence hydroxylase enzymes (of liver and kidney) requirecytochromePa56,NADPH and molecular oxygen for the hydroxylation process. The synthesis of calcitriol is depicted in Figs.7,7 and 7.8.

Ergocalciferol(vitamin D2) is formed from ergosterol and is present in plants (Fi9.7.6). Chol e c a l c i fe ro(v l i ta m i nD 3 ) i s fo u n d i n ani mal s. Both the sterolsare similar in structureexcept that ergocalciferol has an additional methyl group and a double bond. Ergocalciferol and cholecalciferol are sourcesfor vitamin D activitv and are referred to as provitamins. Regulation of the synthesis of 1125.-DHCC During the course of cholesterolbiosynthesis (Chapter l4), 7-dehydrocholesterol is formed The concentrationof 1,25-DHCC is regulated as an intermediate.On exposure to sunlight, by plasma levels of calcium and phosphate. 7-dehydrocholesterolis converted to choleThey control hydroxylationreaction at position c alc i fe ro l i n th e s k i n (d e rm i s a nd epi dermi s) 1. Low plasma phosphateincreasesthe activity (Fig.2.V Vitamin D is regarded as sun-shine iferol 1-hydroxylase.Low of 25-hydroxycholecalc vitamin. pl asma cal ci um enhances the producti on of The synthesisof vitamin D3 in the skin is parathyroid hormone which in turn activates proportional to the exposure to sunlight. Dark 1-hydroxylase.Thus the action of phosphateis skin pigment lm-qianjn)-adversly influences the di rectw hi l e that of cal ci um i s i ndi recton ki d nev s y nth e s i s ' ocf h o l e c a l c i fe ro l . 1-hydroxyl ase.

125

Ghapter 7 : VITAMINS

Biochemical

functions

Calcitriol (1,25-DHCC)is the biologically active (ormof vitaminD.lt tegulatesthe plasma Ievelsof calciumand phospha,fe. Calcitriolacts at 3 differentlevels(intestine, kidneyand bonel to maintainplasmacalcium(normal9-11 mg/dl). 7-Ilehydrocholestercl (animals) Sunlight 4S k i n

1. Action of calcitriol on the intestine: Calcitriof increlses the intestinal absorptionof calciumand phosphate. In the intestinalcells, calcitriolbindswith a cytosolicreceptorto form a calcitriol-receptor complex.Thiscomplexthen approachesthe nucleusand interactswith a specific DNA leading to the synthesisof a specific calcium binding protein.This protein increases the calciumuptakeby the intestine. The mechanismof action of calcitriolon the targettissue(intestine) is similarto the actionof a steroidhormone. 2. Action of calcitriol on the bone : In the osteoblasts of bone,calcitriolstimulates calcium uptakefor deposition as calciumphosphate, Thus calcitriolis essential for boneformation.Thebone is an important reservoir of calciumandphosphate. Calcitriol along with parathyroidhormone increasesthe mobilizationof calcium and phosphate fromthe bone.Thiscauses in elevation the plasnlacalciumand phosphate levels.

25-Hydroxycholecalciferol (Calcidiol)

II Calcidiol1o-hydroxylase |

(kidney)

J

1,25-Dlhydrorycholecalclferol (1,25DHCCor calcitrlol)

3. Action of calcitriol on the kidney : Calcitriolis also involvedin minimizingthe excretionof calciumand phosphate throughthe kidney, by decreasingtheir excretion'and enhancingreabsorption. The sequenceof eventsthat take place in response to low plasmacalciumconcentration and the actionof calcitriolon intestine,kidney and bone, ultimatelyleadingto the increasein plasma calcium is given in Fi9.7.9. 24,25-Dihydroxycholecalcife rol (24,25-DHCC\ is anothermetaboliteof vitamin D, lt is also synthesized in the kidneyby 24-hydroxylase. The exactfunctionof 24,2S-DHCC is not known.lt is believedthat when' calcitriolconcentration is adequate,24-hydroxylase acts leading to the synthesisof a less importantcompound24, 25-DHCC. In this way, to maintain the homeostasis of calcium,synthesis of 24,25-DHCC is alsoimportant.

4

B IOC H E MIS TFI Y

Skin 7-Dehydrocholesterol

!*ro*-t*a*

lntestine calcitriol(r)

JRecentor(J) Calcitriol

ffi8lrr Bone formation and turnover

Calciumbinding protein Plasma

Ca

Ca2*absorption

Chapter 7 : VITAMINS

127

Plasmacalcium{

In countries with good sunlight (like Indi a), the RDA for vitamin D is 200 lU (or 5mg chol ecal ci ferol ).

J t

Dietary sources

,'i

Cood sourcesof vitamin D include fany fish, fish liver oils, egg yolk etc. Milk is not a good source of vitamin D.

Fig. 7.9 : Summary of the action of calcitiol in elevating plasma calcium.

Y it am in D i s a h o rmo n e not a vitamin-justification

and

Vitamin D can be provided to the bodv in three ways '1. Exposureof skin to sunlight for synthesis of vi tami n D ; 2. Consumptionof natural foods; 3. By irradiating foods (like yeast) that contain precursorsof vitamin D and fortification of foods (milk, butter etc.).

Calc i tri o l (1 ,2 5 -D H C C ) i s n o w consi dered Deficiency symptoms an important calciotropic hormone,. while -:.olecalciferol is the prohormone.The following Vitamin D deficiency is relatively less :"aracteristicfeaturesof vitamin D (comparable common/ si nce thi s vi tami n can be synthesi ze d in the body. However, insufficient exposure .r tn hormone)justify its statusas a hormone. to sunlight and consumption of diet lacking I . Vitamin D3 (cholecalciferol)is synthesized vitamin D results in its deficiency. in the skin by ultra-violetrays of sunlight. Vitamin D deficiency occurs in strict l. T h e b i o l o g i c a l l ya c ti v efo rm o f vi tami n D , vegetar.ians, chronic alcoholics,individualswith : a c it r io l i s p ro d u c e di n th e k i d n e y . Iiver and kidney diseasesor fat malabsorption 3. Calcitriol has target organs-intestine, syndromes.In some people,who cover the entire body (purdah)for religiouscustoms,vitamin D lo.e and kidney, where it specificallyacts. deficiencyis also observed,if the requirementis i. Calcitriol action is similar to steroid not met through diet. lnnnones. lt binds to a receptor in the cytosol Deficiency of vitamin D causes rickets in :-"0 ihe complex acts on DNA to stimulatethe children and osteomalacia in adults. , , - : hes i s o f c a l c i u m b i n d i n g p ro te i n . derived from an old English *ord 'rvr-i.kk"n', J . { c ti n o m y c i n D i n h i b i ts th e a cti on of meaning to twist. Osteomalacia is derived :: :ri:'iol. This supportsthe view that calcitriol from Creek (osteon-bone; malakia-softness). :rets its etfecton DNA leadingto the synthesis Vitamin D is often called as antirachitic vitamin. :- R.\ { (transcription). Ricketsin children is characterizedby bone i Calcitriol synthesisis self-regulatedby a deformi ti esdue to i ncompl ete mi neral i zati on , mechanismi.e., calcitriol decreasesits -:e:3ack resultingin soft and pliable bones and delay in r " " . - S \n th e s i s . teeth formation. The weighrbearing bones are bent to lorm howJegs. In rickets, the plasma Recommended dietary level of calcitriol is decreased and alkaline allowan c e { R D A I phosphatase activity is elevated. Alkaline --t iaill' requirementof vitamin D is 400 phosphataseis concerned with the processof Wenntknal Units or 10 mg of cholecalciferol. bone formation. There is an overproductionof

B IOC H E MIS TR Y

128 alkalinephosphatase relatedto more cellular activitv of the bone. lt is believedto be due to a vain attemptto resultin bone formation.

Hsc.

CHn t-

cH2-(cH2-cH2-cH-cH2)3-H

In case of osteomalacia(adult rickets) HO demineralizationof the bonesoccurs (bones becomesofter),increasingtheir susceptibility to fractures. Rena ! ri c k e ts {renal osteodystrophy)

cHs

(5,7,8-trimethyltocol) cr,-Tocopherol p-Tocopherol (5,8-dimethyltocol) (7,8-dimethyltocol) y-Tocopherol

Fiq.7.10 : Structureof a-tocopherol (Note : The tocopherols differ in the substitution of methyl groups, represented in red).

This is seen in patientswith chronic renal failure. Renalricketsis mainly due to decreased tocopherols (vitamin E vitamers) have been synthesisof calcitriol in kidney. lt can be treated identified-a, p, T, 6 etc. Among these, u-tocopherol is the most active. The tocopherols by ad m i n i s tra ti o no f c a l c i tri o l . are derivatives of 6-hydroxy chromane (tocol) Hypervitaminosis D ring with isoprenoid (3 units) side chain. The Vitamin D is storedmostly in liver and slowly antioxidantpropertyis due to the chromanering. metabolised.Among the vitamins, vitamin D is the mosf toxic in overdoses(10-100times RDA). Toxic effects of hypervitaminosisD include demineralization of bone (resorption) and increasedcalcium absorptionfrom the intestine, Ieading to elevated calcium in plasma (hypercalcemia). Prolonged hypercalcemia is associatedwith depositionof calcium in many soft tissuessuch as kidney and arteries.HypervitaminosisD may lead to formationof stonesin k idne y s (re n a l c a l c u l i ). H i g h c o n sumpti on of vitamin D is associatedwith loss of appetite, nausea,increasedthirst, loss of weight etc.

Absorption,

transport

and storage

Vitamin E is absorbedalong with fat in the small intestine.Bile salts are necessaryfor the absorption.In the liver, it is incorporatedinto lipoproteins (VLDL and LDL) and transported. Vitamin E is stored in adipose tissue, liver and muscl e.The normal pl asmal evel of tocoph er ol is less than 1 mg/dl. Biochemical

functions

Most of the functionsof vitamin E are related to its anfioxidant property. lt prevents the nonenzymaticoxidationsof variouscell components (e.g. unsaturated fatty acids) by molecular oxygen and free radicals such as superoxide Vitamin E (tocopherol)is a naturallyoccurring (Otl and hydrogen peroxide (H2O2). Ttie antioxidant.lt is essentialfor normal reproduction element selenium helps in these functions. in many animals, hence known as anti-sterility V i tami n E i s l i pophi l i c i n characterand is vitamin. Vitamin E is described as a 'vitamin in found in association with lipoproteins, fat searchof a disease.'Thisis due to the lack of any depositsand cellular membranes.It protectsthe s pec i fi cv i ta m i n E d e fi c i e n c yd i s e a sei n humans. polyunsaturated fatty acids (PUFA) from Evansand his associates(1936) isolatedthe peroxidation reactions. Vitamin E acts as a compounds of vitamin E activity and named scavengerand gets itself oxidized (to quinone them as tocopherols(Creek : tokos-child birth; form) by free radicals (R) and sparesPUFA, as shown below pheros-tobear; ol-alcohol).

Clremistry Vitamin E is the name given to a group of tocopherols and tocotrienols. About eight

Chapter 7 : VITAMINS

129

The biochemical functions of vitamin E, related either directly or indirectly to its antioxidantproperty,are given hereunder

Vitamin E and selenium

unsaturatedfatty acids in various tissues and membranes.lt protectsRBC from hemolysisby ox idiz ing a g e n ts(e .g .H 2 O 2 ).

Recommended dietary allowance (RDA)

Theelementseleniumis foundin the enzyme glutathione peroxidase that destroys free 1. Vitamin E is essentialfor the membrane radicals. Thus,Seis alsoinvolvedin antioxidant structure and integrity of the cell, hence it is functionslike vitaminE, and both of them act regarded as a membrane antioxidant. synergistically. To a certainextent,Secan spare the requirement vitaminE, and vice versa. 2, lt prevents the peroxidation of poly-

3. lt is closely associatedwith reproductive functions and prevents sterility. Vitamin E preservesand maintainsgerminal epithelium of gonadsfor proper reproductivefunction. 4, lt increases the synthesis of heme by enhancing the activity of enzymes 6am inolev u l i n i ca c i d (A L A) s y n th a s ea nd A LA dehydratase.

Intakeof vitaminE is directlyrelatedto the consumptionof polyunsaturated fatty acids (PUFA)i.e.,requirement increases with increased intakeof PUFA.A daily consumption of about l0 mg (15 lU) of c-tocopherol for man andI mg (12 lU) for womanis recommended. One mg of a-tocopherolis equal to 1.5 lU. Vitamin E supplemented diet is advisedfor pregnantand lactatingwomen.

5. lt is re q u i re d fo r c e l l u l a r re s p i rati on- Dietary sources through electron transport chain (believed to Many vegetableoils are rich sourcesof stabilizecoenzyme Q).

vitamin E. Wheat germ oil, cotton seed oil, peanutoil, corn oil and sunflower oil are the goodsources of thisvitamin.lt is alsopresentin 7. lt is requiredfor proper storageof creatine meat,milk, butterand eggs.

6. Vitamin E prevents the oxidation of vitamin A and carotenes. in s k elet a lm u s c l e .

Deficiency

symptoms

8. Vitamin E is neededfor optimal absorption of amino acids from the intestine.

The symptomsof vitamin E deficiencyvary from one animal speciesto another.In many 9. lt is involved in propersynthesisof nucleic animals, the deficiency is associatedwith ac ids . sterility, degenerativechanges in muscle, anaemiaand changesin central 10. Vitamin E protects liver from being megaloblastic nervous system. Severesymptomsof vitaminE damaged by toxic compounds such as carbon deficiencyare not seen in humans except tetrachloride. increasedfragility of erythrocytes and minor 11, lt w o rk s i n a s s o c i a ti o w n i th v i ta mi nsA , C neurological symptoms, and p-carotene, to delay the onset of cataract.

12. Vitamin E has been recommendedfor the prevention of chronic diseasessuch as cancer and heart diseases.Clinical trials in this regard are rather disappointing,hence it is no more recommended. However, some clinicians continue to use it particularly in subjects susceptible to heart attacks. lt is believed that vitamin E preventsthe oxidation of LDL. (Note : The oxidized LDL have been implicated to promote heart diseases.)

Toxicity of vitamin E Amongthe fat solublevitamins(A, D, E, K), vitamin E is the leasttoxic. No toxic effecthas been repoftedeven after ingestionof 300 mg/ day for 23 years.

Vitamin K is the only fat soluble vitamin with a specific coenzyme function. lt is required for

130

B IOC H E MIS TRY

the production of blood clotting factors,essentialfor coagulation(in Cerman-Koagulation; hence the nam e K fo r th i s v i ta m i n ). Ghemistry

CH? t-

?r,

C H 2-C H :C -C H 2- (C H 2-C H 2-C H -C H 2) 3- H VitamlnK1 (phylloquinone)

Vitamin K exists in different forms (Fig.7.ll). Vitamin K1 ( phyl l o q u i n o n ei)s p re s e n ti n p l a n ts . V it am i n K2 (me n a q u i n o n e ) i s produced by the intestinalbacteria and a l s o fo u n d i n a n i m a l s .Vi ta m i n K3 (menadione)is a syntheticform. All the three vitamins(K1, K2,K3) are naphthoquinone derivatives. ls opre n o i ds i d e c h a i n i s p re s e n ti n vitamins Kt and K2. The three vitamins are stable to heat. Their activity is, however, lost by oxidizing agents,irradiation,strong ac ids a n d a l k a l i e s .

Absorption, transport and storage Vitamin K is taken in the diet or svnthesized by the intestinal bacteria. Its absorption takes place along with fat (chylomicrons) and is dependenton bile salts.Vitamin K is transported alongw i th L D L a n d i s s to re dm a i n l y i n l i ver and, to a lesserextent, in other tissues. Biochemical

CHs

functions

The functionsof vitamin K are concernedwith blood clotting process.lt brings about the posttranslational (after protein biosynthesis in the cell) modification of certain blood clotting factors.The clotting factors ll (prothrombin), Vil, lX and X are synthesizedas inactive precursors (zymogens)in the liver. Vitamin K acts as a coenzyme for the carboxylation of glutamic acid residuespresentin the proteinsand this reaction is catalysedby a carboxylase(microsomal).lt involves the conversion of glutamate (Clu) to (Gla)and requiresvitamin K, y-carboxyglutamate 02 and COz Gig.7.l\. The formation of y-carboxyglutamate is inhibited by dicumarol, an anticoagulantfound in spoilt sweet clover. Wartarin is a syntheticanaloguethat can inhibit vitamin K action (Fig.7.13).

o

VitaminK2 (menaquinone)

VitamlnK3 (menadione) Fig.7.l1 : Structures ol vitaminK.

Vitamin K is also required for the carboxylation of glutamic acid residues of osteocalcin,a calcium binding protein present in the bone. The mechanismof carboxylationis not fully understood. lt is known that a 2,3-epoxide derivative of vitamin K is formed as. an intermediateduring the course of the reaction. Dicumarol inhibits the enzyme (reductase)that convertsepoxide to active vitamin K. Role of Gla in clotting : The lcarboxyglutamic acid (Cla) residuesof clotting factors are negatively charged (COO-) and they combine with positively charged calcium ions (Ca2+)to form a complex. The mechanism of action has been studied for prothrombin. The prothrombin -Ca complex binds to the phospholipidson the membrane surfaceof the platelets(Fig.7,14. This leads to the increased conversionof prothrombinto thrombin. Recommended dietary (RDA) allowance Strictlyspeaking,there is no RDA for vitamin K, since it can be adequatelysynthesizedin the

131

Ghaprer 7 : VITAMINS

H I Protein,^.,,'\,2\,,rN-CHlll

C/^,/"v'\,,\

cH2o Glu--Jl cHz

VitaminK

coz T

H I PrOtein,^:,,T,2:,,rNl- CH- C,^\,4/'\,,\ lll

cH2o

I CH{-Gla - GTP + CoA of oxaloacetate

Regeneration

in TGA cycle The TCA cycle basically involves the oxidation of acetyl CoA to COz with simultaneousregenerationof oxaloacetate.As such,there is no net consumptionof oxaloacetate or any other intermediatein the cycle. Requirement

of O" by TGA cycle

There is no direct participation of oxygen in Krebs cycle. However, the cycle operatesonly under aerobic conditions. This is due to the fact that NAD+ and FAD (from NADH and FADH2, respectively)required for the operation of the cycle can be regeneratedin the respiratorychain only in the presenceof 02. Therefore,citric acid cycle is strictly aerobic in contrastto glycolysis 7. Formation of succinate : Succinyl CoA is which operatesin both aerobic and anaerobic convertedto succinateby succinatethiokinase. condi ti ons. reaction is This coupled with the phosphorylation of CDP to CTP. This is a Energetics of citric acid cycle substrate level phosphorylation. GTP is During the processof oxidationof acetylCoA converted to ATP bv the enzvme nucleoside via citric acid cycle, 4 reducingequivalents(3 as diphosphatekinase. NADH and one as FADH2) are produced. CT P + AD P < + AT P + C D P Oxidation of 3 NADH by electron transport 8. Conversion of succinate to fumarate : chain coupled with oxidative phosphorylation Succinate is oxidized by succinate dehydro- resultsin the synthesisof 9 ATP,whereasFADH2 genaseto fumarate.This reaction resultsin the leadsto the formationof 2 ATP.Besides,there is production of FADH2 and not NADH. Thus, a total one substratelevel phosphorylation. 9. Formation of malate : The enzyme of twelve ATP are producedfrom one acetyl CoA. fumarasecatalvsesthe conversionof fumarateto malate with the addition of H2O. Inhibitors of Krebs cycle 10. Conversion of malate to oxaloacetate : Malate is then oxidized to oxaloacetate by malate dehydrogenase.The third and final synthesisof NADH occurs at this stage. The oxaloacetateis regeneratedwhich can combine with another molecule of acetyl CoA, and c ont inuet he c y c l e . Summary

of TCA cycle

The eventsof Krebscycle may be summarized as given in the next column

The important enzymes of TCA cycle inhibited by the respectiveinhibitorsare listed Enzyme

Inhibitor

Aconitase

Fluoroacetate (non-competitive)

o,-Ketoglutarate dehydrogenase

Arsenite (non-competitive)

Succinate dehydrogenase

Malonate (competitive)

257

Ghapter 13: METABOLISM OF CARBOHYDRATES Fluoroacetate-a suicide substrate : The inhibitor fluoroacetate is first activated to fluoroacetvl CoA which then condenseswith oxaloacetate to form fluorocitrate. TCA cycle (enzyme-aconitase) is inhibited by fluorocitrate. The compound fluoroacetate, as such, is a harmlesssubstrate.But it is convertedto a toxic compound (fluorocitrate)by celIu lar metabolism. T his is a s u i c i d ere a c ti o nc o mmi tte db y the cel l , and thus fluoroacetateis regardedas a suicide substrate. Regulation

of citric

acid

cycle

T he c e l l u l a r d e ma n d so f AT P a re cruci al i n controlling the rate of citric acid cycle. The regulationis broughtabout either by enzymesor the levels of ADP. Three enzymes-namely citrate synthase, isocitrate dehydrogenase and a-ketoglutarate dehydrogenase-regu Iate c itric ac id c y c l e .

The most importantsynthetic(anabolic)reactions connectedwith TCA cycle are given (Fig.l3.l0) 1 . Oxaloacetate and o-ketoglutarate,respectively, serve as precursorsfor the synthesisof aspartate and glutamate which, in turn, are requiredfor the synthesisof other non-essential ami no aci ds, puri nesand pyri mi di nes. 2. Succinyl CoA is used for the synthesisof porphyri nsand heme. 3. Mitochondrial citrate is transportedto the cytosol, where it is cleaved to provide acetyl CoA for the biosynthesisof fatty acids, sterols etc. Anaplerosis

or anaplerotic

reactions

The synthetic reactions described above depletethe intermediates of citric acid cycle. The cycle will cease to operate unless the intermediatesdrawn out are replenished. Ihe 1. Citrate synthase is inhibited by ATP, reactions concerned to replenish or to fill up the intermediates of citric acid cycle are called NADH, acetyl CoA and succinyl CoA. anaplerotic reactions or anaplerosis (Creek : fill 2. lsocitrate dehydrogenase is activated by up). l n Fi g.l3.10, the i mportant synthe t ic A DP , a n d i n h i b i te db y A T P a n d N AD H . pathways that draw the intermediates of TCA is inhibited cycle and the anapleroticreactionsto fill them 3. o-Ketoglutaratedehydrogenase by s uc c i n y lC o A a n d N AD H . up are grven. 4. Availability of ADP is very important for The salient featuresof important anaplerotic the citric acid cycle to proceed.This is due to reactionsare described the fact that unlesssufficientlevels of ADP are 1. Pyruvate carboxylase catalyses the available, oxidation (coupled with phosphoconversion of pyruvate to oxaloacetate.This is rylation of ADP to ATP) of NADH and FADH2 an ATP dependentcarboxylationreaction. through electron transport chain stops. The ac c um u l a ti o no f N AD H a n d F AD H 2wi l l l ead to Pyruvate + CO2 + ATP ------+ inhibition of the enzymes(as statedabove) and Oxal oacetate+ A D P + P i als o limi ts th e s u p p l y o f N AD + a n d FA D w hi ch The details of the above reaction are are essentialfor TCA cycle to proceed. describedunder gluconeogenesis.

Amphibolic nature of the citric acid cycle

2. Pyruvateis convertedto malate by NADP+ (malicenzyme). dependentmalatedehydrogenase

The citric acid cycle provides various Pyruvate + CO2+ NADPH+ H+$ intermediates for the synthesis of many Mal ate+ N A D P H + +Hr O compounds needed by the body. Krebscycle is 3. Transaminationis a orocesswherein an both cataholic and anaholic in nature, hence amino acid transfersits amino group to a keto regarded as amphibolic. acid and itself gets converted to a keto acid. The TCA cycle is actively involved in gluco- formation of a-ketoglutarateand oxaloacetate neoge n e s i s ,tra n s a mi n a ti o na n d d eami nati on. occurs by thi s mechani sm.

258

BIOCHEMISTF|Y

Non-essential amino acids,

punnes, pyrimidines

AsDartate

TransaminationAcetyl CoA

Pyruvate

Fattyacids,sterois Citrate.-'....-..-.-f

Citric acld cycle d,-Ketoglutarate

Glutamate

I J Non-essential aminoacids,purines Fig. 13.10 : Major synthetic and anaplerotic pathways of the intetmediates of citric acid cycle.

4. a-Ketoglutaratecan also be synthesized living system,energy is trapped leading to the from glutamate by glutamate dehydrogenase synthesi of s 38 A TP w hi ch i s equi val entto 1,159 action. KJ (1 ATP has high energy bond equivalentto 30.5 KJ).That is, about 48% of the energy in Clutamate + NAD(P)++ H2O 8 Acetyl CoA + 7 FADH2 + 7 NA DH + 7 H + Palmitoyl CoA undergoes 7 cycles of p-oxidation to yield I acetyl CoA. Acetyl CoA can enter citric acid cycle and get completely oxidized to CO2 and H2O. Energetics

of B-oxidation

The ultimate aim of fatty acid oxidation is to generate energy. The energy obtained from the complete oxidation of palmitic acid (16 carbon) is given in Table 14.2 and Fig.l4.8.

Mechanism

ATP vield

l. p0xidation7 cycles transport byelectron 7 FADH2 [oxidized gives2 ATPI eachFADH, chain(ETC), bYEIC,eachNADH 7 NADH(oxidized 3 ATP) liberates ll. From8 acetylCoA bycitricacid cycle,eachacetyl Oxidized 12ATP CoAprovides CoA Totalenergyfromonemoleof palmitoyl foractivation utilized Energy (tormation CoA) of palmitoyl

14 21

131

-2

129 of palmitate of one molecule Netvieldof oxidation

Chapter 14 : METABOLISMOF LtPtDS

297

o

il R-CH2-CH2-CH2-C-OFatty acid

ott\l-coASH -

Ms"y

Thiokinase I !ro tl R-CH2-CH2-CH2-C-SCoA AMP + PPiy'l

Acyl CoA

|I

tr.

Ca:'nitineti ansportsystem

1a.

I

"yrosol

MrocHoNDRloN

p *o

?

-CH2-CH2-CH2-C-SCoA Acyl CoA

FAD-! (1),1 oerivo'rAd;is€ ^cytc{A Q]..-rnonz+{ oHo W '

ttl -CH2-CH:CH-C-SCoA A2 traneenoyl CoA Hzo._ (2)

OH ttl -CH2-CH-CH2-C-SCoA pHydroxyacyl CoA " IADH+

f"*roxyacyl CoA dehydrogenase

o -cH2-c-cHr-8-s"oo ftKetoacyl CoA

lr

ucAs!-l-l Thiorase\\ (4)l

o+o

-C-SCcn + Cn3-8Acyl CoA (-2C)

Ac€ry

Flg. 14.7 : p-Oxidation of fatty acids : Palmitoyl CoA (16 carbon) undergoes seven cycles to yield I acetyl CoA fl-Activation; ll-Transport; lll-p Oxidation proryr(1) Oxidation, (2) Hydntion, (3) Oxidation and (4) Cleavagel.

Fig. 14.8 : An overview of oxidation of palmitic acid.

SIDS-a disorder due to blockade in j-l.oxidation The sudden infant death syndrome (SIDS) is an unexpecteddeath of healthy infants,usually overnight.The real causeof SIDS is not known. It is now estimatedthat at least 10% of SIDS is due to deficiency of medium chain acyl CoA dehydrogenase. The enzyme defect has a frequencyof 1 in 10,000 births and is, in fact, more prevalent than phenylketonuria. The occurrenceof SIDS is explained as follows Glucose is the principal source of energy, soon after eating or feeding babies. After a few hours, the glucose level and its utilization decreaseand the rate of fatty acid oxidation must simultaneously increase to meet the energy needs. The sudden death in infants is due to a blockade in p-oxidation caused by a deficiency in medium chain acyl CoA dehydrogenase (MCAD). Jamaiean

vomitinE

siekness

This disease is characterized bv severe hypoglycemia,vomiting, convulsions,coma and death. lt is caused by eating unripe ackee fruit which contains an unusual toxic amino acid, hypoglycin A. This inhibits the enzyme acyl

292

BIOGHEMISTF|Y

CoA dehydrogenase and thus p-oxidation of fatty acids is blocked, leading to various complications. Oxldation of odd carbon chain fatty acids The p-oxidation of saturatedfatty acids containing odd number of carbon atoms proceedsin the same manner, as described above for even carbon fatty acids. The only difference is that in the last and final p-oxidation cycle, a three-carbonfragment is left behind (in place of 2 carbon unit for saturated fatty acids). This compound is propionyl CoA which is converted to succinyf CoA as follows (Fig.la.fl

Succinyl CoA

L-MethylmalonylCoA j B,rdeficiency j Methylmalonic acid

TCA cycle

1. PropionylCoA is carboxylatedin the presence of ATP, CO2 and vitamin biofin to D-methylmalonyl CoA. 2. Methylmalonyl CoA racemaseconvertsthe methylmalonyl CoA to L-form. This reaction (D -+ L) is essentialfor the entry of this compound into the metabolic reactionsof the body.

same extent as saturated fatty acids. Therefore, oxidation of unsaturated fatty acids, in general, provides less energy than that of saturated fafty acids.

3. The next enzyme, methylmalonyl CoA mutase, is dependenl on vitamin 812 (deoxyadenosyl cobalamin). lt catalysesthe conversion of methylmalonyl CoA (a branched compound) to succinyl CoA (a straight chain compound), which can enter citric acid cycle.

Most of the reactions involved in the oxidation of unsaturatedfatty acids are the same as found in the p-oxidation of saturated fatty acids. However, the presence of double bonds poses problem for p-oxidation to proceed. This is overcome by two additional enzymes-an isomerase and an epimerase.

Methylmalonie

acidemia

Two types of methylmalonic acidemias are known

F-Oxidation of fatty in peroxisomes

acids

1. Due to deficiency of vitamin 812;

Peroxisomesare organelles present in most eukaryotic cells. The p-oxidation occurs in a modified form in peroxisomes. Acyl CoA (a flavoenzyme) leads to the dehydrogenase In either case, there is an accumulation of formation FADH2, as in p-oxidation. The of methylmalonic acid in body, followed by its reducing from FADH2 are not equivalents increasedexcretion in urine. This causessevere electron transport chain, but transferred to the metabolic acidosis,damagesthe central neryous to This results in the handed over directly 02. systemand retardsthe growth. lt is often fatal in which is by catalase. formation of H2O2, cleaved the early years of life. 2. Due to defect in the enzyme methylmalonyl CoA mutase.

Oxidation

of unsaturated

fatty

acids

Due to the presence of double bonds, the unsaturatedfatty acids are not reduced to the

E-FADH2 + Oz -----) E-FAD + H2O2 Catalase H2O2i::= UrO + !O'

Chapter 14: METABOLISM OF LIPIDS

293

There is no ATP synthesizedin peroxisomal rr-vOxidation of fatty ac;ds p-oxidation of fatty acids, since the reducing This is a minor pathway. lt involves equivalentsdo not passthrough ETC. However, hydroxylation followed by oxidation of or-carbon heat is liberated. present as a methyl group at the other end (at It is now believedthat the peroxisomescarry one end carboxyl group is present)of fatty acid. out the initial oxidation of long chain (C26,C22 This reactionrequirescytochromeP4s0,NADPH etc.) fatty acids which is followed by and 02, besides the enzymes. The overall mitochondrialoxidation. reaction may be representedas follows. Peroxisomal oxidation is induced by high fat diet and administration of hypolipidemic drugs (e.9. clofibrate). Zellweger syndrome : This is a rare disorder characterizedby the absenceof peroxisomesin alnlostall the tissues.As a result,the long chain fatty acids (C26-C36) are not oxidized. They accumulatein tissues,particularlyin brain, liver and kidney. Hence the disorderis also known as cerebroh e pato renal synd ro m e. a"Oxidation

of fatty

acids

p-Oxidationis the most predominantpathway for fatty acid degradation.However, the removal of one carbon unit at a time by the oxidation of cl,-carbon atom of fatty acid is known. a-Oxidation does not involve the binding of fatty acid to coenzymeA and no energy is produced. Refsumts disease is a rare but severe neurologicaldisorder characterizedby cerebral ataxia and peripheral neuropathy.The patients of this diseaseaccumulatelarge quantitiesof an unusual fafty acid, phytanic acid. lt is derived from phytol, a constituentof chlorophyll. Hence it is found mostly in plant foods. However, it is als o pr es e n t i n m i l k l i p i d s a n d a n i mal fats. Phytanicacid cannot undergoB-oxidationdue to the presenceof a methyl group on carbon-3.This fatty acid undergoes initial o-oxidation (to remove c-carbon as carbon dioxide) and this is followed by p-oxidation. Refsum'sdiseaseis caused by a defect in the cr-oxidation due to the deficiency of the enzyme phytanic acid a-oxidase. The result is that phytanic acid cannot be converted to a compound that can be degradedby p-oxidation. The patientsshould not consumedietscontaining chlorophyll (i.e., green leafy vegetables).

cHs-(cH2)n-cooHo-H2c-(6H2)n-coo-

-ooc-16Hry"-cooOxidatian of fatty acids and metabolic urater Fatty acid oxidation (even other forms of aerobic respiration) is accompanied by the production of water, referredto metabolic water. For instance,when one molecule of palmitic acid is oxidized, it releases16 molecules of water. This metabolic water has great significancein some animals. Camel can store Iipids in. its hump which is good source of water, besides energy supply. For this reason, camel can travel in deserts for long periods even without food and water supply. Kangaroo rat is a small animal that is believed to live indefinitely without water. It consumes only oil rich seeds, and the metabolic water produced is adequate to meet its water needs. lt may however, be noted that the use of metabolic water is an adaptation, and is accompanied by reduced output of urine.

The compounds namely acetone, acetoacetate and B-hydroxybutyrate (or 3-hydroxybutyrate)are known as ketone bodies (Fig.la.l0). Only the first two are true ketones while phydroxybutyrate does not possessa keto (C=O) group. Ketone hodies are water-soluble and energy yielding. Acetone, however, is an exception,since it cannot be metabolized.

294

B IOC H E MIS TRY

o

o

cH3-c-cH3

cH3-c-cH2-coo-

Acetone

Acetoacetate

oo

il CH3-C-S-CoA

ll + CH3-C-S-CoA

AcetylCoA

AcetylCoA

---------.---

OH I

I

cH3-cH-cH2-cooftHydrorybutyrate

Col-sHt-J0rc*ottlotase

oi l tlo

Fig. 14.10 : Structures of ketone bodies.

CH3-C-CH2-C-S-CoA Acetoacetyl CoA

Ketogenesis The synthesisof ketone bodies occurs in the Iiver.The enzymes for ketone body synthesisare focated inthe mitochondrial matrix. Acetyl CoA, formed by oxidation of fatty acids, pyruvate or some amino acids, is the precursorfor ketone bodies.Ketogenesis occursthroughthe following reactions(Fig.Ia.l l). 1. Two moles of acetyl CoA condense to form acetoacetylCoA. This reaction is catalysed by thiolase,an enzyme involved in the final step of p-oxidation. Hence, acetoacetatesynthesisis appropriatelyregardedas the reversalof thiolase reaction of fafty acid oxidation.

oHo

ttl -OOC-CH2-C-CH2-C-S-CoA

cHs p-Hydrory-P-methylglutaryl CoA(HMGCoA) I

9l

coAlYase cHr-d-s-coR+-1 HMG AcetylCoA I

+?

-ooc-cH2-c cHs Acetoacetate

2. Acetoacetyl CoA combines with another molecule of acetyl CoA to produce p-hydroxy p-methyl glutaryl CoA (HMC CoA). HMG CoA synthase,catalysing this reaction, regulates the synthesis of ketone bodies. 3. HMG CoA lyase cleaves HMC CoA to produce acetoacetateand acetyl CoA. 4. Acetoacetate can undergo spontaneous decarboxylation to form acetone. 5. Acetoacetate can be reduced by dehydrogeneaseto p-hydroxybutyrate.

a

The carbon skeleton of some amino acids (ketogenic)is degradedto acetoacetateor acetyl CoA and,,therefore, to ketone bodies, e.g. le u c i n e ,l y s i n e ,p h e n y l a l a n i n eetc.

o cH3-c-cH3 Acetone

OH -ooc-cH2-c-cH3 H p-Hydrorybutyrate

sourcesof energy for the peripheral tissuessuch as skeletalmuscle, cardiac muscle/ renal cortex etc. The tissueswhich lack mitochondria (elg. Utillzation of ketone bodies erythrocytes) however, cannot utilize ketone The ketone bodies, being water-soluble,are bodies. The production of ketone bodies and easily transported from the liver to various their utilization become more significantwhen tissues. The two ketone bodies-acetoacetate glucose is in short supply to the tissues, as and p-hydroxybutyrate serve as important observed in starvation, and diahetes mellitus.

METABOLISMOF L|PIDS

295

During prolonged starvation, ketone bodies intermediate in citric acid cycle. Thiophoraseis are the major fuel saurce for the brain and other absent in liver, hence ketone bodies are not pafts of central nervous system. lt should be utilized by the liver.Thiolasecleavesacetoacetyl noted that the ability of the brain to utilize fatty CoA to two moles of acetyl CoA (Fig.l4.l2). acids for energy is very limited. The ketone The summary of ketone body synthesrs, bodies can meet 5O-7Oo/o of the brain's energy utilization and excretion is depicted in needs.This is an adaptationfor the survival of Fig.t4.13. t he or ga n i s m d u ri n g th e p e ri o d s of food .i ' . i :.r ..i ta deprivation. r I n r i t,,i :::, - ;' !gg" ::r :u;+ bfg$!* :g, Reactions of ketone bodies : B-Hydroxybrutyrate is first converted to acetoacetate (reversal of synthesis) and metabolized. Acetoacetate is activated to acetoacetyl CoA by a mitochondrial enzyme thiophorase (succinylCoA acetoacetate CoA transferase). The coenzyme A is donated by succinyl CoA, an

l n normal i ndi vi dual s,there i s a constant production of ketone bodies by liver and their utilization by extrahepatictissues.The concentration of ketone bodies in blood is maintained around 1 ng/dl. Their excretion in urine is very low and undetectableby routine tests(Rothera,s fes0.

BrgntEDrcAL/ cLtnitcf,LcotucEFni

An adult human body contains qbout 10-11 kg of t'at reseruecorrespondingto about 100,000 Cal. This can meet the energg requirements for seueral weeks of t'ood depriuationin man. The sudden infant death syndrome (SIDS)----on unexpectedouernight death oJ healthy infants-is ottributed to a blockade in ftoxidation of Jatty acids,causedby a deftciency of medium chain acyl CoA dehydrogenase(MCAD). Jamaican uomiting sicknessis due to consumptionof unripe ackee fruit contoining hypoglycin A which blocks ftoxidation. Methylmalonic acidemia occurseither due to a deficiencyol the uitamin 812 or o det'ect in an enzyme methyl malonyl CoA mutase. This disorder retards growth and damages central neruoussystem. Zellwegersyndrome is causedby the absenceof peroxisomesin fissues;as a result, the Iong chain fatty acids cannot be oxidized. Refsum's disesse is due to a defect in a-oxidation of lattg acids. The patients are oduisednot to consumediets containingchlorophyll. Ketosis is commonly associated with uncontrolled diabetes mellitus and starvation. Diabetes ketoacidosisis dangerous-moy result in coma or euen death. Starvation, howeue4 is noi accompaniedby ketoacidosis. Insulin promotes fatty acid synfhesisby stimulating the conuersionol pyruuate to ocetyl CaA. The lack of the ability ol the organismsfo introduce double bonds in fatty acidsbeyond C9 and Crc makes linoleic and linolenic acids essentialto mammals.

296

B IOC H E MIS TRY

OH cH3-cH-cH2-coop-Hydrorybutyrate NAD oxybutyrate lrogenase NADH + H*

otl

cH3-c-cH2-cooAcetoacetate

cH2-cooCH2-CO-SCoA

Thiophorase

SuccinylCoA

cH2-coocH2-cooSucclnate

oi l rlo

Diabetes mellitus : Diabetes mellitus is associatedwith insulin deficiency.This resultsin impaired carbohydrate metabolism and increased lipolysis, both of them ultimately leading to the accumulationof acetyl CoA and its conversion to ketone bodies. In severe diabetes,the ketone body concentration in blood plasma may reach 100 mgldl and the urinary excretion may be as high as 500 m{day. of ketogenesis

Regulation

CH3-C-CH2-C-SCoA Acetoac€tyl CoA

I 'o^tt\|nr,rhr" I oYo il tl CH3-C-SCoA + CH3-C-SCoA Acetyl GoA

productionof acetyl CoA which cannot be fully handled by citric acid cycle. Furthermore,TCA cycle is impaired due to deficiency of oxaloacetate, since most of it is diverted for glucose synthesis to meet the essential requirements (often unsuccessful)for tissues like brain. The result is an accumulationof acetyl CoA and its diversion for overproduction of ketone bodies.

Acetyl CoA

Fig. 14.12 : Metabolism (utilization) of ketone bodies to acetyl CoA.

When the rate of synthesisof ketone bodies rate of exceeds the utilization, their concentrationin blood increases,this is known as ketonemia. Ketonemia is predominantly due to incresed production of ketone bodies rather than the deficiency in their utilization.The term ketonuria represents the excretion of ketone bodies in urine. The overall pictureof ketonemia and ketonuria is commonly referredto as kefosis Smell of acetone in breath is a common feature in ketosis. Ketosis is most commonly associated with starvationand severeuncontrolleddiabetes m e l l i tu s . Starvation : Starvation is accompanied by increased degradation of fafty acids (from the fuel reserve triacylglycerol) to meet the energy needs of the bodv. This causes an over-

The ketone body formation (particularly overproduction)occurs primarily due to nonavailabilityof carbohydratesto the tissues.This is an outcome of excessive utilization of fatty acids to meet the energy requirementsof the ceffs. The hormone glucagon stimulates ketogenesis whereas insulin inhibits. The increasedratio of glucagon/insulinin diabetes mellituspromotesketonebody formation.This is due to disturbancescausedin carbohydrateand lipid metabolisms in diabetes, as discussed elsewhere (Chapter 35). Ketogenic

and antiketogenic

substances The dietary compounds are divided into two categoriesdepending on whether they promote ketone body formation (ketogenic) or inhibit (antiketogenic). The ketogenicsubstancesinclude fatty acids and certainamino acids(leucine,lysine,tyrosine etc.). The antiketogenicsubstancesare glucose, glycerol and glucogenic amino acids (".9. I glycine, alanine, serine,glutamateetc.) Ketoacidosis Both acetoacetateand p-hydroxybutyrateare strong acids. Increasein their concentrationin blood would causeacidosis.The carboxyl group

Chapter 14: MEIABOLISMOF LIPIDS

297 BLOOD Fattyacids Ketonebodies

Kldneys

Ketone bodies -

zCO2 Ketonebodies (in urine)

has a pKuaround 4. Therefore,the ketone bodies in the blood dissociate and release H+ ions which lower the pH. Further, the volume of plasma in the body is reduced due to dehydrationcausedby the excretionof glucose and ketone bodies. Diabetic ketoacidosis is dangerous-may resultin coma, and even death, if not treated. Ketosis due to starvation is not usually accompaniedby ketoacidosis.

Acetone (exhaled)

the cytosomal fraction of the cell. Acetyl CoA is the source of carbon atoms while NADPH provides the reducing equivalehts and ATP suppliesenergy for fatty acid formation. The fatty acid synthesismay be learnt in 3 stages l.' Productionof acetyl CoA and NADPH ll. Conversionof acetyl CoA to malonyl CoA lll. Reactionsof fatty acid synthasecomplex.

Treatment of ketoacidosis : Rapid treatment l. Production of acetyl CoA of diabetic ketoacidosisis required to correct the ANd NADPH metabolic abnormalities and the associated Acetyl CoA and NADPH are the prerequisites water and electrolyteimbalance.Administration of insulin is necessaryto stimulate uptake of for fafty acid synthesis.Acetyl CoA is produced glucoseby tissuesand inhibition of ketogenesis. in the mitochondriaby the oxidationof pyruvate and fatty acids, degradation of carbon skeleton of certain amino acids, and from ketone bodies. Mitochondria, however, are not permeable to acetyl CoA. An alternate or a bypass The dietary carbohydratesand amino acids, arrangement is made for the transfer of acetyl when consumed in excess,can be converted to CoA to cytosol. Acetyl CoA condenses with fatty acids and stored as triacylglycerols.De oxaloacetate in mitochondria to form citrate. novo (new) synthesis of fatty acids occurs Citrate is freely transportedto cytosol where it is predominantly in liver, kidney, adipose tissue cleaved by citrate lyase to liberate acetyl CoA and factating mammary glands. The enzyme and oxaloacetate.Oxaloacetate in the cytosol is machinery for fatty acid production is located in convertedto malate (Fig.la.l4.

298

B IOC H E MIS TR Y

Glucose i

Pyruvate

Fig. 14.14 : Transfer oI acetyl CoA from mitochondria to cytosol (HMP shunt-Hexose monophosphate shunt; @-also known as malate dehydrogenase).

Malic enzyme converts malate to pyruvate. lll. $teaetions of fiatty aeld NADPH and CO2 are generatedin this reaction. synthase cor$pleJ( Both of them are utilized for fatty acid synthesis. The remainingreactionsof fatty acid synthesis Advantages of coupled transport of acetyl are catalysed by a multifunctional enzyme CoA and NADPH : The transport of acetyl CoA known as fatty acid synthase (FAS) complex.ln from mitochondriato cytosol is coupled with the eukaryotic cells, including man, the fatty acid cytosomal production of NADPH and COz synthaseexists as a dimer with two identical the activitiesof whic h i s h i g h l y a d v a n ta g e o u sto th e cel l for units. Each monomer possesses seven different enzymes and an acyl carrier optimum synthesisof fatty acids. ll. Forruration of nnalony! GoA Acetyl CoA is carboxylatedto malonyl CoA by the enzyrne acetyl CoA carboxylase Gig.la.lfl. This is an ATP-dependentreaction and requires biotin for COz fixation. The mechanismof action of acetyl CoA carboxylase is similar tci that of pyruvatecarboxylase(Refer Chapter 7, Fi9.7.29).Acetyl CoA carboxylase is a regulatory enzyme in fatty acid synthesis (detailsgiven later).

n i

Cl'i3 C SCon Acetyl CoA coo -.---^\l I AcetvlCoA A'r-J cab

,.OH

,trz

\,,, \ cHz oH

cHz

\,/

.ctlz

\ 6fi, dn, 'o-O-O

OH GoA(HMGCoA) B-Hydroxy B-methylglutaryl

-ooc. ).1

.c.

Dirnethylallylpyrophosphate(5C)

Mevalonate (6C)

cls-Prenyl transferase

9Hs 9Hs ,c. .c.Hz.Q QHz.

.(.\(\(,v[-eGeranylpyrophosphate(10C)

S-Phosphomevalonate

cls-Prenyl transferase

t{t

-ooc. \,.'

.roH

'pa

\,/'

g!12 \

cHz cHz 'o-O-e

S-Fyrophosphomevalonate F,g, 14,27 contd.

next column

-zA-1p\ '\'/ \'/ Farnesylpyrophosphate (15C) F,9.14.27

contd. trext tEg.

312

B IOC H E MIS TR Y (15C) 2 Farnesylpyrophosphate NADPH+ Hl 2L

2!

Mg- , Mn-

Squalene synthase

NADPPP i

R egui ati on

of chol esterol

synthesi s

Cholesterolbiosynthesisis controlled by the rate limiting enzyme HMG CoA reductaset at the beginningof the pathway (Fig.la.28). HMC CoA reductase is found in association with endoplasmic reticulum, and is subjected to different metabolic controls. 1. Feedback control : The end product cholesterol controls its own synthesis by a feedback mechani sm.Increasei n the cel l ular concentration of cholesterol reduces the synthesisof the enzyme HMG CoA reductase. This is achieved by decreasing the transcription of the gene responsiblefor the production of HMC CoA reductase.Feedbackregulation has been investigatedwith regardto LDl-cholesterol taken up by the cells, and the same mechanism is believed to operate whenever cellular cholesterollevel is elevated.

W"

NADPH+ H* NADP'

Oz

Epoxidase Hydtoxylase Cyclase

Hzo

2COz NADPH,Oz

A seriesot (about19) reactions

NADP'

Fig. 14.27 : Biosynthesis of cholesterol.

2. Hormonal regulation : The enzyme HMG CoA reductase exists in two interconvertible forms. The dephosphorylatedform of HMC CoA reductase is more active while the phosphorylated form is lessactive.The hormones exerttheir influencethroughcAMP by a seriesof reactionswhich are comparablewith the control of the enzyme glycogensynthase.The net effect is that glucagon and glucocorticoids favour the formation of inactive HMC CoA reductase (phosphorylated form) and, thus, decrease chofesterolsynthesis.On the other hand, insulin and thyroxine increasecholesterolproduction by enhancing the formation of active HMC CoA form). reductase(dephosphorylated 3. Inhibition by drugs : The drugs compactin and lovastatin (mevinolin)are fungal products. They are used to decreasethe serum cholesterol level in patients with hypercholesterolemia. Compactin and lovastatin are competitive inhibitors of the enzyme HMG CoA reductase and, therefore, reduce cholesterol synthesis. About 50 to 60"/" decreasein serum cholesterol level has been reported by a combined use of these two drugs. 4. HMG CoA reductaseactivity is inhibited by bile acids. Fastingalso reducesthe activity of thi s enzvme.

Ghapter'14: METABOLISM OF LIPIDS

mRNA

t -C Cholesterol

I

>lTranscription

I

DNA Fig. 14.28: Regulationof cholesterolbiosynthesis by HMGCoAreductase(&-Prcmoting effect;Q-lnhibitory effect). F.

DEGRADATIONOF CHOLESTEROL The steroid nucleus (ring structure) of the cholesterol cannot be degraded to CO2 and H2O. Cholesterol (50%) is converted to bile acids (excretedin feces),servesas a precursor for the synthesisof steroidhormones,vitamin D, coprostanoland cholestanol.The latter two are the fecal sterols,besidescholesterol. l. Synthesis

function as surfactants. In the bile, the conjugated bile acids exist as sodium and potassiumsaltswhich are known as bile salts. Cholesterol

of bile acids

The bile acids possess24 carbon atoms,2 or 3 hydroxyl groups in the steroid nucleus and a s ide c h a i n e n d i n g i n c a rb o x y l Bro up. The bi l e ac ids a re a m p h i p a th i c i n n a tu re si nce they possessboth polar and non-polar groups.They serve as emulsifyingagentsin the intestineand ac t iv e l y p a rti c i p a te i n th e d i g e sti on and absorptionof lipids. The synthesisof primarybile acidstakesplace in the liver and involves a series of reactions (Fi9.14.29).The step catalysed by 7 a-hydroxylase is inhibited by bile acids and this is the rafe Iimiting reaction. Cholic acid and chenodeox y c ho l i c a c i d a re th e p ri ma ryb i l e a ci dsand the f or m er i s fo u n d i n th e l a rg e s at mo u n t i n bi l e. On conjugationwith glycine or taurine, conjugated bile acids (glycocholic acid, taurocholic acid etc.) are formed which are more efficient in their

7-Hydrorycholesteror seterv ' ^rra.-"e .-:/ep. g.uuu"=t 'i

GIY

Cholifacid ri ne

Chenodeorycholic acid

_.9Jy-"9:,,x .Ta.uro-. .* chol i c aci d'

chol i c aci cl '

I I lntestinal I bacteria + Deoxycholic acid**

Tauro-or glycochenodeoxycholicx acid I Intestinal bacteria I LithochJic acid**

Fig. 14.29 : Outline ot bile acid synthesis(*-Primary bile acids, **-Secondary bile acids).

BIOCHEMISTFIY

314 In the intestine,a portion of primarybile acids undergoesdeconjugationand dehydroxylationto form secondary bile acids (deoxycholic acid and lithocholic acid). These reactionsare catalysed by bacterialenzymes in the intestine.

(27C) Cholesterol

I

+ (21C) Pregnenolone

I

+

Enterohepatic circulation : The conjugated bile sa l tss y n th e s i z e di n th e l i v e r a ccumul atei n (21C) Progesterone gall bladder. From there they are secretedinto the small intestine where they serve as emulsifying agents for the digestion and (1BC) (21C) Estradiol absorption of fats and fat soluble vitamins. A (21C) Aldosterone Cortisol large portion of the bile nlts (primary and Flg. 14.30: Outlineof steroidhormonesynthesis secondary) are reabsorbed and returned to the from eholesterol(Numbersin the brackets liver through portal vein. Thus the bile saltsare reDresentthe number of carbon atoms). recycledand reusedseveraltimes in a day. This is known as enterohepaticcirculation.About 15(d) Androgens (e.9. testosterone) 30 g of bile saltsare secretedinto the intestine each day and reabsorbed.However, a small (e) Estrogens(e.9. estradiol). portion of about 0.5 g/day is lost in the feces.An A brief outline of steroid hormonal synthesis equal amount (0.5 g/day) is synthesizedin liver given in Fig.l4.30 and more details are is to reolace the lost bile salts.fhe fecal excretion of bile salfs is the only route for the removal of discussedunder 'Hormones' (Chapter 19). cholesterol from the hody. l l l . S ynthesi s of vi tami n D Cholelithiasis: Bile salts and phospholipids an intermediatein the 7-Dehydrocholesterol, are responsiblefor keepingthe cholesterolin bile in a s o l u b l e s ta te . D u e to th e i r defi ci ency synthesisof cholesterol,is converted to chole(particularly bile salts), cholesterol crystals calciferol (vitamin O3) bV ultravioletrays in the precipitatein the gall bladder often resultingin ski n. cholelithiasis-cholesterol gall stone disease. A brief summary of prominent sources and Cholelithiasismay be due to defectiveabsorption the major pathwaysfor utilization of cholesterol of bile salts from the intestine, impairment in with the liver as the central metabolic organ is liver function, obstructionof biliary tract etc. depicted in Fi9.14.31. The patientsof cholelithiasisrespond to the administrationof bile acid chenodeoxy cholic Trarrsport of cholesterol acid, commonly known as chenodiol. lt is Cholesterol is present in the plasma believed that a slow but gradual dissolutionof lipoproteinsin two forms gall stonesoccurs due to chenodiol. For severe 1. About 70-75% of it is in an esterifiedform casesof cholelithiasis,surgical removal of gall with long chain fatty acids. bladder is the only remedy. 2. A bout 25-30% as free chol esterol .This form of cholesterolreadily exchangesbetween different lipoproteins and also with the cell is the precursor for the synthesis membranes. Cholesterol

trl" $ynthesis of steroid hormones from cholesterol

of all the five classes of steroidhormones Role of ICAT : High density lipoproteins (a) Clucocorticoids (e.9.cortisol) (HDL) and the enzyme lecithin-cholesterol (b) Mineralocorticoids (e.9.aldosterone) acyltransferase (LCAT) are responsible for the (e.g.progesterone) (c) Progestins transportand eliminationof cholesterolfrom the

3t5

Chapter 14 : METABOLISM OF LIPIDS

Dietarycholesterol body. LCAT is a plasma enzyme, (500 mg/day) synthesizedby the liver. lt catalyses Bile salts and bile acids the transfer of fattv acid from the CHOLESTEROL (250 mg/day) Cholesterol POOL second position of phosphatidyl synthesis in ( 1 0 0 0m g ) c holin e (l e c i th i n ) to th e h y d ro x y l liver (500 mgiday) Cholesterollost in bile group of cholesterol (Fig.Ia32). (500 mg/day) HDL-cholesterolis the real substrate for LCAT and this reaction is freely is reversible. LCAT activity extrahepatic associatedwith apo-A1 of HDL. tissues(variable) The cholesterol(cholesteryl)ester Majorsourcesof liver l a rt o f H D L . In th i s f or m sa n i n te g ra p Majorroutesof cholesterol cholesterol utilization manner, the cholesterol from the peripheraltissuesis trapped in HDL, Fig. 14.31Summaryof majorsourcesof livercholesterol and its by a reactioncatalysedby LCAT and utilization(valuesgivenin bracketsare variable). then transported to liver for degradation and excretion. This m ec ha n i s m i s c o m m o n l y k n o w n as revefse Carr and Dructor method and, more recently, cholesterol oxidase method. HDL- cholesterol cho Ie sterol t ran sport. can be determined after precipitatingLDL and V LD L by pol yethyl ene gl ycol (P E C ). V LDL Flasma eholesterol.l/5th cholesterol is equivalent to of plasma bionnedical innportance (TC) triacylglycerol in a fasting state. LDLI n h e a l th y i n d i v i d u a l s , th e to tal pl asma cholesterol can be calculated from Friedewald cholesterolis in the range of 150-200 mg/dl. ln formula given below. the new born, it is lessthan 100 mgldl and rises LDl-cholesterol= Total cholesterol- (HDLto about 150 mg/dl within an year. The women have relatively lower plasma cholesterol cholesterol+ TCl5). which is attributed to the hormones-esfrogens. The above formula is not valid if TC Cholesterollevel increaseswith increasingage concentrationis above a)O mg/dl. ( in wo me n p a rti c u l a rl ya fte r m e n o pause),and is about In adults,the normal LDL-cholesterol also in pregnancy. 80-150 mgi dl w hi l e H D L-chol esteroli s around Plasmacholesterolis associatedwith different 30-60 mg/dl. Elevation in plasma HDLlipopro te i nfra c ti o n s(L D L , VL D L a n d H D L). cholesterol is beneficial to the body, since it Total cholesterolcan be estimatedby many protects the body from atherosclerosis and methods such as Libermann-Burchardreaction, coronarv heart diseases(CHD). On the other hand, increasein LDl-cholesterolis harmful to the body as it may lead to variouscomplications, .\f Phosphatidvrch" choresterol i ncl udi ngC H D .

I{YPERCHOLESTEROLEM IA Lecithincholesterol acyltransferase(LCAT)

/ Lysophosphatiavt"notin"

\

a no,"r," rotester

Fig. 14.32 : Reaction catalysedby LCAT.

Increasein plasma cholesterol(> 200 mg/dl) concentrationis known as hypercholesterolemia and is observedin many disorders 1. Diabetes mellitus : Due to increased cholesterol synthesissince the availability of acetyl CoA is increased.

B IOC H E MIS TFIY

I 316

/

tyl d.rsm due,E u"_

:

T h ts IS i n tthe he HD DL L

Due to an

,r€ -t i or cholesterol iev heoaFetion be\ 31.;n "ou;{", [::f;''; I Ev-, -.t

(described earlier) and its excretion from the body. The oi l s w i th ri ch P U FA content i ncl u de cottonseedoil, soya bean oil, sunfloweroil, corn oi l , fi sh oi l s etc. Ghee and coconut oi l are poor sourcesof PUFA. 2. Dietary cholesterol: Cholesterolis found

pl ant food s. 3. oltlj?jtome : Increase in plasma onl v i n ani mal foods and not i n

Dietary cholesterol influence on plasma oSstruc$ofation is the characteristic is minimal. However, avoidance of cholesterol l1rou$hrotic syndrome. Cholesterol foods is advocatedto be on the cholesterol-rich z u e to i n c re a s ei n p l a s mal i poprotei n safe side. n th i s d i s o rd e r. ,crcholesterolemia is associated with .iosclerosis and coronary heart disease. is characterizedby depositionof .nerosclerosis c holes te ryel s te rsa n d o th e r l i p i d s i n the i nti ma of the arterial walls often leading to hardening of coronary arteriesand cerebral blood vessels. A positive correlation between raised plasma lipids w i th a th e ro s c l e ro s ios n o n e ' hand and coronarv heart diseaseon the other has been established.More specifically,LDL-cholesterolis positivelycorrelated,whereasHDL-cholesterolis negatively correlated with cardiovascular diseases.

3. Plant sterols : Certain plant sterols and their esters(e.g.sitostanolesters)reduceplasma cholesterol levels. They inhibit the intestinal absorptionof dietary cholesterol. 4. Dietary fiber : Fiber presentin vegetables decreasesthe cholesterol absorption from the i ntestine. 5. Avoiding high carbohydrate diet : Diets rich in carbohydrates (particularly sucrose) should be avoided to control hypercholesterolemia.

6. lmpact of lifestyles: Elevationin plasma Bad cholesterol and good cholesterol : cholesterolis obseved in people with smoking, Cholesterolis a natural metaboliteperforminga abdominal obesity,lack of exercise,stress,high wide range of functions (membrane structure, blood pressure,consumption of soft water etc. precursorfor steroid hormones,bile acids).The Therefore,adequatechangesin the lifestyleswill usagesgood and bad to cholesterol,although bri ng dow n pl asmachol esterol . inappropriate,are still in use. The cholesterolin Z. Moderate alcohol cosumption : The high concentration,presentin LDL,is considered beneficial effectsof moderatealcohol intakeare had due to its involvement in altherosclerosis masked by the ill effectsof chronic alcoholism. and related complications.Thus, LDL may be particularly beneficial due to its wine is Red regardedas lethally dangerouslipoprotein. On low alcohol content. antioxidants, besides the other hand, HDL cholesterol is good since its high concentrationcounteractsatherogenesis. 8. Use of drugs : Drugs such as lovastatin HDL m a y b e c o n s i d e re da s h i g h l y desi rabl e which inhibit HMG CoA reductaseand decrease fipoprotein. cholesterolsynthesisare used. Statinscurrently in use include atorvastatin, simvastatin and pravastatin. Certain drugs-cholestyramine and col esti pol -bi nd w i th bi l e aci ds an d Several measuresare advocated to lower the Thi s helps decreasethei r i ntesti nalreabsorpti on. plasma cholesterollevel in the conversion of more cholesterol to bile 1. Consumptionof PUFA : Dietary intake of acids and its excretion through feces. Clofibrate polyunsaturatedfatty acids (PUFA) reduces the increases the activity of lipoprotein lipase plasma cholesterol level. PUFA will help in and reduces the pl asma chol esterol an d transoort of cholesterol bv LCAT mechanism triacylglycerols. Gontrol

of hypercholesterolemia

316

B IOC H E MIS TFIY

2. Hypothyroidism (myxoedema) : This is believed to be due to decrease in the HDL receptors on hepatocytes. 3. Obstructive jaundice : Due to an obstruction in the excretion of cholesterol t hr ough b i l e . 4. Nephrotic syndrome : Increasein plasma globulin concentration is the characteristic feature of nephrotic syndrome. Cholesterol elevationis due to increasein plasmalipoprotein fractionsin this disorder. Hypercholesterolemia is associated with atherosclerosis and coronary heart disease. Atherosclerosis is characterizedby depositionof cholesterylestersand other lipids in the intima of the arterial walls often leading to hardening of coronary arteriesand cerebral blood vessels. A positive correlation between raised plasma lipids with atherosclerosison one h4nd and coronary heart diseaseon the other has been established.More specifically,LDL-cholesterolis positivelycorrelated,whereasH DL-cholesteroli s negatively correlated with cardiovascular diseases.

(describedearlier) and its excretion from the body. The oi l s w i th ri ch P U FA content i ncl ude cottonseedoil, soya bean oil, sunfloweroil, corn oi l , fi sh oi l s etc. C hee and coconutoi l are poor sourcesof PUFA. 2. Dietary cholesterol: Cholesterolis found only in animal foods and not in plant foods. Dietary cholesterol influence on plasma cholesterol is minimal. However, avoidance of cholesterol-richfoods is advocatedto be on the safe side. 3. Plant sterols : Certain plant sterols and their esters(e.g.sitostanolesters)reduceplasma chol esterol l evel s. They i nhi bi t the i ntesti na l absorptionof dietary cholesterol. 4. Dietary fiber : Fiber presentin vegetables decreasesthe cholesterol absorption from the i ntesti ne. 5. Avoiding high carbohydrate diet : Diets rich in carbohydrates (particularly sucrose) should be avoided to control hvpercholesterol emi a.

6. lmpact of lifestyles: Elevationin plasma Bad cholesterol and good cholesterol : cholesterolis obseved in people with smoking, Cholesterolis a natural metaboliteperforminga abdominal obesity,Iack of exercise,stress,high wide range of functions (membrane structure, blood pressure,consumption of soft water etc. precursorfor steroid hormones,bile acids).The Therefore,adequatechangesin the lifestyleswill usagesgood and bad to cholesterol,although bring down plasma cholesterol. inappropriate,are still in use. The cholesterolin 7. Moderate alcohol cosumption : The high concentration,presentin LDL, is considered beneficial effectsof moderatealcohol intake are bad due to its involvement in altherosclerosis masked by the ill effectsof chronic alcoholism. and r ela te d c o m p l i c a ti o n s T . h u s , L D L may be particularly beneficial due to its Red wine is regardedas lethally dangerousIipoprotein. On low alcohol content. antioxidants, besides the other hand, HDL cholesterol is good since its high concentrationcounteractsatherogenesis. 8. Use of drugs : Drugs such as lovastatin HDL m a y b e c o n s i d e re da s h i g h l y desi rabl e w hi ch i nhi bi t H MC C oA reductaseand decrease fipoprotein. cholesterolsynthesisare used. Statinscurrently in use include atorvastatin, simvastatin and pravastatin. Certain drugs-cholestyramine and col esti pol -bi nd w i th bi l e aci ds and Severalmeasuresare advocatedto lower the Thi s hel ps decreasethei r i ntesti nalreabsorpti on. plasma cholesterollevel of more cholesterol to bile in the conversion feces. 1. Consumptionof PUFA : Dietary intake of acids and its excretion through Clofibrate pofyunsaturatedfatty acids (PUFA) reduces the increases the activity of lipoprotein lipase plasma cholesterol level. PUFA will help in and reduces the plasma cholesterol and transport of cholesterol by LCAT mechanism triacylglycerols. Gontrol

of hypercholesterolemia

F Ghapter 14 : METABOLISM OF LIPIDS

317 1. Chylomicrons : They are synthesizedin the intestine and transport exogenous (dietary) triacylglycerol to various tissues. They consist of highest(99"/")quantityof lipid and lowest (1%) concentration of protein. The chylomicrons are the leastin densityand the largestin size,among the lipoproteins.

Shell (coat)

Fig. 14.33: A generalstructurcof lipoprcteincomplex. (Note : For the sakeof clarity,only a part of the shell and coreare filled with the constituents).

2. Yery low density lipoproteins (VtDt) : They are produced in liver and intestineand are responsiblefor the transport of endogenously synthesizedtriacylglycerols. 3. Low density lipoproteins(LDt) : They are formed from VLDL in the blood circulation.Thev transportcholesterolfrom liver to other tissues.

a. High densitylipoproteins(HDt) : They are mostly synthesized in liver. Three different Hypoeholesterolemia fractionsof HDL (1, 2 and 3) can be identified A decrease in the plasma cholesterol, by ultracentrifugation.HDL particles transport although less common, is also observed. cholesterol from peripheral tissues to liver p e rn i c i o u s Hy per t hy ro i d i s m , anemi a, (reversecholesteroltransport). m alabs or p ti o ns y n d ro me , h e mo l y ti c j a undi ce 5. Freefatty acids-albumin : Freefatty acids etc., are some of the disordersassociatedwith in the circulation are in a bound form to hypocholesterolemia. al bumi n. E ach mol ecul e of al bumi n can hol d

(-) Cathode

Lipoproteins are molecular complexes that consist of lipids and proteins (conjugated proteins).They function as transportvehiclesfor lipids in bl o o d p l a s ma .L i p o p ro te i n sd e l i ver the lipid components (cholesterol, triacylglycerol etc.) to various tissuesfor utilization. Structure

Origin Chylomicrons

LDL(p-lipoprotein)

of lipoproteins

A lipoprotein basically consistsof a neutral lipid core (with triacylglyceroland/orcholesteryl ester) surrounded by a coat shell of phospholipids, apoproteins and cholesterol The polar portions (amphiphilic)of ffigJa3\. phospholipidsand cholesterolare exposed on the surfaceof lipoproteinsso that lipoprotein is s oluble in a q u e o u ss o l u ti o n . G las s if ic a ti o n

Mobility

VLDL(pre-plipoprotein)

HDL(cr-lipoprotein)

o f l i p o p ro te i n s

Five major classes of lipoproteins are ident if ied i n h u ma n p l a s ma , b a s e d o n thei r separation by electrophoresis(Fig.l 4.34).

(+) Anode

Fig. 14.34: Electrophoresis of plasma(serum) Iipoptoteins.

318

B IOC H E MIS TR Y

about 20-30 moleculesof free fatty acids. This lipoprotein cannot be separated by electrophoresis. Apolipoproteins

(apoproteins!

The protein cornponentsof lipoproteinsare known as apolipoproteins o(, simply, apoproteins. They perform the following functions

Chylomicrons(nascent)are synthesizedin the small intestine during the course of fat absorption. They contain apoprotein Ba6 and mostly triacylglycerols.Apo 846 name is given since this apoproteincontains 48'h of protein coded by apo B gene (apo B1s6is found in LDL and VLDL). Chylomicrons are produced when nascentparticlescombine with apo C ll and apo E, derived from HDL.

The liver synthesizes nascent VLDL contai ni ngapo 8166 w hi ch are ri ch i n tri acyl 2. Recognize the cell membrane surface gl ycerol s and chol esterol . C i rcul ati ng H D L receptors. donatesapo C ll and apo E to convert nascent 3. Activate enzymes involved in lipoprotein V LD L to V LD L. metabolism. Role of lipoprotein lipase : The enzyme The comparative characteristic features of l i poprotei nl i pasei s presenti n the capi l l aryw al l s different lipoproteins with regard to electro- of adipose tissue, cardiac and skeletal muscle, phoretic patterns, size, composition etc. are besidesother tissues.lt hydrolysesa portion of given in Tahle 14.5. triacylglycerols present in chylomicrons and VLDL to liberate free fatty acids and glycerol. Metabolism of lipoprotein$ Lipoproteinlipase is activatedby apo C ll. -a general view Uptake of chylomicron remnants by liver : A generalpictureof lipoproteinmetabolismis As the triacylglycerols of chylomicrons and depicted in Fig.|4.35. VLDL are degraded,they losethe apo C ll which 1 . Act as structuralcomponentsof lipoproteins.

Characteristic

Chylomicrons

VLDL

Electrophoretic mobility

Origin

Pre-p

po

Density

2 mg/dl). The three types of jaundice (hemolytic,obstructiueand hepotic)can be diflerentiollg diognosedb9 biochemicoltests. Thus, unconjugated bilirubin (indirect positiue) is increosed in hemolytic jaundice, conjugated bilirubin (direct positiue) in obstructiuejaundice qnd both ol them (biphasic) are increasedin hepatic jaundice.

6. lmpaired galactose tolerance test, diminished serum olbumin concentration and prolonged prothrombin time are o/so ossociof ed with liuer molfunction.

7. The renal (kidneil t'unction is usuol/y ossessedby eualuatingeither the glomerular (clearoncetests)or tubular function (urine concentration test). This is often guided by blood onalysis(for ureo, creatinine) and/or urine examination.

8. The clearanceis defined as the uolume of the plasma that u)ould be completely cleared of o substanceper minute. Inulin clearancerepresentsglomerular filtrotion rate (GFR). Creotinine clearanceand urea clearance tests are ot'ten used to ossessrenol function. A decreosein their clearance is an indication of renal damoge.

9. Impoirment in renol function is often associatedwith eleuotedconcentration of blood urea, serum creatinine, decreasein osmolality and specific grauitg of urine (by urine concentrotion tesil.

10. The tests to eualuate gastric function include troctional test meql, pentagastrin stimulation test, augmented histamine test and tubelessgostric onolysis. Gastric HCI secretion is eleuoted in chronic duodenal ulcer and gastric hyperplosia. Gastritis and pernicious anemio ore associatedwith decreased gastric HCI. Pancreatic function is ossessedby serum omylaseond lipase.Both of them are eleuatedin acute pancreatitis.

Ghapten 2O : OFIGANFUNCTIONTESTS

467

I. Essayquestions employedto assessliver function. 1. Write brieflyon the differentlaboratoryinvestigations 2. Discussthe biochemicalparameters for the differentialdiagnosisof jaundice. 3. Cive an accountof the serumenzymesderivedfrom liver and their importancein LFT. 4. Describethe renal function tests. 5. Discussthe different laboratoryinvestigationsto evaluategastricfunction. II. Short notes (a)Serumbilirubin,(b) van den Berghreaction,(c) Calactosetolerancetest,(d) Prothrombintime as LFT, (e) Renal threshold substances,(0 Clomerular filtration rate, (g) Creatinineclearance, (h) Standardurea clearance,(i) Urine concentrationtest, (j) Gastricfunction tests. III. Fill in the blanks 1. Bilirubinis the excretoryend productof 2. The laboratoryreactionmostcommonlyemployedto detectthe elevationof serumbilirubinis 3. The serumenzymemost predominantlyelevatedin viral hepatitisis by an increasein the serumenzyme 4. Obstructivejaundiceis characterized 5. The excretoryfunctionof liver can be evaluatedby usinga dye 6. The renalthresholdfor glucoseis 7. The exogenoussubstanceused to measureglomerularfiltrcationrate (GFR)is 8. Standardureaclearanceis calculatedwhen the volumeof urineoutputis lessthan 9. Name the stomachtube usedto aspirategastricjuice 10. Name the syntheticpeptideusedto stimulategastricsecretionfor evaluationof gastricfunction IV. Multiple choice questions 11. In hemolyticjaundice,van den Berghreactionis (a) Indirectpositive(b) Direct positive(c) Biphasis(d) None of these. 12. The serumenzymeelevatedin alcoholiccirrhosisof liver is (c) Alcohol dehydrogenase (a) Alaninetransaminase (b) Aspartatetransaminase (d) y-Glutamyl transpeptidase. 13. B ilir ub i ni s n o t e x c re te di n u ri n e i n (a) Obstructivejaundice(b) Hepaticjaundice(c) Hemolyticjaundice(d) All three. 14. Urea clearanceis lessthan CFR becauseit rs (a) Partiallysecretedby the renaltubules(b) Partiallyreabsorbed by the tubules(c) Only filtered by glomeruli(d) None of these. 15. The serumenzymeusedto evaluatepancreaticfunction is (a) Alkaline phosphatase(b) Amylase (c) Aspartatetransaminase(d) Lactatedehydrogenase.

Watero Electro

The seiil-bsse hsmeostssis speeks : - oK^ + loq -

IHco;1 lunn tr rwv"l

I

"We wtei.utainthe bloodpH nt 7.4! Regulatedby buffe,rs,lwtgs arzdkiineys; fit*eased./tj,rirogeaion causetacitlos,ii;

., .

h.l,rirogen ion leadsto alhal,nsis." Decrensed

organismpossesses tremendouscapacity Jhe I t o s u rv i v e a g a i n s t o d d s a n d mai ntai n homeostasis. This is particularlytrue with regard to water, electrolvteand acid-basestatusof the body. These three are interrelated,hence they are consideredtogetherfor the discussionin this chapter. Kidney actively participates in the regulation of water, electrolyte and acid-base balance. The general functions of kidney have already been described (Chapter 20).

2. Water directly participatesas a reactantin severalmetabolic reactions. 3. lt serves as a vehicle for transport of solutes. 4. Water is closely associated with the regulationof body temperature. D i stri buti on

of w ater

Water is the major body constituent.An adult human containsabout 60% water (men 55-70%, women 45-60%).The women and obese individuals have relativelylesswater which is due to the higher content of storedfat in an anhydrous Water is the solvent of life. Undoubtedly, form. water is more important than any other single A 70 kg normal man containsabout 42 litres compound to life. lt is involved in severalbody of w ater. Thi s i s di stri buted i n i ntracel l ular f unc t io n s . (i nsi dethe cel l s 281)and extracel l ul ar(outsi de the cells 141)compartments,respectivelyknown F unc t i o n s o f w a te r as intracellular fluid (lCF) and extracellular fluid 1. Water provides the aqueous medium to (ECD.The ECF is further divided into interstitial the organismwhich is essentialfor the various fl ui d (10.51)and pl asma(3.51).The di stri bution biochemical reactionsto occur. of water in man is given in Table 21.1.

468

{:iraster- 21 : WATER. ELECTHOLYTE AND ACID-BASEBALANCE

469

of water from the body-urine, skin, lungs and feces. 'Compartment

% Body weight

Volume (l)

Total

60

42

Intracellular fluid(lCF)

40

28

Extracellular fluid(ECF)

20

14

lnterstitial fluid

15

10,5

c

3,5

Plasma

Urine : This is the major route for water loss from the body. In a healthy individual,the urine output is about 'l-2 l/day. Water loss through kidneys although highly variable, is well regulated to meet the body demands-to get rid of water or to retain. lt should, however, be rememberedthat man cannot completely shut down urine production, despitethere being no water intake. This is due to the fact that some amount of water (about 500 ml/day) is essential as the medium to eliminate the waste products from the body.

Hormonal regulation of urine production : lt The body possessestremendous capacity to is indeedsurprisingto know that about 180 litres regulate its water content. In a healthy of water is filteredby the glomeruli into the renal indiv idual, t h i s i s a c h i e v e d b y b a l a n c i ng the tubules everyday. However, most of this is reabsorbedand only 1-2 litres is excreted as daily water intake and water output. urine. Water excretion by the kidney is tightly controlled by vasopressin also known as bllater intake antidiuretic hormone (ADH) of the posterior Water is supplied to the body by exogenous pituitary gland. The secretion of ADH is and endogenous sources. regulatedby the osmotic pressureof plasma.An Exogenous water : Ingested water and increasein osmolalitv promotesADH secretion beverages, water content of solid foods- that leads.to an increasedwater reabsorption constitute the exogenoussource of water. Water from the renal tubules(lessurine output).On the intake is highly variable which may range from other hand, a decreasein osmolality suppresses 0.5-5 litres. lt largely depends on the social ADH secretion that results in reduced water habits and climate. In general,people living in reabsorptionfrom the renal tubules (more urine hot climate drink more water. Ingestionof water output). Plasmaosmolality is largely dependent is mainly controlled by a thirst centre located in on the sodium concentration, hence sodium the hypothalamus.lncrease in the osmolality of indirectly controls the amount of water in the plasma causes increased water intake by bodv. stimulatingthirst centre. Diabetes insipidusis a disorder characterized the deficiency of ADH which results in an by Endogenous water : The metabolic water produced within the body is the endogenous increased loss of water lrom the body. water. This water (300-350 ml/day) is derived Skin : Loss of water (450 ml/day) occurs from the oxidation of foodstuffs. lt is estimated through the body surfaceby perspiration.This is that 1 g each of carbohydrate, protein and fat, an unregulated process by the body which respectively,yield 0.5 ml, 0.4 ml and 1.1 ml of mostly depends on the atmospherictemperature water. On an average/about 125 ml of water is and humidity. The loss is more in hot generatedfor 1,000 Cal consumedby the body. climate. Fever causes increased water loss through the skin. lt is estimatedthat for every W at er out p u t 1oC rise in body temperature, about 15"/" Water losses from the body are variable. increase is observed in the loss of water (through There are four distinct routesfor the elimination ski n).

470 Drinking water andbeverages Foodstuffs (1,500ml) (700 ml)

BIOCHEMISTF|Y Metabolic water

Water intake (2,s00ml)

Urine Lungs Feces Skin (1,500ml) (450ml) (400ml) (150mD

Water output (2,500ml)

instance, NaCl does not exist as such, but it exists as cation (Na+) and anion (Cl-). The concentration of electrolytes are expressedas milliequivalents (mEdl) rather than mi | | igrams. A gram equivalent weight of a compound is defined as its weight in gramsthat can combine or displace 1 g of hydrogen. One gram equivalent weight is equivalent to 1,000 mi l l i equi val ents. The following formula is employedto convert the concentration mgll to mEq/|.

Fig.21.1 : Water balanee in the body, representedby

mEdl = Lungs : During respiration/some amount of water (about 400 ml/day) is lost through the expiredair. The latteris saturatedwith water and expelled from the body. In hot climates and/or when the person is suffering from fever, the water loss through lungs is increased.

I

I

mg per litre x Valency Atomic weight

cortlpos;tion Electrolyte ol body fluids

Electrolytesare well distributed in the body fluids in order to maintain the osmotic equi l i bri umand w ater bal ance.A compari sonof electrolytespresentin extracellular(plasma)and The loss of water by perspiration(via skin) i ntracel l ul ar (muscl e) fl ui ds i s gi ven in and respiration(via lungs)is collectivelyreferred Table 2l .2. The total concentration of cations and anions in each body compartment(ECFor to as insensible water loss. ICF) is equal to maintain electrical neutrality. Feces : Most of the water entering the There is a marked difference in the concengastrointestinal tract is reabsorbed by the intestine.About 150 ml/day is lost through feces tration of electrolytes (cations and anions) in a healthy individual. Fecal loss of water is betweenthe extracellularand intracellularfluids. Na+ is the principal extracellular cation while tremendouslyincreasedin diarrhea. K+ is the intracellular cation. This difference in A summaryof the water intake and output in the concentrationis essentialfor the cell survival the body is depicted in Fig.2l.l,lt may be noted which is maintained by Na+ - K+ pump (for that water balance of the body is regulated details, Refer Chapter 33). As regards anions, predominantlyby controlling the urine output. Cl- and HCOI predominantly occur in This happensafter an obligatory water loss via extracel l ul arfl ui ds, w hi l e H P O;, protei nsand skin, lungs and feces. organicacidsare found in the intracellularfluids. The abnormalities associated with water balance-dehydration and overhydration-will Osrnolarity and osmolalaty be described, following a discussion on of body fluids electrolvte balance.

the concenTherearetwo waysof expressing trationof moleculeswith regardto the osmotic pressure.

Electrolytes are the compoundswhich readily dissociate in solution and exlbf as ions i.e. positively and negativelycharged particles.For

1. Osmolarity: The number of moles(or per literof solution. millimoles) 2. Osmolality: The number of moles (or millimoles)per kg of solvent.

Ghapter 21 : WATER, ELECTFIOLYTE AND ACID-BASEBALANCE

Extracellular fluid (plasma)

Cations

Intracellular fluid (muscle)

Anions 142

Na+

ct-

K+

5

Ca2*

5

HCO; HPO14-

Mgz+

3

soiProteins

471

Cations

Anions

103

K+

150

27

Na*

10

2

Mg'*

40

1

Ca2r

2

16

Organic acids

6

140

HPO42-

Hcot clsoot Proteins Organic acids

10 2 40 5 202

tcc

lf the solventis pure water, there is almost no not be valid in severe hyperproteinemiaand difference between osmolarity and osmolality. l i pemi a. However, for biological fluids (containing molecules such as proteins),the osmolality is Osmolality of EGF and ICF more commonly used. This is about 6% greater Movement of water across the biological than osmolarity. membranes is dependent on the osmotic pressure differences between the intracellular Osmolality of plasma fluid (lCF) and extracellular fluid (ECF).In a Osmolalityis a measureof the soluteparticles healthy state, the osmotic pressure of ECF, pr es entin t h e fl u i d me d i u m . T h e o s m o l a l i tyof mainly due to Na+ ions, is equal to the osmotic plasma is in the rangeof 285-295 milliosmoles/ pressureof. ICF which is predominantlydue to kgffable 21.3).Sodiumand its associatedanions make the largestcontribution (-90%) to plasma osmolality.Osmolality is generallymeasuredby osmometer. For practical purposes, plasmaosmolality can be computed from the concentrations(mn'rol/l) of Na+, K+, urea and glucose as follows 2( Na+)+ 2 (K+ )+ U re a + C l u c o s e The factor 2 is used for Na+ and K+ ions to account for the associatedanion concentration (assumingcompleteionizationof the molecules). S inc e plas m a N a + i s th e m o s t p re d o m i nant contributorto osmolality,the above calculation is further simplified as follows P las m aos mo l a l i ty= 2 x Pl a s ma N a * (mmol/kg) (mmol/l) The above calculation holds good only if plasmaconcentrationof glucoseand urea are in the normal range.This calculation,however,will

Constituent (solute)

Osmolality (mosm/kg)

Sodium Associated anions Potassium Associated anions Calcium Associated anions Magnesium Associated anions Urea Glucose Protein Total

135 135 3.5 3.5 1.5 1.5 1.0 1.0 b.u

5.0 1.0

472

B IOC H E MIS TFI Y

K+ ions.As such,there is no net passageof water m olec u l e s i n o r o u t o f th e c e l l s . due to thi s os m o ti c e q u i l i b ri u m . Regulation

of electrolyte

balance

zA\ ,-'(/ Ansioterisin ',

oj:"ff'

Electrolyteand water balance are regulated togetherand the kidneysplay a predominantrole in this regard.The regulationis mostly achieved through the hormones aldosterone,ADH and renin-angiotensin. AngiotensinI

Aldosterone : lt is a mineralocorticoid produced by adrenal cortex. Aldosterone increasesNa+ reabsorptionby the renal tubules at the expenseof K+ and H+ ions. The net effect is the retentionof Na+ in the bodv.

Aldosterone

\ \nin Angiotensinogen

Fig.2l.2: Hormonalregulationof Antidiuretic hormone (ADH) : An increasein Na' balanceby the kidney. the plasma osmolality (mostly due to Na+) stimulateshypothalamusto releaseADH. ADH effectively increaseswater reabsorption by renal these ions (Na+ and Cl-). Therefore,the amount t ubule s . of N a+ i n the E C F ul ti matel v determi nesit s Renin-angiotensin : The secretion of vol ume. aldosterone is controlled by renin-angiotensin system.Decreasein the blood pressure(due to a Dietary intake and fall in ECFvolume) is sensedby juxtaglomerular electrolyte balance apparatusof the nephron which secreterenin. C eneral l y, the consumpti on of a w ellRenin acts on angiotensinogento produce bal anceddi et suppl i esthe body requi rementof angiotensin l. The latter is then converted to electrolytes.Humans do not possessthe ability angiotensin ll which stimulatesthe releaseof to distinguishbetweenthe salt hunger and water aldosterone. hunger.Thirst,however,may regulateelectrolyte The relation between renin, angiotensinand intakealso. In hot climates,the lossof electrolyte aldosteronein the regulationof Na+ balance is is usuallyhigher.Sometimesit may be necessary depicted in Fig.2l.2. Aldosterone and ADH to supplementdrinking water with electrolytes. coordinate with each other to maintain the nor ma l fl u i d a n d e l e c tro l y teb a l a n ce. Dehydration Atrial natriuretic peptide : This is a Dehydration is a condition characterizedby polypeptide hormone secreted by the right water depletion in the body. lt may be due to atrium of the heart. Atrial natriuretic peptide insufficient intake or excessivewater loss or increases the urinary Na+ excretion. The both. Dehydrationis generallyclassifiedinto two significanceof this hormone, however, is not types. c lear . 1. Due to loss of water alone. Na* concentration

and ECF

It is important to realise that Na+ and its anion s (ma i n l y C l -) a re c o n fi ned to the extracellularfluid. And the retentionof water in the ECFis directlv relatedto the osmoticeffectof

2. Due to electrolytes.

deprivation of

water

and

Causes of dehydration : Dehydration may occur as a resultof diarrhea,vomiting, excessive sweating, fluid loss in burns, adrenocortical

AND ACID-BASEBALANCE Ghapter 21 : WATEB, ELECTFIOLYTE

473

dysfunction,kidney diseases(e.9. renal insuffi- lumen. Theseions collectivelyraisethe osmotic ciency), deficiency of ADH (diabetesinsipidus) pressureand suck the water into lumen. This results in diarrhea with a heavy loss of water etc. (5-10 liters/day). lf not treated in time, the Characteristicfeatures of dehydration : There victims of cholera will die due to dehydration are three degrees of dehydration-mild, and loss of dissolved salts. Thus, cholera and moderate and severe. other forms of severe diarrhea are the major The salient featuresof dehydrationare given ki l l ers of young chi l dren i n many devel opi n g hereunder countries. 1. The volume of the extracellular fluid (e.g. Oral rehydration therapy (ORT) is commonly plasma)is decreasedwith a concomitantrise in used to treat cholera and other diarrheal electrolyteconcentrationand osmotic pressure. diseases. 2. Water is drawn from the intracellularfluid Overhydration t hat r es u l ts i n s h ru n k e n c e l l s a n d di sturbed metabolisme.g. increasedprotein breakdown. Overhydrationor water intoxicationis caused by excessive retention of water in the body. This 3. ADH secretion is increased.This causes may occur due to excessive intake of large increased water retention in the body and vol umes of sal t free fl ui ds, renal fai l ure, consequentlyurine volume is very low. overproduction of ADH etc. Overhydration is 4. Plasma protein and blood urea concentra- observed after major trauma or operation, lung tions are increased. infectionsetc. 5. Water depletion is often accompaniedby a loss of electrolytes from the body (Na+, K+ etc.).

Water intoxicationis associatedwith dilution of ECF and ICF with a decreasein osmolalitv. The cl i ni cal symptoms i ncl ude headache, lethargy and convulsions. The treatment 6. The principal clinical symptomsof severe advocated is stoppage of water intake and dehydration include increasedpulse rate, low administrationof hypertonicsaline. blood pressure,sunkeneyeballs,decreasedskin turgor, lethargy,confusion and coma.

Water tank model

Treatment : The treatment of choice for dehydration is intake of plenty of water. ln the subjectswho cannot take orally, water should be administered intravenously in an isotonic solution (usually5% glucose).lf the dehydration is accompaniedby loss of electrolytes,the same should be administeredby oral or intravenous routes. This has to be done by carefully monitoringthe water and electrolytestatusof the body. Osmotic imbalance in cholera

and dehydlation

The distribution of body water (in the ECF and ICF),dehydrationand overhydrationcan be better understood by a water tank model (Fi9.21.3).The tank has an inlet and outlet, respectively, representing the water intake (mostlyoral) and water output (mainly urine) by the body. Dehydrationis causedwhen the water output exceeds the intake. On the other hand, overhydration is due to more water intake and less output.

Metabolism of electrolytes -r Lnotera rs transmrfleo tnrougn water ano The body distribution,dietaryintake,intestinal foods, contaminated by the bacterium Vihrio produces which absorption cholerae.This bacterium a toxin and biochemical functions of stimulatesthe intestinalcells to secretevarious individual electrolytesare discussedunder the ions (Cf-, Na+, K+, HCOI etc.) into the intestinal section mineral metabolism (Chapter 18). The

B IOC H E MIS TR Y

474 lnlet

t--l tl tl tl \./

O a

Normal

Overhydration Fig. 21.3 : Water tank model representingbody fluid compaftments (N-Normal level; ECF-Extracellular fluid; ICF-lntracellular fluid)-

electrolytedisorders,particularlyhypernatremia the vol ati l e aci ds l i ke carboni c aci d (most and hyponatremia(of sodium);hyperkalemiaand predominent, about 20,000 mEq/day) or nonhypokalemia(of potassium)mustalso be referred. volatile acids (about 80 mEq/day)such as lactic acid, sulfuricacid, phosphoricacid etc. Carbonic acid is formed from the metabolicproduct CO2; lactic acid is producedin anaerobicmetabolism; sulfuric acid is generated from proteins (sulfur phosphori c aci d i s The normal pH of the blood is maintained in contai ni ng ami no aci ds); phosphates (e.9. from organic derived the narrow range of 7,35-7.45, i.e. slightly phosphol i pi ds). aci ds add up H + i ons A l l these r u i d i s rather alk aline .T h e p H o f i n tra c e l l u l a fl proteins rich in animal A diet to blood. the variable. Thus, for erythrocytesthe pH is 7.2, the body that produclion by acid results in more while for skeletalmuscle,it may be as low as 6.0. ultimately leadsto the excretionof urine which Maintenance of blood pH is an important is profoundly acidic. homeostaticmechanismof the body. In normal the regulationis so effectivethat Production circumstances, of bases by the body the blood pH variesvery little. Changesin blood The formation of basic compounds in the pH will a l te rth e i n tra c e l l u l apr H w h i ch, i n turn, body, in the normal circumstances,is negligible. influence the metabolism e.g. distortion in Some amount of bicarbonateis generatedfrom protein structure, enzyme activity etc. lt is the organicacids such as lactateand citrate.The estimatedthat the blood pH compatibleto life is ammoni a produced i n the ami no aci d 6.8-7.8. (For a good understandingof acid-base metabolism is converted to urea, hence its balance, adequate knowledge on acids, bases, contribution as a base in the body is pH and buffers is essential.The reader,therefore, insignificant.A vegetariandiet has a tendency must first refer Chapter 40 tor this purpose. for a net production of bases.This is due to the These basic aspects are not discussed here to fact that vegetariandiet producessaltsof organic avoid repetition). acids such as sodium lactatewhich can utilize H+ ions produced in the body. For this reason,a Production of acids by the body vegetarian diet has an alkalizing effect on the The metabolismof the body is accompanied body. fhis is reflectedby the excretion of neutral by an overall productionof acids.Theseinclude or slightly alkaline urine by these subjects.

Ghapten 21 : WATER, ELECTROLYTE AND ACIIBASE BALANCE

475

By takingthe reciprocals (for and logarithms logs,multiplication becomesaddition). The body has developedthree lines of defense to regulate the body's acid-base balance and maintain the blood pH (around 7.4). L Blood buffers ll. Respiratorymechanism

roe4 = roe** brl:gl Ka lH+t loq 1 = oK^ -K

......(3)

[H2co3J

The equation3 may now be written as

lll. Re n a l me c h a n i s m .

[Hco;l

l. Blood buffers

pH = pK " + l og-.

...... (4)

[H2co3l A buffer may be defined as a solution of a The above equation is valid for any buffer weak acid (HA) and its salt (BA) with a strong base. The buffer resiststhe change in pH by the pair. The general equation referred to as addition of acid or alkali and the buffering Henderson-Hasselbalch equation for any buffer capacity is dependent on the absolute is written as concentrationof salt and acid. lt should be borne in mind that the buffer cannot remove H+ ions Is"t"l p H = p K "+ lo g f . $ . . . . . . .(5) from the body. lt temporarily acts as a shock absorbantto reduce the free H+ ions. The H+ Incid] ions have to be ultimately eliminated by the It is evidentfrom this equationthatthe pH is renal mechanism(describedlater). dependenton ratio of the concentrationof the baseto acid (HCO3and H2CO3in equation4).

The blood contains 3 buffer systems.

Blood pH and the ratio of HCO! to H2CO3 : The plasma bicarbonate(HCO]) 2. Phosphatebuffer conceritration is around24 mmol/ (range22-26 3. Protein buffer. mmol/l).Carbonicacid is a solutionof CO2 in water. lts concentration is givenby the product 1. Bicarbonate buffer system : Sodium bi(arterial pcoz partial of pressureof CO2 = 49 (NaHCO3 carbonateand carbonicacid - H2CO3) is the most predominant huffer system of the mm Hg) and the solubilityconstantof CO2 plasma. (0.03). extracellular fluid, particularly 1. Bicarbonatebuffer

the Carbonic acid dissociatesinto hydrogen and bicarbonateions.

H2co3r^ H* + HCof By the law of mass action, at equilibrium (1) (Ka = Dissociationconstantof H2CO3). The equation may be rewritten as follows

I- corl . = K"H''--'i ["..| I r

[nco;l WeknowthatpH = loc;fi.

(2) ......

ThusH2CO3= 40 x 0.03 = 1.2 mmol/|. The Henderson-Hasselbalch equation for bicarbonatebuffer is [Hco. ]

pH= pK"+ log+ - :+ . [H2co3l Substituting the values(bloodpH = 7.4; pK^ = for H2CO3 6.1; HCOg= 24 mmol/l;H2COJ= 1.2 mmol/ll, in the aboveequation 7.4 = 6.'l * loe 24 - 1.2 = 6.1 + log 20 = 6.1 + 1.3 = 7.4

476 lt is evident that at a blood pH 7.4, the ratio of bicarbonate to carbonic acid is 20 : 1. Thus, the bicarbonateconcentration is much higher (20 times)than carbonic acid in the blood. This is referredto as alkali reserveand is responsible for the effective buffering of H+ ions, generated in the body. In normal circumstances,the concentrationof bicarbonateand carbonic acid determines the pH of blood. Further, the bicarbonate buffer system servesas an index to understand the disturbancesin the acid-base balance of the body.

B IOC H E MIS TR Y are described elsewhere (Chapter 10, Refer Fig.l0.6). The large volumes of CO2 produced by the cel l ul ar metabol i c acti vi ty endangerthe aci dbase equi l i bri um of the body. B ut i n normal circumstances, all of this CO2 is eliminated from the body in the expired air via the lungs, as summarizedbelow. Carbonicanhydrase

H2CO3

C Or+ H rO.

The rate of respiration(or the rate of removal of CO2) is controlled by a respiratorycentre, 2. Phosphate buffer system : Sodium locatedin the medullaof the brain.This centreis dihydrogen phosphateand disodium hydrogen highly sensitiveto changesin the pH of blood. phosphate(NaH2POa- Na2HPOa)constitutethe Any decreasein blood pH causeshyperventilation phosphate buffer. lt is mostly an intracellular to blow off CO2, thereby reducing the H2CO3 bufferand is of lessimportancein plasmadue to concentration.Simultaneously,the H+ ions are its low concentration.With a pK of 6.8 (closeto el i mi natedas H 20. blood pH 7.4), the phosphatebuffer would have Respiratorycontrol of blood pH is rapid but been more effective,had it been presentin high only a short term regulatory process/ since concentration. lt is estimated that the ratio of hyperventilation cannot proceed for long. baseto acid for phosphatebuffer is 4 compared to 20 for bicarbonate buffer. Hemoglobin as a buffer : Hemoglobin of is also important in the respiratory erythrocytes 3. Protein buffer system : The plasma pH. At the tissuelevel, hemoglobin regulation of proteinsand hemoglobintogetherconstitutethe binds to H+ ions and helps to transportCO2 as protein buffer systemof the blood. The buffering HCOt with a minimum change in pH (referred capacity of proteins is dependenton the pK of isohydric transport). ln the lungs, as to as ioniz ableg ro u p so f a m i n o a c i d s .T h e i m i dazol e with H+ ions are hemoglobin combines 02, group of histidine(pK = 6.7) is the most effective with HCOt to form removed which combine contributor of protein buffers. The plasma lafter to release H2CO3. The dissociates CO2 to proteins account for about 2oh of the total (Refer Fig.l0.6). be exhaled bufferingcapacity of the plasma. Hemoglobin of RBC is also an important buffer. lt mainly buffersthe fixed acids, besides being involved in the transportof gases(Oz and CO2). More details on hemoglobin are given underrespiratorymechanismfor regulationof pH. ll. Respiratory mechanism for pH regulation Respiratory system provides a rapid m ec hanis mfo r th e m a i n te n a n c eo f a ci d-base balance. This is achieved by regulating the concentrationof carbonic acid (H2CO3)in the blood i.e. the denominator in the bicarbonate buffer system. The details of CO2 transport and the role of hemoglobin in this process

Generationof HCO3 by RBC : Due to lack of aerobic metabolic pathways,RBC produce very little CO2. The plasmaCO2 diffusesinto the RBC along the concentration gradient where it combines with water to form H2CO3. This reaction is catalysedby carbonic anhydrase(also called carbonate dehydratase).In the RBC, H2CO3 dissociatesto produce H+ and HCOt . The H+ ions are trapped and buffered by hemoglobin. As the concentration of HCOf increasesin the RBC, it diffuses into plasma along with the concentration gradient, in exchange for Cl- ions, to maintain electrical phenomenon, referred neutrality. This to as chloride shift, helps to generate HCOI (Fig.2t.Q.

477

AND ACID-BASEBALANCE Chapter 21 : WATER, ELECTBOLYTE

the renal regulationof pH which occurs by the fol l ow i ng mechani sms.

Erythrocy'te

1. E xcreti onof H + i ons 2. Reabsorptionof bicarbonate

CO2+ H2O

3. Excretionof titratableacid

l"o +

4. E xcreti onof ammoni um i ons.

H2C03

t HHb HCo!+ n.-1 Hb

1. Excretionof H+ ions : Kidney is the only route through which the H+ can be eliminated from the body. H+ excretion occurs in the proxi malconvol utedtubul es(renaltubul arcel l s) and is coupled with the regeneration of HCOj. The process depicted in Fig.2l .5, occurs as follows.

Carbonic anhydrasecatalysesthe production of carbonic acid (H2CO3)from CO2 and H2O in the renal tubular cell. H2CO3then dissociatesto H+ and HCO!. The H+ ions are secretedinto the lll. Renal mechanism for pH tubul ar l umen i n exchangefor N a+ . The N a+ i n regulation associationwith HCOI is reabsorbedinto the This is an effective mechanism to The role of kidneys in the maintenanceof blood. el i mi nate aci ds (H + ) from the body w i th a acid-base balance of the body (blood pH) is generation of HCO3. The latter simultaneous highly significant.The renal mechanismtries to adds up to the alkali reserveof the body. The H+ provide a permanent solution to the acid-base with combines a non-carbonate base and is disturbances.This is in contrastto the temporary in excreted urine. buffering system and a short term respiratory mechanism,describedabove. 2. Reabsorptionof bicarbonate: This mechanism is primarily responsibleto conserve the The kidneys regulate the blood pH by blood HCO3, with a simultaneousexcretion of maintainingthe alkali reserve,besidesexcreting H+ ions. The normal urine is almost free from as or reabsorbingthe acidic or basic substances, HCOI. This is explained as follows (Fi5.21.5). the situationdemands. Fig. 21.4 : Generation of bicarbonate by the erythrocyte (CA-Carbon ic anhydrase; Hb-Hemoglobi n).

Urine pH normally lower than blood pH : T he pH of u ri n e i s n o rma l l ya c i d i c (-6 .0 ) . Thi s clearly indicates that the kidneys have contributedto the acidificationof urine, when it is formed from the blood plasma (pH 7.4). In other words, the H+ ions generatedin the body in the normal circumstances,are eliminated by ac idif ied ur i n e . H e n c e th e p H o f u ri ne i s nor m ally ac i d i c (-6 .0 ), w h i l e th a t o f b l o od i s alk aline ( 7. 4 ). U ri n e p H , h o w e v e r, i s v a ri abl e and may range between 4.5-9.5, dependingon the concentrationof H+ ions. Carbonic anhydrase and renal regulation of pH : T he en z y mec a rb o n i ca n h y d ra s e(i n hi bi ted by acetazolamide) is of central importance in

Blood

Renaltubularcell

Na-

N aHC03

Tubularlumen

HCOJ+ H*

1

H2C03

l"o CO2+ H2O

H*+ B-

II

+ HB ;

Fig. 21.5 : Renal regulationof blood pH-Excretion of ll ions (CA-Carbonic anhydrase).

I

478

B IOC H E MIS TR Y

Renaltubularcell

Tubularlumen

HCOf + H*

1 I H2C03

H2C03

1"u H2O+ CO2

J CO2+ H2O

l"u

FIg.2l.6 : Renalreglulationof btoodpH4eabsorption t t" :::|:::qL:0 E ilt!t!4Ftfeli..,i,:::: .4d,/i9i!ryF!+r, ,'..;, Bicarbonatefreely diffusesfrom the plasma into the tubular lumen. Here HCO3 combines with H+, secreted by tubular cells, to form H2CO3. H2CO3 is then cleaved by carbonic anhydrase(of tubular cell membrane)to form CO2 and H2O. As the CO2 concentrationbuilds up in the lumen, it diffusesinto the tubular cells along the concentrationgradient.In the tubular cell, CO2 again combines with H2O to form H2CO3 which then dissociatesinto H+ and HCOI. The H+ is secreted into the lumen in exchangefor Na+. The HCO3 is reabsorbedinto plasma in associationwith Na+. Reabsorptionof HCOI is a cyclic processwith the net excretion of H+ or generation of new HCOt. This is because the H+ is derived from water. This

Blood

Renaltubular cell

Na*

Na*

mechanism helps to maintain the steady state and will not be effectivefor the elimination of H+ or generationof new HCOJ. 3. Excretion of titratable acid : Titratable aciditv is a measureof acid excreted into urine by the kidney. This can be estimatedby titrating urine back to the normal pH of blood (7.4). ln quantitative terms, titratable acidity refers to the numberof mi l l i l i tersof N /l 0 N aOH requi redto titrate 1 liter of urine to pH 7.4. Titratableacidity reflectsthe H+ ions excreted into urine which resultedin a fall of pH from 7.4 (that of blood). The excreted H+ ions are actually buffered in the urine by phosphate buffer as depicted in Fi9.21.7,and briefly describedhereunder. As alreadydiscussed,H+ ion is secretedinto the tubul ar l umen i n exchangefor N a+ i on. Thi s Na+ is obtained from the base, disodium hydrogen phosphate (Na2HPOa).The latter in turn combines with H+ to produce the acid, sodium dihydrogen phosphate (NaH2POa), in which form the major quantity of titratableacid in urine is present.As the tubular fluid moves down the renal tubules/more and more H+ ions are added, resultingin the acidificationof urine. This causesa fall in the pH of urine to as low as 4.5. A ny further fal l i n the pH w i l l cause depletion of Na+ ions. 4. Excretion of ammonium ions : This is another mechanismto buffer H+ ions secreted i nto the tubul arfl ui d. The H + i on combi nesw i th

Tubularlumen Na2HPOa

HCO3

pH 7.4

Na*+f,+ NaHPOZ

HCOI + H*

I

HzCQ

1'o

CO2+ H2O

NaHzPO+ +

Excreted

Fiq.21.7 : Renal regulationof blood pH-Excretion of titratableacid by phosphate buffer mechanism (CA-Carbonic anhydrase).

pH4.5

479

Ghapter 21 : WATEFI, ELECTFOLYTE AND ACID-BASEBALANCE

Renal tubular cell

Tubularlumen

Glutamine

I Iutaminase

f-*tt,

-+ NH3

aerobic metabolismmay be exhaled via lungs, or converted to HCOt by erythrocytes and kidneys to add up to the alkali reserveof the body. Buffers

of intracellular

fluids

Glutamate

Na' HCO;

HCO!+ H"

T

NH;

H2COg

l"o

CO2+ H2O

The regulation of pH within the cells is as important as that discussed above for the extracellularfluid. The H+ ions generatedin the cells are exchangedfor Na+ and K+ ions. This is particularly observed in skeletal muscle which reducesthe potentialdangerof H+ accumulation i n the cel l s.

Excreted

Fiq.21.8 : Benal regulationof blood pH-Excretion of ammonium ions (CA-Carbonic anhydrase).

The body has developed an efficient system for the maintenance of acid-base equilibrium with a result that the pH of blood is almost NH3 to form ammonium ion (NH;). The renal constant (7.4. The blood pH compatible to life tubular cells deamidateglutamine to glutamate is 6.8-7.8, beyond which life cannot exist. and NH3. This reaction is catalysed by the enz y m eglut a mi n a s eT. h e N H 3 , l i b e ra te di n thi s For a betterunderstandingof the disordersof reaction,diffusesinto the tubular lumen where it acid-base balance, the Henderson-Hasselbalch combines with H+ to form NHi Gig.2l.A. equation must be frequentlyconsulted. Ammonium ions cannot diffuseback into tubular cells and, therefore,are excreted into urine. lHco-] NHf is a major urine acid. lt is estimatedthat about half to two-thirds of body acid load is eliminated in the form of NHf, ions. For this reason, renal regulation via NHf, excretion is very effective to eliminate large quantities of acids produced in the body. This mechanism becomespredominantparticularlyin acidosis. central Garbon dioxide-the molecule of pH regulation

pH=pK"+rosfi,c;t

Lungs (CO, exhaled)

As is observedfrom the foregoingdiscussion, CO2 is of central importance in the acid-base balanceof the body. lt hasthe ability to combine Erythrocytes Kidneys with H2O to from H2CO3 which can dissociate (CO,transported, gene1HCO3 to HCOJ and H+. A summaryof the interaction HCOJgenerated) H+lost) 161sfl, between the lungs, erythrocytesand kidneys in Fig.21.9 : Carbon dioxide-the central molecule of handling CO2 to maintain pH of the blood is blood pH regulation. depicted in Fig.2l.9. The CO2 generated by

I

BIOCHEMISTF|Y

480 It is evident from the above equation that the blood pH (H+ ion concentration)is dependent on the relative concentration (ratio) of bicarbonate(HCOI) and carbonic 26ii (H2CO3). The acid-basedisordersare mainly classifiedas 1. Acidosis-a decline in blood pH (a\ Metabolic acidosis-due to a decrease in bicarbonate. (b) Respiratory acidosis-due increasein carbonic acid.

to

an

2. Alkalosis-a rise in blood pH (a\ Metabolic alkalosis-Aue increasein bicarbonate. (b) Respiratory alkalosis-due decreasein carbonic acid.

to to

Metabolic acidosis

mellitus Diabetes (ketoacidosis)

asthma Severe Pneumonia anest Cardiac

failure Renal

in airways Obstruction

Lacticacidosis

Chestdeformities

diarrhea Severe acidosis Renaltubular

of Depression center(by respiratory drugse.g.opiates)

an a

Respiratory acidosis

Metabolic alkalosis

Respiratory alkalosis

vomiting Severe

Hyperventilation

Hypokalemia

Anemia Highaltitude

administration lntravenous of bicarbonate

poisoning Salicylate

The four acid-base disorders referred above are primarily due to alterations in either bicarbonateor carbonicacid. lt may be observed that the metabolic acid-basebalance disorders are causedby a direct alterationin bicarbonate concentrationwhile the respiratorydisturbances are due to a change in carbonic acid level (i.e. CO2). This type of classification is more theoretical. ln the actual clinical situations, mixed type of disordersare common.

makes every attempt to restorethe pH to normal level (7.4). This is referredto as compensation which may be partialor full. Sometimesthe acidbase disordersmay remain uncompensated.

alkalemia, terms acidemia and The respectively,refer to an increaseor a decreasein tH*] ion concentration in blood. They are, however, not commonly used.

The principal acid-basedisturbances,along with the blood concentration of HCO3 and H2CO3, in acute and compensated states are given in the Table. 21.5.

For the acute metabolic disorders (due to changesin HCO!, respiratorycompensationsets in and regulatesthe H2CO3 (i.e. CO2) by hyperThe most important clinical causes/disease or hypoventilation. As regards acute respiratory statesthat result in acid-basedisordersare listed disorders (due to changes in H2CO3), the in Table 21.4. Metabolic acidosis could occur renal compensation occurs to maintain the due to diabetes mellitus (ketoacidosis),lactic HCOJ level, by increasing or decreasing its acidosis,renal failure etc. Respiratoryacidosisis excretion. common in severe asthma and cardiac arrest. fn the lable 2l ,6, a summary of the acid-base Vomiting and hypokalemia may result in primary changes and metabolic alkalosiswhile hyperventilationand disorders with severeanemia may lead to respiratoryalkalosis. compensatorymechanismsis given.

Glinical causes of acid-base disorders

Gompensation of acid-base disorders To counter the acid-base disturbances,the body gears up its homeostaticmechanism and

Anion

gap

For a better understanding of acid-base disorders,adequateknowledge of anion gap is essential.The total concentrationof cations and

Chapter 21 : WATEFI,ELECTROLYTE AND ACID-BASEBALANCE

48r

anions (expressedas mEq/l)is equal in the body disordersare often associatedwith alterations in f luids . T his is re q u i re d to ma i n ta i n e l e c t ri cal the anion gap. neutrality. Metabolic acidosis The commonly measuredelectrolytesin the The primary defect in metabolicacidosisis a plasmaare Na+, K+, Cl- and HCOJ. Na+ and K+ reduction in bicarbonate concentrafion which together constitute about 95% of the plasma leads to a fall in blood pH. The bicarbonate cations. Cl- and HCO3 are the major anions, concentration may be decreased due to its contributingto about 80"h of the plasmaanions. utilization in buffering H+ ions, loss in urine or The remaining 20"/. of plasma anions (not gastrointestinal tract or failure to be regenerated. normally measured in the laboratory) include proteins, phosphate,sulfate, urate and organic The most important cause of metabolic ac ids . acidosis is due to an excessiveproduction of Anion gap is defined as the difference between the total concentration of measured cations(Na+ and K+)and that of measuredanion (Cl- and HCOj). The anion gap (A-) in fact representsthe unmeasured anions in the plasma which may be calculated as follows, by substituting the normal concentration of efectrolytes (mEq/l). Na*+

K*

=

Cl-

+

HCO, + A-

organicacidswhich combine with NaHCOj and deplete the alkali reserve. NaHCO3 + Organic acids -----+ Na saltsof organic acids + CO2 Metabolic acidosis is commonly seen in severe uncontrolled diabetes mellitus which is associated with excessive production of acetoaceticacid and p-hydroxybutyric acid (both are organic acids).

Anion gap and metabofic acidosis : Increased production and accumulation of organic acids t5 nEql A- causes an elevation in the anion gap. This type T he anion g a p i n a h e a l th y i n d i v i d u a l i s of picture is seen in metabolic acidosis around 15 mEq/l (range8-18 mEo/l).Acid-base associatedwith diabetes (ketoacidosis). 136+ { - 10 0 + 2 5 + A -

Disorder

Blood pH

lHCo;l

IH2CO3]

J J

-)

-)

1 t

Metabolic acidosis Acute (byt ventilation) Compensated

\or-)

Respiratory acidosis Acute (HCOIretained by kidney) Compensated

\0r-+

I

Metabolic alkalosis Acute (byJ ventilation) Compensated

t

t 1

--)

V o( --)

Hespiratory alkalosis Acute by kidney) Compensated fHCO; excretion

1 v or --+

-) J

J

v

A

v

t I

482

BIOCHEMISTRY

Primary change

Disorder

Metabolic acidosis

Decreased olasma bicarbonate

ptasma Metabolic alkalosis Increased oicii6oni6 pCO, Respiratory acidosis Increased

Respiratoryalkalosis DecreasedpCO,

Compensation of metabolic acidosis : The acute metabolicacidosisis usuallycompensated hy hyperventilation of lungs. This leads to an increased elimination of CO" from the bodv (hence H2CO3U. but respiratorycompensation is only short-lived.Renal compensationsets in within 3-4 days and the H+ ions are excretedas NHi io n s . Respiratory

acidosis

Compensatory mechanism

Timescalefor compensation

Hyperventilation (decrease in pCOr)

to hours Minutes

Htd6llhtio;

Minutes to hours

(increase in pCOJ Elevation in plasma increase in bicarbonate; of renalreabsorption bicarbonate in plasma Reduction degease bicarbonate: in renalreabsorption of bicarbonate

Days

Days

sodium bicarbonate for therapeutic purposes (e.g. control ol gastric acidity). Cushing's of aldosterone)causes syndrome(hypersecretion increased retention of Na+ and loss of K+ from the body. Metabolic alkalosis is commonly low K+ concentration associated with (hypokalemia).In severeK+ deficiency,H+ ions are retained inside the cells to replace missing K+ ions. In the renal tubular cells, H+ ions are exchanged (instead of K+) with the reabsorbed Na+. Paradoxically,the patient excretes acid urine despitealkalosis.

The primary defect in respiratoryacidosis is due to a retention of CO2 (H2CO3|. There may be severalcausesfor respiratoryacidosiswhich The respiratory mechanism initiates the include depression of the respiratory centre compensation by hypoventilation to retain CO2 (overdose of drugs), pulmonary disorders (hence H2CO3T).This is slowly taken over by (bronchopneumonia) and breathingair with high renal mechanism which excretes more HCO3 content of CO2. and retains H+. The renal mechanismcomesfor the rescueto compensaterespiratoryacidosis.More HCO3 is Respiratory alkalosis generatedand retained by the kidneys which The primary abnormality in respiratory adds up to the alkali reserveof the body. The alkalosis is a decreasein H2CO3 concentration. excretion of titratable acidity and NHf is prolonged to This may occur due elevated in urine. hyperventilationresultingin increasedexhalation of COz by the lungs. Hyperventilation is M et ab o l i c a l k a l o s i s observed in conditions such as hysteria, primary The abnormality in metabolic hypoxia, raised intracranialpressure,excessive artificial ventilation and the action of certain afkalosis is an increase in HCO3 concentration. This may occur due to excessive vomiting drugs (salicylate) that stimulate respiratory (resultingin lossof H+) or an excessiveintakeof centre.

WATER. ELECTROLYTE AND ACID-BASEBALANCE The renal mechanismtries to compensateby inc r eas ingt he u ri n a rye x c re ti o no f H C O 3 . : r,!:rl j:i,i; ;iqi - if?;E..s *,* gl i * ti.*.'d + dtt

483

pl asmaK + )or hypokal emi a(l ow pl asmaK + )can be Iife-threatening. The relevanceof potassium bal ance i n certai n aci d-base di sorders i s di scussedbri efl y.

Sometimes,the patientmay have two or more Potassiumand diabetic ketoacidosis : Tne ac id- bas edis t u rb a n c eosc c u rri n gs i mu l ta n e o usl y. hormone i nsul i n i ncreasesK + uptake by cel l s y In such instances,both HCO3 and H2CO3 are (parti cul arlfrom skel etalmuscl e).The pati entof (of severeuncontrolleddiabetes(i.e. with metabolic alt er ed.I n gen e ra l ,i f th e b i o c h e mi c a ld a ta blood gas analysis)cannot be explained by a aci dosi s)i s usual l y w i th hypokal emi a.W hen specific acid-basedisorder, it is assumedthat a such a pati ent i s gi ven i nsul i n, i t sti mul atesK + m ix ed dis t ur b a n c ei s o c c u rri n g .M a n y a ti mes, entry into cells.The resultis that plasmaK+ level compensatory mechanisms may lead to mixed is further depleted. Hypokalemia affects heart functi oni ng,and i s l i fe threateni ng. acid-base disorders. Potassium and alkalosis : Low plasma concentrationof K+ (hypokalemia)leads to an increasedexcretion of hydrogen ions, and thus P las m a po ta s s i u m c o n c e n tra ti o n (n o r mal may cause metabol i c al kal osi s. C onversel y, 3.5-5.0 mEq/l) is very importantas it affectsthe metabolic alkalosisis associatedwith increased contractility of the heart. Hyperkalemia (high renal excretion of K+. :i n;ifjl""S,*lt*#'d*aerders j,ilr',r l:l rli +slmlia P@ffi *SE{.C""t.$

B|oMEDTCAL/ CLIIU|CALCONCEPTS

s

Existence ot' life is unimaginable in the absenceof water.

w Kdneys play a predominant role in the regulationof water;electrolyteand acid-basebalance. we Electrolyte and water balanceregulotion occurs through the inuoluementof hormonesaldosterone,ADH and renin-ongiotensin. sE Seueredehydration is characterizedby low blood pressure, sunken egeballs,Iethargy, confusion and coma. re' Sodium is the principal extracellular cation while K+ is intracellular. The maintenance of the differentiol concentration of these electrolytesis essentialfor the suruiualoJ liJe which is brought about by No+-K+ pump. ss The body metabolism is accompaniedby the production of acidssuch os carbonic acid, sult'uric ocid, phosphoric acid etc. w

Vegetariandiet hason alkalizingefiect on the body. This is ottributed to the formation of organicocidssuch os sodium lactate which can depleteH+ ions by combining with them.

s€ The blood pH is maintained by blood buffers, respiratory and renal mechanisms. *q Carbon dioxide is the central molecule of acid-baseregulation. w

Disturbancesin acid-baseregulotion result in acidosis(decreasedblood pH) or alkalosis (raised blood pH).

w

Uncontrolled diabetesmellitus is associatedwith metabolic ocidosis,commonly referred to as ketoacidosis(due to the ouerproduction of ketone bodies)

BIOCHEMISTFIY

484 In view of the importancediscussedabove, the measurementof plasma K+ concentration assumessignificancein the acid-basedisorders. In cases of these disorders associated with hypokalemia, potassiumsupplementation(with carefull monitoring of plasma K+) needs to be considered.

of blood gas is an important The measurement investigationin the laboratoryservice.In certain conditions associatedwith respiratory failure and/or acid-base disorders, blood gas (COz and 02) measurementassumessignificance.Based on the results obtained and the severity of the condition, oxygen treatment or artificial ventilation is carried out. For blood gas analysis,a sample of arterial blood collected from (most commonly) radial artery in the forearm, or (lesscommonly) from the femoral artery in the leg is used. The

biochemical profile measured include POz, pCOz, and pH (H+ ion concentration).The concentration of bicarbonate is calculated by equation. In fact, using Henderson-Hasselbalch the blood gas analysers employed in the hospitals are designed to perform the various calculations automatically and give the final results. The reference ranges of blood gas analysisare given in Table 21 .7.

Parameter

Concentration/value

tH-1

35-43mmoUl

pH

7.3y7.45

PC0z

4.5-6.0kPa

Poz

10.5-13.5 kPa

Bicarbonatex

2_ryqTq9r4

xBianbonate is nfulatd fronpHaN {'Orvafues. con@ntration

chapter 21 : WATEFI,ELECTROLYTE AND ACID-BASEBALANCE

485

1. Water is the soluent of life and constitutes about 600/oof the total body weight, distributedin intracellularand extracellularJluids. The daily water intake (by drinking, from loodstuffs and metabolic water) and output (/oss uio urine, skin, lungs and t'eces) maintain the body balonceof water.

2. Electrolytesare distributedin the intracellularond extracellularfluids to maintoin the osmotic equilibrium and water balance,No+ is the principal extracellularcation while K+ is the intracellularcation As regardsonions, CI- and HCO1 predominantlyoccur in the extracellulor t'luidswhile HPO42-, proteins and organic acidsare present in the intracellularfluids.

3. The osmolality of plasma is about 285 milliosmoles/kg,which is predominontly contributed by No+ and its ossociatedonions. Thus, Jor practical purposes, plosma osmololity can be calculated from Na+ concentration (2 x No+ in mmol/l).

4. Water and electrolyte balance are usually regulated together and this is under the antidiuretichormone and renin. control of hormones---aldosterone,

5. Dehydration of the body may be due to insufficient woter intake or its excessiue/oss or both. Depletion of water in the ICF causes disturbance in metabolism. The manifestationsof seueredehydration include increasedpulse rate, Iow blood pressure, sunken eyebolls,decreasedskin turgor, lethorgy and coma. The normal pH of blood is maintained in the narrow range of 7.35-7.45. The metabolismot' the body is accompaniedby an ouerall production of acids. The body has deuelopedthree lines of det'ense(blood buffers, respiratory and renal mechanisms)to regulatethe qcid-basebalanceand maintain the blood pH.

7. Among the blood buffers, bicarbonatebut't'er(with a rotio ol HCOS to H2CO3 as 20 : 1) is the most important in regulating blood pH. Phosphate and protein buft'er systems also contribute in this regard. The respiratory system regulates the concentration of carbonicacid by controlling the elimination of CO2 uia lungs.

8. The renal (kidney) mechanism regulotes blood pH by excreting H+ and NHy' ions besidesthe reabsorptionof HCO7. The pH of urine is normally acidic which indicates thot the kidneyshaue contributed to the acidificationof urine.

9. The ocid-basedisordersare classit'iedos ocidosis(metabolicor respiratory)and alkalosis (metabolicor respiratory),respectiuely,due to a rise or foll in blood pH. The metabolic are associotedwith olterotionsin HCOS concentrationwhile the respiratory disturbonces disorders ore due to changes in H2CO3 ft.e. CO2).

10. Blood gasmeasurementincludesthe parameterspO2, pCO2, pH and bicorbonate,and it is uery important to eualuateand treat acid-basedisorders.

B IOC H E MIS TR Y

486

I. Essayquestions 1. Describethe role of kidney in the regulationof blood pH. 2. Cive an accountof the water distributionand its balancein the body. fluids.Discussthe and intracellular 3. Camparethe compositionof electrolytes in the extracellular regulationof electrolytebalance. 4. Describethe role of blood buffersin the acid-basebalance. mechanisms. 5. Classifyacid-basedisordersand discussthem with compensatory II. Short notes (a)Dehydration,(b) Vasopressin and water balance,(c) Osmolalityof plasma,(d) Acids produced in the body, (e) Henderson-Hasselbalch equation, (fl Bicarbonatebuffer, (g) Excretionof H+ by kidney,(h) Titratableacidity,(i) Metabolicacidosis,(j) Anion gap. III. Fill in the blanks 1. The hormonecontrollingwater excretionvia kidneysis 2. The principalcation of extracellular fluid is 3. The normal osmolalitvof plasmais 4. Na+ reabsorption by renal tubulesis increasedby the hormone 5. The most predominantvolatileacid generatedin the body is 6. The most importantbuffer systemregulatingblood pH is 7. At a normal blood pH 7.4, the ratio of bicarbonateto carbonicacid is 8. The body acid load is predominantlyeliminatedin the form of 9. The primary defect in metabolicacidosisis a reduction in the plasma concentrationof of 10. The respiratory alkalosisis primarilyassociated with a decreasein the plasmaconcentration IV. Multiple choice questions 11. The metabolic(endogenous) water is derivedby the oxidationof (a) Carbohydrate(b) Protein(c) Fatsd) All of them. 12. The most predominantanion in the extracellular fluids (a) Cl- (b) HCof (c) HPoi- (d) Protein. 13. The only routethroughwhich H+ ions are eliminatedfrom the body (a) Lungs (b) Stomach (c) Kidneys (d) None of them. 1 4 . N a meth e a m i n oa c i dfro m whi ch ammoni ai s deri vedi n the renaltubul arcel l sw hi ch i s fi nallv excretedas NHf, (a) Asparagine(b) Clutamine(c) Clutamate(d) Aspartate. (in the laboratory) plasmaanion concentration and is 15. The anion gap refersto the unmeasured representedby (a) Proteinsand organicacids (b) Phosphate and sulfate(c) Urate (d) All of them.

The ptotein,

collagen;,, speaks t

l

"I am the vnostabundant protei/t in marnmab; Triple helical in structure, with distinct types; Predominantly cortposedof glycine and pralinl; Gry-X-Y-G!-X-Y

Gy

X-Y

I giue strength, support and shapteto tissues,"

-l-hu body possesses a vast number of proteins I designed with specific structuresto perform specialized functions.A selectedfew of the most importantproteinsthat are intimatelyconnected with the tissuestructureand functionsare briefly des c r ibedin th i s c h a p te r.In a d d i ti o n ,th e b ody f luids ar e als o d i s c u s s e d .

of the total body protein. Collagen is the predominant component of the connective tissue,although its distribution varies in different tissues.For instance,collagenforms 90% of the organic matrix of bones, 85"/t of tendons, 707o of skin, and 4oh of liver. Fu;lc t itins t,i i:+:;rl;i,Eeii

1 . B ei ng a maj or component of the connective tissue, collagen gives strength, support and shape to the fissues. The tensile The connective tissue or extracellular matrix strengthof collagenfiber is impressive.To break (ECM) refers to the complex material a collagen fiber of 1 mm in diameter,a load of surroundingthe mammaliancells in tissues.The 104O kg is needed! However, in diseasedstates major protein components of ECM include with altered collagen structure, the tensile c ollagen,elas ti n , fi b ri l fi n , fi b ro n e c ti n , l a m i ni n strengthis reduced. and proteoglycans.Besidesthese proteins, the 2. Collagen contributesto proper alignment structural proteins namely keratins are also which in turn helps in cell proliferation, of cells, described. and their differentiationto different tissuesand orS ans. COLLAGEN Collagen is th e m o s t a b u n d a n t p ro te i n i n nammals, comprising approximately one-third

3. Collagen(that is exposedin blood vessels) contributesto thrombus formation.

487

I

488 Types

B IOC H E MIS TRY of eoltagen

Collagen is not a single homogeneous protein,but a group of structurallyrelated an d g e n e ti c a l l y d i s ti n c t p ro tei ns. In humans, at least 19 different types of 300nm collagens, composed of 30 distinct polypeptidechains (encodedby separate genes),have been identified.The typesof collagen are numbered (by Roman numerals)as l, ll...XlX. The differenttypes of collagen are suited to perform specialized functions in tissues. For instance,collagenstype I and type ll are Gly-X-Y-Gly-X-Y-Gly-X-Y respectivelyfound in skin and bone. Stnueture

of collagen

Collagen molecule

i1.4 nm Triplehelix 7.6 mg/ dl), Iung maturity (lecithin-sphingomyelin ratio > 2 : 1) ond for the prenatal diognosis of congenital disorders(e.9. hemophilia, Down's syndrome).

500

B IOC H E MIS TFI Y

1. The maJor proteins of connectiue tissue are collagen, elastin, fibrillin, Iaminin and proteoglgcans.Among these, collagen is the most abundant, constituting one-third of the total body proteins.

2. Type I mature collagen is a triple helicol structure i.e. contoinsthree polypeptlde chains each with about 1000 omino acids. The repetitiue amino acid sequenceof collagen is (Glv-X-Y)". Glycine constitutes about 7/3 rd of the qmino acids while X and Y represent other amino acids.

3. Kerotins are structural proteins t'ound in hair; skin, nails and horns. The strength oJ the keratins is directly related to the number ol disulfide bonds.

4. Muscle is the single largest tissue of the human body (3040o/o of body weight). lt is composed of t'ibre cells into which myofibrils are embedded. Each myot'ibril contains alternoting A and I bands. Sarcomere is the functional unit of muscle.

5. Actin, myosin, tropomyosin qnd troponin ore the major controctile proteins t'ound in muscles.The muscle contraction and relaxation occur due to the actiue inuoluementof these proteins. ATP is the immediate source ot' energy lor muscle contractlon.

6. Proper folding of proteins is essentialt'or thetr structure. Mislolding ol proteins results in certsin diseosese.g. mad cow disease,Alzheimer's disease.

7. The specialized fluids of the body include milk, cerebrospinalfluid, amniotic t'luid, aqueous humor, sweat and tears.

8. Milk is almost a complete t'ood with uariousnutrients---carbohydrotes,Iipids, proteins, uitamins and minerals. Howeue4 milk is deflcient in uitamin C, iron and copper.

9. Cerebrospinal t'luid is an ultrat'iltrate oJ plosma. ln the disease,tuberculosis meningitis, the total cell count and protein concentrationare increesed,while glucoseconcentration is decreased tn CSF

10. Amniotic fluid is a liquid produced by the t'etus. lts biochemicalonolysis is importont for the diagnostic purpose-ossessment of |etal maturity, diognosis of congenital diseqses.

Ghapten 22 : TISSUEPHOTEINSAND BODY FLUIDS

501

I. Essayquestions 1. Cive an account of the structureand functionsof collagen.Add a note on the abnormalities associated with collagen. 2. Describethe muscleproteins,and musclecontraction. 3. Discussthe proteinmisfoldingand variousdiseasesrelatedto it. 4. Give an accountof the compositionof milk. 5. Describethe functionsand compositionof cerebrospinalfluid. Add a note on the alterationsin CSF in diseasedstates. II. Short notes (a) Biosynthesis of collagen,(b) Collagenand scurvy,(c) Elastin,(d) Light and heavy meromyosins, (e) Priondiseases, (fl Amyloidosis, (g) Hair waving,(h) Vitaminsand mineralsin milk, (i) Collection of CSF,(j) Amniotic fluid. III. Fill in the blanks 1. The most abundantproteinin mammals 2. The amino acid that contributesto one-thirdof the total numberof amino acids in collagen 3. The toxic compound that interfereswith the cross-linkingof lysine in collagen,causing lathyrism 4. Marfan syndromeis a geneticdisorderdue to a mutationof the gene coding for 5. Name the carbohydratesassociatedwith the structureof proteoglycans 6. The region of the muscle fibre betweentwo Z lines is termed as 7. Name the major proteinfound in the structureof thin filamentsof sarcomere 8. The white colour of milk is due to the dispersionof 9. Name the vitamin deficientin milk 10. The fetal lung maturityis evaluatedby measuring

ratio.

IV. Multiple choice questions 11. The numberof polypeptidechainspresentin collagen (a) 1 (b) 2 (c) 3 d\ 4. 12. The functionalunit of muscle (a) Fibrecell (b) Myofibril (c) H band d) Sarcomere. 13. The immediatesourceof energyfor musclecontraction (a) ATP (b) Creatinephosphate(c) CTP d) Phosphoenolpyruvate. 14. O n e o f th e fo l l o w i n gmi n e ra l si s l acki ngi n mi l k (a) Calcium(b) Sodium(c) lron d) Potassrum. 15. One of the following biochemicalparameters is increasedin tuberculosis meningitis (a) Clucose(b) Protein(c) Sodiumd) Chloride.

T|rc nattitlon

spea,hs t

"Sorne eat to liae, And some liae to eat!

Ml,function is Tocaterforall," '

hether a man eats for living or lives for e a ti n g , fo o d i s h i s p ri me concern. Nutrition may be defined as the utilization of food by living organr'srns.Biochemists have largely contributedto the scienceof nutrition.

nutrition, undernutrition and areas-ideal overnutrition ldeal nutrition is the concern of everyone.U ndernutri ti oni s the pri me con cer n of devel opi ngcountri esw hi l e overnutri ti o nis a seriousconcern of developedcountries.

N u tri ti o n s i g n i fi c a n tl y p ro motes man' s development,his healthand welfare.The subject nutrition,perhaps,is the most controversial.This is due to the fact that nutrition is concernedwith food, and everyone feels competent enough to t alk l i k e a n e x p e rt o n n u tri ti o n . Further,hi gh public awarenessand the controversialreports by scientistsalso contributeto the controversy.

A sound knowledgeof chemistryand metabolipids, proteins, lism of foodstuffs(carbohydrates, prereq uisit e vi tami nsand mi neral s)i s an essenti al of nutrition.The reader for a betterunderstanding must, therefore,first refer these chapters.The principlesof nutrition with special referenceto energy demands, carbohydrates,fats, proteins, recommended dietary/daily allowances (RDA), bal anced di et and nutri ti onal di sorders ar e Methodology in nutrition : Most of the i n the fol l ow i ng pages. di scussed ex is ti n g k n o w l e d g e o n n u tri ti o n i s ori gi nal l y der i v e d fro m a n i m a l e x p e ri m e n tati on.Thi s i s despite the fact that there may exist several differences in the biochemical composition between man and animals! For instance,some ani ma l sc a n s y n th e s i z e a s c o rb i caci d w hi l e man Food is the fuel source of the body. The cannot do so. ingestedfood undergoesmetabolismto liberate Study of human nutrition : The study of energy required for the vital activities of the nut ri ti o n m a y b e l o g i c a l l y d i v i ded i nto three bodv.

502

Otraster 23 : NUTFI|TION

ffivtr CrHrydrate h Pr*il Abdd

503

Energy value (Cal/g) In bomb calorimeter In the body

4.1 9.4 5.4 7.1

It must be noted that the nutrients,namely vitamins and minerals,have no calorific value, although they are involved in severalimportant body functions, including the generation of energy from carbohydrates,fats and proteins.

4 I

Respiratory quotient of foodstuffs

4

The respiratoryquotient (R. Q.l is the ratio of the volume of CO2 produced to the volume of 02 utilized in the oxidation of foodstuffs.

'7

Energy content of foods The calorific value (energycontent)of a food ;s calculatedfrom the heat releasedby the total combustion of food in a calorimeter.

Carbohydrates : The carbohydrates are completely oxidized and their R. Q. is close to 1, as representedbelow for glucose.

C6H12O6+ 6C2 ---+ 6COz + 6HzO Unit of heat : Calorie is the unit of heat. One caforie representsthe amount of heat required to = =t=,. R.Q. forcarbohydrate + z rilr the temperature ol one gram of water by loC i. e. f r om 1 5 o to 1 6 " C ).A c a l o ri ei s to o smal l a Fats : Fats have relatively lower R.Q. since unit. Therefore,it is more convenientlyexpressed they have a low oxygen content. For this reason, as kilocalories (1,000 times calorie) which is fats require more 02 for oxidation. The R.Q. represented by kcal or simply Cal (with capital 'C'). for the oxidation of the fat, tristearin is given The joule is also a unit of energy used in below. some countries. The relationship between 2 C57HrgO6+ 16 3O2------+114 CO2+ 110 H2O caloriesand joules (J) is (-(-l tu2 1 Cal (1 kcal) = 4.128 Kl = 114 = 0.7. R. Q. for fat = o" 163 The joule is /ess commonly used by Proteins : The chemical nature of proteins is nutritionists. highly variable, and this cannot 6e represented Calorie value of foods : The energy values of by any specific formula. By indirect the three principal foodstuffs-carbohydrate, fat measurements, the R.Q. of protein is found to be and protein-measured in a bomb calorimeter around 0.8. and in the body are given in the Table23.1.The Mixed diet : The R. Q. of the diet consumed carbohydratesand fats are completely oxidized (to CO2 and H2O) in the body; hence their fuel is dependent of the relative composition of values,measuredin the bomb calorimeteror in carbohydrates,fats and proteins.For a normally the body, are almost the same. Proteins, ingesteddiet, it is around 0.8. however, are not completely burnt in the body as they are convefted to products such as urea/ creatinineand ammonia, and excreted.Due to this reason,calorific value of protein in the body is lessthan that obtained in a bomb calorimeter. Man consumes energy to meet the fuel The energy values of carbohydrateg fafs and demands of the three ongoing processess in the proteins(when utilized in the body) respectively, body. are 4, 9 and 4 Cal/9. 1 . Basal metabolic rate Alcohol is a recent addition to the calorie 2. Specificdynamic action 1ZCal/e)contribution,as it is a significantdietary 3. Physicalactivity. component for some people.

B IOC H E MIS TFIY

504 Besidesthe above three, additional energy supply is neededduring growth, pregnancyand lactation.

BASAL METABOLIC RATE Basal metabolism or basal metabolic rate (BMR) is defined as the minimum amount of energy required by the body to maintain life af complete physical and mental rest in the postabsorptivestate(i.e. 12 hoursafterthe lastmeal). ft may be noted Ihat resting metabolic rate (RMR) is in recent use for BMR. Under the basal conditions, although the body appears to be at total rest, several functions within the body continuously occur. These include working of heart and other nerve impulse, organs, conduction of reabsorption by renal tubules, gastrointestinal motility and ion transport across membranes (Na+-K+ pump consumes about 50% of basal energy).

Measurement of BMR

For the calculationof body surfacearea, the si mpl eformul adevi sedby D u B oi sand D u B ois i s used. A = H0 72sx W0.42sx 71.84 where A = Surfacearea in cm2 H = H ei ght i n cm W = Weight in kg. To convertthe surfacearea into squaremeters (m2),di vi de the above val ue (cm2) by 10,000 . Nomogramsof body surfacearea (directlyin m2) from heightsand weightsare readilyavailablein literature. Normaf values of BMR : For an adult man 35-38 Cal/sq. m/hr; for an adult woman 32-35 Cal/sq.m/hr. A BMR value between -157o and +20o/ois consideredas normal. Some authors continue to representBMR as Cal/day. For an adult man BMR is around 1,600 Cal/day, while for an adult woman around 1,400 Cal/day.This is particularlyimportantfor easily calculatingenergy requirementsper day.

Preparationof the subject : For the Factors measurementof BMR the subject should be awake, at complete physicaland mental rest, in a post-absorptive state and in a comfortable s ur r oun d i n g(a t 2 5 ' C ). Measurement: The BMR is determinedeither by the apparatus of Benedict and Roth (closed circuit device) or by the Douglas bag method (open circuit device). The former is more frequently used.

affecting

BMR

1. Surfacearea : The BMR is directly proportional to the surfacearea. Surfacearea is related to weight and height. 2. Sex : Men have marginally higher (about 5% ) B MR than w omen. Thi s i s due to the hi gher proportion of lean muscle mass in men. 3. Age : ln infantsand growing children,with l ean muscl emass,the B MR i s hi gher.In adul ts, BMR decreases at the rate of about 2o/o per decade of life.

By Benedict-Rothmethod, the volume of 02 consumed (recordedon a graph paper) by the subject for a period of 2-6 minutes under basal 4. Physical activity : BMR is increased in conditions is determined.Let this be A liters for persons(notably athletes)with regular exercise. 6 minutes.The standardcalorific value for one This is mostly due to increasein body surface liter 02 consumed is 4.825 Cal. area. Heat produced in 6 min = 4.825 x A

5. Hormones: Thyroid hormones(T3and Ta) have a stimulatoryeffect on the metabolismof Hea t p ro d u c e di n o n e h o u r = 4 .8 25A x 10 the body and, therefore, BMR. Thus, BMR is Units of BMR : BMR is expressedas Calories raised in hyperthyroidism and reduced in per square meter of body surface area per hour hypothyroidism. In fact, the measurement of BMR was earlier used to assessthvroid function. i.e. Callsq.m/hr.

Ghapter 23 : NUTRITION

505

The other hormones such as epinephrine, However, when 25 g protein is utilized by the cortisol, growth hormone and sex hormones body, 130 Cal of heat is liberated.The extra 30 increaseBMR. Cal is the SDA of protein.Likewise,consumption of 100 C al of fat resul tsi n 113 C al and 100 C a l 6. Environment: In cold climates,the BMR is of carbohydratein 105 Cal, when metabolized higher compared to warm climates. in the body. SDA for protein, fat and Z. Starvation : During the periods oJ carbo)>yltale a/e 32%t /3% and f%, starvation, the energy intake has an inverse respectively. Thus, proteins possess the highest relation with BMR, a decreaseup to 50% has SDA while carbohydrates have the lowest. been reported.This may be an adaptation by the SDA for mixed diet : For a mixed diet, the body. SDA is not an additive value of differentfoods 8. Fever : Fevercausesan increasein BMR. but it is much less. The presenceof fats and An elevation by more than 1O'/' in BMR is carbohydratesreducesthe SDA of proteins.Fats observedfor every 1'C rise in body temperature. are most efficient in reducingSDA of foodstuffs. For a regularlyconsumedmixed diet, the SDA is 9. Diseasestates : BMR is elevated in various around l0o/". infections, leukemias, polycythemia, cardiac Significance of SDA : For the utilization of failure, hypertensionetc. In Addison's disease (adrenal insufficiency), BMR is marginally foods by the body, certain amount of energy is consumedfrom the body stores.This is actually lowered. an expenditureby the body for the utilizationof 10. Racialvariations: The BMR of Eskimosis foodstuffs.lt is the highestfor proteins (30o/') and m uc h hig h e r.T h e BMR o f O ri e n ta lw o men l i vi ng lowest for carbohydrates(5%) and for a mixed in USA is about 10% lessthan the averageBMR diet around 10%. lt is, therefore,essentialthat of American women. an additional 10% caloriesshould be added to the total energy needs (of the body) towards Significance of BMR S D A . A nd the di et shoul d be pl anned, BMR is important to calculate the calorie accordingly. (SDA is quite comparable to the requirement of an individual and planning of handling charges levied by a bank for an diefs. Determinationof BMR is useful for the outstationcheque). function. In assessment of thyroid The higher SDA for protein indicatesthat it is hypothyroidism, BMR is lowered (by about not a good source of energy. Fat is the best -40o/o) while in hyperthyroidismit is elevated source of energy due to its lowering effect on (by about +70%). Starvationand certaindisease SDA. However, excessiveutilization of fat leads c ondit io n s a l s o i n fl u e n c e B M R ( descri bed to ketosis. above). Mechanism of SDA : The exact cause of SDA is not known. lt is generallybelievedthat SDA of SPECIFIC DYNAMIC ACTION foods is due to the energyrequiredfor digestion, The phenomenon of the extra heat absorption,transport,metabolismand storageof production by the body, over and above the foods in the body. cafculatedcaloric value, when a given food is Intravenousadministrationof amino acids or metabolized by the body, is known as specific the oral ingestion of proteins gives the same dynamic action (SDA). lt is also known as SDA. This shows that the SDA of proteinsis not calorigenic action or thermogenic action or due to their digestion and absorption. thermic action (effec0 of food. HepatectomyabolishesSDA, thereby indicating SDA for different foods : For a food that SDA is closelyconnectedwith the metabolic containing25 g of protein, the heat production functions of liver. The SDA of proteins is f r om t he c a l o ri c v a l u e i s 1 0 0 C a l (2 5 x 4 C al ). primarily to meet the energy requirementsfor

505

B IOC H E MIS TRY their physical activity and the requirementof enerSy.

Physical activity

(quietly) Sitting (quietly) Standing g/eating/reading Writin Cardriving Typing Household work(dishwashing) (slow) Wdking Sexual intercourse (slow) Cycling (moderate) Running Swimming Walking upstairs

Energy requirement (Cal164

25 30 30 60 75 80 130 140 150 500 600 800

Light work 3040% of BMR (teachers,office workers, doctors) Moderatework (housewives,students)

-

4O-5O"hof BMR

Heavy work 50-60% of BMR (agriculturallabourers,miners)

Veryheavywork

-

60-100%of BMR

(construction workers, pullers) rickshaw Energy requirements of man

As already stated, the three factors-basal metabolic rate, specific dynamic action and physical activity-determine the energy needed by the body. In an individual with light work, about 50% of the calories are spent towards BMR, about 30% for physical activity and about l0% to take care of the SDA.

The daily requirement of energy is rather deamination,synthesisof urea, biosynthesisof variable which depends on the BMR (in turn proteins,synthesisof triacylglycerol (from carbon dependson age,sex,body size etc.)and physical skeleton of amino acids). lt has been activity. As per some rough calculation,caloric demonstrated that certain amino acids requirementsof adults per day (Cal/day)are in (phenylalanine, glycine and alanine)increasethe the following ranges. SDA. lt is a common experience that Light work 2,200-2,500 consumptionof a protein rich diet makesus feel warm and comfortablein cold weather. This is Moderatework 2,500-2,900 due to the high SDA of proteins. Heavy work 2,900-3,500 The SDA of carbohydratesis attributed to the Very heavy work 3,500-4,000 energy expenditurefor the conversion of glucose to glycogen. As regards fat, the SDA may be due to its storage,mobilization and oxidation.

PHYSICAL ACTIVITY OF THE BODY The physical activity of the individual is highly variable. The amount of energy needed for this depends mainly on the duration and intensityof muscularactivity.The expenditureof energy for the various physical activities has been cafculated (Table 23.2\.

Dietary carbohydratesare the chief source of energy. They contribute to 60-70% of total caloric requirement of the body. lncidentally, carbohydraterich foods cost less.

Carbohydratesare the most abundantdietary constituents, despite the fact that they are not essential nutrients to the body. From the For the sake of convenience,the individuals nutritional point of view, carbohydrates are are grouped into four categorieswith regard to grouped into 2 categories.

507

Ghapter 23 : NUTBITION

6. Synthesis of pentoses : Pentoses (e.g. ribose)are the constituentsof severalcompounds in the body e.g. nucleic acids (DNA, RNA), (NAD+, FAD). These pentosesare coenzymes 2. Carbohydratesnot utilized (not di8ested) produced in carbohydrate metabolism. by the body-cellulose, hemicellulose,pectin, gums etc. 7. Synthesis of non-essential amino acids : The intermediatesof carbohydrate metabolism, Among the carbohydrates utilized by the mainly the keto acids (e.g. pyruvic acid), serve body, starch is the most abundant. The as precursorsfor the synthesisof non-essential consumption of starch has distinct advantages ami no aci ds. due to its bland taste, satiety value and slow

1. Carbohydrates utilized by the bodystarch, glycogen, sucrose/ lactose, glucose, fructose etc.

digestion and absorption. Sucrose (the table can be consumedto sugar),due to its sweetness, a limited extent. Excessive intake of sucrose causes dental caries, and an increase in plasma lipid levels is associatedwith many health complications.

8. Synthesis of fat : Excessconsumption of carbohydratesleadsto the formation of fat which is stored in the adipose tissue.

9. lmportance of non-digestible carbohydrates : These are the carbohydrates not utilized by the body. Yet, they are important since they improve bowel motility, prevent Functions of carbohydrates constipation, lower cholesterol absorption and 1. Major sourcesof energy: Carbohydrates improve glucose tolerance (details discussed are the principalsourceof energy,supplying later).

of the body. 60-80%of the caloricrequirements

2. Proteinsparingaction : Proteinsperforma specializedfunction of body building and growth.The wastefulexpenditureof proteinsto meet the energyneedsof the body should be cometo the rescueand curtailed.Carbohydrates proteins for caloric from being misused sparethe purpose.

Glycemic

index

Thereare variationsin the increaseand fall of blood glucose levels after the ingestion of different carbohydratecontaining foods. These quantitative differencesare assayedby glycemic index which measuresthe fime course of postprandial glucose concentrations from a graph. 3. Absoluterequirementby brain : The brain Glycemic index may be defined as the area and other parts of central nervoussystemare under the blood glucosecurve afterthe ingestion dependenton glucosefor energy.Prolonged of a food compared with the area under the hypoglycemiamay lead to irreversiblebrain blood glucose curve after taking the same amount of carbohydrate as glucose. lt is damage. expressedas percentage.

4. Requiredfor the oxidationof fat : Acetyl CoA is the product formed in fatty acid Area under the blood glucosecurve after oxidation.Foritsfurtheroxidationvia citricacid ingestionof test meal cycle,acetylCoA combineswith oxaloacetate, x 100 the latter is predominantlyderived from Glycemic irdo -.1

d

z

9l

s 9l

546

BIOCHEMISTF|Y

-35 Sequence 5' -TTGAC

Coding strand

Template a, strand

Startof transcription Fiq.25.5: Promoterregionsof DNAin prokaryotes. 3' Codingstrand 5' Templatestrand RNA----------------+s'..--..A U G C A U G G C A........3', Fig. 25.6 : nanscription-Complementary

TRANSGRIPTION

IN EUKARYOTES

RNA synthesisin eukaryotesis a much more complicated process than the transcription describedabove for prokaryotes.As such, all the details of eukaryotic transcription (particularly about termination)are not clearly known. The salientfeaturesof availableinformationare given hereunder. RNA po l y n te ra s e s The nuclei of eukaryotic cells possessthree distinct RNA polymerases(Fi9.25.7).

base pair relationship.

2. RNA polymerase ll synthesizes the precursorsfor mR N A s and smal l nucl earR N A s. 3. RNA polymerase lll participates in the formation of tRNAs and small ribosomal RNAs. Besidesthe three RNA polymerasesfound in the nucleus, there also exists a mitochondrial RNA polymerase in eukaryotes. The latter resembles prokaryotic RNA polymerase in structureand function. Promoter

sites

ln eukaryotes,a sequenceof DNA bases1. RNA polymeraseI is responsiblefor the which is almost identical to pribnow box of synthesisof precursorsfor the large ribosomal prokaryotes-is identified (Fig.25.A. This RNA s . sequence,known as Hognessbox (or TATA box),

s',

3',

3',

5'

I I +

I I RNApolymerase ll RNApolymerase II I

RNApolymerase I

+

e.

r\

r-

I

+

q'l tl

{ a-1 )t-r
2H2O+ 02

(c) Glutathione reductase

/

\*ooPH

+ H+

Fig. 34.2 : The antioxidant enzyme system (G- SHreduced glutathione; GS- gG-oxidized glutathione).

cr-Tocopherolcan directly act on oxyradicals (b) Metabolic antioxidants e.g. glutathione, (e.9.02, OH-, singletoxygen),and thus servesas c er ul o p l a s m i n , a l b u m i n , b i l i rubi n, an importantchain breakingantioxidant. transferrin, ferritin, uric acid Ascorbic acid (vitamin C) : lt is a vitamin that participatesin many normal metabolic reactions of the body. Ascorbicacid is an importantwaterThe antioxidant enzymes are truly the sol ubl eanti oxi danti n bi ol ogi calfl ui ds. V i tami n scavangersof free radicals.The major reactions C efficientlyscavangesfree radicals,and inhibits of these enzymes are depicted in Fig.34.2, some Iipid peroxidation. lt also promotes the regeneration of cr-tocopherol (from o,highlight sare g i v e n b e l o w . tocopheroxyl produced radical during Superoxidedismutase: lt convertssuperoxide scavengingof ROS). (O2) to hydrogen peroxide and 02 ffig3a.2A). Carotenoids : These are the natural This is the first line of defenseto protect cells compounds w i th l i pophi l i c properti es.A bout from the injurious effectsof superoxide. 500 different carotenoidshave been identified, Catalase: Hydrogen peroxide, produced by among them B-carotene is the most important. lt superoxidedismutase,is metabolisedby catalase can act as an antioxidant under low partial ffig3a.2R). pressureof C'-2.p-Caroteneusually functions in association with vitamins C and E. Lycopene,a Glutathione peroxidase: lt detoxifies H2O2 fat soluble pigment is a carotenoid. lt is (G-S H ) t o H2O , whi l e re d u c e d g l u ta th i o n e is responsible for (GS-SG). convertedto oxidized glutathione colour of certain fruits and The reduced glutathionecan be regeneratedby the vegetables (e.g. tomato). Lycopene possesses enzyme glutathione reductaseutilizing NADPH antioxidant propefty. Lutein and zeaxanthin are GigJa.2Q. The hexosemonophosphateshunt is also carotenoid pigmentsthat impart yellow or green colour to fruits and vegetables.These the major source of NADPH. pigmentscan also serve as antioxidants. F{utriesrt antis:cidaerts Selenium : lt is an essentialtrace element, Tocopherols (vitamin E) : Vitamin E is fat and is proved to be a significant antioxidant. soluble, and among the tocopherols, cr-toco- Selenium works with vitamin E in fighting free pherol is biologically the most active. lt is an radicals.lt is also requiredfor the function of an ant iox idantp re s e n t i n a l l c e l l u l a r me mb ranes, important antioxidant enzyme, namely and protects against lipid peroxidation. glutathione peroxidase. The amt$*,w&&nm9*ffieyms* 6:ywk*wn

'**il

B IOC H E MIS TR Y

660 c r - Li p o i ca c i d : l t i s v i ta mi n -l i k ecompound, produced in the body, besidesthe supply from plant an d a n i m a l s o u rc e s a. -L i p o i c a ci d pl ays a key role in recyclingother importantantioxidants such as ascorbic acid, cx,-tocopheroland glutathione. Besides the above, there are many other important nutrient antioxidants,some of them are listed below . Coen z y m eQ ro o f u b i q u i n o n efa m il y

E Vitamin (tocopherols)

vegetable oils Unprocessed (cottonseedoil,peanutoil, oil)wholegrains, sunflower legumes leafyvegetables,

Vitamin C

grapes) fruits(oranges, Citrus guava, green gooseberry (amla), (cabbage, spinach), vegetables melons cauliflower, green fruitsandvegeCanots, turnip, tables, spinach, apricots. Tomatoes, andtheirproducts pink papaya, (tomato sauce),

(ascorbic acid)

. Proanthocyanidins of grape seeds . Catechinsof green tea . Cur c u mi n o i d so f tu rm e ri c r Q uerc e ti no f o n i o n s ln the lable 34.2, some important nutrient antioxidants and their dietary sources are given. Consumption of a variety of nutrient antioxidants is important, since each antioxidant targets certain types of damaging free r adic al s .

.. .. .919Y9:.Y.?!91T.9191,....

greenleafy Eggyolk,fruits, Leutein andzeaxanthin vegetables, corn,greenpeas, Selenium

Seafoods,meats,organmeats, wholegrains

a-Lipoicacid

iedm;;i;iiv;;;t;;st oidiil i6d;i'#ni;6ei; 'eaii chicken.

Coenzyme Q,o

Metsbm&6* xmtisxida*x{s Glutathione : Reduced glutathione (CSH) play s a k e y ro l e i n th e b i o l o g i c a l a nti oxi dant enzyme system(SeeFi9.34.2O.CSH and H2O2 are the twin substrates for glutathione peroxidase.The reduced glutathione(GSH)gets regenerated from the oxidized glutathione (GS-SC) throughthe participationof glutathione reductaseand NADPH. lt is sugestedthat the ability to synthesize GSH decreasesas age

Dietarv Source

Antioxidant

tea redwine,green Onions, pomegranates walnuts, Berries, lemon. fruits(oranges), Citrus

EtgifiEtrleAl 1 ct-lHlcAt corucEFTs

Free radicals haue been implicated in the causation and progressof seueral diseasese.g. atherosclerosisand CHD, canceri respiratory diseases,aging. The estimation of serum malondialdehyde is ot'ten used fo qssessoxidatiue sfressond free radical damage to the body. The respiratory burst oJ macrophages, occompanied by the generation ot' ROS (HFz and HCIO), brings about bactericidal action, ond is beneliciol to the body. Dietary consumption ot' a variety of nutrient antioxidants (uitamins C and E, ft carotene, Iycopenes,Se, ftlipoic acid) is desirable sinceeoch antioxidont targets certain types of damaging free radicals.

Chapter 34: FREEHADICALSAND ANTIOXIDANTS

661

advances,and this has been implicatedin certain . Transferrin binds to iron and prevents ironcatalysed free radical formation. diseasese.g. cataract. There are many more metabolic antioxidants . Albumin can scavange the free radicals formed on its surface. of biological importance.A selectedfew of them are listed below Bilirubin protectsthe albumin bound free fatty . Uric acid, a powerful scavengerof singlet acids from peroxidation. oxygen (rOr) and OH- radicals. Haptoglobin binds to free hemoglobin and prevents the acceleration of lipid peroxi. Ceruloplasmin inhibits iron and copper dependentlipid peroxidation. dati on.

1. Free radicals are the moleculesor molecular speciescontaining one or more unpoired electrons with independent existence.e.g. Ot HzO2, OH-, 1O2.

2. ROS ore constantly formed during the normal cellular metabolism, (e.g. Iipid peroxidotion) and due to uariousenuironmental influences (e.g. ionizing radiotions).

3. Free radicals are highly reactiue and are capable of damaging almost all types of biomolecules(proteins, lipids, corbohydrates,nucleic acids),ond haue been implicated in the cousation of many diseasese.g. cardiouasculardiseoses,cance\ inflammatory diseases.

4. To mitigate the harmJul eft'ects oJ t'ree radicals, the aerobic cells houe developed antioxidont defense mechanisms-enzgmatic antioxidants (superoxide dismutase, catalase)and non-enzymotic antioxidants (glutathione, Se, a-tocopherol, ftcarotene).

Biochemistty

(ai r, normal temperature (despite cold and heat f nv i ro n me n t c o n s ti tu te sth e n o n -l i vi ng and Lwate r, l a n d ,e n e rg ye tc .)a s w e l l as the l i vi ng surroundi ngs)for opti mal physi ol ogi cal functi ons. ( biolo g i c aal n d s o c i a l )s y s te mss u rroundi ngman. bi ochemi cal E nv iro n m e n tabl i o c h e mi s tryp ri ma ri l ydeal sw i th t he me ta b o l i c (b i o c h e m i c a l ) re sponses and EXPOSURETO COLD adaptationsin man (or other organisms)due to Short-termexposureto cold causesshivering the environmentalfactors. (mainly due to skeletalmuscle)to produce extra A h e a l th y e n v i ro n me n t i s re qui red for a heat. Heat is generatedby the hydrolysisof ATP' healthylife which is however,not really possible or pr a c ti c a b l e .T h i s i s m a i n l y b e causeof the tw,+. g,,:t:t;j,i+'r e"t:94 r:li:*s;ie (c l i m a ti c ) and c h a nges at m o s p h e ri c Chronic exposure to cold results in nonp o l l u ti o n . env ir o n m e n ta l shi veri ng phase w hi ch i s characteri zed by Environmental biochemistry is a very vast severalmetabolic adaPtations. subject. The basic concepts regarding the Energy metabolism : Heat generation by a atmospheric changes and environmental processcalled chemical thermogenesisoccurs oollu ti o n o n h u ma n sa re d e a l t w i th here. in non-shiveringphase.The foodstuffsundergo oxidation to generateheat at the expenseof growth and other anabolic processes. Elevation in BMR, and increased intake oi foods are observed. T h e c l i m a ti c c h a n g e si n c l u d e col d, heat etc. The body makes every effort to maintain its

. Lipid metabolism : Storedfat (triacylglycerol) i n the adi poseti ssue i s mobi l i zed to supply

662

BIOCHEMISTRY ENVIHONMENTAL free fatty acidsfor oxidationand productionof energy. Brown edipose tissue, particularly in neonat al lif e , s i g n i fi c a n tl y c o n tri b u te s to t her m ogenes i s . . Hormonal changes : Thyroxine, a hormone closely associatedwith energy metabolism,is elevated.Further,corticosteroidsare increased on exoosureto cold.

EXPOSURETO HEAT There is a continuous generationof heat by the body due to th e o n g o i n g b i o c h e mi cal processes,referred to as metabolic heat. This heat has to be exchangedwith the environment to maintain a constant body temperature.On exposureto heat in surroundings,as happensin sLrmmer, the body is subjected to an uncomfortablesituation(sincetemperatureof the s ur r oundingsis m u c h h i g h e r th a n th a t o f th e body). However, heat is still lost from the body throughsweatingand evaporation.Normally,the body (thermoregulation)gets acclimatized to highert em per a tu rew i th i n 3 -5 d a y s .

t

663 The term pollutant refers to a substance w hi ch i ncreasesi n quanti ty due to human activity and adversely affects the environment (e.9.carbon monoxi de,sul fur di oxi de, l ead).A substancewhich is not present in nature but rel eased duri ng human acti vi ty i s the contaminant (e.g. methyl isocyanate, DDT, malathion).A contaminanthowever, is regarded as a pollutant when it exertsdetrimentaleffects. E nvi ronmentalpol l uti on may be consi deredi n different ways-industrial pollution; agricultural pollution;pollution due to gaseouswastes,liquid wastesand solid wastes.Environmentalpollution with reference to air, water and foodstuffs is briefly discussed. A IR P OLLU TION

The maj or componentsof ai r i ncl udeni trogen (78.1'/.), oxygen (20.93%) and carbon dioxide (0.03%),along with water vapour and suspended particles.The rapid growth of industriescoupled w i th changi ng l i festyl esof man (urbani zati on, smoking, use of motor vehicles etc.) largely contri buteto ai r pol l uti on.The maj or chemi cal constituentsof air pollution are sulfur dioxide, Heat stroke : lt is characterizedby the failure oxides of carbon (CO2 and CO), oxides of of the heat regulatorysystem(thermoregulation) nitrogen, hydrocarbons and particulates.The of the body. The manifestationsof heat stroke biochemical affects of air pollution are i n c lude high bo d y te mp e ra tu re ,c o n v u l s i o ns, described. partial (some times total) loss of consciousness. In extreme cases, heat stroke may cause $ulfur elFoxrs!+; irreversibledamage to brain. The treatmentfor Sulfur dioxide (SO2) is the most dangerous the heat stroke involves rapid cooling of the pollutantgas to man. lndustrialactivitiessuch as body. burni ng of coal and oi l emi t l arge quanti ti esof The milder form of heat stroke is referredto soz. as heat syncope. Although the body temperature S ul fur di oxi de pol l uti on pri mari l y affects i s not r ais edm u c h i n th i s c o n d i ti o n ,th e b l o o d respiratory system in man. lrritation of the pressure falls and the person may collapse respiratory tract and increasingairway resistance suddenly. Heat syncope is easily reversible. (breathingdifficulty) are observed. Lung tissue may get damaged due to acidic pH. Further, di pal mi tyl l eci thi n, the phosphol i pi dacti ng as the lung surfactant,gets affected. Continuous exposure to SO2 (> 1 ppm) for several days Environmentalpollution may be regardedas causesbronchi ti sand i n some i ndi vi dual sl ung the addition of extraneous(foreign)materialsto cancer.AtmosphericSO2when dissolvedin rain air, water or land which adverselyaffects the water becomesvery acidic (acid rain) damaging q ualit y of lif e. P o l l u ti o n ma y b e c a u s e d by soil, plantsand vegetables.Exposureof plantsto phy s ic al,c hem ic a lo r b i o l o g i c a lp ro c e s s e s . SO2 destroys leaves.

664 earbon

BIOCHEMISTFIY

rnonoxide

N i trogendi oxi de (i n the form of H N O3) along with SO2 (as H2SOa)contributes to acid rain.

Carbon monoxide (CO) is mostly produced by incomplete combustion of fuel or carbonH ydrocarbons c ont a i n i n g c o m p o u n d s .Au to mo bi l es,ai rcrafts, Many hydrocarbonspollutingthe environment r ail e n g i n e s a n d b u rn i n g o f c o al i n factori es affect human life. The aromatic hydrocarbons c ont ri b u teto C O o o l l u ti o n . causei rri tati onto i nj uri es. C a rb o nm o n o x i d ec o m b i n e sw i th hemogl obi n to form carboxyhemoglobin (Refer Chapter 10). P arti cul ates This causesa drastic reduction in the supply of The solid dust particles suspended in the 02 to tissues.At a CO concentrationaround l ppm , i mp a i rm e n t i n m e n ta l p e rf ormanceand atmosphereconstitute particulates.The sources visual perception take place. With a further of particulatesare grinding, spraying, erosion, increasein CO level, headache,dizzinessand smoki ngetc. los s o f c o n s c i o u s n e s so c c u r. D eath may be inevitablein personsexposedto above 750 ppm of CO. Car h o n

disxide

The particulateshave ill-affectson humans. Theseinclude interferencein respiratoryfunction (coughing,sneezing)and toxicity causedby the absorpti on parti cul atechemi cal s. Further , t he dust particles carry microorganismsand other infectiveagentsto spreaddiseases.

C a rb o n d i o x i d e (C Oz ), c o n s tituti ngonl y a fraction (0.03%)of the atmosphericgases,plays a s ig n i fi c a n ro t l e i n c o n tro l l i n gth e cl i mate.Thi s Ozsne layer is done by trapping the heat radiationfrom the Ozone is formed from atmosphericoxygen earth's surface.Without the presenceof CO2, duri ng hi gh energy radi ati ons of el ectr ical the earth would be as cold as moon! discharges.This ozone forms a layer above the (15-35 km). lt absorbs harmful Carbon dioxide is often referred to as earth's surface radiations of sun which would ultraviolet greenhouse Bas. The term greenhouse and mutations, cause skin diseases effect refers to an elevation in CO2 near earth's otherwise of earth. increasing the temperature surface that traps sunlight and increases besides atmospheric temperature. Deforestation, bur n i n g o f c o a l , o i l s e tc ., e l e v a teatmospheri c CO2 resulting in greenhouseeffect. Hence the global propagandafor increased plantation of trees!

In recentyears,a decreasein the ozone layer i s observeddue to chemi calpol l uti on i n the air . Nitrogen oxides (releasedfrom enginesof aeroplanes)and chlorofluorocarbons(usedin refrigerators and air conditioners)deplete the ozone laver.

F o rtu n a te l yma , rg i n avl a ri a ti o n si n atmospheri c CO2 are tolerated by the cells. The body gets r:dATElt POLLUTIOH adapted to prolonged exposure to higher concentrations of CO2 (evenupto 1%) with minor Water is the most predominantconstituentof alterationsin electrolytebalance. living matter. The very existence of life is uni magi nabl ew i thout w ater. fltlEtrogen dioxide As such, pure water does not exist in nature. N i tro g e nd i o x i d e(N O 2 )l i k e c a rb onmonoxi de The avai l abl e w ater contai ns di ssol vedgases, (CO), combines with hemoglobin and reduces mi neral s and some suspended particles. the supply of 02 to the tissues.NO2 is more Pollution of water occurs due to waste disposal harmfulto human healththan CO. lt is fortunate from i ndustri es,agri cul tureand muni ci palit ies. that the atmosphericconcentrationof NO2 is The pol l utants may be organi c, i norganic, relativelvlower. sediments,radioactive,thermal etc., in nature.

." .JIi . ENVIFIONMENTALBIOCHEMISTHY

66s

i r?G4$Sdf P0&I'{,t7-ArVfS

rodenticides.Basedon their structure,pesticides are classifiedas follows. The organicpollutantsinclude agentscarrying (a) Chlorinated hydrocarbons : e.g. aldrin, water borne diseases,oxygen demandingwastes di el dri n,endri n,di chl orodi phenyl tri chl oroand or ganicc h e mi c a l s . ethane(D D T). ! ' f ; r ler . bor n e d i s e a s e a g e n ts (b) Organophosphates : e.g. malathion, Severalpathogenicorganismsfind their entry di azi non. into water and cause diseases.The water borne (c) Carbamatese.g. baygon, carbaryl (sevin) disease include typhoid, paratyphoid, cholera, (d) Chforophenoxy e.g. 2,4-dichlorophenoxy am oebias is g , i a rd i a s i sa n d i n fe c ti o u sh e p ati ti s. acetic acid. Thesediseasescan be preventedby disinfection techniquesemployed for the treatmentof water. The useof pesticideshas helped in controlling certain diseases (malaria, typhus), besides 'l}'clr_err du'rmanding waste$ boosting food production. However, pesticides Sewage, and wastes from industries and pollute water and cause several health agricultureprovide good nutrientsfor algae. As compl i cati ons to humans, besi des damagi ng the algae grow utilizing the wastes, oxygen acquati cl i fe. depletion occurs. This phenomenon of water Dichloro-diphenyltrichloroethane(DDT) is a deoxygenation is technically referred to as widely used pesticide to control cotton and eutrophication. As a consequence of peanut pests, besides malaria. However, eut r ophic at i o nfi, s h a n d o th e r a c q u a ti ca ni mal s conti nuoususeof D D T l eads i ts to accumul ati on die (due to lack of O2), causingfoul smell. in foods causing ill effects (hence banned in countri esl i ke U S A ). some #r gar *i* c h e m Ec a l s DDT, being fat soluble, accumulates in the T he or ga n i c c h e m i c a l p o l l u ta n ts o f w ater adipose fissue and is not excreted. Thus, its include pesticides and several synthetic concentration in the body goes on increasing. compounds (detergents, paints, plastics, DDT causes nervous irritability,muscletwitching pharmaceuticals,food additivesetc.) and convul si ons. F es t ic ides A l dri n and di al dri n are al so fat sol ubl e and Pesticides is a broad term used for their effectson humansare comoarablewith that herbicides, fungicides insecticides, and of DDT.

BIOMEDICAL/CLINICALCONGEPTS

The body makeseuergeflort to malntqin its normal temperature,despite cold and heat surroundings,t'or optimal physiologicaland biochemical functions. Failure of heat regulatory system (thermoregulation) Ieads to heot stroke chorocterized by high body temperature, conuulsions etc. Sulfur dioxide (SOz)is the most dangerouslndustriol pollutant gas to man. lt primarilg affects

the respirotory

system, and

mov

result

in bronchifis,

and euen lung

cancen

Corbon monoxide (CO) combines uifh hemoglobin to form carboxyHb. This reduces 02 supply to fissues. rs Pollution ol water with pathogenic organisms couses many diseases e.g. typhoid, cholera, omoebiasis. r* Lead toxicity at'fectscentrol neruous system-learning disabilities, mental retqrdation etc.

]

666

Organophosphates and carbamates are powerful neurotoxic agents. They prevent the t r ans m i s s i o n o f n e rv e i m p u l s e b y i n hi bi ti ngthe enzyme cholinesterase.

BIOCHEMISTFIY

consumi ngfi sh contai ni ng methyl mercury, a s i ndustri alpol l utant). i -3 4 4

: r: -

rrr

r!

rruoFcAntc pof[urAryrs

The outbreak of cadmium toxicity was reported in Japan in the form o( itai itai or ouch Hea v y me ta l s (l e a d , me rc u ry , cadmi um, disease. Cadmium poisoning causes fragile alum in i u m ,a rs e n i ce tc .)a re th e mo st dangerous bones, anemia, bone marrow disorders and am ong th e i n o rg a n i cp o l l u ta n ts . ki dney damage. B i ochemi cal l y, cadmi um replaceszinc and adversely influencesseveral ilean€j metabolic reactions. Lead is the most common inorganicpollutant found in water, air, foods and soils.The sources of lead p o l l u ti o ni n c l u d ep e tro l ,g a s o l i ne,pai nts, cigarettes,news papers, lead pipes and xerox copies. The plasma concentrationof > 25 1tg/dl in adu l ts a n d > 1 0 p g l d l i n c h i l d ren resul tsi n toxic manifestations. The principal targetof lead toxicity is central nervous system. In the growing children, Pb c aus esl e a rn i n gd i s a b i l i ti e sb, e h a v i o uralchanges (hyperexcitability)and mental retardation. In adult s, c o n fu s i o n , i rri ta b i l i ty ,a b d o mi nal col i c and severe anemia are associatedwith lead toxicity. Lead inhibits several enzymes, particularly, 6-aminolevulinate (ALA) synthase, ALA dehydrataseand ferrochelatase of heme synthesis (Refer Chapter 10 also). This results in severe anemia.There has been an increasingawareness worldover on the toxic manifestationsof lead. This has lead to the supply of unleaded petrol in most countries. flfler*rlryt M erc u ry i s a c o m m o n i n d u s tri al (pl asti c, paints,electricalapparatus,fungicides)pollutant. Acute mercuric poisoning causes gastritis, v om it in g a n d p u l mo n a ry e d e m a . C hroni c manifestations of Hg include emotionalchanges, loss of memory and other neuropsychiatric dis t ur b a n c e sl n. a d d i ti o n ,d e p o s i ti o nof mercuri c salts mav cause renal failure. O r g a n i c me rc u ri c p o i s o n i n g i s commonl y referred to as rninamata disease (as it first oc c ur re d i n M i n a ma ta , J a p a n i n 1953-60 by

+!(irYI,ftr:;;,ir;* The sourcesof al umi ni um i ncl ude cooki ng vessels,building materials,food additives and cosmetics.Aluminium toxicity is associatedwith Alzheimer'sdisease,anemia and osteomalacia. r'rirtrd.

fi : i'

A rseni c, commonl y found i n many insecticidesand fungicides,is toxic to the body. Arsenic binds with-SH groups of several enzymesand i nhi bi tsbi ochemi calreacti onse. g. pyruvatedehydrogenase. Further,arseniccauses coagulation of proteins and blockage of ATP generation(functionsas an uncoupler). N OIS E P OI.LU TIGN The unw anted sound i s noi se, w hi ch i s a major urban environmentalpollutant. Man can tol eratenoi se upto 100 deci bel s(speaki nB - 60 decibels;telephonebell 70 decibels;motor cycle 110 deci bel s; rockets 170 deci bel s).A noi se above 150 deci bel si s uncomfortabl e. The affects of noise pollution include headache,increasedblood pressure,irritability, neuromuscul artensi on, confusi on, di sturbed vi si on and di gesti on,depressi onand l oss o f heari ng. RADIGAGTIVE

POTLI'TilOI$

The pollution due to radioactivesubstancesis the most dangerousto human life. The health hazards of radi oacti ve ool l uti on i ncl ude gene mutatrbns, cancer, destruction of living cells etc.

I:-It::.!" i;'i , ENVIRONMENTALBIOCHEMISTRY

TOXiE COMPOUNDS lid FGOE}STUFFS

667 Anti-vitamins : Avidin of raw egg is a good example of anti-vitaminof biotin.

The foodstuffsconsumedby humans contain g r uf f t* T.t' rr F;;"':gl*rflf *ur a irit r; erf ri*i.,ol.d severaltoxic compounds which may be either The foodstuffsmay get polluted with several normally presentor enter foodstuffsduring the toxi c chemi cal s w hi ch mi ght occur duri ng course of cultivation, processingor storage. cultivation, processingor storage. :',.1 ril t':;-'lti! {i:: 4i;,ri::: 1,r",f t:,,,;,tie;*qgffq-:

Neurotoxins : Kesari dal (Lathyrussativus) is pulse grown in some partsof Madhya Pradesh, a Bihar and Uttar Prodesh.Excessive consumption of kesari dal causes paralysis of lower limbs referred to as lathyrism. This is due to a p-oxalylaminoalanine neurotoxin namely (BOAA). BOAA damagesupper motor neurons, and inhibits the enzyme lysyl oxidase (reduces c ollagen c ro s s -l i n k i n g ).C o o k i n g o f k e s ari dal 2-3 times and removal of the supernatantwater will elim ina teth e to x i n .

Cultivation : Pesticidesand other unnatural chemicals used during cultivation do find an entry into the foodstuffs.lt is fortunate that most of these chemicals can be removed by peeling the outer layersof vegetablesand fruits, besides repeatedwashings. Processing: Defectsin freezing,and packing provide a suitableenvironmentfor the growth of severalorganismswhich releasetoxic products e.g. milk contaminationby Salmonella.

Several food additives are in use for preservationand enchancingflavour. Not all of Proteaseinhibitors : Certain legumes (soya them are safee,g. ani l i nedyes usedas col ouri ng bean, peanut) contain inhibitors of protease agents are carcinogenic; sweetening agent enzymesparticularlytrypsin. Normally, protease cyclamate may cause bladder cancer. inhibitors are destroyedby cooking. However, Storage : Contamination of stored foods partial cooking does not totally destroythem. In occurs mostly due to fungal infections. s uc h a c as e , p ro te a s e i n h i b i to rs c a n inhi bi t Aflatoxins are produced by Aspergillus favus digestionand proteins. when ground nuts or coconuts are stored in Goitrogens : These compounds prevent moist conditions.Aflatoxinsare heoatotoxicano upt ak eand u ti l i z a ti o no f i o d i n e b y th y ro i dgl and. carcinogenic. Goitrogens are found in cabbage and turnips (thioglycosides),mustard and rape seed oils ( t hioc y ana te s ),g ro u n d n u ts a n d a l m onds ( poly phenoi cl g l y c o s i d e s ). Biogenic amines : Bananas and cheese c ont ain bio g e n i c a m i n e s n a m e l y h i s t ami ne, tryptamine,tyramine serotoninand epinephrine. In normal metabolism, they are degraded by m onoam ine o x i d a s e (M AO). H o w e v er, i n personstaking MAO-inhibitors, the foodstuffs with amines may cause hypertension.

The group of chemi cal sthat causecancer i n man and animals are collectively referredto as carcinogens(Refer Chapter 3V. Environmental pol l uti on i s undoubtedl y associ ated w i th increasedrisk of cancer.The topic 'cance/ may be considered as a part of environmental bi ochemi stryfor l earni ngpurpose.

568

B IOC H E MIS TR Y

1. Enuironmentol biochemistry deals with the biochemical responsesand odaptations in man (ond other organisms)due to enuironmental foctors. 2. The atmospheric (climottc) changes like cold and heat inJluence the body. Seuerol metabolic adaptotions occur to ouercome the aduerseoffects. 3. The major chemical constituentsof oir pollution include SO2, CO, Ca2 ond oxides of nitrogen. Among these, sulfur dioxide is the most dongerous.

4. Water pollution occurs mainly due to r.oostedisposal lrom industries,agriculture and municipalities. The pollutants may be organic (pathogenic organisms, pesticides),or inorganic (leod, mercurg).

5. The Joodstuffsconsumed by humans may contain seueraltoxic compounds. These may be normally present (e.g. BOAA causing lothyrtsm)or enter the loodstulls during the course of cultiuation (e.9. pesticides),or storage (e.g. aflatoxins).

andDiabetes Mellitus

iabetes mellitus is the third leading cause ll lJ of death (after heart diseaseand cancer) in many developedcountries.lt affectsabout 2 to 3% of the generalpopulation.The complications of diabetes affect the eye, kidney and nervous system.Diabetesis a major cause of blindness, renal failure, amputation, heart attacks and stroke.(The term diabetes,whenever used, refers to diabetes mellitus. lt should, however, be noted that diabetes insipidus is another disorder characterized by large volumes of urine excretion due to antidiuretic hormone deficiency).

An important feature of diabetes is that the body cells are starved of glucose despite its very high concentrationaround i.e. scarcityin plenty. For a comprehensiveunderstandingof diabetes,the relevanthormones,namely insulin and glucagon, homeostasisof blood glucose, besidesthe biochemicalaspectsof diabetes,are di scussedi n thi s chaoter.

lnsufin is a polypeptide hormone produced by the B-cells of islets of Langerhans of Diabet es me l l i tu s i s a c l i n i c a l c o ndi ti on pancreas. lt has profound influence on the characterized by increasedblood glucose level metabolism of carbohydrate,fat and protein. (hyperglycemia)due to insufficient or inefficient Insul i ni s consi deredas anabol i chormone,as i t synthesis of glycogen, ( inc om pet e n t)i n s u l i n .l n o th e r w o rd s , i n sul i n i s promotes the triacylglycerols proteins. and This hormone has produced either not in sufficient quantity or inefficientin its action on the targettissues.As a been implicated in the developmentof diabetes consequence,the blood glucoselevel is elevated mel l i tus. whic h s pillso v e r i n to u ri n e i n d i a b e te smel l i tus Insul i noccupi esa speci alpl ace i n the hi story (Creek : diabetes-a siphon or running through; of bi ochemi stryas w el l as medi ci ne.Insul i nw as mellitus-sweet). the first hormone to be isolated, purified anq

669

670

BIOCHEMISTF|Y

synthesized;first hormone to be sequenced;first hormone to be produced by recombinantDNA technology.

Structure of insulin contains Humaninsulin(mol.wt. 5,7341 5l amino acids, arranged in two polypeptide c hains .T h e c h a i n A h a s 2 1 a mi n o a c i dsw hi l e B has 30 amino acids. Both are held together by two interchaindisulfide bridges,connectingA7 to 87 and A2s to 819. In addition, there is an intrachaindisulfide link in chain A betweenthe am ino a c i d s 6 a n d 1 1 .

Preproinsulin

Biosynthesis of insulin of theislets Insulinis produced by thep-cells The genefor this of Langerhans of pancreas. protein synthesisis locatedon chromosome1 1. The synthesisof insulin involvestwo precursors, nam ely p re p ro i n s u l i nw i th 1 0 8 a m i no aci ds ( m ol. wt. 1 1 ,5 0 0 )a n d p ro i n s u l i nw i th 86 ami no acids (mol. wt. 9,000). They are sequentially degraded (Fig.36.l) to form the active hormone insulin and a connecting peptide (C-peptide). Insulinand C-peptideare producedin equimolar concentration. C-peptide has no biological activity, however its estimation in the plasma serves as a useful index for the endogenous pr oduc ti o no f i n s u l i n . I n t h e p -c e l l s , i n s u l i n (a n d a l s o p roi nsul i n) combines with zinc to form complexes.In this form, insulin is stored in the granules of the cytosol which is releasedin responseto various stimuli (discussedbelow) by exocytosis.

Regulation of insulin secretion About40-50unitsof insulinis secreted daily Thenormalinsulinconcenby humanpancreas. trationin plasmais 20-30pUlml. The important factorsthat influencethe releaseof insulinfrom the p-cellsof pancreas arediscussed hereunder.

B-chain Ineulin C-peptide

meal).A risein bloodglucoselevelis a signal for insulinsecretion. 1. Factors stimulating insulin secretion : These include glucose, amino acids and . Amino acids inducethe secretionof insulin. gastrointestinal hormones. the ingestion observed.after Thisis particularly mealthat causestransientrise of protein-rich . Glucoseis the most importantstimulusfor Among in plasmaamino acid concentration. insulin release.The effect is more predothe amino acids, arginineand leucine are minantwhen glucoseis administered orally (eitherdirect or througha carbohydrate-rich potentstimulators of insulinrelease.

C:'apter sE : INSULIN,GLUCOSEHOMEOSTASIS, AND DIABETESMELLITUS

*letaholism

Net effect

Crbotrydrate metabolism ' Glycolysis

677

Effect on important enzyme(s)

Increased

Z Gluconeogenesis

Decreased

3. Glycogenesis 4. Glycogenolysis 5. HMP shunt

Increased Decreased Increased

Glucokinase 1 Phosphofructokinase t Pyruvate kinase t carboxylase J Srruvate pyruvate Phosphoenol carboxykinase J Glucose o-phosphatase J Glycogen synthetase t phosphorylase Glycogen J Glucose 6-phosphate dehydrogenase'f

Increased Decreased Decreased

Acetyl CoAcarboxylase t Hormone sensitive lioase J HMGCoAsynthetase J

Increased Decreased

RNApolymerase t Transaminases J Deaminases J

Li*l metabolism 6. Lipogenesis 7. Lipolysis 8. Ketogenesis Protein metabolism 9. Protein synthesis 10. Protein degradation

. Gastrointestinal hormones (secretin, gastrin, pancreozymin) enhance the secretion of insulin. The GIT hormonesare releasedafter the ingestionof food. 2. Factors inhibiting insulin secretion : Epinephrine is the most potent inhibitor of insulin release. In emergency situations like stress, extreme exerciseand trauma, the nervous system stimulates adrenal medulla to release epinephrine. Epinephrine suppresses insulin release and promotes energy metabolism by mobilizingenergy-yieldingcompounds-glucose from liver and fatty acids from adiposetissue. Degradation

of insulin

F#etabolic

cffects

of insulin

lnsulin plays a key role in the regulationof carbohydrate, lipid and protein metabolisms (Table 35.1). lnsulin exerts anabolic and anticatabolicinfluenceson the body metabolism. 1 . Effectson carbohydrate metabolism : In a normal individual, about half of the ingested glucose is utilized to meet the energy demands of the body (mainly through glycolysis).The other half is converted to fat (- 40%) and glycogen (- 10%). This relation is severely i mpai redi n i nsul i ndefi ci ency.Insul i ni nfl uences glucosemetabolismin many ways. The net effect is that insulin lowers blood glucose level (hypoglycemic promoting effect) by its utilization and storage and by inhibiting its production.

ln the plasma,insulin has a normal halfJifeof 4-S minutes. This short half-life permits rapid metabolic changes in accordance to the alt er at ion si n th e c i rc u l a ti n g l e v e l s o f i nsul i n. . Effect on glucose uptake by tissues z Insulin is This is advantageous for the therapeutic required for the uptake o{ glucose by muscle (skeletal, cardiac and smooth), adipose tissue, purposes.A proteaseenzymef namely insulinase (mainly found in liver and kidney), degrades leukocytesand mammary glands.Surprisingly, ins ufin. about 80% of glucose uptake in the body is

672

B IOC H E MIS TR Y

utilization of acetyl CoA for oxidation (Krebs not d e p e n d e n to n i n s u l i n .T i s s u e si nto w hi ch glucosecan freely enter include brain, kidney, cycle) and lipogenesis. Therefore, the availability of acetyl CoA for ketogenesis,in erythrocytes,retina, nerve, blood vesselsand the normal circumstances,is very low intestinal mucosa. As regards liver, glucose entry into hepatocytes does not require 3. Effectson protein metabolism : Insulin is ins u l i n . H o w e v e r, i n s u l i n s ti mu l a tesgl ucose an anabolic hormone. lt stimulatesthe entry of ut ili z a ti o n i n l i v e r a n d , th u s , i ndi rectl y amino acids into the cells, enhances protein promotes its uptake. synthesisand reducesprotein degradation. . Effect on glucoseutilization : Insulin increases Besides the metabolic effects described glycolysis in muscle and liver. The activation above, insulin promotes cell growth and as well as the quantities of certain key repl i cati on.Thi s i s medi ated through certa in enz y m e s o f g l y c o l y s i s ,n a me l y g l ucoki nase factors such as epidermal growth factor (EGF), (not hexokinase) phosphofructokinaseand platelet derived growth factor (PDGF) and py r u v a te k i n a s e a re i n c re a s e d b y i nsul i n. prostagland ins. Clycogen production is enhanced by insulin by increasing the activity of glycogen Mechani sm sf aeti on of i nsul i n synthetase. lt is now recognized that insulin binds to . Effect on glucose production : Insulin plasma membrane receptorspresenton specific decreasesgluconeogenesisby suppressingthe target tissues,such as muscle and adipose. the enzymes pyruvate carboxylase,phosphoenol in a series of reactions ultimately This results pyruvate carboxykinase and glucose 6to the biological action. Three distinct leading pho s p h a ta s e .In s u l i n a l s o i n h i b i ts gl ycoi nsul i n acti on are know n. On e of mechani sms genolysisby inactivatingthe enzymeglycogen induction of transmembrane with the concerned phosphorylase. (si gnal transducti on),second w i th the si gnal s 2. Effectson lipid metabolism : The net effect glucosetransportacrossthe membraneand the of ins u l i n o n l i p i d m e ta b o l i s mi s to reducethe third with induction of enzyme synthesis. release of fatty acids from the stored fat and 1. Insulin receptor mediated signal trans' decrease the production of ketone bodies. duction Among the tissues,adipose tissue is the most sensitiveto the action of insulin. Insulin receptor : lt is a tetramerconsistingof . Effect on lipogenesis : Insulin favours the synthesis of triacylglycerols from glucose by providing more glycerol 3-phosphate (from gly c o l y s i s )a n d N AD PH (fro m HMP shunt). Insulin increasesthe activity of acetyl CoA carboxylase, a key enzyme in fatty acid synthesis. . E f f e c t o n l i p o l y s i s : l n s u l i n d e creasesthe activity of hormone-sensitivelipase and thus reduces the releaseof fany acids from stored fat in adiposetissue.The mobilizationof fatty ac id s fro m l i v e r i s a l s o d e c re a s e dbv i nsul i n. I n t h i s w a y , i n s u l i n k e e p sth e c i rc ul ati ngfree fatty acids under a constantcheck.

4 subunits of two types and is designatedas a2p2. The subunitsare in the glycosylatedform. They are held togetherby disulfidelinkages.The . w t. 135,000)i s extracel l ul ar and cx-subuni(mol t i t contai ns i nsul i n bi ndi ng si te. The p-subunit (mol. wt. 95,000) is a transmembraneprotein w hi ch i s acti vatedby i nsul i n.The cytopl asmic domain of p-subunithas tyrosinekinaseactivity. The insulin receptoris synthesizedas a single polypeptide and cleaved to a and p subunits The i nsul i n recep t or w hi ch are then assembl ed. has a hal f-l i feof 6-12 hours. There are abo ut 20,000 receptorsper cell in mammals.

Signaltransduction : As the hormone insulin . Effect on ketogenesis : Insulin reduces binds to the receptor,a conformationalchange ketogenesisby decreasingthe activity of HMG of i nsul i n receptor . i s i nduced i n the cr-subuni ts CoA synthetase.Further,insulin promotesthe Thi s resul tsi n the generati onof si gnal sw hi ch

Ghapter 36 : INSULIN,GLUCOSEHOMEOSTASIS, AND DIABETESMELLTTUS

acti vi ty of i ntracel l ul ar p-subuni t of i nsul i n receptor.This causesthe autophosphorylation of tyrosineresidueson B-subunit.lt is believedthat receptor tyrosine kinase also phosphorylates insulin receptor substrate(lRS). The phosphorylated lRS, in turn, promotesactivationof other protein kinasesand phosphatases, finally leading to biological action (Fig.36.2).

It,i-

Cytoplasm

Cytoplasm

673

\,rr_-u, ,l

2. Insulin-mediated glucose transport : The bi ndi ngof i nsul i nto i nsul i nreceptorssi gnal sthe transl ocati on of vesi cl es contai ni ng gl ucose transporters from intracellular pool to the plasma membrane. The vesicles fuse with rne membrane recruiting the glucose transporters. The glucosetransportersare responsiblefor the i nsul i n-medi ated uptakeof gl ucoseby the cel l s. As the insulin level falls,the glucosetransporters move away from the membrane to the intracellular pool for storage and recycle (Fig.s6.3).

3. Insulin mediated enzyme synthesis : Insulin promotesthe synthesisof enzymessuch as gl ucoki nase, phosphofructoki nase and pyruvate kinase. This is brought about by are transducedto p-subunits.The net effect is i ncreased transcri pti on (mR N A synthesi s), that insulinbindingactivatestyrosinekinase followed by translation(protein synthesis). Flg. 36.2 : lnsulin receptormediatedsignal tnnsduction (IRS-l nsulin receptorsubstrate).

Flg. 36.3: lnsulinmediatedglucosetrunsport.

.-t

674

Clucagon, secretedby a-cells of the pancreas, opposesthe actionsof insulin.lt is a polypeptide hormonecomposedof 29 amino acids (mol. wt. 3, 500) i n a s i n g l e c h a i n . C l u c a g o n i s actual l y synthesizedas proglucagon (mol. wt. 9,000) which on sequentialdegradationreleasesactive glucagon. Unlike insulin, the amino acid sequence of glucagon is the same in all mammalian species(so far studied).Clucagon has a short half-life in plasma i.e. about 5 minutes. f, ++qu[eti tr ! af q! Nle;tg€'re li€rcrr!.fidi*l

B IOC H E MIS TR Y

Glucose is carbohydrate curuency of the body. A n adul t human body contai nsabout 1B g free glucose. This amount is just sufficient to meet the basalenergy requirementsof the body for one hour! The liver has about 100 g stored glycogen. Besidesthis, it is capable of produci ngabout 125-150mg gl ucose/mi nute or 180-220 {24 hrs.

Expressionof glucose concentration : In most developed countries,plasma glucose(insteadof The secretion of glucagon is stimulated by glucose) blood is estimatedand expressedas Sl Iow blood glucose concentration, amino acids (mmol/l). units This is not however so, in protein derived from dietary and low levels of developing countries for practical reasons. lt epinep h ri n e . In c re a s e d b l o o d g l u cose l evel may plasma concentration be noted that the markedly inhibits glucagon secretion. of glucose is slightly higher (about 15%) than blood glucose. Further, a glucose ftiletahe;iit: ei'fe:lts oi qgir.l*;:r';nn concentrationof 180 mg/dl (plasma or blood) Clucagon influencescarbohydrate,lipid and corresponds to 10 mmol/|. In this book, protein metabolisms.In general, the effects of expressionof blood glucose as mg/dl is more this hormone oppose that of insulin. frequently used. 1. Effects on carbohydrate metabolism : A heal thyi ndi vi duali s capabl eof mai ntai ni n g Glucagon is the most potent hormone that the blood glucoseconcentrationwithin a narrow enhancesthe blood glucoselevel(hyperglycemic). range. The fasting blood glucose level in a postPrimarily, glucagon acts on liver to cause absorptive state is 70-100 mddl (plasmaglucose increasedsynthesisof glucose(gluconeogenesis) 80-120 me/dl ). Fol l ow i ng the i ngesti on of a and enhanced degradation of glycogen carbohydrate meal, blood glucose may rise to (glycogenolysis). The actions of glucagon are '120-'l40mg/dl. The fastingblood glucosevalue mediatedthrough cyclic AMP (Chapter t3). i s comparati vel y l ow er i n rumi nant ani mals 2. Effects on lipid metabolism : Clucagon (sheep 30-aOmg/dl; cattle 50-60 mg/dl), while it promotes fatty acid oxidation resulting in is higher in birds (250-300 mg,/dl). energy production and ketone body synthesis (ketogenesis). The term hyperglycemia refers to an increase in the blood glucose above the normal level. 3. Effectson protein metabolism : Glucagon Hypoglycemia represents a decreased blood increasesthe amino acid uptake by liver which, glucose concentration.Excretionof glucose in in turn, promotes gluconeogenesis. Thus, urine is known as glycosuria. The concentration glucagon lowers plasma amino acids. of blood glucoseis dependenton the quantityof glucosethat entersthe circulation from various Meehanisrn of ;.icti*"rri #[ g{e.E$fil{g}f, sources (dietary carbohydrates,glycogenolysis, Clucagon binds to the specific receptorson gluconeogenesisetc.) and the amount that is the plasma membrane and acts through the utilized for different metabolic purposes mediationof cyclic AMP, the secondmessenger. (glycolysis,glycogenesis,fat synthesisetc.) as The details are given elsewhere(Chapter l9). illustratedin Fig.35.4.

Ghapter 35 : INSULIN,GLUCOSEHOMEOSTASIS, AND DIABETESMELLTTUS Dietarycarbohydrates (starch,sucrose,glucose) \ \ Digestionandabsorption \ Glycogenolysis \ in muscle Glucosein liver

Horrrlonal requlatior,'

l

+ BI-OODGLUCOSE Fasting70-100mg/dl

675

Glycolysis and TCAcycle Glucose-+ COr,HrO Glycogenesis in liverandkidney Synthesisof other monosaccharides and aminosugars

Post-prandial 120-140mg/dl

Glycogenolysis in liver

I + Excretedinto urine(>'180mg/dl bloodglucose)

HMPshuntfor pentoses andNADPH Synthesisof fat i J t;l ,z ai i J n , j f DioL-r(lgi-rcr)srl

Sourcesof blood glucose

Sources of bloo7.0

(< 1 1 0 )

(126)

200)

(t

J-

Activation (cytoplasmic kinases)

I DNAsynthesis

andcellmultiplication Fig. 37.3 : Model for the mechanism of action of ras P^ protein (GRF-Guanine nucleotide releasing factor; GAP-GTPase activating proteins).

Non-receptor tyrosine kinases : These proteins are found on the interior of the inner plasma membrane. They phosphorylate the cellular targetproteins(involvedin cell division) in responseto externalgrowth stimuli. Mutations in the protooncogenes(e.9. abl) encoding nonreceptor tyrosine kinases increase the kinase activity and, in turn, phosphorylationof target protei nscausi ngunl i mi tedcel l mul ti pl i cati on. Antioncogcnes A special category of genes, namely cancer suppressor genes (e.g. pFs gene) ot, more commonly, antioncogenes,have been identified. The products of these genes apply breaks and regulate cell proliferation. The loss of these

Chapter 37 : CANCEFI

Oncogenic v,ruses

Environmental tactors(physical and chemical) I

\

J ONCOGENEACTIVATION

.)/ .//

(+ ./l

691

CARCJNOGENESIS

Fig. 37.4 : A simplified hypothesis for the development of cancer.

The biochemical indicators employed to detect the presenceof cancersare collectively referred to as tumor markers. These are the abnormally produced molecules ol lamor ce,//s such as surface antigens, cytoplasmic protdns. enzymes and hormones. Tumor markers can be measuredin serum (or plasma). In theory, the tumor markers must ideally be useful for screeningthe population to detect cancers. In practice,however,this has not been totallv true. As such,the tumor markerssupportthe diagnosis of cancers,besidesbeing useful for monitoring the response to therapy and for the earlv detectionof recurrence.

suppressorgenes removes the growth control of cells and is believed to be a key factor in the A host of tumor markershave been described development of several tumors, e.g. retinoblastoma, one type of breast cancer, and the list is evergrowing.However,only a few of them have proved to be clinically useful. A carcinoma of lung, Wilms' kidney tumor. selectedlist of tumor markersand the associated With the rapid advancesin the field of genetic cancers are given in Table 57.4. engineering, introducing antioncogenes to a A couple of the most commonly used tumor normal chromosome to correct the altered growth rate of cells may soon become a reality. markersare discussedhereunder. 1. Carcinoembryonicantigen (CEA)z This is Genes that regulate apoptosls a complex glycoprotein,normally produced by A new category of genes that regulate the embryonic tissueof liver, gut and pancreas. programmed cell death (apoptosis) have been The presenceof CEA in serum is detected in discovered.These genes are also important in severalcancers(colon,pancreas,stomach,lung). In about 67'/. of the patients with colorectal the developmentof tumors. cancert CEA can be identified. Unfortunatery, The gene, namely bcl-2, causes B-cell serum CEA is also detected in several other lymphoma by preventing programmed cell disorders such as alcoholic cirrhosis (70o/o), death. lt is believedthat overexpression of bcl-2 emphysema(57%) and diabetesmellitus (38"/.). allows other mutationsof protooncogenesthat, Due to this, CEA lacks specificity for cancer ultimately, leads to cancer. detection. However, in established cancer patients (particularlyof colon and breast),the Unified hypothesis serum level of CEA is a usefulindicatorto detect of careinogenesis the burden of tumor mass, besides monitoring The multifactorial origin of cancer can oe the treatment. suitably explained by oncogenes.The physical and chemical agents,virusesand mutationsall lead to the activation of oncogenes causing carcinogenesis. The antioncogenes and the genes regulatingapoptosisare intimately involved in development of cancer. A simplification of a unified hypothesisof carcinogenesisis depicted in Fi9.37.4.

2. Alpha-fetoprotein(AFP): tt is chemicallya glycoprotein,normally synthesizedby yolk sac in early fetal life. Elevation in serum levels of AFP mainly indicatesthe cancers of liver ano germ cells of testis and, to some extent, carcinomasof lung, pancreasand colon. As is the case with CEA, alpha-fetoproteinis not specific for the detection of cancers. Elevated

692

BIOCHEMISTRY

Tumormarker

Associatedcancer(s)

antigens Oncofetal (CEA) antigen Carcinoembryonic (AFP) Alpha tetoprotein antigen-1 25(CA-1 25) Cancer

lung,pancreas andbreast ofcolon, stomach, Cancers ofliverandgermcellsoftestis Cancer Ovarian cancer

Hormones gonadotropin (hCG) Human chorionic Calcitonin Catecholamines andtheir (mainly metabolites vanillyl mandelic acid)

Choriocarcinoma thyroid Carcinoma ofmedullary and Pheochromocytoma neuroblastoma

Enzymes Prostatic acidphosphatase Neuron specific enolase

Prostate cancer Neuroblastoma

proteins Specilic (PSA) Prostate specific antigen lmmunoglobulins

Prostate cancer Multiple myeloma

levelsof AFP are observedin cirrhosis,hepatitis and pregnancy.However/measurement of serum AFP provides a sensitiveindex for tumor therapy and detection of recurrence.

The morphological and biochemical changes in the growingtumorcellsare brieflydescribed here.Theseobservations are mostlybasedon the in vitro culturestudies.Knowledgeon the in the biochemicalprofileof tumor alterations cellsguidesin the selectionof chemotherapy of cancers.

Further,they cannot they form monolayers. moveawayfrom eachother.The cancercells form multilayersdue to loss of contact As a result,the cancer inhibition(Fig.37.5). in anypart cellsfreelymoveandgetdeposited of the body, a property referred to as metastasis. Lossof anchoragedependence: The cancer cells can grow without attachmentto the This is in contrastto the normalcells surface. which firmly adhereto the surface. Alteration in permeabilityproperties : The tumor cells have altered permeabilityand transport.

1. Generaland morphologicalchanges Shapeof cells : The tumor cells are much rounderin shapecomparedto normalcells. Alterationsin cell structures: Thecytoskeletal structureof the tumor cells with regardto actinfilamentsis different. . Lossof contactinhibition: The normalcells are characterized bv contact inhibition i.e.

(B) Flg. 37,5 : Growthcells in culture (A) Normal cells formingmonolayer(exhibitingcontact inhibition);(B) Cancer cells forming multilayers(loss of contact lnhibition).

Chapter 37 : CANCER

693

2. Biochemicalchanges Increased replication and transcription : The synthesisof DNA and RNA is increasedin c anc erc ell s . Increased glycolysis : The fast growing tumor cells are characterizedby elevationin aerobic and anaer o b i c g l y c o l y s i s d u e to i n c re ased ener gydem a n d so f m u l ti p l y i n gc e l l s .

known. lt is believed that the morphological changesi n tumor cel l s,l ossof contacti nhi bi ti on, lossof anchoragedependenceand alterationsin the structure of certain macromolecules are among the important factors responsible for metastasis.

. Reduced requirement of growth factors : The tumor cells require much less quantities of growth factors. Despite this fact, there is an increased production of growth factors by t hes ec ells .

Chemotherapy,employing certain anticancer drugs, is widely used in the treatmentof cancer. ln the lable 37.5, a selected list of the most commonly used drugs,and their mode of action of anticancerdrugs is . Synthesisof fetal proteins : During fetal life, is given. The effectiveness certain genes are active, leading to the inverselyproportionalto the size of the tumor synthesisof specificproteins.Thesegenesare i .e. the number of cancer cel l s. The maj or suppressedin adult cells. However,the tumor limitation of cancer chemotherapy is that the cells synthesize the fetal proteins e.g. rapi dl y di vi di ng normal cel l s (of hematopoi eti c system, gastrointestinaltract, hair follicles) are carcinoembryonicantigen, alfa fetoprotein. also affected.Thus,the use of anticancerdrugs is . Alterations in the structure of molecules : associatedwith toxic manifestations. Changesin the structureof glycoproteinsand glycolipids are observed. For the treatmentof solid tumors,surgeryand radiotherapy are very effective. M et as t as is Metastasisrefersto the spreadof cancer cells from the primary site of origin to other tissuesof the body where they get depositedand grow as secondarytumors. Metastasisis the major cause ln recent years, certain precautionary of cancer related morbidity and mortality. The measuresare advocatedto prevent or reduce the biochemical basis of metastasisis not clearly occurrence of cancer. The most imoortant

B|oMEDICAL/ CHNTCALCONGEPTS

6 r€

s€

About 800/oof the human cancers are caused by chemical carcinogens. The products ol oncogenes(growth factors, GTP-binding proteins) have been implicated in the deuelopment ol cancer. Antioncogenes apply breaks and regulate the cell proltferation. The physical and chemlcal agents, uiruses and mutatlons result in the actiuqtion of oncogenes causing carctnogenests, The abnormal products of tumor cells, referred to as tumor markers (CEA, AFe PSA) ore useful far the dlognosis and prognosis of cancer. Anttconcer drugs (e.9. methotrexate, clsplatin) are commonly used tn the treotment of cancer. Antloxldants (ultamlns E and C, ftcarotene, Se) decreqse the risk of carclnogenesisond hence their increosed consumption ls adwcated.

694

B IOC H E MIS TR Y

Anticancerdrug

Chemical naturc

Methotrexate

Folicacidanalogue

6-Mercaptopurine

Mode of action

(inhibits the Blocks theformatin oftetrahydrofolate THFisrequired for enzyme dihydrofolate reductase). nucleotide synthesis. Inhibits theformation ofAMPfromlMP.

6-Thioguanine

Mitomycin C

Antibiotic

Actinomycin D Vinblastine andvincrisline

Antibiotic

Cisplatin

Platinum compound

Alkaloids

Blocks reaction. lhymidylate synthase Results intheformation ofcrossbridges between DNA pairs. base Blocks transcriotion (ofcelldivision) lnhibit spindle movement andinterfere withcytoskeleton formation Results intheformation of intrastrand DNAadducts.

among them, from the biochemicalperspective, system, and promote detoxification of various are the antioxidants namelv vitamin E, carcrnoSens. p-carotene,vitamin C and selenium. In general, most of the vegetablesand fruits The antioxidants prevent the formation or are rich in antioxidants. Their increased detoxify the existing free radicals (free radicals consumption'is advocated to prevent cancer. are known to promote carcinogenesis).In (For more dethils on free radicals and addit io n ,a n ti o x i d a n tss ti m u l a teb o d y' s i mmune antioxidants, Refer Chapter 34).

1.

Cancer is characterizedby uncontrolledcellular growth and deuelopment,Ieading to excessiueproliferotion ond spread of cells. Cancer is the second largest killer diseose (next to heart disease)in the deueloped world.

2.

Regulatorygenes-namely oncogenes, antioncogenes and genescontrollingcell deathare inuolued in the deuelopment ot' cancer. Actiuatlon ol oncogenesis a /undomental step in corcinogenesis. This may occur by insertion of uiral DNA into host chromosome, translocationof chromosomes,gene amplilication ond point mutation.

3. The products of actiuated oncogenessuch os growth t'actors,growth factor receptors, GTP-binding proteins, non-receptor tyrosine kinoses haue all been implicoted in the deuelopment of cancer.

4.

Tumor markers of cancers include carcinoembryonicantigen (CEA), alpha fetoprotein (AFP), cancer antigen-721 and prostote specilic antigen (PSA). They are mainly uselul to support diognosis, monitor therapy and detect recurrence.

5.

There are seueral morphologicol and biochemical chonges in the tumor cells which distinguish them from the normal cells. The cancer cells ore chorqcterizedb9 loss oJ contact inhibition, altered membrane transport, increosed DNA ond RNA synthesis, increased glycolysis,alteration in the structure of certain molecules etc.

syndrome(AIDS) Epidemiology A cquiredimmunodeficiency first reported in 1981 in'homosexual Awas AIDS was first describedin USA and this men. AIDS is a retroviraldiseasecaused by country hasthe majorityof reportedcases.The human immunodeficiencyvirus (HlV). The prevalenceof AIDS has been reported from diseaseis characterizedby immunosuppression, almostevery country.The numberof people secondary neoplasma and neurological living with HfV worldwide is estimatedto be manifestations. AIDS is invariably fatal since around40 million by the end of the year2005. there is no cure. In the USA, it is the fourth (lndia alone has about 5 million persons). At leadingcauseof deathin men betweenthe ages least5 milliondeathsoccurredin 2005,due to 15 to 55 years. AIDS.AIDS is truely a globaldiseasewith an No other disease has attracted as much alarmingincreasein almosteverycountry. publicand aftention asAIDSby thegovernments, Transmission of HIV : Transmission of AIDS scientists. AIDShasstimulated an unprecedentedessentiallyrequires the exchangeof body fluids amountof biomedicalresearchwhich led to a (semen, vaginal secretions, blood, milk) majorunderstanding of thisdeadfydiseasewithin containingthe virusor virus-infected celfs.There a short periodof time.So rapidis the researchon are three major routes of HIV transmissionAIDS(particularly relatingto molecularbiofogy), sexualcontact,parenteralinoculation,and from any review is destinedto be out of date by the infectedmothersto their newborns. time it is published! The distributionof riskfactorsfor AIDStransThe isolationof human immunodeficiencymissionare as follows. virus (HIV) from lymphocytesof AIDS patients (homosexuals) - 60"/" wasindependently achievedby Gallo(USA)and Sexbetweenmen Sex betweenmen and women Montagnier(France) in 1984. -15% 695

696 Intravenous drug abusers

B IOC H E MIS TR Y

- 15"/"

Transfusionof blood and blood products-

6%

All others

4o/o

-

predominant methods of The HIV transmission(about 75o/") are through anal or vaginal intercourse. The risk for the transmission is m uc h h i g h e r w i th a n a l th a n wi th vagi nal intercourse.The practice of 'needle sharing' is mainly responsiblefor the transmissionof HIV in drug abusers.PediatricAIDS is mostlycausedby vertical transmission(mother to infant). It should, however,be noted that HIV cannot be transmitted by casual personal contact in the household or work place. Further, the transmissionof AIDS from an infectedindividual to health personnel attending on him is extremelv rAre.

The lipid membrane of the virus is studded with two glycoproteins Bprzo and gpot. The Virology of HIV surface antigeir 8p126 is very important for the AIDS is causedby a retrovirus,namely human viral infection and the detection of AIDS. immunodeficiency virus (HlY), belonging to Genome and gene products of HIV : The HIV lentivirus family. Retrovirusescontain RNA as genome contains 3 structural Benes-gag,pol and the geneticmaterial.On entry into the host cell, they transcribeDNA which is a complementary env that, respectively, code for core proteins, copy of RNA. The DNA, in turn is used, as a reverse transcriptase and envelop proteins. On either side of the HIV genome are long terminal template to produce new viral RNA copies. repeat (LTR)genes which control transcription. Two different forms of HlV, namely HIV-I Besides the structural genes/ HIV contains and HIV-2 have been isolated from AIDS several regulatory genes including vif, vprt tat, patients.HIV-1 is more common, being found in rev, vpu and nef (Fi9.38.4. These genes control AIDS patients of USA, Canada, Europe and the synthesisand assembly of infectious viral Central Africa while HIV-2 is mainly found in proteins. In fact, the regulatorygenesof HIV play West Africa. Both the virusesare almost similar a key role in the developmentof AIDS. except they differ in certain immunological properties. HIV-1 is describedin some detail. Structureof HIV : The viruse is sphericalwith a diameterof about 110 nm. lt containsa core, surroundedby a lipid envelop derived from the host pfasma membrane (Fig.3fl.l). The core of the HIV has two strandsof genomic RNA and four core proteins, PZq,PtB, reversetranScriptase (poolpsr)and endonuclease(p32).Note that the namingof the proteinsis basedon the molecular weight. For instance,a protein with a molecular weight of 24,0OOis designated as p2,4.

lmmunological abnormalities in AIDS

As is evident from the name AIDS, (or immunosuppression) is immunodeficiency primarily AIDS the haflmark of this disease. affectsthe cell-mediatedimmunesystemwhich protectsthe body from intracellularparasites protozoaand mycobacteria. This suchasviruses, (cluster is caused by a reduction in CD+ antigen4) cells of T-lymphocytes, determinant besidesimpairmentin the functionsof surviving CDa cells.

SYNDHOME(AIDS) IMMUNODEFICIENCY Chapter 3a: ACGIUIRED CDa cells may be regardedas 'naster cells of cell mediated rnmunity.They producecytokines, rracrophage chemotactic factors, remopoietic growth factors, and others involved in the bodv im m unit y .

Core proteins transcriDtase

Entryof HIV and lysisof CDacells: Thevirus HIV bindsto the entersthe CDaT-lymphocytes. specificreceptorson CDa cells by using its surface membrane glycoprotein (gprzo). Followingthe entry into the hostcells,RNA of HIV is transcribed into DNA by theviralenzyme reverse transcriptase.The viral DNA gets incorporated into the host genomicDNA. The virusmay remainlockedin the hostgenomefor monthsor yearsand this is consideredas the latent period. The viral DNA may undergo replication , and translation, respectively, producingviral R'NAand viral proteins.The resultin new viruses. lattertwo, on assembly, virusesleavethe host The newly synthesized cells by forming buds on plasmamembrane. Extensive viral buddingis associated with lysis The new viral and deathof CDa cells(Fi9.38.3). particlesinfectother host cells and repeatthe wholeprocess, ultimatelyresulting in a profound loss of CDa cells from the blood. Most of the immunodeficiency symptoms of AIDS are associatedwith the reduction in CDa cells. Other immunological

697

Envelope glycoproteins

Fiq.38.2: Genomeof HlV.

CDncell

J

abnormalities

The viral membraneprotein gp12s binds with normal T-helper cells and kills them. AIDS patientsalso display abnormalitiesin (humoral antibodyproductionby B-lymphocytes immunity). Abnormalitiesof central nervous system : HIV also infectsthe cells of central nervous system. lt is believed that HIV infected monocytesenterthe brain and causedamage, the mechanism of which remainsobscure. Consequences of immunodeficiency: The various clinical symptoms (fever, diarrhea, multiple weightloss,neurological complications, opportunisticinfections,generalizedlymphadenopathy,secondaryneoplasma etc.)of AIDS

Extensiveviral multiplication

Lysisof CDocells Fig. 38.3 : lmmunological abnomalities in CD4 cells on HIV infection.

698

BIOCHEMISTF|Y Acute phase

Crisis phase

Chronic phase

1,200 E

8 1,100 E 1,000 E

=g

eoo

8.

800

;f

700

1 :512 1 :256

Clinicallatency --1

1 :128 o

J--

1 :64 Opportunistic diseases 1 :32

[]- eoo

.g E o '5

E o

soo

*tr 5 *o o o

400

1:16 E o

300 2oo 100

1:8

(! 6

E

1i 4

1:2 0 2

4

6 8 1 0 1 ,2 Weeks-4

1294

s

67 I 91011121314 Years.l

FIg. 38.4: Graphicrepresentationof a typicalcourseof HIV infection.

are directly or indirectly related to the immunosuppression causedby HlV. Due to the deficiencyin the immune system,the body of AIDS patient is freely exposedto all sorts of infections(viral,bacterial,fungal). Natural course of AIDS Threedistinctphasesof HIV interaction with the immunesystemof infectedbody havebeen identified.Theseare the early,acutephase;the intermediate,chronic phase;the final, crisis phase(Fi9.38.4).

hence this phase is also referred to as seropositiveperiod. 3. Crisis phase : A failure in the defense system of the ,body, caused by immunothe crisisphase. suppression by HlV, represents -tremendously The plasma level of virus i5 increased.CD+ T-lymphocyteconcentration drasticallyfalls.A patientwith lower than 200 CDa T-lymphocytes/plblood is consideredto have developed AIDS. Crisis phase is characterizedby opportunisticinfectionsand In Western the relatedclinical manifestations. countries, a cancer-Kaposis sarcoma-is with AIDS. associated

1. Acute phase: This represents the initial body response to HIV infection. lt is characterizedby high rate of production of In general,AIDS patientsdie between5-10 viruseswhich are lodgedin the lymphoidtissues years after HIV infection. Treatment ffidy, and the antiviralimmuneresponse of the body. however,prolongthe life. This periodmay lastfor about8-12 weeks. 2. Chronic phase: During this periodthat may last for 5 to 10 yearsor even more, the body tr:iesto contain the virus. The immune systemis largelyintact.The personobviously appearsnormal,althoughhdsheis the carrierof HIV which can be transmittedto others. Antibodiesto HIV are found in the circulation,

Laboratory

diagnosis

of AIDS

The following laboratorytestsare employed to diagnosethe HIV infection. 1. The detection of antibodies in the immunocirculationby ELISA(enzyme-linked sorbantassay).

SYNDHOME(AlDSl Ghapter 38 : ACGIUIRED IMMUNODEFICIENCY

3'-Azldo2',3'dlod€oxythymldlne(AZT)

699

2',3'-Dldeorylnoslne(DDl)

Fi,.38.5: Structureof anti-AIDSdrugs

2. Westernblot technique,a more specific to the T-lymphocytessince cellular DNA test for the HIV antibodies,is employedfor pofymerasehas low affinity for AZT. However, AZT is found to be toxic to the bone marrow of ELISApositivecases. confirmation the patientsdevelopanemia. PCRcan cells,therefore, 3. A morerecentand sophisticated be used to detect the presenceof the HIV Themechanism is of actionof dideoxyinosine genomein the peripheralblood lymphocytes. almostsimilarto that of AZf . Drugs for the treatment

of AIDS

Althoughthere is no cure for AIDS, use of certain drugs can prolong the life of AIDS patients. Zidovudine or AZT (3'-azido 2', 3'-dideoxythymidine),a structuralanalog of was the first drug used and deoxythymidine continuesto be the drug of choice for the treatmentof Al DS. Didanosine(dideoxyionosine, DDt) is anotherdrug employedto treat AIDS. The structuresof AZf and DDI are shown in Fig.38.s.

Vaccine against AIDS -a failure so far

HIV exhibitsgeneticheterogenecity with a resultthatseveralspecies of virusmay be found in the sameAIDSpatient.Theprincipalcausefor the geneticvariationis the lack of proof-reading activityby the enzymereversetranscriptase. This leads to very frequent alterations in the DNA base sequence synthesized from viral RNA which, in turn, influencesthe amino acid sequenceof proteins.Thus,the proteinproducts Mechanismof action : AZT is takenup by the of HIV are highly variablein the amino acid fymphocytes and convertedto AZt triphosphate composition and, therefore, the antigenic which inhibits the enzyme HIV reverse properties.For this reason, it has not been AZT triphosphatecompeteswith possibleto develop a vaccine againstAIDS. transcriptase. dTTPfor the synthesisof DNA from viral RNA. However,there have been some encouraging which raisefresh Further,AZI is addedto the growingDNA chain animaland in vitroexperiments is halted.Thisdrugis not toxic hopesfor a vaccinein the nearfuture. andthe synthesis

B IOC H E MIS TR Y

700

BIOMEDICAL/ CLINIGAL GOIUCEFfS

u1? AIDS is a global diseasewith an alarming increosein the incidenceof occurrence.By the year 2005, more than 40 million people were globally ot't'ectedby AIDS. Lg

Homosexuality (predominantly in men) qnd intrauenous drug abuse ore the major foctors in the risk of AIDS fronsmission.

G+- The patientsol AIDS are destined to die (within 5-70 gearsaJter infection), since there is no cure. Howeue4 administration of certain drugs (AZT, DDI) prolongs life. E-' The clinicol manifestations ol AIDS are directly or indirectlg related to immunosuppression (mostly due to reduced CDa cells). AIDS patients are lreely exposed to all sorts ol infections (uiral, bacterial, fungal).

1. A/DS is a retoruiral disease coused by human immunodet'iciency uirus (HIV)' It is charocterized by immunosuppression, secondary neoplasms and neurological manifestations. Tronsmission of HIV occurs by sexual contact (more in male homosexuqls),parental inoculation (intrauenous drug abusers) and from infected mothers to their newborns.

2.

HIV enters CDa Tlymphocytes where its genetic material RNA is transcribedinto DNA by the enzyme reuerse transcriptose.The virol DNA gets incorporated into the host genomeultimately leodingto the multiplicationof the uirusand the destructionof CD4 cells. This is the root causeof immunosuppressionleading to opportunistic inlections in A/DS.

chronic ond crisis. A potient 3. The natural course ol AIDS has 3 distinct phases----acute, with lower than 200 CDa Tlymphocytedltl is consideredto haue deueloped AIDS. The sensitiuelaborotory testsfor AIDS detection are-ELISA, Western blot technique and, recently PCR.

4.

There is no cure t'or AIDS. The potients generally die within 5-10 years aJter HIV infection. Administration of drugs (zidouudineand didanosine),howeuer,prolongs the life oJ AIDS patients. These drugs inhibit the uiral enzyme reuerse tronscriptaseand halt the multiplication of the uirus. due to the uoriations The attempts to produce uoccinefor AIDS haue been unsuccesst'ul in the genome (and, thereJore,the protein products)ol the HlV.

Introduction to Bior Chemistry

,,

Overview of Biophysical

chemistry

lli ,,,ii:,

-a

lS

fools of Biochemistry

Bioorganic Chemistry

I s life comes from previous life, it was vitamins are the most common organic A believed for a longthatthecarboncompounds compoundsof life. Their chemistryhas been (hence in Section| (Chapters1-V. the nameorganic)arosefrom discussed of organisms force thury. This is referred to as vital fife onfy. that urea Comnnon funetiona! groups FriedrichWohler(1825)firstdiscovered (NH2-CO-NH2),the organiccompound,could in bEochemistry be preparedby heating ammonium cyanate Most of the physicaland chemicalproperties (NH4NCO), Thereafter, thousands of organiccompoundsare determinedby their in the laboratory. of organiccompoundshavebeen functionalgroups.Biomolecules and thousands possess certain synthesized outsidethe livingsystem. functional groups which are their reactive Organic chemistry broadly deals with the centres.The common functional groups of chemistry of carhon compounds, regardlessof importancein biomoleculesare presentedin their origin. Biochemistry, however, is Table39.1. concerned with the carbon chemistry of life Gomrmon ring structures of organicchemistry only.Thegeneralprinciples in biochemistry provide strongfoundationsfor understanding Thereare manyhomocyclicand heterocyclic exclusively However,biochemistry biochemistry. rings, commonlyencountered in biomolecules. that occur in the living dealswith the reactions given A list is in Fig.S9.l. selected of them systemin aqueousmedium. Homocyclicrings : Phenylring derivedfrom Most commoR organie eonnpounds benzene is found in several biomolecules found in living systenr (phenylalanine, tyrosine, catecholamines). The organic compounds,namely carbo- Phenanthreneand cyclopentaneform the hydrates,lipids, proteins,nucleic acids and backboneof steroids(cholesterol, aldosterone).

703

704

E}IOCHEMISTRY

Functional group Name Group

Hydroryl

General structural Type of formula compound

o

Aldehyde

-c-H

tl R _ C _H

Keto

o -c-

tl R1-C-R2

il

o Carboxyl

Sulfhydryl

Ester

tl

Aldehyde

glucose Glyceraldehyde,

Ketone

Fructose, sedoheptulose

o

o ll

-c-oH

R-C-OH

acid Carboxylic

Aceticacid,palmitic acid

-NHz

R-NH2

Aminoacid

Alanine, serine

H I -N-

H I R_N-

lminoacid

Proline, hydroryproline

_ SH

R _ SH

-o-

R1-O-R2

o

o

tl

-c-o-R1

o

Amido

Glycerol, ethanol

R_OH

o

Examplesof biomolecule(s)

-d--