M E D I C I N EKINGSAUDUNIVERSITY

SiCl4CuCl2CCl4HCN

MgCl2

KMnO4

H2O

H2O2

KClO3

Co2

Cl2O7

COOH

NH2HbA

CH2OPO4

SO2

NAOH

ImportantExtra Information

DoctorsslidesDoctors notes

BiochemistryEditing file

Start where you areUse what you haveDo what you canUrea Cycle

O B

J E

C T

I V

E S Upon completion of this lecture, the

students should be able to :

Understand the reactions for removal of α-amino group of amino acids and formation of ammonia

Identify the importance of blood transport of ammonia to the liver in the form of glutamine/alanine

Understand the importance of conversion of ammonia into urea by the liver through urea cycle

Identify urea as the major form for the disposal of amino groups derived from amino acids

Identify the causes (hereditary & acquired), clinical manifestations and management of hyperammonemia

Overview

• Unlikeglucoseandfattyacids,aminoacidsarenotstoredbythebody.

• Aminoacidsinexcessofbiosyntheticneedsaredegraded.

• Degradationofaminoacidsinvolves:

• Removalofα-aminogroupAmmonia(NH3)

• RemainingcarbonskeletonEnergymetabolism

Background

UreacycleisbasicallytheformationofureafromtheAmmoniaoftheAminoacids(occursintheliver)

SoourmaingoalistogettheAmmonia(whichistoxic)fromaminoacidsofdifferenttissuestoreachtheliverwhereitwillbeconvertedtourea(whichisnottoxic)

FirsttheaminoacidundergoestransaminationreactionandoxidativedeaminationtoformthisammoniaSecond,thistoxicammoniaistransportedtotheliverintheformofglutamineandAlanineThird,uponreachingtheliver,ureacyclecanbeginandwecanconvertammoniatourea.

UreacyclehappensonlyinthehepatocytesbecausewehaveuniqueenzymesuchasArginasethatispresentonlyinliver,that’swhyptswithlivercirrhosishavehighlevels

ofammonia

α-aminogroupRemovalofα-aminogroup,formationof

ammoniaanditstransporttoliver

1. Transaminationtoglutamate

2. Oxidativedeaminationofglutamate

A:Removalofα-aminogroupofaminoacidsandformationof

ammonia1. intheformofglutamine (most

tissue)

2. intheformofalanine(muscle)

B:Bloodtransportofammoniaintoliver

Removalofaminogroupmakeitactivebecauseitspresenceonthecarbonskeleton(withintheaminoacid)

stabilizeitandmakeitnotactive.

A:α-aminogroupremoval&ammoniaformation• Aminogroupsofaminoacidsarefunneledto

glutamate(Why?)bytransaminationreactionswithα-ketoglutarate

• BecauseGlutamateisunique.Itistheonlyaminoacidthatundergoesrapidoxidativedeamination

• OxidativedeaminationofglutamatewillreleaseNH3andre-generateα-ketoglutarate

ThereasonwhyAminogroupsofaminoacidsarefunneledtoglutamateistheuniquefeaturethatithaswhichistherapid

oxidativedeamination

Transamination

PLP:Pyridoxalphosphate,aco-enzymethatisderivedfromvitaminB6

Transaminationinthispicturewhereaminoacidsdonatetheiraminogrouptoalpha-ketoglutaratetobecomeglutamateandthereforetheaminoacidbecomealphaketoacid(ketoacidis

ageneralterm)eachaminoacidhasitsownketoacidforexamplealanine’sketoacidispyruvate

Important These reactions are bidirectional

TransaminationHereisanexampletoclarify

Glutamate

After the loss of the amino group the amino acids

becomes an alpha - keto acid ( carbon skeleton)

Alpha-Keto acid

Glycine (amino acid) donates its amino group (NH3+) to alpha-ketoglutarate

Gives

Mainly:Theketoglutaratehereisthethereceiver,itreceivesthe(NH3)fromaminoacidsbyanenzymecalledaminotransferase.Thealphaketoglutaratewith theaminogroupiscalled(Glutamate).Theaminoacidthatlostit’saminogroupisgenerallycalled(alphaketoacid)

A:α-aminogroupremoval&ammoniaformation

TransaminationbyALT&AST

ALT& PLP

& PLP

& PLP

Alanineisconvertedintopyruvateaftertheremovalofanaminogroupbyalanineaminotransferaseenzyme

___________________________________________

Theenzymenameconsistofthenameofthesubstratethatdonatealanineand“aminotransferase”whichisthe

functionoftheenzyme___________________________________________

Ifweremovedaminogroup:1. AlaninebecomesPyruvate

2. AspartatebecomesOxaloacetate

Glutamate here can donate amino group toOxaloacetate to give Aspartate

Oxaloacetate Glutamate

Becomes Aspartate

Becomes alpha-

ketoglutaratePyruvateistheketoacidofAlanine.

Oxaloacetateistheketoacidofaspartate

Normallyallaminoacidsgothroughthe

formationofglutamatedirection,exceptinthisreactiontheglutamate

isthedonator.

Important

A:α-aminogroupremoval&ammoniaformation

OxidativeDeamination

Glutamate α-ketoglutarate

GlutamateDehydrogenase

NAD NADH

NH3

Thepurposeofthisreactionistoproducefreeammonia(NH3)anditsreallyimportanttoknowthe

enzymethatmediatethisreaction (Glutamatedehydrogenase)

Theaminogroups(NH3+)fromaminoacidsarefunneledintoglutamateandaswementioneditis

uniquebecauseitcanberapidlyoxidativedeaminated

Theregenerationofalpha-ketoglutarateinthisreactionbecauseweneeditasanrecipientofaminogroupintheprevioustransaminationreactions.

NH3groupthatresultsistoxicbecauseitisnotpositivelynornegativelychargedwhichmeansit's

freeandcandiffusefreelyinthebloodandcancrossthebloodbrainbarrierwhichleadstoCNStoxicity

BytheactionofglutamatedehydrogenasetheglutamategetsoxidizedintoAlphaketoglutarate and

NADgetsreducedtoNADH

A:α-aminogroupremoval&ammoniaformation

Summary

Question:HowcanweremovetheaminogroupfromaminoacidandformAmmonia?

Answer:ByTransaminationandoxidativedeamination

Thisprocessoccursin2steps:1- aminoacidsdonateaminogrouptoa-

ketoglutaratetoformglutamate(Transamination)2- GlutamatebytheactionofGlutamate

dehydrogenasewillreleaseAmmonia(NH3)andbecomesa-ketoglutarate(Oxidativedeamination)

B:TransportofNH3fromperipheraltissuesintotheliverv NH3needstoreachtheliverbecausetheliver

containstheenzymerequiredtoconvertammoniatourea

v Ammoniaisproducedbyalltissuesandthemaindisposalisviaformationofureainliver

v BloodlevelofNH3mustbekeptverylow,otherwise,hyperammonemia andCNStoxicitywilloccur(NH3istoxictoCNS)

v Tosolvethisproblem,NH3istransportedfromperipheraltissuestotheliverviaformationof:

ü Glutamine(mosttissues)ü Alanine(muscle)

TransportofNH3fromperipheraltissuesintotheliver

Frommostperipheraltissues NH3 istransportedIntotheliverthroughforming glutamine

byglutaminesynthetase

AmmoniashouldnotbeleftaloneinthebloodbecauseitcancauseCNStoxicityThereforeit

istransportedtotheliverintheformofGlutamineandalanine

ExplanationTransportofNH3from

peripheraltissuesintotheliver

Sonowtheammoniaisaddedtoglutamatewiththeactionofglutaminesynthetase willformglutamine

Glutamate+NH3 (Glutaminesynthetase)=Glutamine

Nowitissafetotransporttheammoniatotheliver(thishappensinmostperipheraltissues)

آمنة نخلي نقل االمونیا للكبد یتم بصورة تصیرعشانھذه العملیة

Frommusclestotheliver

Fromjust themusclesFirst,NH3 willbetransferredintoα-ketoglutaratetoformglutamate

Then,glutamatewillgiveits aminogrouptopyruvate toformalaninebyALT

Therefore,NH3 istransportedfrommuscleintotheliverthroughformingalanine

B:TransportofNH3fromperipheraltissuesintotheliver

Herewewillpackagetheammoniaintoalanineinorderforittobesafely transportedfromthemusclestothe

liver,butthisrequires2steps1- thisammonia iscombinedtoa-ketoglutarateto form

glutamate2- glutamate givesofitsamino grouptopyruvate and

formsAlanine

B:TransportofNH3fromperipheraltissuesintotheliver

Intheliver

1.Glutamine isconvertedintoglutamatebyglutaminase.

2.Alanine willgiveitsaminogrouptoα-ketoglutaratetoformglutamateby

ALT.

3.Glutamate isconvertedintoα-ketoglutarateandreleasingNH3byglutamatedehydrogenase.

SummaryBloodtransportofNH3fromperipheraltissues (intheformofglutamineandalanine)intotheliverandthereleaseofNH3backinthelivertostarttheurea

cycle

Theliverisjustinterestedinammonia SonowtheGlutamine

andAlaninewillbe brokendowntoreleaseammonia.

Theactionofglutaminase releasesammoniaalso.

Inthethirdstepammoniaisreleasedfromglutamatebyoxidativedeamination..

Andnowafterammoniareachedtheliverureacyclebegins

UreaCycle

Ø Ureaisthemajorformfordisposalofaminogroupsderivedfromaminoacids

Ø Ureacycleoccursintheliver

Ø OnenitrogenofureaisfromNH3andtheothernitrogenfromaspartate

Ø Ureaistransportedinthebloodtothekidneysforexcretioninurine

Thefiveenzymesinvolvedinureacycleare:1. Carbamoylphosphatesynthetase I2. Ornithinetranscarbamoylase (OCT)

3. Argininosuccinate synthase4. Argininosuccinate lyase

5. Arginase(Unique)

CPSI

OCT

ASS

ASL Arginase

Enzyme1&2arepresentinthemitochondriawhiletherestinthecytosol

Ureahastwoaminogroups,onefromtheaspartateandtheotherfromtheammonia

UreaCycle:importantexplanationIthasseveralstepsstartingfromammoniapresenceintheliver

1-NH3+CO2+2ATPCarbamoylphosphate

2-Carbamoylphosphate+L-Ornithine L-Citrulline

3-L-Citrullinediffusesthroughmitochondrialwalltothecytosol

4-L-CitrullinecombineswithAspartatetoformArgininosuccinate byArgininosuccinate synthetase

5-ArgininosuccinatebyArgininosuccinate lyase givesL-Arginine

6-L-ArgininebyArginasegivesurea

CPSI

OCT

UreaCycle

AllostericactivatorofCPSI:N-Acetylglutamate

N-Acetylglutamate issynthesizedby:N-Acetylglutamate synthetase(NAGS)inpresenceofarginine

Rate-limitingenzymeofureacycle:Carbamoylphosphatesynthetase I

(CPSI)

NAGSdeficiencyisefficientlytreatedwithCarbaglue,aCPS1

activator

NAGS

N-Acetylglutamate

Glutamate

HereitshowsyouhowtheN-AcetylglutamateisformedwhichisanallostericactivatorforCPSI

______________________________________________

Glutamate+AcetylCoAinthepresenceofArginineformsN-acetylglutamatebytheactionofN-acetyl

glutamatesynthetase (NAGS)

TheactionofintestinalureasetoformNH3isclinicallysignificantinrenalfailure:

FateofureaMajorpathway

Smallamountgoestotheintestineintheintestinethebacterialflorahaveureaseactivitywhichbreaksureainto

Ammonia(NH3)andCO2

Innormalhealthconditions,theNH3

resultswhichisinsmallamountswon'taffectus

Inrenalfailure,themajorpathwaytothe

kidneyswillbeinhibitedandmoreureawillgototheintestine,thisleadstolargeamountsofammoniathatarereabsorbedintotheblood(acquired

hyperammonemia).ThisNH3candiffuseintoBBBandcauseCNStoxicity

SourcesandFatesofAmmonia

Normalbloodlevelofammonia:5– 50µmol/L

Hyperammonemiav Acquiredhyperammonemia:

1.Liverdiseases:Acute:ViralhepatitisorhepatotoxicChronic:Cirrhosisbyhepatitisoralcoholism2.Renalfailure

v Inheritedhyperammonemia:

A.Geneticdeficienciesofanyofthe5enzymesofureacycleortheactivatorenzymeforCPSI:

CPSI,OTC,ASS,ASL,arginaseorNAGS

B. Ornithinetranscarbamoylase deficiency:X-linkedrecessiveMostcommonofcongenitalhyperammonemiaMarkeddecreaseofcitrulline andarginine.

C. Others:Autosomalrecessive

CNSproblemsmainly

v Lethargy and somnolence

v Tremors

v Vomiting and cerebral edema

v Convulsions

v Coma and death

Clinicalpresentationofhyperammonemia

Ureacyclehappensintheliversoifwehaveliverdysfunctionforanyreason,wewon'tbeabletogetridofammonia

Management&treatmentofhyperammonemia

1. Protein restriction

2. Volume repletion to maintain renal functionUse 10% dextrose in water but limit the use ofnormal saline

3. Ammonia removal by hemodialysis &/or drugs

4. Avoid drugs that increase protein catabolism (eg,glucocorticoids) or inhibit urea synthesis (eg,valproic acid), or have direct hepatotoxicity

Management Treatment

A.Drugsthatscavengeammoniabycreatingan

alternatepathwaytoexcreteN2- precursors:

1.I.V.Sodiumphenylacetate &sodium

benzoate(Ammonul)

2.Oralsodiumphenylbutyrate (Buphenyl)

3.I.V.Arginine:forallUCDsexceptUCDdueto

arginasedeficiency(argininemia)

B.ActivatorstoCPSI(Carglumic acid“Carbaglu”):

Forhyperammoniemia duetoNAGSdeficiencyIfN-acetylglutamate synthetase (NAGS)isdeficient,wecan'tformN- acetylglutamate (NAG)whichisanallostericactivatorofCPSI.WecangiveamedicationcalledCarglumic acid(carbaglu)thatcandirectly

activateCPSItoenhancetheureacycle

Sodiumphenylbutyrate (Buphenyl)

Sodiumphenylbutyrate (Buphenyl):Pro-drugthatisconvertedto

phenylacetate.

Phenylacetate condenseswithglutamineformingphenylacetylglutamine

thatisexcretedinurine

Noureaformationhere,thereisdirectexcretionofammoniaintourineinthe

formofphenyl-acetyl-glutamine

Summarysummary

Removalofα-aminogroup ,formationofammoniaTransamination :By:ALT& AST

Aminogroupsofaminoacidsarefunneled toglutamatebytransaminationreactionswith α-ketoglutarate

B-Oxidative deamination: inliverBy:Glutamate dehydrogenase.IMP- Theglutamate willreleaseNH3- Regenerate α-ketoglutarate

Transport of NH3 from peripheral tissues into the liver

Glutamine (frommosttissues à liver)

Glutamine formedby glutaminesynthetase

Alanine(frommusclesà liver)

Releaseofammonia fromglutamineandalanineintheliver

Glutamine isconvertedbackintoglutamate

by glutaminase

Alanine willgiveitsaminogroup to α-ketoglutarate toformglutamate by ALT.

Glutamate isconvertedinto α-ketoglutarate andreleasingNH3by

glutamate dehydrogenase

Summary

Enzymeofurea:1.CarbamoylphosphatesynthetaseI

(CPSI) 2.Ornithinetranscarbamoylase(OCT)

3.Argininosuccinatesynthase (ASS) 4.Argininosuccinatelyase (ASL) 5.Arginase

Regulationofureacycle

- RATE-LIMITINGENZYMEofureacycleCPSI

AllostericactivatorofCPSI:N-Acetylglutamate.

N-Acetylglutamate issynthesizedby:N-Acetylglutamate synthetase (NAGS) inpresenceofarginine.

TreatmentofNAGSdeficiency:Carbaglue,aCPS1activator

Fateofurea:A- Tothekidneys(Mostly)excretedinurine.B- Totheintestineby ureasegivesNH₃(lostinfecesorreabsorbed intoblood +CO₂

HyperammonemiaA- Aquired (liverdiseases– Renalfailure)

B- Inherited (all5enzymesareautosomal recessiveexceptOCTwhichisX-linked recessive!!!!)Treatement:Oralsodiumphenylbutyrate(Buphenyl) - aProdrug thatisconvertedtophenylacetate

QUIZQ1 : NH3istransportedfrommusclestoliverby?A. GlutamateB. GlutamineC. AlanineD. B&C

Q2:Glutamineisconvertedintoglutamateby?A. Alanineaminotransferase(ALT)B. GlutamatedehydrogenaseC. ArginaseD. Glutaminase

Q3: Oneofthisenzymesarenotincludedinureacycle?A. Ornithinetranscarbamoylase(OCT)B. GlutaminaseC. ArgininosuccinatesynthaseD. CarbamoylphosphatesynthetaseI

Q4:Themostcommonenzymedeficientandcausecongenitalhyperammonemiais?A. ArgininosuccinatelyaseB. CarbamoylphosphatesynthetaseIC. OrnithinetranscarbamoylaseD. Glutaminase

Q5:Carbaglue isatreatmentofwhichofthefollowing?A. Hyperammonemiasecondarytorenalfailure.B. HyperammonemiasecondarytoCPS1deficiency.C. HyperammonemiasecondarytoOCTdeficiencyD. HyperammonemiasecondarytoNAGSdeficiency.

Q6:AminoGroupofAminoAcidsarefunneledtoGlutamineby?A. TransaminationB. OxidativeDeaminationC. DecarboxylationD. Hydrolysis

QUIZ

Suggestions and recommendations

Q7:MentiontheconditionswhichcancauseHyperammonemia?

Inherited:OrnithinetranscarbamoylasedeficiencyandothersAutosomaldominantAcquired:LiverdiseaselikeViral

Hepatitis,HepatotoxicexAlcohol,CirrhosisRenalFailure

Q8:What isthenormalbloodlevelofAmmonia?

5– 50μmol/L

Q9:Interpretsignsandsymptomsofhyperammonemia?

AmmoniaisatoxicproductanditcancrosstheBloodbrainbarrier,causing:

ü Lethargyandsomnolence,Tremors,Vomitingandcerebraledema,Convulsions,Comaanddeath

Q10:Whatistheenzymerequiredfortheoxidativedeaminationofglutamate?

Glutamatedehydrogenase

1)C2)D 3)B4)C5)D 6)A

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