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
• 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
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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