nucleotides: synthesis and degradation javad zavar reza ph.d in clinical biochemistry department of...

Nucleotides:Nucleotides: Synthesis and Synthesis and DegradationDegradation

Javad Zavar RezaPh.D in Clinical Biochemistry Ph.D in Clinical Biochemistry

Department of Biochemistry Department of Biochemistry School of Medicine School of Medicine

11

Nitrogenous BasesNitrogenous Bases

Planar, aromatic, and heterocyclicPlanar, aromatic, and heterocyclic

Derived from Derived from purinepurine or or pyrimidinepyrimidine

Numbering of bases is “unprimed”Numbering of bases is “unprimed”

22

Nucleic Acid BasesNucleic Acid Bases

Purines Pyrimidines

33

SugarsSugars

Pentoses (5-C sugars)Pentoses (5-C sugars)

Numbering of sugars is “primed”Numbering of sugars is “primed”

44

SugarsSugars

D-Ribose and 2’-DeoxyriboseD-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group

55

NucleosidesNucleosides

66

Phosphate GroupsPhosphate Groups

Mono-, di- or triphosphatesMono-, di- or triphosphates

Phosphates can be bonded to either C3 or C5 Phosphates can be bonded to either C3 or C5 atoms of the sugaratoms of the sugar

77

NucleotidesNucleotides

Result from linking one or more phosphates with a Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through nucleoside onto the 5’ end of the molecule through esterificationesterification

88

NucleotidesNucleotides

Monomers for nucleic acid polymersMonomers for nucleic acid polymers

Nucleoside Triphosphates are important Nucleoside Triphosphates are important energy carriers (ATP, GTP)energy carriers (ATP, GTP)

Important components of coenzymesImportant components of coenzymes– FAD, NADFAD, NAD++ and Coenzyme A and Coenzyme A

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Naming ConventionsNaming Conventions

Nucleosides:Nucleosides:– Purine nucleosides end in “-sine” Purine nucleosides end in “-sine”

Adenosine, GuanosineAdenosine, Guanosine

– Pyrimidine nucleosides end in “-dine”Pyrimidine nucleosides end in “-dine”Thymidine, Cytidine, UridineThymidine, Cytidine, Uridine

Nucleotides:Nucleotides:– Start with the nucleoside name from above and add Start with the nucleoside name from above and add

“mono-”, “di-”, or “triphosphate”“mono-”, “di-”, or “triphosphate”Adenosine Monophosphate, Cytidine Triphosphate, Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine DiphosphateDeoxythymidine Diphosphate

1010

Nucleotide Biosynthesis Nucleotide Biosynthesis

De novo BiosynthesisDe novo Biosynthesis

Salvage BiosynthesisSalvage Biosynthesis

1111

Nucleotide MetabolismNucleotide Metabolism

PURINE RIBONUCLEOTIDES: DPURINE RIBONUCLEOTIDES: De novoe novo– i.e., purines are i.e., purines are notnot initially synthesized as free bases initially synthesized as free bases– First purine derivative formed is Inosine Mono-phosphate First purine derivative formed is Inosine Mono-phosphate

(IMP)(IMP)

1212

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Steps 1 Steps 1

Step 1Step 1:Activation of ribose-5-phosphate:Activation of ribose-5-phosphate

– product: 5-phosphoribosyl-a-pyrophosphate product: 5-phosphoribosyl-a-pyrophosphate (PRPP)(PRPP)

– PRPP is also a precursor in the biosynthesis of:PRPP is also a precursor in the biosynthesis of:

pyrimidine nucleotidespyrimidine nucleotides

HistidineHistidine

Tryptophan Tryptophan

1414

Step 1: purine synthesisStep 1: purine synthesis

1515

Step 2: purine synthesis:Step 2: purine synthesis:commited stepcommited step

1616

Step 3 : purine synthesisStep 3 : purine synthesis

1717

1818

Acquisition of purine atom C8 & purine atom N3

Step 6: purine synthesisStep 6: purine synthesis

1919

Step 7: purine synthesisStep 7: purine synthesis

2020

Acquisition of C6 introduced as HCO3-

Steps 8 thru 11Steps 8 thru 11

Step 8Step 8: acquisition of N1: acquisition of N1– N1 is acquired from aspartate in an amide N1 is acquired from aspartate in an amide

condensation reactioncondensation reaction– enzyme: SAICAR synthetaseenzyme: SAICAR synthetase– product: 5-aminoimidazole-4-(N-product: 5-aminoimidazole-4-(N-

succinylocarboxamide)ribotide (SAICAR)succinylocarboxamide)ribotide (SAICAR)– reaction is driven by hydrolysis of ATPreaction is driven by hydrolysis of ATP

2121

Step 8: purine synthesis

2222

Step 9: purine synthesisStep 9: purine synthesis

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Step 10: purine synthesis

2424

Step 11Step 11

cyclization or ring closure to form IMPcyclization or ring closure to form IMPwater is eliminatedwater is eliminatedin contrast to step 6 (closure of the in contrast to step 6 (closure of the imidazole ring), this reaction does not imidazole ring), this reaction does not require ATP hydrolysisrequire ATP hydrolysisonce formed, IMP is rapidly converted to once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in AMP and GMP (it does not accumulate in cellscells

2525

Step 11: purine Step 11: purine synthesissynthesis

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2727

HN

N N

N

O

Ribose-P

N

N N

N

NH

Ribose-P

COO--OOC

HN

N N

N

O

Ribose-P

O

H

Adenylosuccinate xanthine monophosphate XMP

IMPIMP dehydrogenase

NAD+

NADH

A.S. synthetase

Aspartate + GTP

GDP

N

N N

N

NH2

Ribose-P

HN

N N

N

O

H2N

Ribose-P

A.S. lyase

fumarate

glutamine + ATP

Glutamate + AMP+ PPi

AMP GMP

Synthesis of adenineand guanine nucleotides

28

Purine nucleoside diphosphates and triphosphates: - to be incorporated into DNA and RNA, nucleoside monophosphates (NMP’s) must be converted into nucleoside triphosphates (NTP’s)

- nucleoside monophosphate kinases (adenylate & guanylate kinases)

- nucleoside diphosphate kinase

AMP + ATP 2 ADP

GMP + ATP GDP + ADP

accomplished by separate enzymes

GDP + ATP GTP + ADP

same enzyme acts on all nucleotide di & triphosphatesnucleoside diphosphate kinase is an enzyme which playsa key role in the activation of antiviral nucleosides such as Retrovir/AZT

29

30

Ribose-5-P PRPP

ribose-P-pyrophosphokinase5-P-ribosylamine

amidophosphoribosyltransferase

AMP + GMP

ADP + GDP

activation

3131

Salvage of Purines

32

O

HH

OHOH

CH2

H O

OP

O

OH

OH

H

P

O

O-

O P

O

O-

O-

O

HH

OHOH

CH2

H H

OP

O

OH

OH N N

NN

NH2

adenine

PPi

Adenine phosphoribosyltransferase (APRT)

Salvage is needed to maintain the purine pool Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in (biosynthesis is not completely adequate, especially in neural tissue)neural tissue)

Hypoxanthine-guanine phosphoribosyltransferase Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)(HGPRT)

Hypoxanthine + PRPP Hypoxanthine + PRPP IMP + Ppi IMP + Ppi

Guanine + PRPPGuanine + PRPP GMP + PpiGMP + Ppi

Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cellsmetabolized to uric acid, which damages cells

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Salvage of PurinesSalvage of Purines

Salvage of purine bases

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Salvage of purines

Lesch-Nyhan syndromeLesch-Nyhan syndrome

there is a defect or lack in the HGPRT enzymethere is a defect or lack in the HGPRT enzymethe rate of purine synthesis is increased about the rate of purine synthesis is increased about 200X200Xuric acid level rises and there is gouturic acid level rises and there is goutin addition there are mental aberrationsin addition there are mental aberrationspatients will self-mutilate by biting lips and patients will self-mutilate by biting lips and fingers offfingers off

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Lesch-Nyhan syndromeLesch-Nyhan syndrome

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Purine AutismPurine Autism

25% of autistic patients may 25% of autistic patients may overproduce purinesoverproduce purines

To diagnose, must test urine over 24 To diagnose, must test urine over 24 hourshours– Biochemical findings from this test Biochemical findings from this test

disappear in adolescencedisappear in adolescence– Must obtain urine specimen in infancy, Must obtain urine specimen in infancy,

but it’s difficult to do!but it’s difficult to do!

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Origin of atoms in pyrimidine ringOrigin of atoms in pyrimidine ring

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Step 1: synthesis of carbamoyl Step 1: synthesis of carbamoyl phosphatephosphate

Condensation of glutamine, bicarbonate in the Condensation of glutamine, bicarbonate in the presence of ATPpresence of ATP

Carbamoyl phosphate synthetase exists in 2 Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme biosynthesis) and CPS-II, a cytosolic enzyme used hereused here

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Step 1: pyrimidine synthesisStep 1: pyrimidine synthesis

CPS-II is the major site of regulation in animals: UDP andUTP inhibit the enzyme and ATP and PRPP activate itIt is the committed step in animals

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Step 2: synthesis of carbamoyl Step 2: synthesis of carbamoyl aspartateaspartate

enzyme is aspartate transcarbamoylase (ATCase)enzyme is aspartate transcarbamoylase (ATCase)catalyzes the condensation of carbamoyl phosphate catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Piwith aspartate with the release of PiATCase is the major site of regulation in bacteria; it ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTPis activated by ATP and inhibited by CTPcarbamoyl phosphate is an “activated” compound, so carbamoyl phosphate is an “activated” compound, so no energy input is needed at this stepno energy input is needed at this step

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Step 2: pyrimidine synthesisStep 2: pyrimidine synthesis

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Step 3: ring closure to form Step 3: ring closure to form dihydroorotatedihydroorotate

enzyme: dihydroorotaseenzyme: dihydroorotase

forms a pyrimidine from carbamoyl forms a pyrimidine from carbamoyl aspartateaspartate

water is released in this processwater is released in this process

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Step 3: pyrimidine Step 3: pyrimidine synthesissynthesis

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Step 4: oxidation of dihydroorotate Step 4: oxidation of dihydroorotate to orotateto orotate

an irreversible reactionan irreversible reaction

enzyme: dihydroorotate dehydrogenaseenzyme: dihydroorotate dehydrogenase

oxidizing power is derived from quinones oxidizing power is derived from quinones (thru coenzyme Q)(thru coenzyme Q)

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Step 4: pyrimidine synthesisStep 4: pyrimidine synthesis

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Step 5: pyrimidine synthesisStep 5: pyrimidine synthesis

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Step 6: pyrimidine Step 6: pyrimidine synthesissynthesis

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The big picture again

5252

Orotic aciduriaOrotic aciduria

an inherited human disease caused by a an inherited human disease caused by a deficiency in the multifunctional enzyme that deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine catalyzes the last 2 steps in the pyrimidine synthesissynthesislarge amounts of orotic acid in urinelarge amounts of orotic acid in urineretarded growth and severe anemiaretarded growth and severe anemiatreat by administration (injection) of uridine treat by administration (injection) of uridine and/or cytidineand/or cytidine

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Leflunomide (Arava)Leflunomide (Arava)

Leflunomide is an isoxazole immunomodulatory Leflunomide is an isoxazole immunomodulatory agent agent which inhibits dihydroorotate dehydrogenasewhich inhibits dihydroorotate dehydrogenase)) and has antiproliferative activity. Several and has antiproliferative activity. Several in vivoin vivo and and in vitroin vitro experimental models have experimental models have demonstrated an anti-inflammatory effect. demonstrated an anti-inflammatory effect.

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Leflunomide (Arava)Leflunomide (Arava)

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Synthesis of uridine and cytidine triphosphate

56

UMP + ATP UDP + ADP

UDP + ATP UTP + ADP

nucleoside diphosphate kinase

CTP synthase (cytidylate synthetase)

N

N

O

H

O

Ribose 3 phosphate

N

NO

Ribose 3 phosphate

NH2glutamine +ATP

Glutamate +ADP +Pi

CTPUTP

(in bacteria, ammonia donates the amino group)

Regulation of pyrimidine nucleotide biosynthesis

57

Glutamine +HCO3

- +ATP

Carbamoylphosphate

carbamoyl phosph.

synthetaseOrotate

OMPUMPUTP + CTP

orotatephosphoribosyltransferase

UTP and CTP are feedback inhibitors of CPS II

Formation of deoxyribonucleotides

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O

HH

OHOH

CH2

H H

OP

O

OH

OH Base

O

HH

HOH

CH2

H H

OP

O

OH

OH Base

ribonucleotide reductase

dADP, dGDP, dUDP and dCDP are all synthesized by the same enzymeSynthesized from nucleoside diphosphate (not mono or triphosphate) byribonucleotide reductase

Synthesis of dTMPSynthesis of dTMP

Methylation of d-UMP via NMethylation of d-UMP via N55,N,N1010-methylene -methylene THFTHF

Reaction inhibited by 5-fluorouracil Reaction inhibited by 5-fluorouracil (Efudex)(Efudex)

5959

OCH2

H

OH

H

H

H

OPHO

O

OH

N

HN

O

O

F

H

H

HN

N

O

O

F

H

H

5-fluorouracil

Activation of 5-fluorouracil

60

dUMP dTMP

NADPH + H+

NADP+

SERINE

GLYCINE

Regeneration of N5,N10 Methylen THF

DHFN5,N10 – METHYLENE-THFN5,N10 – METHYLENE-THF

THF

dihydrofolate reductaseserine hydroxymethyl transferase

thymidylate synthase

Inhibitors of N5,N10 Methylene THF Regeneration

DHF

THF

FdUMP

dUMP dTMP

NADPH + H+

NADP+SERINE

GLYCINE

N5,N10 – METHYLENE-THF

dihydrofolate reductaseserine hydroxymethyl transferase

METHOTREXATE AMINOPTERIN TRIMETHOPRIM

thymidylate synthase

Hydroxyurea (Hydrea)Hydroxyurea (Hydrea)inhibits the enzyme ribonucleotide reductaseinhibits the enzyme ribonucleotide reductase– DNA synthesis cannot occurDNA synthesis cannot occur– Cell are killed in the S phaseCell are killed in the S phase– Drug holds other cells in the GDrug holds other cells in the G11 phase phase

Primarily used to treat chronic myelogenous Primarily used to treat chronic myelogenous leukemialeukemiaCancer cell develop resistance by:Cancer cell develop resistance by:– increasing quantity of inhibited enzymeincreasing quantity of inhibited enzyme– decreasing sensitivity of enzyme for inhibitordecreasing sensitivity of enzyme for inhibitor

used orallyused orallymajor side effect is leukopeniamajor side effect is leukopenia

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H2N

N OH

O

H

HYDROXYUREA

64

N

N

NH2

O

OHOCH2

OH F

F

2',2'-DIFLUORODEOXYCYTIDINE

Another inhibitor of ribonucleotide reductase:indicated for non-small cell lung cancer (usually with cisplatin) also first line treatment for non-resectable pancreatic cancer

65

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Digestion: RNA + DNA Nucleotides Nucleosides

Base + 1-P-ribosenucleosidase

base + ribose

N

N N

N

Ribose-P

NH2

N

N N

N

Ribose-P

OH

H2O NH3

AMP deaminase

N

N N

N

Ribose

NH2

Nucleotidase

H2O

Pi

HN

N N

N

Ribose

O

Adenosine deaminase

H2O NH3

Nucleotidase

H2O

Pi

Purine nucleosidephosphorylase

HN

N N

N

H

O

may be reusedthroughsalvage pathway

Pi

Ribose-1-P

hypoxanthine

Degradationof AMP

68

PENTOSTATIN

69

N

HOH

N

NN

OHOCH2

OH H

H

H

H

H

H

2'-DEOXYCOFORMYCIN

previously called deoxycoformycin (DCF)

a purine analog with a 7-membered-ring

potent inhibitor of adenosine deaminaseADA is a key enzyme which regulatesadenosine levels in cells

indicated for refractory hairy cell leukemia

other uses: chronic lymphocytic leukemiaand lymphomas

ADA deficiencyADA deficiency

In the absence of ADA lymphocytes are destroyedIn the absence of ADA lymphocytes are destroyeddeoxyadenosine is not destroyed, is converted to deoxyadenosine is not destroyed, is converted to dAMP and then into dATPdAMP and then into dATPdATP is a potent feedback inhibitor of dATP is a potent feedback inhibitor of deoxynucleotide biosynthesisdeoxynucleotide biosynthesisthis leads to SCID (severe combined this leads to SCID (severe combined immunodeficiency disease)immunodeficiency disease)Infants with this deficiency have a high fatality rate Infants with this deficiency have a high fatality rate due to infectionsdue to infections

7070

ADA deficiencyADA deficiency

treatment consists of administering treatment consists of administering pegylated ADA which can remain in the pegylated ADA which can remain in the blood for 1 – 2 weeksblood for 1 – 2 weeks

more efficient is gene therapy: replacing the more efficient is gene therapy: replacing the gene that is missing or defectivegene that is missing or defective

gene therapy has been performed on gene therapy has been performed on selected patientsselected patients

7171

N

N N

N

O

H

Ribose-P

H2N

N

N N

N

O

H

Ribose

H2N

H2O

Pi

N

N N

N

O

H

H

H2N

PNPPi

Ribose-1P

N

N N

N

O

H

Ribose-P

O

H

H2O

Pi

N

N N

N

O

H

Ribose

O

HPi

Ribose-1P

N

N N

N

O

H

H

O

H

H2O NH3

PNP

nucleotidase nucleotidase

Degradation ofGMP and XMP

72

N

N N

N

NH2

H

N

N N

N

OH

H

H2N

N

N N

N

OH

H

N

N N

N

OH

H

HO

ADENINE GUANINE(6-AMINOPURINE) 2-AMINO-6-OXYPURINE)

HYPOXANTHINE(6-OXYPURINE)

XANTHINE(2,6-DIOXYPURINE)

N

N N

N

H

PURINE

N

N N

N

OH

H

HO

OH

URIC ACID(LACTIM FORM)

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ADENINE + H2O HYPOXANTHINE + AMMONIA

GUANINE + H2O XANTHINE + AMMONIA

HYPOXANTHINE + O2 + H2O

adenase

guanase

XANTHINE + H2O2

XANTHINE + O2 + H20 URIC ACID + H2O2

xanthineoxidase

xanthineoxidase

CATABOLISM OF PURINES

74

N

N N

N

O

H

H

O2 H202

N

N N

N

O

H

H

O

HHYPOXANTHINE

XANTHINE

N

N N

N

O

H

H

O

H

O

H

N

N N

N

O

H

H

O

H

OH

acidic proton

URIC ACID

O2

H202

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GOUTGOUT

Associated with abnormal amounts of urates in Associated with abnormal amounts of urates in the bodythe bodyEarly stage: recurring acute non- articular Early stage: recurring acute non- articular arthritisarthritisLate stage: chronic deforming polyarthritis and Late stage: chronic deforming polyarthritis and eventual renal complicationeventual renal complicationDisease with rich history dating back to Disease with rich history dating back to ancient Greeceancient Greece

7676

GOUTGOUT

Once fashionable to associate gout with Once fashionable to associate gout with intelligenceintelligence

People with gout:People with gout:– Isaac NewtonIsaac Newton– Benjamin FrankinBenjamin Frankin– Martin LutherMartin Luther– Charles DarwinCharles Darwin

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GoutGout

prevail mainly in adult malesprevail mainly in adult malesrarely encountered in premenopausal womenrarely encountered in premenopausal womensymptoms are cause by deposition of crystals symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be of monosodium urate monohydrate (can be seen under polarized light)seen under polarized light)usually affect joints in the lower extremities usually affect joints in the lower extremities (the big toe is the classic site)(the big toe is the classic site)

7878

GoutGout

7979

Diagnostic featuresDiagnostic features

usually affect joints in the lower extremities usually affect joints in the lower extremities ( 95%)( 95%)

onset is fast and suddenonset is fast and sudden

pain is usually severe; joint may be swollen, pain is usually severe; joint may be swollen, red and hotred and hot

attack may be accompanied by fever, attack may be accompanied by fever, leukocytosis and an elevated ESRleukocytosis and an elevated ESR

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Drugs which may induce Drugs which may induce hyperuricemiahyperuricemia

niacinniacin

thiazides and other diureticsthiazides and other diuretics

low dose aspirinlow dose aspirin

pyrazinamidepyrazinamide

ethambutolethambutol

cyclosporinecyclosporine

cytotoxic drugscytotoxic drugs

8181

Non-pharmacological Non-pharmacological approachesapproaches

Avoid purine rich foods:Avoid purine rich foods:– red meat and organ meat (liver, kidneys)red meat and organ meat (liver, kidneys)– shellfish, anchovies, mackerel, herringshellfish, anchovies, mackerel, herring– meat extracts and graviesmeat extracts and gravies– peas and beans, aspargus, lentilspeas and beans, aspargus, lentils– beer, lager, other alcoholic beveragesbeer, lager, other alcoholic beverages

Weight lossWeight lossControl alcohol (binge drinking)Control alcohol (binge drinking)

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Pharmacological management of Pharmacological management of goutgout

based on the premise that the based on the premise that the hyperuricemia is due to both hyperuricemia is due to both overproduction and underexcretion of uric overproduction and underexcretion of uric acidacidsymptomatic relief of pain is also achieved symptomatic relief of pain is also achieved with analgesics (i.e. indomethacin)with analgesics (i.e. indomethacin)drugs used:drugs used:– analgesics (NSAIDs)analgesics (NSAIDs)– uricosuric agentsuricosuric agents– xanthine oxidase inhibitorsxanthine oxidase inhibitors

8383

Colchicine

84

OCH3

CH3O

CH3O

OCH3

O

N

C CH3

H

O

COLCHICINE

a non-basic alkaloid from the seeds and corms of Colchicum autumnale (Meadow Safron)

COLCHICINECOLCHICINEused in the symptomatic treatment of acute used in the symptomatic treatment of acute attacks of goutattacks of goutdecreases leukocyte motility, decreases decreases leukocyte motility, decreases phagocytosis and lactic acid productionphagocytosis and lactic acid productionnot used in other forms of arthritisnot used in other forms of arthritisa very potent druga very potent drugcan cause severe GI distress and abdominal can cause severe GI distress and abdominal painpain

8585

Inhibits the tubular reabsorption of uric acid Inhibit the tubular excretion of certain organic acid

via the transporter Also used to enhance plasma concentration of certain

anti-infective (beta lactams)

86

SHO2C N

O

O

C3H7

C3H7

PROBENECID (BENEMID)

ALLOPURINOL (Zyloprim)ALLOPURINOL (Zyloprim)

prevention of attacks of gouty arthitis prevention of attacks of gouty arthitis and nephropathyand nephropathyalso used during chemotherapy of also used during chemotherapy of cancer and to prevent recurrent calcium cancer and to prevent recurrent calcium oxalate calculioxalate calculimetabolized to oxypurinol (also an metabolized to oxypurinol (also an inhibitor of xanthine oxidase)inhibitor of xanthine oxidase)inhibits the metabolism of certain inhibits the metabolism of certain anticancer drugs (6-MP, azathioprine)anticancer drugs (6-MP, azathioprine)

8787

88

N

N N

N

OH

H

ALLOPURINOL (ZYLOPRIM)

An inhibitor of xanthine oxidase; prevents the formation of uric acid fromprecursorial purines

8989

Catabolism of a pyrimidine

9090

9191

9292

9393