Post on 25-Dec-2015
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IV: Mitochondrial Function (cont’d). HEPATIC DETOXIFICATION OF a) monoamines, alcohol, toluene b) dietary or endogenous purines
(meat), heme (red meat) & bilirubin,
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a) Detoxification of monoamines catalysed by mitochondrial outer membrane MAO
R-CHNH2
O2
H2O2
R-CH=NH
NH3
R-CHO
R-COOH-oxidation
CO2
ATP, HCO3
-
UREAurea cycle
MONOAMINE OXIDASEFlavin-containing amine oxidase
(imine)
inner membrane
outer membrane
e.g.,dopaminenorepinephrinetyraminephenethylamineoctylamineserotonin
aldehyde dehydrogenase
NAD+
NADH
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Amine specificity for the two isoforms (A & B) in humansMAO A preferentially metabolizes serotonin.MAO B preferentially metabolizes phenethylamine, dopamine.
MAO inhibitor + dietary amines or proprietary drugs
MAOB inhibitor deprenyl (selegiline) similar to phenethylamine and increases brain dopamine levels. This is used to treat Parkinson’s disease.
But “Hypertensive crisis” is a hyperadrenergic state induced by MAO inhibitors + pressor amines (e.g., tyramine in cheese,beer,wine or soya sauce) or proprietary drugs (e.g., L-DOPA, mazindol, ephedrine, etc).
Cheese Tyramine (g/g)
English Stilton 1157
Blue 998
Mozzarella 158
Feta 76
Processed cheese slice nil
Cheese Tyramine (g/g)
Cheddar cheese, old 1530
Beer 2-11
Sherry Wine 3
Chianti Wine 25
Perry, 1996. http://www.vh.org/adult/provider/psychiatry/CPS/19.html
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b) Detoxification of alcohols by matrix ALDH2
ETHANOL acetaldehyde
cytosolic alcoholdehydrogenase
NAD+NADHMethylpyrazole
Covalently binds to protein-NH2
NAD+
NADH
aldehydedehydrogenase
acetate ATP
CoASHacetylCoA
CO2
TCAcycle
ANTABUSE (disulfiram) or cyanamide
ADH ALDH Suscept. to alcoholism Appearance social drinking
Chromosome # 4 9, 12, 17
Caucasians 99% normal 90% normal 10% (>) --
Japanese 90% atypicial 40% deficient < 10% flushing
(ADH)
(ALDH)
CONHCH2COOH
Hippuric acid
URINE
glycine
CH3 CH2OH CHO
COOH
Toluene Benzyl alcohol Benzaldehyde
Benzoic acid
ADH
ALDH1/ALDH2
Cyt P-450
Teratogen Update: Toluene teratology. 55, 145-51, (1997)
Benzoyl-CoA synthetase
ATPCoA
Benzoyl-CoA
N-acyltransferase
c) Benzoic acidosis induced by toluene glue sniffing
ER CYTOSOL
MITOCHONDRIA
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8. Hemoprotein toxicity diseases caused by heme or bilirubin
A) Rhabdomyolysis (drug induced) releases myoglobin and heme and causes kidney mitochondrial damageB) Kernicterus:- bilirubin causes neonatal brain mitochondrial damage
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A) Myoglobin mediated rhabdomyolysis rarely caused by statins STATIN Drug BAYCOL withdrawn by BAYER, 2001
Mb Fe accumulates in kidney renal tubuleAcid lipid peroxidation (proximal tubule) mitochondrial toxicity KIDNEY Failure (7% of all cases of acute renal failure)
Muscle Cell Drug reactive metabolites Mitochondrial/cytotoxicity
CRUSH INJURYOr if LAIN IMMOBILE for hrs (e.g. EtOH, heat stroke)
Myoglobin releaseMassive muscle breakdown
a) x10 creatine phosphokinaseb) Dark brown granular casts in urine
Spontaneous myalgiasMuscle tenderness, Weakness, malaise, feverDEATH
THERAPY1.) NaHCO3 base2.) Desferoxamine
CMAJ 165(5) pg 632 (2001); J. Biol. Chem. 273, 31731-7 (1998)
Acid
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ie. Kernicterus (brain damage caused by bilirubin in the newborn (hyperbilirubinemia))
• re-emergence (due to shorter hospital time, breastfeeding)• danger signs onset of jaundice 3 days following birth, vomiting, lethargy, poor feeding, fever, high pitched crying.• plasma bilirubin > 340 uM = exchange transfusion therapy plasma bilirubin > 240 uM = Phototherapy (blue light) Drug therapy Sn protoporphyrin (inhibits heme oxygenase).
Hemoglobin bilirubin
• Bilirubin binds to released brain mitochondrial serum albumin by competing toxicity (hearing
fatty acids or loss? brain damage drugs encephalopathy)
Heme oxygenase of reticuloendothelial cellsof spleen, liver, kidney
GlucuronylTransferase Appears at 7dafter birthGlucuronide
conjugatedbilirubin.• 0.3% mortality (yellow staining of brain)• family history, hemolytic diseases, Gilbert’s disease, diabetes (erythrocyte fragility), G6PD deficiency (Pediatrics 106, 1478-80 (2000), sickle cell.
B) Hemoglobin/heme mediated brain damage
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Metabolic pathway of heme degradation to bilirubin and detoxification of bilirubin by glucuronidation.
NADPH O2
NADP+
H2O
NADPH: P450 Reductase
COHemeOxygenase
NADPH NADP+
Fe2+
NADPH
NADP+
NADPHBiliverdinReductase
UDP-glucuronic acidUDP
UDP-glucuronosyl transferase
(M=methyl,V=vinyl and P=propionate represent heme side chains
JAUNDICEAntioxidant, but at high concn. TOXIC TO NEONATALBRAIN MITOCHONDRIA
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9. HEME BIOSYNTHESIS BYMITOCHONDRIA AND GENETIC
DISEASES
A) Heme biosynthesis for cytochromes, etc.B) Genetic diseases: Porphyrias and porphyrin toxicityC) Oxidative degradation of heme to bilirubin
Heme synthesis
• Heme required for synthesis of mitochondrial cytochromes and endoplasmic reticular P450s.
• Heme required for bone marrow synthesis of hemoglobin and muscle myoglobin
• STEP 1 for heme synthesis is the synthesis of aminolevulinic acid (ALA) from succinyl CoA of the citric acid cycle and glycine
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Heme biosynthesis in bone marrow (hemoglobin) and liver (cytochromes)
R.L.S. ALA synthetaseCOO
CH2
CH2
C
O
S CoA
Succinyl CoA
COO
CH2
COO
COO
CH2
CH2
C CH2
NH3
O
H+ CO2 + CoA
Feedback inhibition by heme(also induces heme oxygenase) Aminolevulinate
(ALA)
Glycine
HEME BIOSYNTHESISSTEP II
(CYTOSOL)
Fe, O2
COO
CH2
CH2
C CH
O
O
4,5-dioxovaleric acid (DOVA)
1) Brain Neuropsychiatric problems in acute porphyria, 2) Liver necrosis / cancer particularly if Fe overload
1) HEME arginate2) High carbohydrate
+
glucose-6-P NADPH
GSH reductaseGSH peroxidase
H2O2
-ketoglutaratesuccinate
citricacidcycle
CO2 + CoA
dehydrogenasecomplex
Therefore ANAPLEROTICreaction required to replacesuccinyl CoA in citric acidcycle.
ALA synthetase induced by:1) Heme deficiency due to excess P450 synthesis/induction e.g., by barbiturates Sulfonamides: Therefore an increase in urine porphyrins2) ERYTHROPOIETIN (formed by kidneys) improves quality of life of patients on kidney dialysis.3) Acute intermittent porphyria or ALA dehydratase deficiency.4) LEAD
Mitochondrial Matrix
pyridoxal
-ketoglutarateALA toxicity
Arch Biochem Biophys. 373, 368-74, (2000)
ALA SYNTHESIS AND TOXICITY
ALA accumulatesa) Acute intermittent porphyriab) Lead
NH4+, O2
ROS
DNA strand breaks
DNA ADDUCTSLIVER CANCER
Therapy
1
or leadNH2
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Erythropoietin synthesised by kidney induces hemoglobin synthesis in bone marrow (replaces blood transfusion!)
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Heme Biosynthesis - STEP 2 in the cytosol (4 enzymes 2-5)
NH HN
NH HN
COOH
COOH
COOH
COOH
HOOC
HOOC
HOOC
coproporphyrinogen I
CYP1A2
NH HN
N HN
PA
PA
A
P P
A
NH HN
NH HN
HO
COOH
COOH
COOH
COOH
HOOC
HOOC
HOOC
non-enzymatic
COOH
COOH
NH
HOOC
HOOC NH2
COOH
H2N
O
NH HN
NH HN
COOH
COOH
COOH
HOOC
HOOC
HOOC
COOH
COOHUroporphyrinogen III synthase
NH HN
NH HN
COOH
COOH
COOHCOOH
CYP1A2
LIVER/BONE MARROW cytosol
ALA
Uroporphyrin
Coproporphyrinogen III
Uroporphoryrinogen decarboxylase
-4CO2
Uroporphyrinogen I
Symmetrical
Porphobilinogen (PBG)
PBGSYNTHASE
r.l.s polymorphism
Hydroxymethylbilane
ALA DehydrataseDeficiencyPorphryia *
phosphobilinogendeaminase
Acute IntermittentPorphyria*
CongenitalErythropoieticPorphyria
PorphyriaCutaneaTarda
PorphyriaCutaneaTarda
(UROPORPHYRIA)
Cutaneous Porphyria
* Acute Porphyria
2
3
4
5
5
STEP 1
STEP 3
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Heme Biosynthesis - STEP 3 - mitochondrial final steps (3 enzymes)
NH N
N HN
COOHCOOH
NH HN
NH HN
COOHCOOH
N N
N N
COOHCOOH
Fe2+
MITOCHONDRIA
O2 CO2 mitochondrialintermembrane space
Protoporphyrinogen IX
Protoporphyrinogen oxidase
i.m.
O2
Protoporphyrin IX
Ferrochelatase
i.m.r.l.s
HEME
Coproporphyrinogen oxidase
NH HN
NH HN
COOH
COOH
COOHCOOH
Coproporphyrinogen III
HereditaryCoproporphyria *
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VariegatePorphyria *
Protoporphyria
Acute Porphyria
* Cutaneous Porphyria
STEP 2
Fe
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I. ENZYME INHIBITORS CAUSING PORPHYRIA
PORPHYRIA - Greek “porphyros” (purple urine) an over-production disease. Often neuropsychiatric (syphilis much more so).
2) ALA dehydratase - lead
5) Uro’gen decarboxylase - PCBs, dioxin, lead, cadmium, hexachlorobenzene(fungicide - seeds). Mitochondrial oxidase - steroids (birth control pills)and estrogens
8) Ferrochelatase – lead
II. GENETIC PORPHYRIAS (Emerg Med Clin.N Am 23 (2005)885-899)
2) ALAD deficiency porphyria (Seminars in Liver Disease, 18, 95-101, 1998) excrete ALA not PBG, symptoms like (neurological).
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3) Acute intermittent porphyria (i.e. phosphobilinogenin deaminase deficiency) excrete ALA and PBG , the most common hepatic porphyria)- MEDALERT bracelet1. Symptoms - excrete PBG and ALA in the urine , high plasma PBG/ALA
- severe abdominal pain, nausea, vomiting, hypertension(cardiovascular)
then CNS - anxiety, insomnia, confusion, hallucination, paranoia then peripheral neuropathy - fatal respiratory paralysis. Liver cancer.
2. a. PBG deaminase deficiency - gene locus 11q24 (>100 mutations), autosomal dominant 1:10,000-50,000 but 1:1000 in N. Sweden (Lapland) b. Precipitated by phenytoin, phenobarbital drugs, alcohol, fasting, hormones estrogen, stress, infection or lead poisoning
3. Biochemistry of neurotoxicity a. heme deficiency P450 drug metabolism tryptophan dioxygenase brain tryptophan brain serotonin mitochondrial cytochromes (mitoch.disease) b. ALA “ROS”, mitochondrial DNA damage.4. Therapy –ALA with heme arginate (taken up by liver not b.m.), high glucose5. Mouse model (PBG knockout) - called Vincent! - behavioural studies Suspects - Van Gogh, King George III(?) (Absinthe for insomnia) 6.Diagnosis :- Patients urine exposure to sunlight (reddish brown fluorescence)
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4) Congenital erythropoietic porphyria (Erythroid, Photosensitive)-Fe overload of b.m., uroporphyrin overload of skin and erythrocytes hemolysis
1. Symptoms: skin lesions (light) , risk of infection in early infancy Anemia (hemolysis)
Teeth reddish brown (fluorescence)2. Uroporphyrinogen III synthase deficiency
• Uroporphyrinogen I and coproporphyrin I accumulation in the skin, bone marrow, erythrocytes, urine, plasma
3. Treatment: activated charcoal (oral); b.m. transplant; gene therapy for bone marrow; blood transfusion to stop erythropoiesis in b.m.4. Only 200 diagnosed so far; werewolf, vampires (early childhood sensitiveto light skin blisters, infection disfigures) is not porphyria
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5) Porphyria Cutanea Tarda - “heme deficiency and uroporphyrinoverload of liver” (Photosensitive; commonest porphyria;readily treated).
1. Symptoms: lesions on backs of hands and face due to photosensitization byuroporphyrin I, III or uroporphyrinogen. Abdominal pain, neural,psychiatric. ALA,porphobilinogen. 2a. Diagnosis :- urinary uroporphyrin >> coproporphyrin. b. Uroporphyrinogen decarboxylase deficiency (liver but also erythrocyte in type II): or hexachlorobenzene or dioxin induced CYP 1A2. (Turkey bread disaster). Also Fe uroporphyrinogen III synthase - uroporphyrin I.c. Candidate hemochromatosis gene leads to diagnosis of hemochromatosis
3. Precip. by alcohol, hepatitis C virus, estrogen (birth control, post-meno- pause, prostate, pregnancy), Fe. 4. Biochemistry - inactivation of decarboxylase (-SH enzyme) by “ROS” from reduced P450/P450 reductase/Fe5. Therapy:- Fe removal (phlebotomy or desferoxamine and ascorbic acid)
- chloroquine endocytosis and releases uroporphyrin chloroquine complex from hepatocytes - heme?
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6) Hereditary Coproporphyria - coproporphyrinogen oxidase deficient, chronic fatigue syndrome. - hepatic porphyria Symptoms: liver cancer
7) Porphyria variegata - protoporphyrinogen oxidase 50% deficient. –hepatic and skin porphyria Gene locus 1q2320. 20,000 S. Africans descended from a Dutch lady in 1688 who went to S. African for marriage to another Dutch settler . Now 3/1000 S.Africans have this! Symptoms: skin and acute porphyria if exposed to sunlight,neurovisceral symptoms (abdominal pain),liver cancerDiagnosis :- plasma fluoresces when exposed to UV.Precipitated by sunlightTreatment: increase ALA synthetase by heme arginate, glucose ( NADPH) ?
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8) Erythropoietic protoporphyria - associated with liver cancer, red urine “porphyrin and Fe overload disease” “Skin photosensitive” a. Ferrochelatase decreased 75-90% b. Symptoms: photosensitive skin (infancy),velvet knuckles, black liver c. Biochemistry - protoporphyrin IX accumulates in erythrocyte membranes and liver; intramitochondrial Fe accumulates in bone marrow - skin photo- sensitivity results from protoporphyria IX effluxing erythrocytes into plasma. d. Treatment – topical sunscreens and oral beta-carotene (ROS scav.?) e. Diagnosis –incr. protoporphyrin in erythrocytes,plasma,bile,feces I. Liver transplant; operation is difficult as operating room must be kept dark (only yellow acetate filters) II. Abdominal burns; transfused blood is destroyed as blood difficult to store.
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Detoxification function of the livera) heme toxin oxidation to bilirubinb) detoxification of bilirubin by glucuronidation or albumin
phagosomeHemeoxygenase
albumin LIVER
CO
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Degradation of heme STAGE 1Hemoglobin of old erythrocyte trappedin spleen
NH
NH
N NH
CH
CH
CH
OO
NH
NH
NH
NH
CH
CH2
CH
OO
M V M P P M M V
VMMPPMVM
BiliverdinNADPH + H+
NADP+
Biliverdin Reductase
Excreted by Reptilesand birds
HEMEO2 + NADPH
H2O + NADP+
Fe3+
BILIRUBIN(Neurotoxin to babies)
Bone marrow
transferrin
Plasma Serum Albumin Complex LIVER
Modified fromFig. 28-31 Stryer4th Ed.
NEONATAL JAUNDICEBilirubin accumulates in newborns.(destroy with sunlight).
Heme oxygenase (ER)
CO
H2O soluble
exhaled
ANTIOXIDANT
Fat soluble
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STAGE II - ligandin in hepatocyte surface membrane traps bilirubinfrom plasma and helps transport it into the liver where it is glucuronidated
Albumin- bilirubin
(a GSH transferase)
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*mutated gene in Gaucher’s disease(carried by 15% ofpopulation)
NH
NH
NH
NH
CH
CH2
CH
OO
VMMMVM
COOCOO
OH
OH
H
UDP
H
OHH
OH
COO
UDP-glucuronate
+
Bilirubin
Endoplasmic reticulumUDP-glucuronosyltransferase (UGT 1A1*)
NH
NH
NH
NH
CH
CH2
CH
OO
VMMMVM
COOC
OH
OH
H
O
H
OHH
OH
COO
O
BILIRUBIN DIGLUCURONIDE (Soluble bilirubin diglucuronide secreted
into the bile)
BILIRUBIN MONOGLUCURONIDE
UDP
UDP gluc. + UGT 1A1
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10. a) N-Catabolism of Amino Acids (The Urea Cycle) and the Detoxification of Ammonia (very toxic)
Mitochondrial Function (Con’t).
b) N-Catabolism of Purines• reperfusion injury and drug therapy• Genetic diseases: HGPRT• Gout, Lesch-Nyhan syndrome
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Disposal of nitrogen by mitochondria a) The urea cycle: Detoxification of NH3
ALANINE
Alanineaminotransferase
Pyruvate
-ketoglutarate
glutamate
aspartateaminotransferase
aspartate
oxaloacetate
citrulline +
arginine +fumarate
fumarase
malatemalate
dehydrogenase
UREA
phospho-enol-pyruvate
INNER MITOCHONDRIAL MEMBRANE transporter
Pi
Cytosol
MitochondrialMatrix
Pyruvate
Alanine
malate
NAD+
glutamatedehydrogenase
NADH
citrulline
NH3
ornithinetranscarbamoylase
2 ATP
+ HCO3- carbamoyl phosphate
synthetase
H+
-ketoglutarate
glutamate
oxaloacetate
arginosuccinatesynthetase argininosuccinase
H2Oarginase
ornithine
ornithine
carbamoylphosphate
transporter
Alanineaminotransferase
CO2
(note: urea cycle enzyme in blue)
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Formation of urea from amino acids
Glutamate -ketoglutarate
Mitochondrial Function (Con’t).
transaminase
dehydrogenase
Urea cycle
dehydrogenase-amino acid -ketoglutarate NADH + NH3
+
NAD++ H2OGlutamate-Keto acid
H2N C NH2
O
UREA
+ NAD(P)+ H2O+ NH4+
+ + NAD(P)HH3N CH
COO
CH2
CH2
COO
O CH
COO
CH2
CH2
COO
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IV: Peroxisomes Function• β-oxidation of fatty acids but oxidase forms H2O2
• Glyceraldehyde is metabolised to D-glycerate and glucose via glycolate (glycolate oxidase forms H2O2 and glyoxylate)
• Catalase detoxifies H2O2 to form oxygen and H2O and cooxidises other substrates, including phenols, formic acid, formaldehyde, and methanol.
• Glyoxylate detoxified by peroxisomal alanine:glyoxylate transaminase to form glycine & pyruvate. Otherwise glyoxylate would be oxidised by lactate dehydrogenase to oxalic acid which causes oxalate stone formation & kidney damage.
• Biosynthesis of bile acids and ether phospholipids
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Disposal of nitrogen (Con’t): b) N-catabolism of purines in the cytosol & peroxisomes
HG
PR
T +
PR
PP
HG
PR
T +
PR
PP
Voet pg. 714.
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1) N-catabolism of purines can be lethal Reperfusion Injury
e.g. Myocardial infarction or Paraplegia
a) ISCHEMIC STAGE
• Lack of O2no ATP synthesis by heart muscle mitochondria. ADPAMPhypoxanthine accumulates and leaves muscle cell to enter endothelial cell.• Acidosis causes endothelial cell xanthine dehydrogenasexanthine oxidase• Acidosis results from marked increase in glycolysis to form ATP + lactic acid (to keep cells alive by maintaining ATP levels)
b) Reperfusion of O2
• O2 is the substrate for xanthine oxidase
•Damaged endothelial and muscle cells (ischemic toxicity) release factors which recruit neutrophils (inflammatory reaction activated NADPH oxidaseO2*-.
Accumulated xanthine and hypoxanthine O2 (Endothelial cell)
Cytosolic Fraction
Xanthineoxidase
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Drugs which prevent reperfusion injury
1) Allopurinol – inhibitor of xanthine oxidase2) Desferal – Complexes Fe2+
3) Antioxidants e.g. Lazeroids (upjohn) to prevent membrane lipid peroxidation4) Removal of H2O2 by NADPH (e.g. glucose) or GSH (e.g. methionine) or dietary Se.
H2O2 OH
H2O
GSSG + H2O
GSH Peroxidase
Catalase (activated by NADPH)
2 GSH
5) Anti-inflammatory drugs (e.g. indomethacin or phenylbutazone)6) Ca2+ Blockers
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Treatment or to prevent Ischemic damage (reperfusion injury)
HC
NC
C
CN
HN
CH
N
OH
HC
NC
C
CN
HC
N
N
OH
HYPOXANTHINE ALLOPURINOL
Xanthine Oxidase
C
NC
C
CN
HC
N
N
OH
HO
ALLOXANTHINE (oxypurinol)
The synthesis of urate from hypoxanthine and xanthine oxidase decreases soon after the administration of allopurinol.
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Figure 22.22 (Voet) – By James Gilroy• Defects in excretion of uric acid and/or partial deficiency in HGPRT activity due to mutation in HGPRT gene• Uric acid precipitation in joints – crystals activate leukocytes H2O2 and O2
*- arthritis.
• Adults (0.55%) in males• Often suffered by over-achieving males with too much drive and ambition (e.g. pharmacists).
2) Purine catabolism can be painful and cause joint damage
Drugs which trigger gout• Anticancer drug treatment of acute leukemia resulting in destruction of nucleic acids and accumulation of uric acid.• Long term diuretic treatment (impair glomerular filtration)Risk Factors• Eating too much meat (purines e.g. xanthine)• Alcohol ingestion increases hypoxanthine • age, male gender, high body mass index, hypertension.
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Hypoxanthine + PRPP PPi + IMP Guanine + PRPP PPi + GMP
3) Genetic Deficiency of HGPRT LESCH-NYHAN SYNDROME – X-linked recessive (X-chromosome gene) 50% of cells have a complete deficiency of salvage enzyme HGPRT (Hypoxanthine – guanine phosphoribosyl transferase) or lyase (ASLI) which catalyses:
Cytosolic Fraction
XMP
adenylsuccinate
AMP
Lyase(ASLI)
synthetase
Nucleotidesynthesis
adenosine
PRPP (5-phosphoribosyl-a-pyrophosphate)
Results in enormous overproduction of PRPP,hypoxanthine,uric acid.
Ribose-5-phosphate
ATP ADP
Pyrophosphokinase
O
OHOH
HH
H
OH
CH2OP3O2
P O P O
O
O
O
O
HGPRT
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Children • Neurological dysfunctions ( serotonin metabolism)• Aggressive or self mutilating but affectionate, quick to laugh, likeable and open.• Tend to chew their lips and finger tips• Mental retardation• Cerebral palsy and spasticity• Obscene gestures• Flinging their feces• Die before reaching the age of 10 due to renal failure.• If mild enough, gout will develop into adulthood.
Children with LESCH-NYHAN SYNDROME(Incidence 1:380,000) (HGRPT deficiency)
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DRUG THERAPY
ALLOPURINOL is the most useful drug for treatment of GOUT.
Inhibition of uric acid production because xanthine oxidase (terminal step to uric acid production) is inactivated by allopurinol.
However, neurological problems associated with LESCH-NYHAN SYNDROME which have been attributed to PRPP and de novo purine synthesis can not be alleviated by allopurinol. Allopurinol will prevent uric acid stones accumulating in the kidney and will prevent kidney infections and hematuria.
NEUROLOGICAL TOXIC MECHANISM UNKNOWN HGPRT, which is required for purine synthesis is still functionally deficient andPRPP accumulates.Dopamine neuron function is still lost and neurological problems persist.