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IV: Mitochondrial function (e.g. hepatocytes) 1) citric acid cycle as an energy source a) pyruvate or a-ketoglutarate dehydrogenase b) lipoic acid therapy 2) the respiratory chain as an energy source 3) oxidative phosphorylation and uncouplers 4) membrane transporters and shuttles a) cytosolic NADH oxidation b) acetyl CoA (NADPH export) c) transport systems in the mitochondria d) gluconeogenesis and glucose transport 5) mitochondrial diseases and treatment a) creatine therapy b) coenzyme Q10 therapy 6) b-oxidation of fatty acids as an energy source a) starvation/diabetes/endstage renal disease b) carnitine therapy c) ketogenic diet therapy d) drug induced fatty liver and NASH e) alcohol induced fatty liver and ASH 7) hepatic detoxification of a) monoamines b) alcohols c) toluene 8) hemoprotein mediated diseases a) rhabdomyolysis b) kernicterus 9) Heme biosynthesis & porphyria a) Heme biosynthesis b) Porphyria c) Oxidative degradation of heme to bilirubin 10) Nitrogen metabolism a) N-catabolism of amino acids b) N-catabolism of purines 1 1. CITRIC ACID CYCLE AS AN ENERGY SOURCE A) Pyruvate or a-ketoglutarate dehydrogenase Requires Thiamine (Vit B1) and lipoic acid B) Lipoic acid therapy 2 An overview of the citric acid cycle Stryer 3 Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA toxic! 120uM plasma citrate complexes Fe 4 Pyruvate dehydrogenase complex . Babies born with mitochondrial diseases usually have cytochrome deficiencies but some have low pyruvate dehydrogenase E1 that causes lactic acidosis . Therapy includes thiamine (B1) or CoQ suppl, dichloroacetate or a ketogenic diet. Pyruvate decarboxylase (pyruvate to OAA)inhibited by branched chain aminoacids that accumulate in maple syrup disease, inborn error of metabolism. 5 Pyruvate dehydrogenase complex R CoA O O COO- Thiamine pyrophosphate (TPP) O R S L SH R E2 E1 OH TPP SH As(III) POISON S Lipoamide SH L Stryer Fig. 20-12. Summary of the reactions catalyzed by the pyruvate dehydrogenase complex. L. refers to the lipoyl group. CoA L L R S SH S CO2 SH As SH FADH2 NAD E3 + FAD Arsenite complex NADH + H+ Pyruvate + CoA + NAD+ acetyl CoA + CO2 + NADH (Thiamine (Vitamin BI) required) 6 Lipoic acid (thioctic acid) therapy • • • Scavenges reactive oxygen and nitrogen oxide species restores other cellular antioxidants decreases oxidative stress toxicity • prevents diabetes complications, cataracts and alcoholic liver damage • prevents Cis Pt chemotherapy induced nephrotoxicity, hypertension and aging in rats Free Rad. Biol. Med. 24, 1023-39 (1998). 7 The citric acid cycle is a source of biosynthetic precursors Glucose Pyruvate ATP, CO2 Phosphoenolpyruvate Acetyl CoA ADP, Pi Amino acids Oxaloacetate Succinyl CoA Porphyrins Citrate Stryer Fig. 20-17. Biosynthetic roles of the citric acid cycle. Intermediates drawn off for biosyntheses are replenished by the formation of oxaloacetate from pyruvate. (Anaplerotic) aketoglutarate Amino acids 8 Control of the citric acid cycle Stryer Fig. 20-22. Control of the citric acid cycle and the oxidative decarboxylation of pyruvate: * indicates steps that require an electron acceptor (NAD+ or FAD) that is regenerated by the respiratory chain. 9 2. THE RESPIRATORY CHAIN AS AN ENERGY SOURCE 10 The mitochondrial respiratory chain NADH Diagram of a mitochondrion FMNH2 complex I NADH-Q reductase 2Fe-2S 4Fe-4S Q FADH2 in flavoproteins succinate:Q reductase (complex II) complex III Cytochrome reductase Chemiosmotic theory of oxidative phosphorylation cyt c complex IV Cytochrome oxidase O2 Sequence of electron carriers in the respiratory chain 11 O H3C C H3C C H C C H C C N C C N N O NH C CH2 e- + + C H3C C C H C C H N C C N N O NH C e- + CH2 H O + H3C C H3C C H H C C H C C H N C CH N CH2 H C OH H C OH H C OH H C OH H C OH H C OH H C OH H C OH H C OH a v i n 2- CH2O PO3 m 2- o n o n u c l Se eo mt i id qe u i ( F M N ) n o n e i C N H CH2OPO3 F l O H3C H C NH C O CH2OPO32n t e r m e d i a t e R e d u c e d f l a v ) 2 Iron-sulfur complexes The reduction of flavin mononucleotide (FMN) to FMNH2 proceeds through a semiquinone intermediate Molecular models of iron-sulfur complexes: iron = red; cysteine sulfur = yellow; 12 inorganic sulfur = green NADH coenzyme Q reductase: complex I FM N NADH NAD+ eQ R O FM NH2 x QH2 N A D O O OH C H 3 CO C C H 3 CO C C C CH CH 3 ( C H 2C H C e 3 C H2 ) 10 - + C H 3 CO C H 3 CO C H e- + C CH C R + + C 3 C H 3 CO C H 3 CO C C C C O OH C ( 1 o U 0 e B S I OH n I eR n z Q m t The reduction of ubiquinone to ubiquinol proceeds through a semiquinone anion intermediate. 13 Model of NADH-Q reductase Stryer Fig 21-9 14 Q:Cytochrome c reductase (Complex III) Q cyt b (+2) QH Fe-S(+2) cyt c1(+3) cyt c(Fe+2) QH cyt b (+3) QH2 Fe-S(+3) cyt c1(+2) cyt c(Fe+3) Stryer p. 537 cytochrome c reductase Stryer Fig. 21-11 Model of a portion of Q: cytochrome c reductase 15 Cytochrome oxidase (Complex IV) Lodish Fig. 17-30 16 Electron transport can be blocked by specific inhibitor poisons NADH NADH-Q Reductase QH2 Blocked by rotenone and amytal Cytochrome b Blocked by antimycin Cytochrome c1 Sites of action of some inhibitors of electron transport Cytochrome c Cytochrome Oxidase Blocked by CN- , N3 -, and CO O2 17 Cytochrome C - catalytic site CH3 + R C CH2 HS C R ' R C S C R ' H H2 C y s t e i n e r e s iH d u e H2 V i n y l g r o u p o f t h e p r o t e Ti hn i o e t h e r o f t h e h e m e The heme in cytochromes c and c1 is covalently attached to 2 cysteine side chains by thioether linkages The iron atom of the heme group in cytochrome c is bonded to a methionine 18 sulfur atom and a histidine nitrogen atom l Spectroscopic characteristics of cytochrome c Http://www.princeton.edu/~macase/cytochrome_c.html 19 Evolutionary Tree of Cytochrome C 20 Cytochrome C - soluble NOT membrane bound 1. 26/104 amino acids residues have been invariant for > 1.5 x 109 years. 2. Met 80 and His 18 - coordinate Fe. 3. 11 residues from number 70 - 80 lining a hydrophobic crevice have remained virtually unchanged throughout all cytochrome c regardless of species or even kingdom. 4. A number of invariant arginine and lysine clusters can be found on the surface of the molecule. Cytochrome c has a dual function in the cell. Electron transport for ATP production AND the major cause of most programmed cell death (apoptosis) is initiated by the release of cytochrome c into the cytosol! 21 Origin of mitochondria: the endosymbiont hypothesis The endosymbiont hypothesis suggests that mitochondria have evolved from anaerobic bacteria which were phagocytosed by eukaryote cells at the time oxygen appeared on earth, Similarities between mitochondria and bacteria include the presence of: • cardiolipin •transporters • ribosomes • circular RNA and DNA Therefore mitochondria protein synthesis should be inhibited by: • TETRACYCLINE • CHLORAMPHENICOL. E.g. The extensive use of these drugs can inhibit 1. Bone marrow mitochondrial protein synthesis leading to a decline in the production of white or red cells. 2. Intestinal epithelial cells causing them to cease dividing. 22 3. OXIDATIVE PHOSPHORYLATION AND UNCOUPLERS 23 Oxidative phosphorylation 24 Uncouplers: Physiological use of uncoupling oxidative phosphorylation Brown fat which is very rich in mitochondria/chloroplast supply heat for newborns, hibernating animals, flowering plants. Thermogenin is a 33Kd protein which uncouples mitochondria to generate heat. thermogenin 25 MATRIX CYTOSOL pH = 7 Uncouplers of oxidative phosphorylation O H+ pH = 8 H+ OH NO2 OH O NO2 NO2 NO2 NO2 NO2 NO2 NO2 2,4 - Dinitrophenol ADP H+ H+ ATP Valinomycin OO+ O OK OO i.e. six valine residues K+ Loss of electromotive force. Energy used for K+,Na+ transport and not for the production of ATP Gramicidin 15 amino acids long which forms a transmembrane channel for Na+ entry into the matrix. Na+ Na+ 26 a - ketoglutarate O 2 level Oligomycin (inhibits ATPase) ADP in medium O Oligomycin however is an ATPase inhibitor. a - ketoglutarate 2 level Oxygen electrode shows DNP is a protonophore (short circuits proton gradient) & markedly increases oxygen uptake causing heat without ATP formation. in medium 2,4 - dinitrophenol a - ketoglutarate ADP ADP 2 level O in medium Coupled control cells Peter Mitchell 1978 Nobel Prize ATP 27 Time 4.Mitochondrial MEMBRANE TRANSPORTERS A) Cytosolic NADH oxidation B) Acetyl CoA (NADPH export) C) Transport systems in the mitochondria D) Gluconeogenesis and glucose transport 28 Compartmentalization of the major pathways of metabolism 29 a) Cytosolic NADH oxidation: membrane transporters glycerol phosphate shuttle (Bucher shuttle) 30 Vit.B6 requd. for Malate-aspartate shuttle NADH can’t permeate mitochondrial membrane but cytosol NADH must be oxidised by mitochondria to sustain glycolysis Vitamin B6 31 b) Acetyl CoA/NADPH export to cytosol for fatty acid synthesis/ drug metabolism G l u c o s e C y t P A y c r u v a t C i a t C i t t S y y l n e O x a A r a h t i g i o t a h c C + + e r a t e t C h o a As l o l e t M o s o i A C n t T o e c r A P A O o c h o n d r i c e t a t c e aT x n tP a a ml e CO2 y r u v a t P e t e a y r c l f o ( a i o a NADH lM a a C t o t Ay r d r N a c t i r t c e ta te t e z o a t a r id y m e t e t eP at y 4 ca t e n t t e e x e h s5 eb a u0 to NAD+ m P t l M ADP e a y l r i u a l a NADP+ e n c v a t e NADPH ATP CO2 T C h i e t r r e a f t o e r e L P m a y e n t a e s o l s i s e c u p P e p n l h NADPH o s f a o y mr em s z i p h a P32 a Isocitrate as an NADPH shuttle for drug metabolism G l u c o s e P y r u v a t e M O x F o u a c c e t e t u H a C O C i o A C Y T O R I A L N D t r a t S O M e e R I CI D e L NAD+ E s o c i t rI i d s e o h c y i d t r L A T R e n c l t a t NADH S y C I T C I a t Y C i t O c C A a r C m c T NAD+ l a a u S I a l NADH M A c C i o NADH e a- as to ec i t r a t NADP+ r a t e i s o o g e n ca i s t e r d e h y d r o NADPH CO2 k e t o g la- uk t e at r o ag t l eu t NAD+ n A y l P D 4 R 5 U 0 G c a M CO2 33 c) Transport systems in the mitochondria Pyruvate transporter inhibited by hydroxycinnamate Malate transporter inhibited by butylmalonate Harper’s Biochemistry. 34 d) Gluconeogenesis and glucose export by the liver ! Mitochondria/ cytosolic enz., & 6 ATP,2NADH required by pyruvate carboxylase, PEP carboxykinase, Pglycerate kinase,GAPDH for 2pyruvateglucose 35 Glucagon 51aa & Insulin 29aa • Pancreas synthesises both peptide hormones • Glucagon hepatocyte receptors signals glycogenolysis (glycogen breakdown to glucose then increases gluconeogenesis pyruvate -- glucose) • Drugs. Dipeptidyl peptidase-4 inhibitor (Januvia, new anti type 2 diabetes) increases incretin , a GI hormonal peptide inhibitor of glucagon which lowers plasma glucose. • Metformin,major antidiabetic but also can inhibit mitoch.complex I causing lactic acidosis (rare). • Insulin required for cells (e.g.liver,muscle,fat) to take up glucose and synthesise glycogen. 36 5. MITOCHONDRIAL DISEASES (e.g. DEFECTIVE ELECTRON TRANSPORT) AND TREATMENT A) Creatine therapy B) Coenzyme Q10 therapy 37 Mitochondrial Myopathies • Genetic defects in mitochondrial structure & function leading to defective aerobic energy transduction and resulting in: exercise intolerance, lactic acidosis, stroke/seizure, headaches. 38 a) CREATINE THERAPY Creatine supplementation (an ergogenic aid effective against mitochondrial myopathies?) ATP ADP k i d n e y g u a n i d o a c e t a ta er g i n i n e l i v e r Pi, 2OH S AM • daily requirement is 2g • daily C r e dietary a t i n eintake is 1g (meat, c r e a tfish, i n eanimal p h o sproducts) p h a t e c r e a t i n i ( p h o s p h o c r e a t i n e ) ( u r i n e • also formed in liver, kidneys, pancreas from the amino acids glycine, E NE RGY S T ORE a n a e r o b i c e x e arginine and methionine ir nc i rs ee s t> i n3 g0 ms e uc s c l e • 5-7g x 4 per day for 5-7 days increases muscle creatine stores by 18% (particularly in vegetarians); enhances performance in certain repetitive, high intensity, short-term exercise tasks in healthy individuals, offsets fatigue in mitochondrial myopathy patients and improves the mobility of the elderly. J. Amer. Coll. Nutr. 17, 216-234 (1998). n ) 39 b) Ubiquinone (Coenzyme Q10) as a Food Supplement or Therapy • An essential electron and proton carrier in the mitochondrial respiratory chain. • Found in all intracellular membranes (acts as a mobile lipid soluble antioxidant that prevents membrane lipid peroxidation) • Better antioxidant if reduced to ubiquinol (UQH2) by NADH dehydrogenase of the respiratory chain. • Synthesised in mitochondria • Contributes to the fluidity of the phospholipid bilayer in membranes • Prevents plasma lipoprotein oxidation • Is a dietary supplement that protects liver from hepatotoxins (e.g. ethanol) and partly prevents mitochondrial myopathies (J. Neurol. Neurosurg. Psych. 50, 1475-81) • Deficiency may occur in patients taking cholesterol lowering drugs (the statins) which act by inhibiting HMG-CoA reductase (e.g. lovastatin) Proc. Nat. Acad. Sci. 87, 8931 (1990) 40 6. b-OXIDATION OF FATTY ACIDS AS AN ENERGY SOURCE a) Starvation/diabetes/endstage renal disease b) Carnitine therapy c) Ketogenic diet therapy d) Drug induced non alcoholic steatohepatitis , NASH e) Alcohol induced steatohepatitis , ASH 41 Stages in the extraction of energy from food stuffs. 42 b-Oxidation of fatty acids - transport of acyl carnitine into the mitochondrial matrix Stryer Fig 24-4 43 Fatty acid Metabolism • Fatty acids are linked to coenzyme A (CoA) before they are oxidised • Carnitine carries long-chain activated fatty acids into the mitochondrial matrix Carnitine therapy for mitochondrial diseases 44 The b-oxidation pathway as an energy source O O R C C C C S CoA H2 H2 H2 Acyl Co A oxida tion R C C C C S CoA H2 H H trans - -En oyl C oA FAD FADH 2 H 2O O H O R C C C C S CoA H2 H b-Ketoacyl CoA H + + NAD H NAD + oxida tion Hyd ration OHH O R C C C C S CoA H2 H H 3-L-hyd ro xyacy l CoA CoA-SH Th io lys is O R + C C S CoA H2 Acyl Co A s hortened by 2 carbon ato ms O H 3C C S CoA Acetyl Co A Citric acid cycle 45 a) Starvation/Diabetes/Endstage renal disease Fat breaks down to acetyl CoA which form ketone bodies • Under low carbohydrate condition, oxaloacetate is converted to glucose (gluconeogenesis). 2 D -b- H y d r o x y b u t y r CH3 HC OH CH2 COO NAD+ H+ + N A D H Ac e t y l Co A CoA O O Ac e t y l + Co A C S CoA H2O CoA C S CoA CH3 CH2 CH2 C O Ac e t y l Co A HO CH C O Ac e t o a c H M G C oCH A HM G- Co A t h i o l a s e 2 l y a s e CH3 s y n t h a s e CH2 COO COO Ac e t o a c e t y l b- H y d r o x y - H+ Co A b- m e t h y l g l u t a r y l Co A s u c c i n a t e c i t r i c c y c l e CO2 a c i d Co A t r a n s f e r a s e CH3 C KETOGENESIS s u c c i n y l Ac e t o a c e t a t e ( M E T ABOLI S M i . e . , a c t a s Co A O CH3 Ac e t o n e b- h y d r o x y b u t y r a t e o f k e t o n e b o d i e s ) f u e l a n d s p a r e s g l u c o s e 46 Diabetic ketoacidosis weakness, dehydration, thirst, drowsiness,coma • Usually precipitated by infection • lipolysis is the major energy source increases acetyl CoA levels which increases ketone body formation.Acetone excreted by the lungs/kidney. e.g. by starvation or diabetes mellitus (insulin-stimulated glucose entry into cells is impaired fatty acids are oxidised to maintain ATP levels. • if citric acid cycle is slowed by thiamine deficiency. • disease state plasma ketone levels: 10-25 mM (normal <0.5mM) and acetone breath smell( rotten apples or pear-drop smell) • LIFE THREATENING: ketogenesis faster than ketone body metabolism b-hydroxybutyric acid ↑↑> acetoacetic acid ↑& causes severe ACIDOSIS. Antidote – insulin , water, base therapy (bicarbonate?), carnitine • urinary excretion of Na+, K+, Pi, H2O, H+ dehydration, blood volume 47 b) Carnitine Therapy Carnitine alleviates acetyl-CoA mediated inhibition of pyruvate dehydrogenase. • Both glycolysis and fatty acid metabolism produce acetyl CoA • Accumulation of acetyl CoA can inhibit pyruvate dehydrogenase, the enzyme responsible for producing acetyl CoA from pyruvate. • Pyruvate will then be converted to lactic acid • Carnitine can temporarily scavenge acetyl CoA to form acetylcarnitine thus alleviating lactic acidosis in the muscle. C H 3 H O C CCC H2 H 2 O C H O H + H N 3C G L 3 C F O p d A A A X y e48 T Carnitine supplement Uses 1. Improves quality of life and walking performance in patients with limited walking capacity e.g., from end-stage renal disease and peripheral arterial disease. 2. Neurodegenerative diseases and recovery from cerebral ischemia. 3. Possible ergogenic aid but can cause an unpleasant body odour likened to rotting fish. 4. Improves memory of old rats (PNAS 99, 1876-81 (2002)) Biochemistry 1. Increases carnitine content, carries activated fatty acids across mitochondrial membrane and required for mitochondrial fatty acid oxidation. 2. Prevents acetyl CoA accumulation which inhibits pyruvate dehydrogenase. 3. Chelates iron and stabilizes membranes (antioxidant properties) 49 Carnitine supplement (cont) Sources Meat and dairy products exported and synthesized by liver > kidney from lysine + methionine. Highest levels in skeletal muscle, heart, adrenal gland but can’t synthesise it so take it up from the plasma. - total body store = 20-25gms. Oral Bioavailability 5-15% But over-the-counter formulations have low carnitine content and poor dissolution. - plasma acylcarnitines accumulate Journal of the American College of Nutrition, 17, 207-215 (1998) Progress in Cardiovascular Diseases, 40, 265-286 (1997) 50 c) Ketogenic diet therapy (results 10-25% seizure free & 60% better) for epileptic children resistant to phenytoin or valproate Energy Source Normal Diet Ketogenic Diet Protein 27% 10.4% adequate Carbohydrate 56% Fat 17% 89.6% Ketogenic diet consists of an egg nog that tastes like a mild shake (or frozen like ice cream) Supplying the body with fuel in the form of fat and proteins but not carbohydrates. fasting, diabeties Ketogenic diet Ketone Bodies Brain uses either glucose or ketone bodies as fuel Liver produces ketone bodies 51 d) Drugs that inhibit fatty acid oxidation cause fatty liver (steatosis) and 10% get NASH (nonalcoholic steatohepatitis & liver cancer) Steatosis (fatty liver) in 33% population & 80% of obese patients. Higher also in diabetes , high plasma triglycerides. NASH in 2-9% patients undergoing routine liver biopsy. Hepatocellular carcinoma. Drugs that inhibit mitochondrial β-fatty acid oxidation 1)Tetracycline, valproic acid,oestrogens,glucocorticoids 2) Amiodarone,perhexiline are charged lipophilic drugs concentrate in liver mitochondria & inhib. β-fatty acid oxidn & respiration, cause lipid peroxidn. & reactive oxygen species (ROS). Steatosis and steatohepatitis are independent. Fibrosis occurs. 3) Drugs induce sporadic events of both e.g. carbamazepine 4) Latent NASH e.g. tamoxifen 52 e) Ethanol induced steatohepatitis (ASH) proposed endotoxin mechanism 1) Ethanol causes lipogenesis and fatty liver (caused by inhibition of LDL synth. & export). 2) Ethanol oxidised by CYP2E1 to form hydroxyethyl radicals AND ethanol oxidised by ADH to form acetaldehyde which cause oxidative stress and hepatocyte & gut cytotoxicity. 3) Oxidative stress disrupts intestinal mucosal cell actin cytoskeleton (prev. by oats supplement). 4) Intestine becomes leaky & endotoxin enters blood & liver which causes liver inflammation and ASH. 53 JPET 329,952-8(2009) Drugs causing weight gain in some patients Tricyclic antidepressants imipramine,amitriptyline,nortryptyline, Antipsychotics haloperidol,clozapine,olanzapine,risperidone,chlorpromazine,paxil Corticosteroids prednisone. Also steroids estrogen,tamoxifen Antidiabetics rosiglitazone,pioglitazone,insulin Antirheumatism etanercept,enbrel Anti-seizure valproate,depakote,carbamazepine Lithium MAO inhibitors phenelzine,tranylcypromine (not a hydrazine) 54 THE END • Don’t memorize the following slides 12,13,14,15,16,19,21 55