1
IV: Mitochondrial function (e.g. hepatocytes)
1) citric acid cycle as an energy source a) pyruvate or -ketoglutarate dehydrogenase b) lipoic acid therapy2) the respiratory chain as an energy source3) oxidative phosphorylation and uncouplers4) membrane transporters and shuttles a) cytosolic NADH oxidation b) acetyl CoA (NADPH export) c) transport systems in the mitochondria d) gluconeogenesis and glucose transport5) mitochondrial diseases and treatment a) creatine therapy b) coenzyme Q10 therapy
6) -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) toluene8) hemoprotein mediated diseases a) rhabdomyolysis b) kernicterus9) Heme biosynthesis & porphyria a) Heme biosynthesis b) Porphyria c) Oxidative degradation of heme to bilirubin
2
Stryer
An overview of the citric acid cycle
CITRIC ACID CYCLE AS AN ENERGY SOURCECITRIC ACID CYCLE AS AN ENERGY SOURCE
3
Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA
120uM plasma citrate120uM plasma citratecomplexes Fecomplexes Fe
toxic!toxic!
4
The citric acid cycle is a source of biosynthetic precursors
Stryer Fig. 20-17.Biosynthetic roles of thecitric acid cycle. Intermediates drawn offfor biosyntheses are replenished by the formation of oxaloacetatefrom pyruvate.(Anaplerotic)
Pyruvate
Acetyl CoA
Citrate
keto-glutarate
Amino acids
SuccinylCoA
Porphyrins
Oxaloacetate
Aminoacids
ADP, Pi
ATP, CO2
Phosphoenolpyruvate
Glucose
5
Control of the citric acid cycle
Stryer Fig. 20-22.Control of the citric acid cycle andthe oxidative decarboxylation ofpyruvate: * indicatessteps that require anelectron acceptor (NAD+ or FAD) that is regenerated by therespiratory chain.
6
2. THE RESPIRATORY CHAIN AS AN ENERGY SOURCE
7
The mitochondrial respiratory chain
Sequence of electroncarriers in therespiratory chain
Chemiosmotic theory of oxidative phosphorylation
Diagram of a mitochondrionNADH
NADH-Q reductase
Q
Cytochrome reductase
cyt c
Cytochrome oxidase
O2
complex I
complex III
complex IV
FADH2in flavoproteinssuccinate:Q reductase (complex II)
FMNH2
2Fe-2S
4Fe-4S
8
NADH coenzyme Q reductase: complex I
The reduction of ubiquinone to ubiquinol proceeds through a semiquinoneanion intermediate.
NADH
NAD+
FMN
FMNH2
Reduced Fe-S
Oxidised Fe-S
Q
QH2
NADH-Q reductase
C
CC
C
CC
O
O
H3CO
H3CO
CH3
(CH2CH
C
CH3
CH2)10 HC
CC
C
CC
O
OH
H3CO
H3CO
CH3
R
C
CC
C
CC
OH
OH
H3CO
H3CO
CH3
R
e- + H+ e- + H+
Coenzyme Q10(UBIQUINONE
Semiquinone Intermediate (Q )
Reduced Coenzyme Q10 ( UBIQUINOL)
9
Model of NADH-Q reductase
Stryer Fig 21-9
10
Q:Cytochrome c reductase (Complex III)
Q
QH
cyt b (+2)
cyt b (+3)
QH
QH2
Fe-S(+2)
Fe-S(+3)
cyt c1(+3)
cyt c1(+2)
cyt c(Fe+2)
cyt c(Fe+3)
cytochrome c reductase
Stryer Fig. 21-11Model of a portion ofQ: cytochrome c reductase
Stryer p. 537
11
Cytochrome oxidase (Complex IV)
Lodish Fig. 17-30
12
Electron transport can be blocked by specific inhibitorpoisons
NADH
NADH-QReductase
QH2
Cytochrome b
Cytochrome c1
Cytochrome c
Cytochrome Oxidase
O2
Blocked by rotenone and amytal
Blocked by antimycin
Blocked by
CN-, N3-, and CO
Sites of action of someinhibitors of electron transport
13
Cytochrome C - catalytic site
The heme in cytochromes c and c1 is covalently attached to 2 cysteine side chainsby thioether linkages
The iron atom of the heme group incytochrome c is bonded to a methioninesulfur atom and a histidine nitrogen atom
R CH
CH2
HSCH2 R'R C
H
CH3
CH2
R'S+
Vinyl groupof the heme
Cysteine residueof the protein Thioether linkage
14
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!
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Origin of mitochondria: the endosymbiont hypothesis
The endosymbiont hypothesis suggests that mitochondria have evolvedfrom 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.
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3. OXIDATIVE PHOSPHORYLATION AND
UNCOUPLERS
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Oxidative phosphorylation
18
4.Mitochondrial MEMBRANE TRANSPORTERS
A) Cytosolic NADH oxidation
B) Acetyl CoA (NADPH export)
C) Transport systems in the mitochondria
D) Gluconeogenesis and glucose transport
19
Compartmentalization of the major pathways of metabolism
20
a) Cytosolic NADH oxidation: membrane transporters glycerol phosphate shuttle (Bucher shuttle)
Glucose
Glyceraldehyde - 3 - phosphate
1,3 - Bisphosphoglycerate
NAD+
NADH, H+Dihydroxyacetone phosphate
Glycerol - 3 - phosphate
Glycerol - 3 - phosphate
Dihydroxyacetone phosphate
Cytosol Outer membrane Inner Membrane
FAD
FADH2
RE
SP
IRA
TO
RY
C
HA
IN
Q
QH2
b
c1
c
a, a3
Glycolysis
1
2
1. Glycerol phosphate dehydrogenase2. Glycerophosphate oxidase
H2C
CHH2C
HO
OH
OP
H2C
CH2CO
OH
OP
See figure 21-30 Stryer 4th Ed.
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b) Acetyl CoA/NADPH export to cytosol for fatty acid synthesis/drug metabolismGlucose
Pyruvate
Acetyl CoA
Oxaloacetate
Pyruvate
Citrate
CitrateSynthase
Citrate
Oxaloacetate
Acetyl CoA fatty acid synthesisor drug metabolism(N-acetylation)
+ATP+CoA
ATP citrate lyaseat high concentration
Malate
Pyruvate
NADH
NAD+
NADP+
NADPH
CO2
malate dehydrogenase
malic enzyme
fatty acid synthesisor P450 catalyzed drug metabolism
Pentose Phosphate Pathway NADPH
Therefore malic enzyme supplies NADPHCitrate Lyase supplies acetyl CoA.
ATP
ADP CO2
Mitochondrial Matrix
Cytosol
22
Isocitrate as an NADPH shuttle for drug metabolismGlucose
Pyruvate Acetyl CoA
Citrate
Isocitrate
-ketoglutarate
NAD+
NADH
isocitratedehydrogenase
CO2
Succinyl CoA
NAD+NADH
CO2
Succinate
Fumarate
Malate
Oxaloacetate
NAD+
NADH
Isocitrate
-ketoglutarate
NADP+
NADPH
P450 catalyzedDRUG METABOLISM
isocitratedehydrogenase
CITRICACIDCYCLE
MITOCHONDRIAL MATRIXCYTOSOL
23
d) Gluconeogenesis and glucose export by the liver ! 3 irreversible steps
Major antidiabetic drug METFORMINInhibits gluconeogenesisDecr Hepatic Glucose Synth.
24
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, antidiabetic drug inhibits gluconeogenesis but also can inhibit mitoch.complex I causing lactic acidosis.
• Insulin required for cells (e.g.liver,muscle,fat) to take up glucose and synthesise glycogen.
25
5. MITOCHONDRIAL DISEASES(e.g. DEFECTIVE ELECTRON
TRANSPORT) AND TREATMENT
A) Creatine therapyB) Coenzyme Q10 therapy
26
Mitochondrial Myopathies
• Genetic defects in mitochondrial structure & function leading to defective aerobic energy transduction and resulting in: exercise intolerance, lactic acidosis, stroke/seizure, headaches.
27
CREATINE THERAPY (an ergogenic aid effective againstmitochondrial myopathies?) stored in muscles (makes ATP)
• daily intake is 2g including1g (meat, fish, animal products)• 1g formed in liver, kidneys, pancreas from glycine,arginine,methionine• plasma levels incr. in kidney,heart,liver damage or rhabdomyolysis • 5-7g x 4 per day for 5-7 days increases muscle creatine stores by 18%
(bigger increase 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).
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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)
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6. -OXIDATION OF FATTY ACIDS AS THE BEST ENERGY SOURCE
a) Starvation/diabetes/endstage renal diseaseb) Carnitine therapyc) Ketogenic diet therapyd) Drug induced non alcoholic steatohepatitis , NASH e) Alcohol induced steatohepatitis , ASH
30
Stages in theextraction ofenergy fromfood stuffs.
FATS POLYSACCHARIDES PROTEINS
Fatty acids and glycerol
Glucose andother sugars
Amino Acids
Acetyl CoA
CoA
ATP ADP
O2
CitricAcidCycle
2 CO2
e
STAGE I
STAGE II
STAGE III Oxidative
Phosphorylation
31
-Oxidation of fatty acids - transport of acyl carnitine into themitochondrial matrix
Stryer Fig 24-4
32
The -oxidation pathway as an energy source
R CH2
CH2
CH2
C S CoA
O
Acyl CoA
R CH2
CH
CH
C S CoA
O
R CH2
C C C S CoA
OOHH
HHR C
H2C C C S CoA
OO H
H
R CH2
C S CoA
O+
H3C C S CoA
O
trans- -Enoyl CoA
3-L-hydroxyacyl CoA-Ketoacyl CoA
Thiolysis
CoA-SH
H2OFAD FADH2
oxidation
Hydration
NAD+H+ + NADH
oxidation
Acyl CoA shortenedby 2 carbon atoms
Acetyl CoA
Citric acid cycle
33
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
R C
O
O
ATP HS-CoA R C
O
S CoA AMP PPi+ + + +acyl CoA synthetase(aka fattyacid thiokinase)
Outer Mitochondrial Membrane
R C
O
S CoA + H3C N
CH3
CH3
CH2
C
OH
H
CH2
C
O
OHS-CoA + H3C N
CH3
CH3
CH2
C
O
H
CH2
C
O
O
C O
R
Acyl CoA Carnitine acyl carnitine
carnitine acyl transferase I
Carnitine therapy for mitochondrial diseasesCarnitine therapy for mitochondrial diseases
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The -oxidation pathway as an energy source
R CH2
CH2
CH2
C S CoA
O
Acyl CoA
R CH2
CH
CH
C S CoA
O
R CH2
C C C S CoA
OOHH
HHR C
H2C C C S CoA
OO H
H
R CH2
C S CoA
O+
H3C C S CoA
O
trans- -Enoyl CoA
3-L-hydroxyacyl CoA-Ketoacyl CoA
Thiolysis
CoA-SH
H2OFAD FADH2
oxidation
Hydration
NAD+H+ + NADH
oxidation
Acyl CoA shortenedby 2 carbon atoms
Acetyl CoA
Citric acid cycle
35
a) Starvation/Diabetes/Endstage renal diseaseFat breaks down to acetyl CoA which formketone bodies• Under low carbohydrate condition, oxaloacetate is converted to glucose (gluconeogenesis).
KETOGENESIS
2 Acetyl CoA
CoA
C
CH2
C
CH3
O
S CoA
Acetyl CoA
+H2O CoA
CH3
C
CH2
COO
O
C
CH2
CH
CH2
COO
O
S CoA
O HO
AcetylCoA
CH3
HC
CH2
COO
OH
CH3
C
CH3
O
H+ + NADH
NAD+
H+
CO2
D--Hydroxybutyrate
Acetone
Acetoacetate
-Hydroxy--methylglutaryl CoA
Acetoacetyl CoA
thiolase HMG-CoAsynthase
HMG CoAlyase
citric acidcycle
succinyl CoA
succinate
CoA transferase
Acetoacetate -hydroxybutyrate
(METABOLISM of ketone bodies)i.e., act as fuel and spares glucose
36
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-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
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b) Carnitine TherapyCarnitine alleviates acetyl-CoA mediated inhibition of pyruvatedehydrogenase.• 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.
FATTY ACIDOXIDATION
GLYCOLYSIS
ACETYL CoAPYRUVATE
pyruvatedehydrogenase
Carnitine
Acetylcarnitine + Coenzyme A
LACTATE
H3C N+
CH3
CH3
CH2
C
H
OH
CH2
C
O-
O
TCA (Krebs)
38
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)
Carnitine supplement
39
Sources
Meat and dairy products exported and synthesized byliver > kidney from lysine + methionine. Highest levels inskeletal muscle, heart, adrenal gland but can’t synthesise itso take it up from the plasma.
- total body store = 20-25gms.
Oral Bioavailability 5-15%
But over-the-counter formulations have low carnitine content andpoor dissolution.- plasma acylcarnitines accumulate
Journal of the American College of Nutrition, 17, 207-215 (1998)Progress in Cardiovascular Diseases, 40, 265-286 (1997)
Carnitine supplement (cont)
40
c) Ketogenic diet therapy (results 10-25% seizure free & 60% better) for epileptic children resistant to phenytoin or valproate
Energy Source Normal Diet Ketogenic DietProtein 27% 10.4% adequateCarbohydrate 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 notcarbohydrates.
Ketone Bodies
fasting, diabetiesKetogenic diet
Brain uses eitherglucose or ketone bodies as fuel
Liver produces ketonebodies
d) Drug induced Fatty liver by inhibiting fatty acid oxidation.
Liver (steatosis) and NASH (nonalcoholic
steatohepatitis & whilst 5% of these get 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 rarely.
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 41
e) Ethanol induced steatohepatitis (ASH) proposed endotoxin fatty liver 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.
JPET 329,952-8(2009) 42