Maria G Lane

◦ Mitochondria are tiny cellular organelles responsible for energy production via oxidative phosphorylation

◦ Complexes I-IV form the electron transport chain and create a proton gradient

◦ Complex V uses this gradient to make ATP from ADP

The liver is a vital organ ◦ Has a high energy requirement

◦ Has a role in metabolism of macromolecules, protein biosynthesis and clearing of toxic compounds

◦ Highly dependent on ATP and functioning mitochondria

◦ Mitochondrial disease affects about 1 in 5,000 to 1 in 10,000 of the population and the liver can be affected in as many as 20% of these cases

Many types of liver disease including alcoholic liver disease, hepatitis, acute liver failure

Symptoms are varied – lactic acidosis, hypoglycaemia, jaundice, anaemia, coagulopathy

Treatment is symptomatic

Transplantation is life saving in severe cases

Often unknown

Drugs such as valproate

Viruses such as hepatitis B and C

However it is speculated that mitochondrial dysfunction has a role in liver disease

Several studies have associated liver disease with mitochondrial defects

Defects in assembly of respiratory chain complexes

◦ BCS1L Missense mutations

Chaperone for Rieske Fe-S subunit of Complex III

Hepatopathy, tubulopathy and encephalopathy

◦ SCO1 Copper insertion into Complex IV

Hepatopathy and ketoacidic coma

Alpers’ Syndrome◦ Polymerase gamma mutations

◦ Affects the liver and nervous system

DGUOK deficiency◦ Deoxyguanosine kinase maintains a stable

ribonucleotide pool for mtDNA synthesis

◦ Affects the liver and nervous system

TSFM◦ Encodes elongation factor EFTs

◦ A mutation causes a severe hepatopathy with a respiratory chain deficiency

TRMU◦ Encodes a tRNA modifying enzyme

◦ Required for thiol addition to tRNAs for lysine, glutamate and glutamine

◦ A mutation causes acute liver failure with reduced activity of complexes I, III and IV

Isolation of mitochondria from frozen liver tissue

◦ Homogenisation of tissue Cut and washed Homogenised in a glass Elvehjem potter

◦ Differential centrifugation Sucrose gradient

◦ Addition of detergents and sample buffer containing aminocaproic acid for BN PAGE only

Blue native polyacrylamide gel electrophoresis

◦ Native separation of protein complexes on a gradient gel

◦ Uses Coomassie G250 Attaches a negative charge to protein complexes Doesn’t dissociate complexes into subunits

◦ Digitonin and n-dodecyl B-D-maltoside Detergents Break down mitochondrial membrane Dissociate some supercomplexes

◦ Immunodetection of proteins with antibodies to complexes I-V

◦ Biochemical assays of the catalytic activity of enzymes

◦ Complex II reduction of ubiquinone at 595 nm

◦ Complex IV oxidation of cytochrome c at 550 nm

◦ Citrate Synthase formation of citrate from oxaloacetate at 405 nm

Indicator of number of functional mitochondria

Confocal microscopy

Immunocytochemistry

Study mtDNA transcription or translation

Sequencing of genes commonly affected in mitochondrial disease e.g. TRMU, DGUOK, GFM1

Whole exome sequencing

Chinnery PF, DiMauro S. Mitochondrial hepatopathies. Journal of hepatology. 2005 Aug; 43(2):207-9

Fernandez-Vizarra E, Ferrin G, Perez-Martos A, Fernandez-Silva P, Zeviani M, Enriquez JA. Isolation of mitochondria for biogenetical studies: An update. Mitochondrion. 2010 Apr; 10(3):253-62

Lee WS, Sokol RJ. Liver disease in mitochondrial disorders. Seminars in liver disease. 2007 Aug; 27(3):259-73

Rahman S. Gastrointestinal and hepatic manifestations of mitochondrial disorders. Journal of inherited metabolic disease. Epub 2013/05/16

Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical biochemistry. 1991 Dec; 199(2):223-31