There are many causes of liver disease however the aetiology is often undetermined. It is speculated that mitochondrial dysfunction has a role in liver disease and this presentation explores possible causes and techniques for investigating mitochondrial dysfunction in liver.

1. Maria G Lane

2. ◦ 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

3.  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

4.  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

5.  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

6.  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

7.  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

8.  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

9.  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

10.  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

11. ◦ 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

12.  Confocal microscopy
 Immunocytochemistry
 Study mtDNA transcription or translation
 Sequencing of genes commonly affected in
mitochondrial disease e.g. TRMU, DGUOK, GFM1
 Whole exome sequencing

13.  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