Fat oxidation and defects of the mitochondrial respiratory chain - - PowerPoint PPT Presentation

▶

Feb 28, 2024 166 likes •513 views

Fat oxidation and defects of the mitochondrial respiratory chain Dr. Simon Olpin august - mid 20030022.jpg Clinical Chemistry Sheffield Childrens Hospital H + acyl-CoA dehydrogenases ETF to CoQ NAD NADH + H + hydroxyacyl-CoA

SLIDE 1

august - mid 20030022.jpg

Fat oxidation and defects of the mitochondrial respiratory chain

Dr. Simon Olpin

Clinical Chemistry Sheffield Children’s Hospital

SLIDE 2

15/06/2005 FAO & RES 2 acyl-CoA dehydrogenases ETF to CoQ hydroxyacyl-CoA dehydrogenases NADH to complex I H+

NAD NADH + H+

SLIDE 3

15/06/2005 FAO & RES 3

FAO & RES

EFAD EFADH2

ETFox ETFred Acetyl- CoA

II

ETFQO

Q

Cytc TCA

I III IV

H+ H+ H+

V

H+

acyl-CoA dehydrogenases 3-hydroxyacyl-CoA dehydogenases

SLIDE 4

15/06/2005 FAO & RES 4

Diagnosis of a respiratory chain defect

Clinical Histological - staining, EM Biochemical - complexes I, II/III, IV (?)V

muscle, liver, heart, fibroblasts Polarography - only a very few centres

Mitochondrial mutations

NARP, MELAS, MERRF, tRNA’s

Nuclear mutations

SCO2, SURF1, Cox10, Cox15

SLIDE 5

15/06/2005 FAO & RES 5

Diagnosis of a respiratory chain defect

In practise

ften a balance of probabilities

SLIDE 6

15/06/2005 FAO & RES 6

Are fibroblasts worthwhile?

Many older children / adults

mtDNA mutations eg. MELAS

Neonates and infants ~ 95% due to nuclear defects BUT

Isoforms in different tissues Defect may not be “limiting” in fibroblasts

SCO2 - normal IV in fibroblasts

SLIDE 7

15/06/2005 FAO & RES 7

Fatty acid oxidation assay

3 substrates

[9,10-3H]myristate, [9,10-3H]palmitate, [9,10-3H]oleate

Incubate monolayers in multiwell plates

24 wells - 3 substrates in duplicate (110 µmol/l)

2 hours at 37oC

Separate & count released 3H2O Assay protein / well

Activity ~ 5 nmol/mg/hr

expressed as % of 3-5 simultaneous controls

SLIDE 8

15/06/2005 FAO & RES 8

Definition of abnormal FAO

Mean of duplicate ≤ 70% (2 full assays) If all results not ≤ 70% ≥ 5/9 duplicate results ≤ 70%

SLIDE 9

15/06/2005 FAO & RES 9

Fibroblast FAO

Probable / definite respiratory chain defects Clinical, biochemical, histological 50 patients over 10 years - all had a skin bx Complexes measured in M or L - 32 Complexes (F) only - 14 Not measured - 4 (NARP, awaiting)

SLIDE 10

15/06/2005 FAO & RES 10

FAO and probable respiratory chain disease

FAO low in 37/50 cases (74%) Complexes low in muscle/liver in 22/32 cases (69%) Complexes low in fibroblasts 7/21 cases (33%)

– Complex I & V not measured – overall function not assessed by complexes

SLIDE 11

15/06/2005 FAO & RES 11

34/50 most probable/definite RES

Clearly low complexes Muscle and / or liver = 22 Fibroblasts (only) = 6 Mutation data or other = 6 (NARP/ tRNA & low PDH)

SLIDE 12

15/06/2005 FAO & RES 12

FAO & RES

FAO low in 19/34 highly probable/definite cases (56%)

FAO low in 7/7 cell lines with low complexes

– Low II/III - 2 – Low III - 1 – Low IV - 4

SLIDE 13

15/06/2005 FAO & RES 13

FAO & RES

2 patients - isolated low I (M)

– both low fat oxidation (F)

3 patients had depletion

– 2 normal FAO – 1 abnormal FAO

SLIDE 14

15/06/2005 FAO & RES 14

Patient TR

Died ~ 10 months (suddenly) Dilated cardiomyopathy Excess fat deposition in heart and liver Microscopically ↑fibrous tissue

SLIDE 15

15/06/2005 FAO & RES 15

Patient TR

Fibroblast fatty acid oxidation Myristate % Palmitate % Oleate % 64±19 73±20 70±18 (n=5) sl.↓ complex IV in fibroblasts (0.006) (0.007 - 0.036)

SLIDE 16

15/06/2005 FAO & RES 16

Mutant tRNAIle

Known tRNA isoleucine point mutations that

can cause isolated cardiomyopathy

A4317G
A4269G
A4300G
C4320T
A4295G

SLIDE 17

15/06/2005 FAO & RES 17

Mutant tRNAIle

A4317G ↓ complex I & IV in heart A4269G ↓ complex IV in skeletal muscle & brain

SLIDE 18

15/06/2005 FAO & RES 18

G (4300) T (4320) G (4295)

SLIDE 19

15/06/2005 FAO & RES 19

G (4325)

G (4300) T (4320) G (4295)

SLIDE 20

15/06/2005 FAO & RES 20

Mutant tRNAIle

A4317G (T loop)

– reduces isoleucylation by 70%

– reduction in mt protein synthesis

A4269G (acceptor stem)

– isoleucylation not affected – conformational changes leading to premature mt translation

termination - isolated low complex IV

A4325G - (?) effect possibly similar to A4269G

SLIDE 21

15/06/2005 FAO & RES 21

Patient OB

Gross motor delay at 14 months CSF lactate 6 mmol/l Fatty acid oxidation Myristate % Oleate % 51 ± 7.4 54 ± 9.0

(3 assays in duplicate)

SLIDE 22

15/06/2005 FAO & RES 22

Patient OB

Fibroblasts complex II/III 0.173 (0.07-0.243) complex IV 0.003 (0.007- 0.036) Low IV (muscle) SURF1 mutation

– complex IV assembly

SLIDE 23

15/06/2005 FAO & RES 23

Patient JT

Leigh’s encephalopathy at 1 year CSF lactate 3.9 mmol/l Plasma lactate 2.9 mmol/l Myristate % Palmitate% Oleate % 57% 70% 59%

(mean of 2 assays in duplicate)

SLIDE 24

15/06/2005 FAO & RES 24

Patient JT

Muscle complexes complex I 0.215 (0.104-0.268) complex II/III 0.093 (0.04-0.204) complex IV 0.17 (0.14-0.034) PDH - normal T8993G present (NARP)

SLIDE 25

15/06/2005 FAO & RES 25

Patient JT

Fibroblasts
T8993G present at high level

– ATPase-6 gene

Why low FAO?

SLIDE 26

15/06/2005 FAO & RES 26

Patient NM

Female 24/40 gestation

pericardial effusion / cardiomegaly

Neonate

hypotonia, muscle weakness, poor feeder hypertrophic cardiomyopathy

MRI

white matter, basal ganglia and cerebellar abnormalities

Plasma lactate

5-6 mmol/l

SLIDE 27

15/06/2005 FAO & RES 27

Patient NM

Muscle biopsy

– Myopathic findings – Normal complexes, no mutations

Urine OA

– (?) suggestive of GAII

– DCA, 3(OH)isovalerate, isovalerylglycine 2(OH)glutarate

SLIDE 28

15/06/2005 FAO & RES 28

Patient NM

Myristate % Palmitate % Oleate % 49 ± 4 50 ± 6 50 ± 5

(3 assays in duplicate)

Fibroblasts complexes

II/III 0.048 (0.07 - 0.243) IV 0.004 (0.007-0.036)

ATP production on digitonised fibroblasts 15% of controls

SLIDE 29

15/06/2005 FAO & RES 29

Fibroblast fat oxidation in detection of RES (?)

Can detect a significant % of neonates / infants
(?) 20 - 60%
False positive rate - (?)
Low fat oxidation seen in:-

– Alstrom’s – Laminopathy – Severe neonatal GSD IV – (?) overlaying !

SLIDE 30

15/06/2005 FAO & RES 30

Disadvantages

Defect not always expressed / present Defect may be lost in culture Difficulty of measuring Complex I ?!

SLIDE 31

15/06/2005 FAO & RES 31

Advantages of fibroblasts

Easy to obtain and grow-repeat assays, storage, PM

– no excuse for muscle bx without skin!!!!

Less subject to secondary factors

– deterioration, nutrition, clinical state

Flux assays (intact cells)

– overall measure of energy metabolism

Complexes

– (?) I II, III, IV & ubiquinone

Digitonised fibroblasts

– ATP production (great potential !!)

SLIDE 32

15/06/2005 FAO & RES 32

SLIDE 33

15/06/2005 FAO & RES 33