A1258150166yXxfS

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METAL IONS IN HEART DISEASE Michael J.
Davies The Heart Research Institute 145
Missenden Road, Camperdown, Sydney, Australia
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The Heart Research Institute

• Independent Research Institute
• Opened in 1989
• Bicentennial initiative of
• National Heart Foundation
• Cardiologists of Royal Prince Alfred Hospital
• NSW State Government
• Non-profit, registered charity
• Affiliated to
• Royal Prince Alfred Hospital
• University of Sydney

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HRI Research Groups

• Cell Biology (Ken Rodgers)
• Clinical Research (David Celermajer / Len
  Kritharides)
• Endothelial Cell Biology (Philip Barter)
• Free Radical (Mike Davies)
• Lipid Research (Kerry-Anne Rye)
• Molecular Biology (Alison Death)
• Vascular Immunology (Annemarie Hennessy)

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Mission statement

• Develop new scientific knowledge which will
  lead to prevention, early detection and reversal
  of atherosclerosis (the cause of cardiovascular
  disease).

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What is atherosclerosis?

• A process that clogs arteries with deposits of
  cholesterol and lipids.

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What is atherosclerosis?

• A process that clogs arteries with deposits of
  cholesterol and lipids.
• This reduces the blood supply to the organ that
  the artery supplies, which can result in serious
  organ damage.
• Rupture of lesions can release particulate matter
  that blocks other blood vessels

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(No Transcript)
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Consequences of atherosclerosis

• Angina
• Heart attack (myocardial infarction)
• Stroke
• Intermittent claudication
• Gangrene

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The cost of atherosclerosis

• Major health problem and cost to society
• 1 person dies every 10 minutes
• 41 of all deaths arise as a result of
  atherosclerosis
• Coronary heart disease is largest single cause of
  death (more than 29,000/year)
• Stroke second greatest killer (more than
  12,000/year)
• Atherosclerosis is a leading cause of long term
  disability in adults
• 3.7 billion a year (12 of health budget)

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Areas of expertise within HRI

• Lipid metabolism
• Free radical chemistry and biochemistry
• Stem cells
• Cell signalling
• Protein modification, metabolism and turnover
• Cytokines
• Endothelial cell function
• Diabetes

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Specialised equipment / facilities

• Cell isolation and culture (primary and cell
  lines)
• Isolation and handling of lipoproteins (LDL /
  HDL)
• Enzyme kinetics and assays
• Quantification of DNA oxidation (HPLC, agarose
  gels)
• Quantification of normal and oxidised lipids and
  cholesterol
• Quantification of normal and oxidised / modified
  proteins (HPLC, 1D and 2D gel electrophoresis,
  proteomics)
• Quantification of antioxidants (HPLC, GC/MS)
• Radioactive tracer work
• Identification and quantification of radical
  formation (EPR, NO / O2 /H2O2 electrodes)

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Quantification of oxidised DNA bases
X.
H2O
8-oxodG
2-deoxyguanosine
Presence of 8-oxodG disrupts base-pairing and
gives G-C to A-T transversions
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Specialised equipment / facilities

• Cell isolation and culture (primary and cell
  lines)
• Isolation and handling of lipoproteins (LDL /
  HDL)
• Enzyme kinetics and assays
• Quantification of DNA oxidation (HPLC, agarose
  gels)
• Quantification of normal and oxidised lipids and
  cholesterol
• Quantification of normal and oxidised / modified
  proteins (HPLC, 1D and 2D gel electrophoresis,
  proteomics)
• Quantification of antioxidants (HPLC, GC/MS)
• Radioactive tracer work
• Identification and quantification of radical
  formation (EPR, NO / O2 /H2O2 electrodes)

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Quantification of protein oxidation measurement
ofspecific side-chain oxidation products
Valine hydroperoxides, alcohols, carbonyl
compounds. Lysine hydroperoxides,
alcohols, carbonyl compounds.
Proline hydroperoxides, alcohols, 5-hydroxy
-2-aminovaleric acid, carbonyl
compounds. Arginine 5-hydroxy-2-aminovaleric
acid Isoleucine hydroperoxides,
alcohols, carbonyl compounds
Glycine Aminomalonic acid Methionine Methionine
sulphoxide Cysteine Cystine, Oxy acids
Tyrosine DOPA 3-chlorotyrosine 3-nitrotyrosin
e di-tyrosine Phenylalanine o-,
m-tyrosine dimers Tryptophan N-formylkynurenine,
kynurenine, 5-hydroxytryptophan, 7-hydroxyt
ryptophan Histidine 2-oxo-histidine Glutamic
acid hydroperoxides Leucine hydroperoxides, alc
ohols, ?-ketoisocaproic acid, isovaleric
acid, isovaleraldehyde, isovaleraldehyde
oxime, carbonyl compounds.
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Quantification of oxidised proteins in tissue
samples
Proteins extracted from tissue samples (normal
and diseased), delipidated, and hydrolysed to
free amino acids. HPLC with UV, fluorescence and
multi-channel electrochemical detection used to
quantify both oxidised species and parent amino
acids. Results expressed as ratio of oxidised
species to parent amino acid. Radiometric and
chemiluminescence detectors also available.
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Oxidised proteins are present in diseased human
arteries
Proteins extracted from human artery samples
(normal and advanced carotid lesions)
delipidated, and hydrolysed to free amino acids,
and analysed by HPLC. Results expressed as ?moles
oxidised species / mole parent. Elevated levels
of oxidised lipids and decreased antioxidant
concentrations also detected.
Fu et al, Biochem. J., 1998, 333, 519-525
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Specialised equipment / facilities

• Cell isolation and culture (primary and cell
  lines)
• Isolation and handling of lipoproteins (LDL /
  HDL)
• Enzyme kinetics and assays
• Quantification of DNA oxidation (HPLC, agarose
  gels)
• Quantification of normal and oxidised lipids and
  cholesterol
• Quantification of normal and oxidised / modified
  proteins (HPLC, 1D and 2D gel electrophoresis,
  proteomics)
• Quantification of antioxidants (HPLC, GC/MS)
• Radioactive tracer work
• Identification and quantification of radical
  formation (EPR, NO / O2 /H2O2 electrodes)

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Oxidation as a cause of atherosclerosis and its
complications

• Oxidative events postulated to be important both
  in the initiation of atherosclerosis and in
  plaque rupture.
• Some evidence to support this hypothesis, but
  also considerable evidence for other factors
  being as, or more, important.
• Potential catalysts of oxidative damage
• Enzyme reactions (lipoxygenase, peroxidases,
  oxidases, heme protein reactions)
• Activated inflammatory cells
• Nitric oxide / peroxynitrite
• Trace redox-active metal ions

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Oxidative modification model of atherosclerosis
Blood vessel
LDL (bad cholesterol)
White blood cells
Endothelial cells
L
D
L
Vessel wall
Oxidative modification of LDL particles
L
D
L
L
D
L
L
D
L
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Are trace metal ions present at elevated levels
in lesions ?
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EPR analysis of human lesions
Non-destructive technique of EPR spectroscopy
used to examine presence of organic radicals and
metal ions, particularly Fe(III) and Cu(II) in
advanced human lesions.
Iron signal
Organic radicals
Advanced Human Plaque
Healthy Human Intima
Magnetic field (G)
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Correlation of iron levels detected by EPR and
ICPMS with tissue status
Mean iron values detected by EPR in lesions
significantly higher than that detected in
healthy intima samples.
Mean iron values detected by ICPMS in lesions
significantly higher than that detected in
healthy intima samples.
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Comparison of iron levels detected by EPR and
ICPMS
Good correlation between the two measurements
with EPR measuring ca. 70 of the total iron
present
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ICPMS detection of copper in atherosclerotic
lesions
Elevated levels of Cu detected in diseased intima
samples compared to normal intima samples.
Cu(II) signals were not detected by EPR.
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Correlation of metal ion levels with other
parameters

• No correlation of metal ion levels with
• Age of tissue donor
• Gender
• Protein levels in lesion
• Calcium levels in lesion

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Correlation of iron levels with cholesterol
accumulation EPR-detectable Fe(III) shows a
positive correlation with cholesterol levels in
lesions. Is iron accumulation a cause or
a consequence of cholesterol accumulation and
hence lesion development? Presence of elevated
levels of non-heme, non-ferritin iron consistent
with presence of elevated levels of protein and
lipid oxidation products and depletion of
antioxidants.
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Unanswered questions

• What are these Fe and Cu complexes ?
• Where are they located - intra- or
  extra-cellularly ?
• Where do they come from ?
• Heme protein degradation ?
• possible role of oxidative damage or heme
  oxygenase
• Micro hemorrhage into plaques ?
• Smooth muscle cell death ?
• Is it possible to modulate metal ion levels in
  the artery wall in vivo ?
• Does modulation of metal ion levels affect
  disease initiation, progression or plaque rupture
• Is it all a secondary artifactual event ?