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Identifying new biomarkers


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SHAPE Society

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Identifying new biomarkers

  1. 1. L.Kritharides June 2003 Identifying New Biomarkers of Coronary Disease Dr. Len Kritharides MBBS,PhD, FRACP, FAHA Centre for Vascular Research University of New South Wales and Department of Cardiology Concord Hospital, University of Sydney, Australia.
  2. 2. L.Kritharides June 2003 Diagnostic markers of atherosclerosis • Strategies for narrowing the field of candidate biomarkers has not kept up with developments in analytical technology/genomics/proteomics • A simple strategy may improve our potential to identify disease specific molecules • Key premise: – molecules continuous enter and leave the artery wall – those which leave more readily are more likely to be useful markers- • eg small MW protein vs matrix bound large protein aggregate – Narrow the focus of investigation by identifying molecules which leave the artery wall
  3. 3. L.Kritharides June 2003 identifying molecules which leave the artery wall • Hypothesis: – That molecules which leave the artery wall most readily during simple perfusion will also leave the artery wall more readily in vivo – Differences in protein elution from atherosclerotic arteries and non-atherosclerotic arteries will correlate with differences in vivo • Problem: – How to perfuse human arteries?
  4. 4. L.Kritharides June 2003 Coronary perfusion prior to bypass surgery • Routine perfusion gives cardioplegia (saline buffer with high potassium) • Can be given antegradely with collection of perfusate coming through coronary sinus (venous system) • Collected at start of operation using cuffed catheter • Successive fractions contain decreasing amounts of blood • Lowest blood contamination fractions used for analysis
  5. 5. L.Kritharides June 2003 Coronary perfusates – AIMS • Is there non-plasma protein derived from coronary tree? • Identify at least one such protein • Quantify in atherosclerotic and non-atherosclerotic circulations • Quantify relative to contaminating blood • Relate to disease status- proof of principle
  6. 6. L.Kritharides June 2003 Patients 30 CAD (23M/7W) 7 no CAD (1M/6W) Age 63 v 61 153 perfusates total protein then SDS page PVDF transfer sequencing Total protein in coronary perfusates
  7. 7. L.Kritharides June 2003 Total protein in coronary perfusates Key points: • at high plasma protein contamination, protein is almost all plasma-derived • at low plasma protein contamination (low Hb in unspun sample) total protein is not plasma-derived • Non-linearity after correction for Hb (plasma protein contamination) implies derivation from coronary circulation
  8. 8. L.Kritharides June 2003 N-tmnl sequencing 40 kDa- Band ILGGHLDA 100% homology with Haptoglobin β-chain Quantified by Western
  9. 9. L.Kritharides June 2003 Hpt (µg/ml) 6.8±0.89 µg/ml vs 3.29±1.56 mg/ml (p=0.039) Hpt/Hb (µg/µg) 0.099±0.017 µg/µg Vs 0.016±0.008 µg/µg (p=0.003)
  10. 10. L.Kritharides June 2003 Quantification Of plasma haptoglobin by Automated Nephelometry •Pre-op plasma •Same cohort of CABG patients •Haptoglobin results Not explained by CRP
  11. 11. L.Kritharides June 2003 New cohort Angiography patients Femoral artery samples N=187 • 147 +CAD • 62.8y • 100M • 47W • 40 -CAD • 60.8y • 20M • 20W L.Kritharides June 2003
  12. 12. L.Kritharides June 2003 Conclusions • Proteins do elute from human coronaries • These proteins potentially distinguish between atherosclerotic and non- atherosclerotic vessels • Co-ordination of elution strategies with proteomic technology may facilitate identification of new markers of arterial pathology which may be disease specific L.Kritharides June 2003
  13. 13. L.Kritharides June 2003 Acknowledgements • Heart Research Institute Sydney • Centre for Vascular Research UNSW • Vincent Fairfax Family Trust L.Kritharides June 2003