1. Metabolomic Profile of Human
Myocardial Ischemia by NMR
Spectroscopy of Peripheral Blood
Serum
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2. contents
• Introduction
• Method
• Results
• Discussion
• conclusion

3. INTRODUCTION

4. Introductions
• The challenge of Diagnosing Myocardial Ischemia
in the Emergency Department..
• Why did currently discovered biomarkers haven't
reached clinical practice yet?
• Benefits and challenges in metabolomics..
• Advantages of NMR + multivariate analysis:
– Powerful, reproducible, inexpensive.
– Simultaneous measurement of small concentrations
– Ability to detect the physiochemical status of the
metabolites

5. Aim of the study
• developing a metabolic profile of acute MIS in
peripheral blood serum.
– Swine and patients, highly controlled models of
MIS based on transient angioplasty balloon–
induced coronary occlusion were used for this
purpose,
– clinical validation was carried out in patients with
spontaneous acute chest pain and normal
electrocardiographic results and troponin values

6. METHODS

8. • The animal model:
– 12h fasting juvenile pigs were sedated and
anaesthesized.
– transient angioplasty balloon–induced MIS in the
proximal left anterior descending coronary artery was
induced
– MIS confirmed by ECG..
– Balloon deflated after 5 minutes
– Blood samples withdrawn after
10 and 120 minutes
– Animal recovered
– Comparison was done using blood from own animal
before the procedure as control

9. • Controlled MIS models in Patients:
– Non ACS pts scheduled for PCI for stable angina
– Balloon inflation time was 1 minutes then stent was
implanted
– No complications were recorded
– Blood samples withdrawn after 10 and 120 minutes
– Controls were patients with normal coronary arteries
diagnosed by angiography
– Blood samples of controls were also withdrawn after 10
and 120 minutes
• Study group with spontaneous chest pain
– To determine accuracy of the method
– Pts presented with ST elevation or high tropnin I were
excluded

11. • NMR spectroscopy:
– 20ul of plasma + 2ul of D2O and measured at 37°C
– Signal of selected metabolites were quantified 
PCA/PLS-DA were applied to the NMR spectral data base
 leave one out cross validation (LOOCV) of results was
then applied
• Statistical analysis:
– mean±SD in tables and as box plots in figures
– Comparison models:
• Before vs. after ischemia (in humans and swine)
• Patients vs. controls (at 10 minutes samples)
• No MIS vs. MIS (in pts with chest pain at the ER)
– Continuous data  t-Test, Mann-Whitney 2-tailed test
– Categorical data  chi-square, Fisher exact test
– PLS-DA accuracy test  ROC analysis

12. RESULTS

13. Before vs. after ischemia (experimental)

15. Before vs. after ischemia (humans)

17. Patients vs. controls

18. Patients vs. controls: Time validity

19. Patients vs. controls: reliability
PLS-DA MODEL
Pts Ctrls
ROC curve
Training data Cross
validation
Validation error =10%
LOOCV F.negatives= 5%
Sensitivity: 95%
Specificity: 88%

20. Study group with spontaneous chest pain
PLS-DA MODEL
ROC curve
Cross
validation
Detection error= 13%
F.negatives= 10%
Sensitivity was 90%
specificity 85%

21. DISCUSSION

22. • under highly controlled conditions of coronary flow
restriction, immediate and striking changes in the
metabolic profile of peripheral blood serum can be
detected using NMR spectroscopy.
• metabolomics in a swine model provided very
reproducible, consistent, and robust markers of
severe MIS. Afterward, this information was
validated in human subjects.
• Advantages of using animal models:
– Although animal model showed low sensitivity
biomarkers, rather potential high specificity in a severe
ischemia scenario was detected
– Reduction in human subjects needed

23. • The cardiac tissue undergoes dramatic
metabolomic changes during ischemia, main
receptors involved are:
– peroxisome proliferator-activated receptor alpha,
– peroxisome proliferator-activated receptor gamma
coactivator 1-alpha,
– estrogen-related receptor alpha transcription
factors

24. • Glucose/FA metabolism and acute ischemia..
• Other changes found:
– Lactate release
– Interruption in TCA cycle activity
– down-regulation in enzymes involved in FA oxidation and
Glucose metabolism
– Liver mediated TCA activity through vassopressin
– Increased oxidation of FA though release of catecholamines
– Increase circulation of TGs during MIS as suggested by this
study.
• The delayed increase in creatine, could be explained by
an increased ischemia-related transport of ATP
between cytoplasm and the mitochondria of
cardiomyocytes.

25. Clinical Validation
• This metabolic biosignature showed high accuracy
in discriminating those patients with and without
MIS.
• In most cases, the final diagnosis was not achieved
until 12 hours of thorough evaluation at the
cardiology department!

26. Study limitaions
• Although this method showed potential in
reaching clinical practice in the future the
author recommends wider studies with larger
groups of patients with chest pain.

27. Conclusion
• Metabolomics, based on the model presented
here, represents a robust, minimally invasive and
cost effective bioprofile for the detection of acute
MIS with potential clinical application.
• This is the first study to apply metabolomics in the
field of diagnostic medicine for CVD which shows
potential in future clinical practice

28. My Personal Thoughts
• Using animal models to induce
controlled pathological environments
can be useful in detecting more specific
biomarkers of CVD
• Using metabolomics combined with
statistical cross validation between
patients and control is helpful in
detecting more CVD specific
metabolites.

29. Thank you for your attention
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