Transcript Διαφάνεια 1

Investigation of the presence of Ischemic Heart
Disease by the 1H NMR-based lipidomics of red
blood cell membranes
Kastani I(1), Kostara C(1), Baltogiannis J(2), Tsimihodimos V(2), Elisaf M(2),
Goudevenos J(2), Bairaktari E(1)
(1)Laboratory
of Clinical Chemistry and (2) Department of Internal Medicine, Medical School,
University of Ioannina, 451 10, Ioannina, Greece
Red blood cells (RBCs)
the major cell component of blood
Composition of RBCs membrane (w/w):
• 20 % water
• 40 % proteins
• 34 % lipids
• 6 % carbohydrates
Main lipid components of RBC membrane:
• Phospholipids (60%)
Phosphatidylcholine 30%
Sphingomyelin 24%
Phosphatidylethanolamine 26%
Phosphatidylserine 15%
• Cholesterol (30%)
mainly non-esterified
• Glycolipids (10%)
Protein and lipid composition of the RBCs is subject to change during its life-time
RBC Membrane Lipid Composition and Cardiovascular Disease
Cholesterol  Ischemic Heart Disease Patients vs Controls
cholesterol of erythrocyte membrane
cholesterol of erythrocyte membrane
Tziakas D.N et al, Coron Artery Dis. 2008 Dec;19(8):583-90
SAP : Stable Angina Pectoris
ACS: Acute Coronary Syndrome
NSTEACS : non-ST-segment elevation Acute Coronary Syndrome
STEMI : ST-segment elevation Myocardial Infraction
CEM : total Cholesterol content of Erythrocyte Membrane.
Zhong et al Ups J Med Sci 2012, 117(4):390-8
Zhang J et al. Coron Artery Dis. 2013, 24(5):361-7
RBC Membrane Lipid Composition and Cardiovascular Disease
Omega-3 Index
EPA: eicosapentaenoic acid
DHA: docosahexaenoic acid
A measure of the amount of % (EPA+DHA) / total fatty acids
in RBC membranes
risk factor of death from coronary heart disease
R.Block,W. Harris et al,Arteriosclerosis 197(2008)821-828
Conventional methods for lipid analysis
 Thin Layer Chromatography (TLC)
 High Performance Liquid Chromatography (HPLC)
 Gas Chromatography (GC)
 Mass Spectrometry (MS)

Without chemical modification

Fast and overall analysis of lipid profiling
Chol
PL
SM/PC
Triglycerides
PL , TG
DU
Fatty acid residues
FC/EC
Aim Of The Study
Investigation of the ability of the 1H NMR-based lipid profiling
of red blood cell membranes to detect the presence
of Ischemic Heart Disease (IHD)
Patients
 11 patients with severe IHD (triple vessel disease, TVD)
 18 patients with normal coronary arteries (NCA)
Groups studied:
Age- and conventional lipid
parameters matched
Sample Collection
After an overnight fast
Red blood cells
Hypotonic Lysis
Extraction with
washing
of red blood cells
Methanol: Chroloform
Evaporation under N2, redissolved in CD3OD:CDCl3 (2:1)
NMR Spectrum
NMR spectrum NMR Center, University of Ioannina
Spectrometer:
Bruker Avance DRX-500
(Magnetic Field Strength 11.7 Tesla)
Temperature: 298 Κ
Time spectrum: 10 λεπτά
NMR Lipidomic Data
Statistical Analysis: Pattern Recognition Methods
PL , TG
Phospholipids
Sphingomyelin/Phosphatidylcholine
Triglycerides
nFA
E+A
LA
DU
ω-3
Free Chol –Esterified Chol
Cholesterol (Chol)
Saturated Fatty acids
Unsaturated Fatty acids
Fatty acid residues
1H
NMR-OSC/PLS-DA analysis
Class 1
Class 2
a) Scores plot
b) Regression coefficient plot
20
0,010
Increased in NCA
DU
PC
NCA
SM DIAL
10
Coefficients
t[2]
0,005
0,000
TVD
ω-3
UFA
E+A
0
-0,005
-0,010
-10
Chemic al
0.745
1.015
1.285
1.555
1.825
2.095
2.365
S h i f t (p p m)
SIMCA-P 10.5 - 5/3/2015 2:07:18 ìì
SIMCA-P 10.5 - 5/3/2015 2:03:44 ìì
TVD: Triple Vessel Disease
NCA: Normal Coronary Arteries
2.635
2.905
3.175
3.685
3.955
4.225
4.495
5.155
t[1]
5.425
0,01
5.695
0,00
5.965
-0,01
chol
Increased in TVD
UFA: Unsaturated Fatty Acids
DIAL: Diallylic Fatty Acids
PC:Phosphatidylcholine
SM:Sphingomyelin
1H
NMR-based lipidomic analysis of red blood cell membranes:
Identification of lipid biomarkers
•Early evaluation of the presence of IHD
•Establishment an appropriate therapeutic option