Lipid Mass Spectrometry in the Cardiovascular System Plasmalogen Targeting by Reactive Chlorinating Species Derived from Phagocytes in Infarcted Myocardium.
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Lipid Mass Spectrometry in theCardiovascular System
Plasmalogen Targeting by Reactive Chlorinating Species
Derived from Phagocytes in Infarcted Myocardium
David Ford, Ph.D. Department of Biochemistry and Molecular Biology
St. Louis University School of Medicine
DIACYL(Phosphatidylethanolamine)
PLASMALOGEN(Plasmenylethanolamine)
ALKYL ETHER(Plasmanylethanolamine)
MOLECULAR SUBCLASSES OF ETHANOLAMINE GLYCEROPHOSPHOLIPIDS
CO C15H31
O C
O
C19H31
P
O
O CH2 NHO
O
O
CH2
+
-
CO
O C
O
P
O
O
O
C C14H29
C19H31
O CH2 NH3CH2
+
-
H H
CH2O
O C
O
P
O
O
O
O CH2 NH3CH2
+
-
C19H31
C15H31
3
O O
O
O
P (CH2)2 N
CH3
CH3
CH3
O C
O
R1
OH
+
O O
O
O
P (CH2)2 N
CH3
CH3
CH3
O C
O
R1
O C C C14
+
HH
C C C14O
Cl
H
+
Myeloperoxidase
+H2 , Cl
Plasmenylcholine(Plasmalogen)
Lysophosphatidylcholine
2-Chlorohexadecanal(-Chloro-fatty aldehyde)
O2
H29
H29
H Schiff base adducts
LPC chlorohydrins
Plasmalogen Targeting by Reactive Chlorinating Species
Derived from Phagocytes
m/z0 300 600
35
37
178196
288
290414
m/z289
288
290
m/z36
35
37
0
50
100
F
F
F
F
F
C O N C C (CH2)13 CH3H
H
H
196(-H)
288 (290)
H
Cl
167
469 = M- (Parent Ion)449 = M- -HF414 = M- -HF - Cl
178 =
F
F
F
F
O
FIGURE 19
GC-MS analysis by negative ion electron capture of the PFB oxime of 2-chlorohexadecanal produced from RCS attack
of plasmalogens in infarcted myocardium
1 day LAD Occlusion
1 day sham surgery
Mid-Ventricle
Mid-Ventricle
D
E
2-Chlorohexadecanal in infarcted myocardium
1 day LAD OcclusionA Zero Time LAD Occlusion
C1 day sham surgeryB
Concomitant accumulation of neutrophils and 2-chlorohexadecanal
0
70
35
0 Day 1 Day 2 Day
Infarct
Infarct (Neutropenic)
Sham Surgery
0 Day 1 Day 2 Day
0
6
Schiff Base Formation
+
O
O P-
O
O
O
NH2O
O
R1R2
O
Phosphatidylethanolamine (PE)
C14H29
O
Cl
2-Chlorohexadecanal (2-ClHDA)
29C14H
Cl
O
O P-
O
O
O
NO
O
R1R2
O
N-2-ClHDA-PE
H2O
2-ClHDA Forms Schiff Bases with di16:0-PE
600 700 800 900 1000m/z
0
50
100 912
674951898
m/z
912
100 200 300 400 500 600 700 800 9000
50
100 255
674362 418 656
+NaCNBH3
CID
Full spectra
600 700 800 900 1000m/z
0
50
100 690
762
946748
910
- NaCNBH3
100 200 300 400 500 600 700 800 900m/z
0
50
100255
391 647672
409153 708634
360416
946910
C14
H29
O
O
O
C15
H31
O
P
O
O
N H
H31
C15
O
O-
O
O
O
C15
H31
O
P
O
O
N
Cl
C14
H29
H31
C15
O
O-
255 Da255 Da255 Da255 Da
362 Da362 Da --HClHCl
910 Da910 Da
360 Da360 Da
N-2-ClHDA-PE Schiff Base Adducts in HCAEC
Incubate HCAEC with 25 M 2-ClHDAStabilize Schiff bases with NaCNBH3
Detect and quantify Schiff base adducts by LC-MS/MS (SRM)
O C
O
C15H31
OC
O
C15H31
O P
O
O
O
NH C14H29
m/z 362
[M - H]- = m/z 912
SRM = [M-H]- m/z 362
N-2-ClHDA-PE Schiff Base Adducts in Human Coronary Artery Endothelial Cells
0
0.5
1
1.5
2
2.5
3
3.5
4
control 1h 2h 4h
time
N-2-ClHDA ethanolamine glycerophospholipid reduced
Schiff base adducts(pmol/nmol Pi )
di14:0 int.std.
p16:0-20:4
p18:1-20:4
p18:0-20:4
18:0-20:4
18:0-22:6
0
100
450 550 650 750 850m/z
0
450 550 650 750 850m/z
100
590 610 825 845m/zm/z
834
836
834
836
596
598
598
596
794782
782
766
744
760
544
PANEL A:16:0-18:1 Phosphatidylcholine
(m/z 760 and 782)+
16:0-18:1 Plasmenylcholine (Plasmalogen) (m/z 744 and 766)
NO Treatment
PANEL B:16:0-18:1 Phosphatidylcholine +16:0-18:1 Plasmenylcholine (Plasmalogen)
After RCSTreatment
574
812
O OO
OP (CH2)2 N
CH3
CH3CH3
O CO
O C C
+
HH
Plasmenylcholine with esterified 18:1 at the sn-2 position
(Plasmalogen) Unsaturated Lysophosphatidylcholine(18:1 Lysophosphatidylcholine)
R1
RCS(CH2)7 C8H17C CH H
O OO
OP (CH2)2 N
CH3
CH3CH3
O CO
OH
+
(CH2)7 C8H17C CH H
LPC Chlorohydrins
RCS Attack
Secondary
O OO
OP (CH2)2 N
CH3
CH3CH3
O CO
OH
+
(CH2)7 C8H17C CH H
HO Cl
Secondary RCS Attack of Plasmalogens: Production of LPC Chlorohydrins
100
0100 200 300 400 500
147104
86
377
321
413
501
537596
391
355337
600
m/z
m/z 537 = (M + Na+ ) - N(CH3)3
m/z 413 = (M + Na+ +H+ ) - m/z 184
m/z 391 = (M +H+ ) - m/z 184
m/z 501 = (M + Na+ ) - N(CH3)3 - H35Cl
m/z 377 = (M + Na+ +H+ ) - m/z 184 - H35Cl
m/z 355 = (M +H+ ) - m/z 184 - H35Cl
m/z 337 = (M +H+ ) - m/z 184 - H35Cl -H20
N+CH3
PO
O
O
OO
OH
O-Na+
CH3
CH3
35Cl OH
CH3
(M + Na+) = m/z 596
CID of m/z 596 (18 carbon LPC Chlorohydrin)
0
100
6000
100
6000
100
600
582 582
582
610
610
610
596
596
568 568
m/zm/z m/z
624
Detection of LPC chlorohydrins in HCAEC treated with HOCl:Neutral Loss Scanning of m/z 95
17C (int.std.)
19C (int.std.)
17C (int.std.)
19C (int.std.)
No Treatment 500 M HOCl
Summary of reactive chlorinating species attack of plasmalogens
PhagocyteChemoattractant,
Schiff base adducts
Cell Death
LPC Chlorohydrin
PKA activationGene activation?
Effects on EndothelialCells
Cell Death?O O
O
O
P (CH2)2 N
CH3
CH3
CH3
O C
O
R1
OH
+
O O
O
O
P (CH2)2 N
CH3
CH3
CH3
O C
O
R1
O C C C14
+
HH
C C C14O
Cl
H
+
Myeloperoxidase
+H2 , Cl
Plasmenylcholine(Plasmalogen)
Lysophosphatidylcholine
2-Chlorohexadecanal(-Chloro-fatty aldehyde)
O2
H29
H29
H
ACKNOWLEDGEMENTS
Saint Louis University:Carolyn AlbertArun ThukkaniKristin WildsmithMaria MessnerViral BrahmbhattDhanam AnbukumarGeorge VoglerBrad Martinson
Washington University Mass Spectrometry Resource:John TurkFong HsuJan Crowley
NIH and American Heart Association
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