Lipid Mass Spectrometry in the Cardiovascular 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 COC 15 H 31 OC O C 19 H 31 P O OCH 2 NHO O O CH CO OC O P O O O CC 14 H 29 C 19 H 31 OCH 2 NH 3 CH HH 2 O OC O P O O O O 2 NH 3 CH C 19 H 31 C 15 H 31 3

OO O O P(CH 2 ) 2 N CH OC O R 1 OH + OO O O P(CH 2 ) 2 N CH OC O R 1 OCCC 14 + HH CCC O Cl H + Myeloperoxidase +H 2, Cl Plasmenylcholine (Plasmalogen) Lysophosphatidylcholine 2-Chlorohexadecanal ( -Chloro-fatty aldehyde) O 2 H 29 H H Schiff base adducts LPC chlorohydrins Plasmalogen Targeting by Reactive Chlorinating Species Derived from Phagocytes

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 D E 2-Chlorohexadecanal in infarcted myocardium

Concomitant accumulation of neutrophils and 2-chlorohexadecanal

Schiff Base Formation + O OP - O O O NH 2 O O R 1 R 2 O Phosphatidylethanolamine (PE) C 14 H 29 O Cl 2-Chlorohexadecanal (2-ClHDA) 29 C 14 H Cl O OP - O O O N O O R 1 R 2 O N-2-ClHDA-PE H2OH2O

2-ClHDA Forms Schiff Bases with di16:0-PE

N-2-ClHDA-PE Schiff Base Adducts in HCAEC Incubate HCAEC with 25 M 2-ClHDA Stabilize Schiff bases with NaCNBH 3 Detect and quantify Schiff base adducts by LC- MS/MS (SRM) OC O C 15 H 31 OC O C 15 H 31 OP O O O NH C 14 H 29 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

Secondary RCS Attack of Plasmalogens: Production of LPC Chlorohydrins

CID of m/z 596 (18 carbon LPC Chlorohydrin)

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 Phagocyte Chemoattractant, Schiff base adducts Cell Death LPC Chlorohydrin PKA activation Gene activation? Effects on Endothelial Cells Cell Death? OO O O P(CH 2 ) 2 N CH OC O R 1 OH + OO O O P(CH 2 ) 2 N CH OC O R 1 OCCC 14 + HH CCC O Cl H + Myeloperoxidase +H 2, Cl Plasmenylcholine (Plasmalogen) Lysophosphatidylcholine 2-Chlorohexadecanal ( -Chloro-fatty aldehyde) O 2 H 29 H H

ACKNOWLEDGEMENTS Saint Louis University: Carolyn Albert Arun Thukkani Kristin Wildsmith Maria Messner Viral Brahmbhatt Dhanam Anbukumar George Vogler Brad Martinson Washington University Mass Spectrometry Resource: John Turk Fong Hsu Jan Crowley NIH and American Heart Association