LC‐MS/MS determination of phosphatidylcholine hydroperoxide and its truncated products in human blood plasma (1001.9)

Abstract

Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine (PC), in the blood plasma has been observed in vascular pathological conditions, including inflammation and atherosclerosis. Here, we developed a method for quantification of PCOOH (1‐palmitoyl‐2‐hydroperoxyoctadecadienoyl‐sn‐glycero‐3‐phosphocholine, 16:0/HODE PC), especially isomers such as 16:0/13‐HODE PC and 16:0/9‐HODE PC that might be present in human plasma, together with their breakdown products (truncated PC such as 16:0/azelaoyl PC), by using LC‐MS/MS. Collision‐induced dissociation of sodiated PCOOH isomers ([M+Na]+, m/z 812) produced not only a fragment ion (m/z 147) but also characteristic neutral losses (88 Da for 16:0/13‐HODE PC and 169 Da for 16:0/9‐HODE PC). Three multiple reaction monitorings (MRMs) of sodiated PCOOH could be performed under optimum conditions. MRM (812/147) enabled determination of 16:0/HODE PC, and MRM (812/541 and 812/388) allowed measurement of 16:0/13‐HODE PC and 16:0/9‐HODE PC, respectively. By using the method, we could determine plasma PCOOH concentrations in healthy subjects (260nmol/L of total PCOOH, 50‐80nmol/L of truncated PC, 36nmol/L of 16:0/13‐HODE PC and 33nmol/L of 16:0/9‐HODE PC) and in patients with angiographically significant stenosis (45nmol/L of 16:0/13‐HODE PC, 51 nmol/L of 16:0/9‐HODE PC). The PCOOH detected is formed with lipoxygenase and free radical reaction. Oxidized PC with singlet molecular oxygen was not found. The newly developed LC‐MS/MS method appears to be a powerful tool for understanding in vivo membrane lipid peroxidation.