Plasma PCOOH Research Articles

Determination of Phosphatidylcholine Hydroperoxide in Human Plasma by Chemiluminescence - High Performance Liquid Chromatography

Abstract To determine phosphatidylcholine hydroperoxide (PCOOH) in human plasma lipids, a chemiluminescence -high performance liquid chromatography (CL-HPLC) system was newly developed and applied. This system * To whom correspondence should be addressed. consisted of separation of phosphatidylcholine derivatives with normal phase HPLC and detection of hydroperoxide-specific chemiluminescence of PCOOH. By use of this system, the PCOOH in normal human plasma was quantitatively assayed to be 28-431 pmol hydroperoxide-02 per ml of plasma. The high sensitivities, reproducibilities and specificities of the CL-HPLC assay were shown for determining plasma PCOOH.

Presence of phosphatidylcholine hydroperoxide in human plasma.

A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was newly developed and used for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH) in human plasma. The method involves separation of phosphatidylcholine derivatives from plasma lipids by normal phase HPLC and subsequent detection of hydroperoxide-dependent chemiluminescence (CL) of PCOOH. CL was produced through luminol oxidation during the reaction of the hydroperoxide and cytochrome c-heme. The high specificity for the hydroperoxide allows the sensitive assaying of a large PCOOH range over a concentration range of 50-2,000 pmol of hydroperoxide-O2. Using this method, the occurrence of PCOOH in normal human plasma was strongly suggested and was confirmed quantitatively.

Chemiluminescence-high performance liquid chromatography of phosphatidylcholine hydroperoxide in human blood plasma.

Normal phase HPLC (high performance liquid chromatography) with a UV detection has been used frequently for analyzing phospholipid classes. (1, 2) However, hydroperoxide and its hydroxy derivative usually give almost the same retention time as that for corresponding unoxidized species. (3) We have shown previously that chemiluminescence (CL) detection is very advantageous in its specificity and sensitivity for hydroperoxides. (4, 5) To analyze the phosphatidylcholine hydroperoxide (PCOOH), we newly devised the chemiluminescence — HPLC (CL-HPLC) system, in which luminol-cytochrome с solution is used as a hydroperoxide-specific CL reagent. We applied this new method to confirm the presence of PCOOH in blood plasma of healthy donors.