Identification of short-chain oxidized phosphatidylcholine in human plasma.
Oxidized phospholipids have been recognized as potentially important compounds that carry biological activities similar to the platelet-activating factor, but their presence in biological tissue has not been firmly established. We developed a novel technique for the quantitative analysis of phospholipids with oxidized acyl chains. The method involves 1) lipid extraction, 2) chromatographic enrichment of phospholipids with short acyl chains, 3) derivatization with 9-(chloromethyl)anthracene, 4) solid-phase extraction of the derivatives, and 5) reversed-phase HPLC with fluorescence detection. The technique was capable of measuring dicarboxylate-containing phosphatidylcholines (PCs) at the picomole level. The method was suited to monitor the generation of oxidized phospholipids from 1-palmitoyl-2-arachidonoyl-PC in the presence of Fe21/ascorbate. The new procedure was used to isolate lipids from human plasma that were identified as anthracene derivatives of short-chain oxidized PC on the basis of chromatographic enzymatic, and spectroscopic evidence. The plasma concentration, determined with an internal standard (1-palmitoyl-2-suberoyl-PC), was 0.6 +/- 0.2 microM (n = 11). The analytical method did not produce oxidation antifacts in significant amount. We concluded that human blood contains oxidatively fragmented PC in submicromolar concentration.
Schlame
M
M
Department of Anesthesiology and Intensive Therapy, University Hospital Charité, Humboldt University, Berlin, Germany.
Haupt
R
R
Wiswedel
I
I
Kox
W J
WJ
Rüstow
B
B
D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006801:Humans; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000097:blood* / Q000302:isolation & purification
D005456:Fluorescent Dyes; D010713:Phosphatidylcholines