Strategies for the analysis of chlorinated lipids in biological systems

Abstract

Myeloperoxidase-derived HOCl reacts with the vinyl ether bond of plasmalogens yielding α-chlorofatty aldehydes. These chlorinated aldehydes can be purified using thin-layer chromatography, which is essential for subsequent analysis of extracts from some tissues such as myocardium. The α-chlorofatty aldehyde 2-chlorohexadecanal (2-ClHDA) is quantified after conversion to its pentafluorobenzyl oxime derivative using gas chromatography-mass spectrometry and negative-ion chemical ionization detection. 2-ClHDA accumulates in activated human neutrophils and monocytes, as well as in atherosclerotic lesions and infarcted myocardium. Metabolites of 2-ClHDA have also been identified, including the oxidation product, 2-chlorohexadecanoic acid (2-ClHA), and the reduction product, 2-chlorohexadecanol. 2-ClHA can be quantified using LC-MS with selected reaction monitoring (SRM) detection. 2-ClHA can be ω-oxidized by hepatocytes and subsequently β-oxidized from the ω-end, leading to the production of the dicarboxylic acid, 2-chloroadipic acid. This dicarboxylic acid is excreted in the urine and can also be quantified using LC-MS methods with SRM detection. Quantitative analyses of these novel chlorinated lipids are essential to identify the role of these lipids in leukocyte-mediated injury and disease.

Fig. 1

Structures of the members of the chlorinated lipidome derived from HOCl oxidation of plasmalogens and abbreviated strategies for their quantification. PX, LPX, PFB-HA, and PFB-Cl are the abbreviations for phospho-head group (e.g., phosphorylcholine), lysophospholipids, pentafluorobenzyl hydroxylamine, and pentafluorobenzoyl chloride.

Fig. 2

Flowchart of strategies to quantify members of the chlorinated lipidome.

Fig. 3

50 μg of human LDL was incubated with (A) 0, (B) 125, or (C) 500 μM HOCl in 50 μl of PBS for 30 min at 37 °C. Reaction products were extracted in the presence of 50 ng of 2-Cl-[d₄]–HDA, and α-chlorofatty aldehydes were converted to their PFB oximes and subsequently subjected to GC–MS as described. a and s indicate anti and syn isomers of the PFB oximes, and the identity of peak * is unknown.

Fig. 4

25 μl of human serum was subjected to base hydrolysis in the presence of 105 fmol of 2-[d₄]-ClHA and subjected to LC–MS as described under Protocols. The concentration of 2-ClHA in this sample was 4.4 nM. This example shows results with a scan time of 0.5 s. The resolution of peaks can be further enhanced with shorter scan times and by limiting the number of SRMs in an analysis.

Fig. 5

100 or 50 μl of human or rat urine spiked with indicated amounts of 2-ClAdA was extracted in triplicate in the presence of 10 ng of [d₄]-AdA and subjected to LC–MS as described under Protocols. The response of 2-ClAdA to [d₄]-AdA is plotted.