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Leukocytes are key cellular mediators of human diseases through their role in inflammation. Identifying unique molecules produced by leukocytes may provide new biomarkers and mechanistic insights into the role of leukocytes in disease. Chlorinated lipids are generated as a result of myeloperoxidase-containing leukocyte-derived hypochlorous acid targeting the vinyl ether bond of plasmalogens. The initial product of this reaction is α-chlorofatty aldehyde. α -Chlorofatty aldehyde is both oxidized to α-chlorofatty acid and reduced to α-chlorofatty alcohol by cellular metabolism. This review focuses on the separation techniques and quantitative analysis for these chlorinated lipids. For α-chlorofatty acid the negative charge of carboxylic acids is exploited to detect the chlorinated lipid species of these acids by electrospray ionization mass spectrometry in the negative ion mode. In contrast, α-chlorofatty aldehyde and α-chlorofatty alcohol are converted to pentafluorobenzyl oxime and pentafluorobenzoyl ester derivatives, which are detected by negative ion-chemical ionization mass spectrometry. These two detection methods coupled with the use of stable isotope internal standards and either liquid chromatography or gas chromatography provide highly sensitive analytical approaches to measure these novel lipids.

A method for the determination of the relative amounts of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipids by benzoolysis is described. The procedure consists of simply heating the phospholipid with benzoic anhydride in the presence of boric acid for 5 h at 100 degrees C, followed by reaction with 4-dimethylaminopyridine for 2 h at room temperature and HPLC analysis of the lipidic products formed. With model compounds of the three subclasses it is shown that diacyl- and alkylacylglycerophosphocholines are completely dephosphorylated by this procedure, yielding quantitatively the corresponding diacyl- and alkylacylglycerobenzoates. The same procedure applied to a model alk-1-enylacylglycerophosphocholine gives only 53.5% of dephosphorylation, while the dephosphorylated products in turn are quantitatively converted into the corresponding acylglycerodibenzoates. The latter figure is shown to be fairly reproducible. The reduced dephosphorylation rate of plasmalogens appears to be due to complete disruption of the vinyl ether bond. The liberated fatty aldehyde gives rise to an addition product with benzoic anhydride, which was identified by gas chromatography-mass spectrometry. It is demonstrated that acyl migration occurring during the benzoolysis does not interfere with the HPLC separation of the glycerobenzoates and dibenzoates derived from the three distinct subclasses. Results of subclass determinations by benzoolysis of several natural diradylglycerophosphocholines are in good accordance with literature values. The agreement between the plasmalogen contents, determined by benzoolysis and by phosphorus determination following exposure to HCl and separation by thin-layer chromatography, is satisfactory. The reliability of the benzoolysis method is generally discussed.

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography–electrospray ionization–tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25 μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC50 value of 52.4 μM.

The cleavage of the specific vinyl ether linkage at the sn-1 position of plasmalogens leads to the formation of two products: the 1-lyso-2-acyl glycerophospholipid and a long-chain fatty aldehyde. Plasmalogens are measured by quantifying one of these two products. In this paper, we describe a rapid and sensitive procedure for measuring plasmalogens via quantification of long-chain fatty aldehydes. After lipid extraction, the sn-1 vinyl ether bond of plasmalogens is cleaved by acidic hydrolysis. The produced aldehydes are then derivatized with (pentafluorobenzyl)hydroxylamine hydrochloride and analyzed by gas chromatography/mass spectrometry in selected-ion mode. Plasmalogens are then indirectly quantified by subtracting the free aldehydes obtained without prior HCl treatment from the total aldehydes obtained after acidic hydrolysis. This method is applied to three rat brain areas selected for this study. Two of these are affected in neurodegenerative diseases (cerebral cortex and hippocampus) and one is rich in white matter (cerebellum). In comparison to other procedures, the advantages of this method are not only its usefulness in plasmalogen quantification but also the identification of aldehydic breakdown products.