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Oxidation of low density lipoproteins (LDL) results in changes to the lipoprotein particle that are potentially pro-atherogenic. To investigate mechanisms contributing to the formation of cholesteryl ester (CE)–core aldehydes (9-oxononanoyl- and 5-oxovaleroyl-cholesterol; 9-ONC and 5-OVC, respectively) LDL was incubated in the presence of mouse macrophages (J774 cells) under different culture conditions. Here we demonstrate that the formation of core aldehydes occurs only in transition metal-containing HAM's F10 medium but not in Dulbecco's modified Eagle's medium (DMEM), independent of supplementation with iron and copper at concentrations up to ten times higher than present in HAM's F10. The antioxidative properties of DMEM could be ascribed to the higher amino acid and vitamin content as compared to HAM's F10 medium. Supplementation with these components efficiently inhibited LDL oxidation in HAM's F10. Stimulation of J774 cells with phorbol ester (PMA) resulted in significantly enhanced 9-ONC and 5-OVC formation rates that were accompanied by increased consumption of LDL cholesteryl linoleate (Ch18:2) and cholesteryl arachidonate (Ch20:4) in the cellular supernatant. In PMA (10 ng/ml) activated cells, approximately 5% of Ch18:2 contained in LDL was converted to 9-ONC and 4% of Ch20:4 was converted to 5-OVC. With respect to core aldehyde formation, lipopolysaccharide (LPS, 10 μg/ml) was a less effective stimulant as compared to PMA. Part of the core aldehydes accumulated within the cells. Our study demonstrates that i) J774 macrophages are able to promote/accelerate core aldehyde formation in HAM's F10 medium, and ii) that core aldehyde formation rates can be increased by stimulation of the cells with PMA, and, although to a lesser extent, with LPS. Finally we could show that iii) a small amount of the core aldehydes is internalized by J774 macrophages.—Karten, B., H. Boechzelt, P. M. Abuja, M. Mittelbach, and W. Sattler. Macrophage-enhanced formation of cholesteryl ester–core aldehydes during oxidation of low density lipoprotein. J. Lipid Res. 1999. 40: 1240–1253.

A new route for the preparation of 9-oxononanoyl cholesterol (5) and its stable dimethylacetal (4) is described. The core aldehyde 5 is one of the major products formed during lipid peroxidation. The synthesis starts with the ozonization of oleic acid in methanol and further reduction with dimethyl sulfide to yield 9,9-dimethoxy nonanoic acid (2a). The condensation of 2a with cholester ol is achieved with N,N′-dicyclohexylcarbodiimide in dichloromethane to give 4. Further hydrolysis of 4 with the help of an acidic ion exchange resin yields 9-oxononanoyl cholesterol.—Boechzelt, H., B. Karten, P. M. Abuja, W. Sattler, and M. Mittelbach. Synthesis of 9-oxononanoyl cholesterol by ozonization. J. Lipid Res. 1998. 39: 1503–1507.

9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during lipoprotein oxidation, was recently identified in advanced human atherosclerotic lesions. Here we present a rapid and sensitive HPLC method for 9-oxononanoyl cholesterol analysis. 9-Oxononanoyl cholesterol was converted to the corresponding fluorescent decahydroacridine derivative by reaction with 1,3-cyclohexanedione. The derivatives formed were purified by solid-phase extraction on C-18 columns, separated by reversed phase HPLC with isocratic elution, and detected by their fluorescence. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were stable for at least 160 h. The limit of quantitation of the method presented is at the low (≈50) femtomole level, with an absolute limit of detection (signal: noise = 6) of 15 fmol. Intra-assay variation was ≤5%, while inter-assay variations were between 5 and 15%, depending on the concentration of the analyte. Standard curves were linear over nearly three orders of magnitude (50 fmol–12.5 pmol). 9-Oxononanoyl cholesterol proved to be the major cholesterol core-aldehyde formed during t-BuOOH/FeSO4 oxidation of cholesteryl linoleate and Cu2+-induced LDL oxidation, findings confirmed by atmospheric pressure chemical ionization–mass spectrometry. Analysis of lipid extracts obtained from advanced human atherosclerotic lesions revealed the presence of 9-oxononanoyl cholesterol in all tissue samples analyzed (28 ± 14 μmol/mol cholesterol, n = 9) despite the presence of α-tocopherol (4 ± 1.2 mmol/mol cholesterol, n = 9).—Karten, B., H. Boechzelt, P. M. Abuja, M. Mittelbach, K. Oettl, and W. Sattler. Femtomole analysis of 9-oxononanoyl by high performance liquid chromatography. J. Lipid Res. 1998. 39: 1508–1519.