Direct electrospray tandem mass spectrometry of the unstable hydroperoxy bishemiacetal product derived from cholesterol ozonolysis

Abstract

Cholesterol is the most abundant neutral lipid in the epithelial lining fluid of the lower airways of the lung also known as pulmonary surfactant and a potential target for reaction with ambient ozone when inspired into the human lung. The isolated double bond of cholesterol has been shown to be susceptible to attack by ozone, but the major reaction product from cholesterol ozonolysis had been remarkably difficult to structurally characterize. Recently, NMR and X-ray crystallography have been used to suggest the formation of a hydroperoxy, hydroxy hemiacetal product, using various derivatives and models of cholesterol to stabilize this chemically reactive product. Electrospray ionization mass spectrometry was used to study the somewhat unstable ozonolysis product of cholesterol which was found to display unique ionization and fragmentation properties when collisionally activated. The electron-deficient carbon atoms of this highly oxygenated product permitted covalent attachment of an acetate anion during negative ion electrospray ionization, leading to the formation of abundant adduct ions at m/z 511. Surprisingly, positive ions were not readily formed. Collision induced dissociation of the adduct anion yielded a major ion at m/z 477, corresponding to the loss of hydrogen peroxide. The most abundant fragment ion following collisional activation was observed at m/z 93, resulting from a complex rearrangement subsequent to the attack of the hydroperoxide anion on the carbon center of the acetate adduct. Based on the interpretation of the tandem mass spectral data, the major cholesterol ozonization product was characterized as a hydroperoxy, hydroxy hemiacetal derivative, which was consistent with the NMR and X-ray crystallographic studies which were carried out on the more stable methyl ether derivative.

Cholesterol, Reactive Oxygen Species, and the Formation of Biologically Active Mediators

“…2). Some of the more stable ozonide products have been recently structurally characterized (21). Peroxidation of polyunsaturated fatty acyl groups is also initiated by reaction of ozone, leading to the formation of abundant phospholipid hydroperoxides.…”

“…It was not possible to analyze species such as the hydroperoxides of Ch directly by gas chromatography/mass spectrometry. Now even reactive hydroxy, hydroperoxy, and intact ozonides can be directly analyzed using electrospray ionization (21). This capability has changed both types of oxysterol studies because it is now possible to detect those oxysterol intermediates and to focus attention on additional oxysterol entities that could mediate biological activities.…”

Characterization of O2 (1Δg)-derived oxidation products of tryptophan: A combination of tandem mass spectrometry analyses and isotopic labeling studies

“…For instance, this technique has been employed to analyze oxidized lipids derived from the Fenton reaction, such as oxidation products of linoleic acid [9], phospholipids [10,11], and also cholesterol ozonolysis derived products [12]. In DNA, a representative example is O 2 ( 1 ⌬ g ) oxidation of 2=-deoxyguanosine, which has been shown to yield 8-oxo-7,8-dihydro-2=-deoxyguanosine in double stranded DNA and also other oxidized products upon reaction with an isolated base [13,14].…”

Studying Interfacial Reactions of Cholesterol Sulfate in an Unsaturated Phosphatidylglycerol Layer with Ozone Using Field Induced Droplet Ionization Mass Spectrometry

“…Alternatively, the vinyl hydroperoxide may be generated for product III [26,30]. In addition, what we assume to be hydroxyhydroperoxide (HHP), methoxyhydroperoxide (MHP) products are observed at m/z 531 and 545, respectively [29,31]. The proposed structures of products and reaction mechanisms are shown in Scheme 2.…”

“…Proposed structures of products I, II, and III are adopted from Reference [25][26][27][28][29]. Proposed structures of products IV and V are adopted from Reference [29,31] aerosol reported bound multimeric products, which are formed through aggregated gas phase clusters [26]. (Figure 2a).…”