Electrospray ionization tandem mass spectrometry of sodiated adducts of cholesteryl esters

Abstract

Cholesteryl esters (CE) are important lipid storage molecules. The present study demonstrates that sodiated adducts of CE molecular species form positive ions that can be detected in both survey scan mode as well as by exploiting class-specific fragmentation in MS/MS scan modes. A common neutral loss for CE is the loss of cholestane (NL 368.5), which can be used to specifically quantify tissue CE molecular species. Using this MS/MS technique, CE molecular species were quantified in mouse monocyte-derived macrophages (J774 cells) incubated with either linoleic (18:2) or arachidonic acid (20:4). These studies revealed that arachidonic acid was not only incorporated into the CE pool, but also was elongated resulting in the accumulation of 22:4 and 24:4 CE molecular species in macrophages. Additionally, this technique was used to quantify CE molecular species present in crude lipid extracts from plasma of female mice fed a Western diet, which led to an enrichment in CE molecular species containing monounsaturated fatty acids compared to female mice fed a normal chow diet. Last, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species containing polyunsaturated fatty acids. Taken together, these studies demonstrate the utility of using sodiated adducts of CE in conjunction with direct infusion electrospray ionization tandem mass spectrometry to rapidly quantify CE molecular species in biological samples.

Figure 1

Survey, CAD, and parent ion scans of sodiated cholesteryl palmitate (16:0 CE) and linoleate (18:2 CE) using direct-infusion ESI-MS analysis. Survey scans (A & B) were acquired for five min over the m/z range of 550 – 750. The CAD analysis (C & D) was performed at a collisional energy of 25 eV. Parent ion scans (E & F) were acquired from the sodiated fatty acyl fragment (25 eV). The neutral loss of cholestane (NL 368.5) was also performed at 25 eV (D). The concentrations of 16:0 and 18:2 CE were 10 µM. The NaOH added prior to ESI analysis was 10 µM.

Figure 2

Neutral loss of 368.5 MS/MS analyses of an equimolar mixture of the cholesteryl esters (each at 2 µM) as sodiated adducts. Spectra were obtained using the neutral loss 368.5 at a collisional energy of 25 eV as described in “Materials and methods”. Horizontal dashed lines highlight the disparate ionization efficiencies associated with CE molecular species containing different levels of unsaturation.

Figure 3

MS and MS/MS analyses of CE and DAG sodiated adducts. 16:0 CE (m/z 647) and 17:0 CE (m/z 661) (A and D), 18:0-18:0 DAG (m/z 647) and 20:0-20:0 DAG (m/z 703) (B and E), and a mixture of both CE and DAGs (C and F) (all lipids present at a concentration of 5 µM) were subjected to direct-infusion ESI-MS analysis. Panels A–C are survey scans of each lipid mixture over an m/z range of 600 – 725 for five min. Panels D–F are scans for the neutral loss of 368.5, which were acquired for three min. The collisional energy for the MS/MS analysis of the CE molecular species was 25 eV. The NaOH added prior to ESI analysis was 10 µM.

Figure 4

NL 368.5 scans of sodiated CE present in J774 cells. J774 cells were cultured in the presence of either no fatty acid supplementation (A), linoleic acid supplementation (B) or arachidonic acid supplementation (C). At the end of the cell culture treatment interval, lipids were extracted from J774 cells with 17:0 CE added as an internal standard as described in detail in “Materials and methods”. Neutral loss scans were acquired for five min at a collisional energy of 25 eV. NaOH added prior to ESI analysis was 10 µM.

Figure 5

CE molecular species present in J774 cells. J774 cells were cultured in the presence of either no fatty acid supplementation (open bars), linoleic acid supplementation (black bars) or arachidonic acid supplementation (hatched bars). At the end of the cell culture treatment interval, lipids were extracted from J774 cells with 17:0 CE added as an internal standard, and were subsequently subjected to ESI-MS using NL 368.5 scanning as described in detail in “Materials and methods”. Values are the means + S.E.M. for n = 3. All values were corrected by calibration constants in Table 1 with the exception of those indicated (‖), which were corrected with the response factor (slope) of the corresponding shorter chain fatty acid with the same degree of unsaturation. For example, 22:4 and 24:4 CE were corrected using the 20:4 response factor. These elongated CE molecular species were not available to derive response curves.

Figure 6

Survey and NL 368.5 scans of sodiated cholesteryl esters present in mouse plasma. Female mice were fed a diet of either normal chow (A & B) or Western diet chow (C & D) for 14 weeks, and plasma was subsequently collected for the analyses of CE molecular species as described in detail in “Materials and methods”. Survey scans (A & C) and NL 368.5 scans (B & D) at a collisional energy of 25 eV were each acquired for five min. The NaOH added prior to ESI analysis was 10 µM.

Figure 7

Plasma CE molecular species in female mice fed either normal chow diet or Western diet. Female mice were fed a diet of either normal chow (open bars) or Western diet chow (black bars) for 14 weeks, and plasma was subsequently collected for the analysis of CE molecular species using NL 368.5 scans as described in detail in “Materials and methods”. All values were corrected by calibration constants in Table 1 with the exception of 22:5 CE (‖), which was corrected with the response factor (slope) of 20:4 CE. Values are the means + S.E.M. for n = 3. * and ** indicate p < 0.05 and p < 0.005 for comparisons between normal chow diet and the Western diet.

Figure 8

Comparative oxidation of saturated and unsaturated CE. Indicated CE molecular species (18:0, 18:2, 20:4, and 22:6 molecular species) were dried individually and exposed to ambient air for 15 h. Treatments were terminated by resuspending CE in methanol/chloroform (4/1) containing equimolar 17:0 CE added as an internal calibrant. Each treated CE was subsequently subjected to direct-infusion ESI-MS analyses using neutral loss 368.5 scanning of sodiated adducts as described in “Materials and methods”. In the absence of oxidizing conditions, NL 368.5 analyses showed precursor CE were not decomposed and no oxidized products were observed.