Int. J. Mol. Sci. 2014, 15, 23283-23293; doi:10.3390/ijms151223283 OPEN ACCESS
International Journal of
Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article
Lipidome of Atherosclerotic Plaques from Hypercholesterolemic Rabbits Lazar A. Bojic, David G. McLaren, Vinit Shah, Stephen F. Previs, Douglas G. Johns and Jose M. Castro-Perez * Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA; E-Mails: [email protected] (L.A.B.); [email protected] (D.G.M.); [email protected] (V.S.); [email protected] (S.F.P.); [email protected] (D.G.J.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-908-740-5033. External Editor: Mario Ollero Received: 23 October 2014; in revised form: 9 December 2014 / Accepted: 10 December 2014 / Published: 15 December 2014
Abstract: The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance
Int. J. Mol. Sci. 2014, 15
23284
of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome. Keywords: lipidomics; atherosclerosis; plaque; lipids; mass spectrometry
1. Introduction For the past century, cardiovascular disease (CVD) has been the leading cause of death in the industrialized world and is projected to soon achieve this status worldwide [1,2]. At the core of most cardiovascular events is atherosclerosis, a chronic inflammatory condition of the macrovasculature initiated by the subendothelial retention of apolipoprotein B (apoB)-containing lipoproteins [3]. The trapping of lipoproteins by proteoglycans within the arterial intima increases the propensity for various lipoprotein modifications (oxidation, hydrolysis, aggregation) which trigger maladaptive immune responses that potentiate lesion development [3]. Eventually, atherosclerotic lesions reach an unstable state that is prone to rupture, ultimately resulting in acute cardiovascular events [3,4]. Monogenic disorders in which plasma low-density lipoprotein cholesterol (LDL-C) levels are substantially elevated (such as familial hypercholesterolemia) result in significantly increased cardiovascular event rates, and a number of randomized controlled trials of LDL-C lowering interventions, namely statins, consistently demonstrate reductions in CVD risk [5]. As a result, this evidence places beyond a reasonable doubt that LDL-C is a causative biomarker of atherogenesis. Given this breadth of existing data surrounding the link between plasma cholesterol and atherosclerotic cardiovascular disease [5], a logical assumption would be that cholesterol predominates as the lipid species within atheromatas. However, two recent studies have suggested that in a hypercholesterolemic rabbit model of atherosclerosis, triacylglycerols (TGs) actually represent a major constituent of atherosclerotic plaques [6,7]. Despite these studies, a detailed lipidomic analysis of rabbit plaque composition has not yet been reported. Such information may highlight novel therapeutic strategies to induce the regression of established atherosclerotic lesions, which current LDL-lowering therapies have only modestly achieved [8]. The goal of this study was to construct a detailed framework of the lipids that comprise atherosclerotic lesions that could help us to identify novel treatment strategies that promote lesion regression. Here we report a comprehensive lipidomic analysis of atherosclerotic plaques that develop as a consequence of hypercholesterolemia. We took advantage of ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry to gain insight into lesion composition. We quantified relative amounts of lipid subclasses based on acyl chain length and degree of acyl chain saturation within major lipid fractions detected in aortic plaques. 2. Results Male New Zealand White rabbits were administered either regular chow (RC) or regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Rabbits fed the HC diet developed hypercholesterolemia compared to rabbits fed the RC diet (43.65 ± 3.67 versus 0.93 ± 0.15 mmol/L) without an elevation in plasma TGs
Int. J. Mol. Sci. 2014, 15
23285
(0.65 ± 0.06 versus 0.53 ± 0.07 mmol/L) These data are consistent with recently published studies from our laboratory [9]. In order to assess plaque lipid content, we took advantage of high resolution time of flight (ToF) mass spectrometry which can delineate analytes with a mass accuracy of less than 5 parts per million (Tables S1–S14). Thus, we were able to accurately measure the relative quantities of lipids within the atherosclerotic plaque. We isolated lipids from the full-length of the aorta beginning with the aortic arch through the descending aorta to the iliac bifurcation, and subjected the plaque to LC-MS/MS on ToF. Our lipidomic analyses detected most of the major lipid classes including: Cholesteryl esters (CEs), TGs, phosphatidylcholines (PCs), sphingomyelins (SMs), diacylglycerols (DGs), fatty acids (FAs), phosphatidylserines (PSs), lysophosphatidylcholines (LPCs), ceramides (Cers), phosphatidylglycerols (PGs), phosphatidylinositols (PIs) and phosphatidylethanolamines (PEs). Herein, we present a relative quantitation of the fatty acyl composition of these lipid fractions, with particular attention to CEs, TGs, and PCs, the three plasma lipid classes that account for greater than 75% of total plasma lipid [10]. We additionally focused on the lipidomics of plaque SMs, as we found this lipid class to be relatively abundant in lesions. The remaining lipidomic analyses of the lipid classes mentioned above have been cataloged in the supplemental material (Tables S5–S14). Figure 1. Fatty acid (FA) biosynthetic pathways and rabbit diet fatty acyl composition. (A) Schematic representation of three major FA biosynthetic pathways: Nonessential FAs (grey), ω-3 FAs (yellow), ω-6 FAs (lavender). Enzymes are depicted in light green; and (B) FA composition of rabbit diets. Regular chow (RC) and high cholesterol (HC).
(A)
Int. J. Mol. Sci. 2014, 15
23286 Figure 1. Cont.
(B) Fatty acids identified in plasma lipids are often categorized as: (i) Nonessential FAs, namely saturated FAs (SFAs) and mono- or polyunsaturated FAs (MUFAs and PUFAs); (ii) Essential FAs that are metabolites from either the ω-3 pathway (linolenic acid derivatives) or the ω-6 pathway (linoleic acid derivatives). Nonessential FAs can originate from the diet or from de novo lipogenesis (DNL), whereas essential FAs can only originate from the metabolism of essential ω-3 or ω-6 FAs obtained from diet (Figure 1A and [9]). We have recently shown that in cholesterol-fed rabbits, over 75% of the FAs within plasma CEs are SFAs and MUFAs, suggesting a strong dietary influence on the plasma lipidome [9]. Given the dietary composition of the diet in this study (Figure 1B), the CE 18:2 and 18:3 species that predominate as the CE species within rabbit plaque (Figure 2A, Table S1) are most likely CE 18:2n6 and CE 18:2n3, both of which are derived from diet. Together with CE 20:1, 18:1, 24:1 and 22:1, over 60% of lesion CEs can be attributed to dietary or nonessential FA contributions (Figure 2A, Table S1). Similar to plasma [9], ω-6 FAs are the next major constituents of plaque CEs, with CE 22:4, 20:3 and 22:2 accounting for another 25% of fatty acyl CEs within rabbit lesions (Figure 2A, Table S1). Rabbits administered the HC diet had identical FA composition of plasma TGs compared to rabbits administered the RC diet, with nearly 80% of FAs within TGs being derived from nonessential FAs [9]. Here, we find that the most abundant TGs in plaque are comprised of FAs obtained from the diet, accounting for over 80% of TGs in plaque (Figure 2B, Table S2). Akin to our findings in the plasma, metabolites of the ω-3 and ω-6 pathways are likely only minor contributors of TG deposition to the plaque (Figure 2B, Table S2). In our previous report, 55% of FAs in PCs came from nonessential FAs, 40% came from the ω-6 pathway and the remaining 5% came from the ω-3 pathway [9]. Interestingly, 91% of the FAs in plaque PCs stemmed from dietary nonessential FAs and ~8% stemmed from metabolites of the ω-6 pathway (Figure 2C, Table S3), suggesting that the dietary FAs in circulating PCs are highly prone to deposition within atherosclerotic plaque PCs.
Int. J. Mol. Sci. 2014, 15
23287
Figure 2. Fatty acyl composition of plaque lipids: Targeted analysis of lipids was conducted on full-length aortae isolated from rabbits fed the HC diet by UPLC/TOF-MS. (A) Plaque cholesteryl esters (CE); (B) Plaque triacylglycerols (TGs); (C) Plaque phosphatidylcholines (PCs); and (D) Plaque sphingomyelins (SMs). All data is presented as the percent of the given lipid species of the total lipid class.
In relative terms, CEs, TGs and PCs constitute over 65% of the lipid classes detectable in rabbit plaque. However, another relatively abundant lipid class detected in plaque, which is not appreciably detected in plasma, is the SMs [9]. As shown in Figure 3, SMs account for roughly 20% of plaque lipids. Within this lipid class, SM 16:0 is unequivocally the most abundant SM, most likely originating from PC 34:2 (Figure 2D, Table S4). SM 16:0, together with the next five most abundant SMs (SM 24:0, SM 24:1, SM 16:1, SM 22:0 and an SM 24:1 variant) account for about 85% of detectable plaque SMs (Figure 2D, Table S4).
Int. J. Mol. Sci. 2014, 15
23288
Figure 3. Summary of aortic lipid class distribution: Targeted analysis of lipids was conducted on full-length aortae isolated from rabbits fed the HC diet by UPLC/TOF-MS. Data is expressed as the percent of the lipid class of the total lipid content in the aorta, calculated as the total peak area of each lipid class, divided by the total peak area of all lipid classes. TG: Triacylglycerol; SM: Sphingomyelin; PC: Phosphatidylcholine; DG: Diacylglycerol; FA: Fatty acid; PE-plas: Phosphatidylthanolamine; PC-plas: Phosphatidyl choline; CE: Cholesteryl ester; Cer: Ceramide; PS: Phosphatidylserine; LPC: Lyso-phosphatidic acid; PE: Phosphatidylethanolamine; PG: Phosphatidylglycerol; and PI: Phosphatidylinositol.
3. Discussion Whole body lipid homeostasis is regulated through the exogenous uptake and the endogenous synthesis of fatty acids and cholesterol, as well as the trafficking of these lipids to the appropriate anatomical depots. The perturbation of any number of mechanisms in these pathways can lead to the development of dyslipidemia, thereby increasing the propensity for lipid delivery and retention within the artery wall. Although the macromolecular components and major neutral lipid species of atherosclerotic plaques have been extensively defined [3,4,11], the lipidome of atheromatas has not been reported. Our goal was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression, with particular attention to the most abundant lipid classes found in plasma. Lipoprotein lipase (LPL) is the primary enzyme responsible for the liberation of FAs from lipoprotein particles, whereas hepatic lipase (HL) is the key enzyme responsible for the rapid removal of the resultant lipoprotein remnant particles from the circulation [12]. It is well established that rabbits are HL deficient, which causes extreme cholesterol accumulation within chylomicron remnant and β very low-density lipoprotein (β-VLDL) particles as a result of lowered metabolic clearance rate, when rabbits are challenged with a HC diet [12]. Although the balance between LPL and HL activities regulates lipoprotein clearance, the lipid composition of lipoprotein particles is primarily owing to the lecithin:cholesterol acyltransferase (LCAT) and acyl-coenzyme A:cholesterol acyltransferase (ACAT) enzymes, which are responsible for the conversion of FC to CE. Lipoprotein-bound LCAT cleaves sn2 position FAs (preferentially PUFAs) of PCs and transfers them to the 3-β-hydroxyl group
Int. J. Mol. Sci. 2014, 15
23289
of FC, thereby generating CE [13]. ACATs use MUFAs as their preferential substrate in the addition of exogenous dietary FAs or endogenous FAs generated through DNL, to FC [14]. The fact that we found predominantly dietary FAs (namely SFAs and MUFAs) within CEs of rabbit atherosclerotic lesions, suggests that ACAT is the major enzyme for the biosynthesis of CEs deposited in plaque. Moreover, our findings are consistent with the verity that ACAT-derived CE is the major atherogenic lipid in plasma [15], whereas LCAT-derived CE is the major atheroprotective lipid in blood, as it is mainly carried within high-density lipoprotein (HDL) particles [13]. Interestingly, our previous study demonstrated a higher dietary influence on plasma CE than on the plaque CE we observed in this study [9]. This discrepancy could potentially be due to macrophage metabolism of CE-derived FAs to other FA subspecies within the site of the lesion [16], warranting further lipidomic analyses of models of atherogenesis. The data presented in this study are consistent with two recent reports which have demonstrated that TGs constitute a relatively abundant lipid species within rabbit atherosclerotic plaques [6,7]. Although challenging these animals with a HC diet does not elevate their plasma TG levels [9], TG accumulation within the atherosclerotic lesion has long been suggested to occur as a consequence of chylomicron and β-VLDL remnant retention in the artery wall [17], as well as DNL in the vascular intima from precursors such as acetate, glucose, and long-chain free fatty acids [18–20]. Our results are perhaps more consistent with the former, as we found that over 80% of the FAs within plaque TGs were nonessential FAs. Thus, our data suggest that lipoprotein-derived TGs are the major driver of plaque TG deposition. While in situ lipogenesis may certainly contribute at least part of the remaining portion of lesion TG, further studies would be required to delineate the exact contributions of lipoprotein versus lipogenic TG sources. Phospholipids are major precursors to a host of signaling lipids, including eicosanoids and SMs [21], both of which are known to be involved in proinflammatory responses in atherogenesis [22,23]. In turn, their FA composition within atherosclerotic plaques is of particular interest. According to the Lands cycle, the ratio of saturated to unsaturated FAs within PCs in particular, is tightly regulated, resulting in a saturated FA occupying the sn1 position and an unsaturated FA occupying the sn2 position [24,25]. The result, at least in plasma, is an approximately equal distribution of FAs from nonessential and ω-6/ω-3 pathways in PCs [9]. In contrast, the distribution of plaque PCs is heavily slated towards the nonessential FA class, suggesting that FAs from other lipid classes within the lesion may influence plaque PC composition. For example, the generation of sphingomyelin results from the transfer of phosphorylcholine from PC to ceramide, which liberates diacylglycerols as a byproduct [26]. Given our finding that sphingomyelins and diacylglycerols are relatively abundant lipid classes within the lesion (Figure 3), the interplay between PCs, SMs and DGs may have influenced the observed distribution of FAs in plaque PCs. Furthermore, the relatively equal abundance of PCs and SMs (~20% of plaque, each) further supports the relationship between these lipid classes within the plaque. Therefore, the complex interactions between lipid classes in plaque warrants further study, as biomarkers of atherosclerosis progression, and perhaps changes in lipid profiles observed during plaque regression, may point towards biomarkers which can be used to validate therapeutics that modulate the disease.
Int. J. Mol. Sci. 2014, 15
23290
4. Materials and Methods 4.1. Animals and Diets Male New Zealand White rabbits (n = 20) were obtained from Covance (Princeton, NJ, USA) and were individually housed in a temperature- and humidity-controlled environment with a 12 h light/dark cycle. Animals were fed purified chow (Purina Mills LabDiet 5326, LLC, St. Louis, MO, USA) with 0.5% added cholesterol ad libitum for 12 weeks and had free access to water. At the end of the study, rabbits were euthanized with 1 mL/kg of Beuthanasia-D solution (Merck, Whitehouse Station, NJ, USA) via the marginal ear vein. Plasma lipid levels were determined on EDTA-plasma as described previously [9]. Full-length aortae were excised from each animal beginning with the aortic root through the aortic arch and the descending aorta to the iliac bifurcation. Aortae were dissected free of fat and connective tissue, flash-frozen in foil packets using liquid nitrogen and stored at −80 °C until future analyses. All animal experiments, procedures and protocols, were reviewed and approved by the Merck Research Laboratories’ Institutional Animals Care and Use Committee. 4.2. Lipid Extraction and Reagents All organic solvents used were of chromatographic grade or better. Leucine enkephalin (Sigma Aldrich, St. Louis, MO, USA) solution was used for lock mass correction. Lipids were extracted from full-length aortae as per a modified Bligh and Dyer method as described previously [27,28]. During the homogenization and extraction process, samples were treated with anti-oxidants (0.5 mg of 2,6-di-tert-butyl-4-methylphenol). Extracts were evaporated to dryness and re-constituted in isopropanol/ acetonitrile/water (65%:30%:5% (v/v/v)). 4.3. Analytical Instrumentation for Relative Quantitation of Lipid Fractions Rabbit plaque lipid extracts were analyzed using an Acquity UPLC system coupled to a Synapt G2-HDMS mass spectrometer system (Waters Corp., Milford, MA, USA). For analyses, 5 µL of extract was injected. Chromatographic separation was achieved using a mobile phase A solution of 60% acetonitrile/40% water with 10 mM ammonium formate solution, and a mobile phase B solution of 90% isopropanol/10% acetonitrile with 10 mM ammonium formate. An HSS T3 C18 reverse-phase column was utilized (1.7 µm, 2.1 mm × 100 mm; Waters Corp.) coupled to the following chromatographic conditions: A linear gradient from 40% B to 100% B in 0 to 10 min followed by a 2 min hold at 100% B. The mass spectral data was acquired in electrospray positive and negative ion modes using nitrogen as the desolvation gas with a flow rate of 700 L/h. The source and desolvation temperatures were maintained at 120 and 450 °C, respectively. The capillary voltage, source cone voltage, and extraction cone voltage were maintained at +/−3 kV, +/−30 V, and 4 V, respectively. Mass spectra were acquired over the m/z range of 50 to 1200 at a resolution of 25,000 FWHM (full width half height mass resolution).
Int. J. Mol. Sci. 2014, 15
23291
5. Conclusions The observations presented in this study demonstrate the ability to obtain specific diet-induced lipid phenotypes from directly within the atherosclerotic plaque. However, these measurements do not provide any specific insights into the pathophysiology of the disease. For example, it is possible that in situ DNL and/or the metabolism of CE-, TG-, PC- and SM-derived FAs gives rise to signaling lipids such as oxylipin eicosanoids; known mediators of onset and progression of atherosclerosis. Although this example is speculative based on the present study, the data presented here provide the first complete characterization of the atherosclerotic plaque lipidome of a widely used in vivo model of the disease. This detailed framework of the lipids within the vessel wall provides a powerful lipid profile to enable future studies to expand on the lipidomic characterization of atherosclerotic lesions. Supplementary Materials Supplementary tables can be found at http://www.mdpi.com/1422-0067/15/12/23283/s1. Acknowledgments We thank the Merck Research Laboratories’ Postdoctoral Fellowship Program for financial support. Lazar A. Bojic is supported by a research fellowship provided by the MRL Postdoctoral fellowship program. Author Contributions Lazar A. Bojic conducted data analyses and drafted, edited and finalized the paper; David G. McLaren conducted analytical experiments and contributed to editing the paper; Vinit Shah conducted analytical experiments and contributed to editing the paper; Stephen F. Previs assisted in study design and execution, and contributed to editing the paper; Douglas G. Johns assisted in study design and execution, and contributed to editing the paper; and Jose M. Castro-Perez contributed to study design and execution, conducted analytical experiments, conducted data analyses, and contributed to editing the paper. Conflicts of Interest The authors declare no conflict of interest. References 1.
2. 3. 4.
Go, A.S.; Mozaffarian, D.; Roger, V.L.; Benjamin, E.J.; Berry, J.D.; Borden, W.B.; Bravata, D.M.; Dai, S.; Ford, E.S.; Fox, C.S.; et al. Heart disease and stroke statistics—2013 update: A report from the American Heart Association. Circulation 2013, 127, e6–e245. Libby, P. The forgotten majority: Unfinished business in cardiovascular risk reduction. J. Am. Coll. Cardiol. 2005, 46, 1225–1228. Moore, K.J.; Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011, 145, 341–355. Libby, P.; Ridker, P.M.; Hansson, G.K. Progress and challenges in translating the biology of atherosclerosis. Nature 2011, 473, 317–325.
Int. J. Mol. Sci. 2014, 15 5. 6.
7.
8. 9.
10.
11. 12. 13.
14. 15.
16. 17. 18. 19. 20. 21.
23292
Laufs, U.; Weintraub, W.S.; Packard, C.J. Beyond statins: What to expect from add-on lipid regulating therapy? Eur. Heart J. 2013, 34, 2660–2665. Lattermann, A.; Matthaus, C.; Bergner, N.; Beleites, C.; Romeike, B.F.; Krafft, C.; Brehm, B.R.; Popp, J. Characterization of atherosclerotic plaque depositions by raman and ftir imaging. J. Biophotonics 2013, 6, 110–121. Matthaus, C.; Dochow, S.; Bergner, G.; Lattermann, A.; Romeike, B.F.; Marple, E.T.; Krafft, C.; Dietzek, B.; Brehm, B.R.; Popp, J. In vivo characterization of atherosclerotic plaque depositions by Raman-probe spectroscopy and in vitro coherent anti-stokes Raman scattering microscopic imaging on a rabbit model. Anal. Chem. 2012, 84, 7845–7851. Williams, K.J.; Feig, J.E.; Fisher, E.A. Rapid regression of atherosclerosis: Insights from the clinical and experimental literature. Nat. Clin. Pract. Cardiovasc. Med. 2008, 5, 91–102. Yin, W.; Carballo-Jane, E.; McLaren, D.G.; Mendoza, V.H.; Gagen, K.; Geoghagen, N.S.; McNamara, L.A.; Gorski, J.N.; Eiermann, G.J.; Petrov, A.; et al. Plasma lipid profiling across species for the identification of optimal animal models of human dyslipidemia. J. Lipid Res. 2012, 53, 51–65. Quehenberger, O.; Armando, A.M.; Brown, A.H.; Milne, S.B.; Myers, D.S.; Merrill, A.H.; Bandyopadhyay, S.; Jones, K.N.; Kelly, S.; Shaner, R.L.; et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 2010, 51, 3299–3305. Libby, P.; Ridker, P.M.; Hansson, G.K. Inflammation in atherosclerosis: From pathophysiology to practice. J. Am. Coll. Cardiol. 2009, 54, 2129–2138. Chang, S.; Borensztajn, J. Hepatic lipase function and the accumulation of β-very-low-density lipoproteins in the plasma of cholesterol-fed rabbits. Biochem. J. 1993, 293, 745–750. Rousset, X.; Vaisman, B.; Amar, M.; Sethi, A.A.; Remaley, A.T. Lecithin: Cholesterol acyltransferase—From biochemistry to role in cardiovascular disease. Curr. Opin. Endocrinol. Diabetes Obes. 2009, 16, 163–171. Chang, T.Y.; Chang, C.C.; Lin, S.; Yu, C.; Li, B.L.; Miyazaki, A. Roles of acyl-coenzyme a: Cholesterol acyltransferase-1 and -2. Curr. Opin. Lipidol. 2001, 12, 289–296. Lee, R.G.; Kelley, K.L.; Sawyer, J.K.; Farese, R.V., Jr.; Parks, J.S.; Rudel, L.L. Plasma cholesteryl esters provided by lecithin:cholesterol acyltransferase and acyl-coenzyme a:cholesterol acyltransferase 2 have opposite atherosclerotic potential. Circ. Res. 2004, 95, 998–1004. Norris, P.C.; Dennis, E.A. A lipidomic perspective on inflammatory macrophage eicosanoid signaling. Adv. Biol. Regul. 2014, 54, 99–110. Vost, A. Uptake and metabolism of circulating chylomicron triglyceride by rabbit aorta. J. Lipid Res. 1972, 13, 695–704. Howard, C.F., Jr. De novo synthesis and elongation of fatty acids by subcellar fractions of monkey aorta. J. Lipid Res. 1968, 9, 254–261. Stein, Y.; Stein, O. Incorporation of fatty acids into lipids of aortic slices of rabbits, dogs, rats and baboons. J. Atheroscler. Res. 1962, 2, 400–412. Vost, A. Lipid accretion in the perfused rabbit aorta. J. Atheroscler. Res. 1969, 9, 221–238. Zhou, L.; Nilsson, A. Sources of eicosanoid precursor fatty acid pools in tissues. J. Lipid Res. 2001, 42, 1521–1542.
Int. J. Mol. Sci. 2014, 15
23293
22. Buczynski, M.W.; Dumlao, D.S.; Dennis, E.A. Thematic review series: Proteomics. An integrated omics analysis of eicosanoid biology. J. Lipid Res. 2009, 50, 1015–1038. 23. Li, Z.; Fan, Y.; Liu, J.; Li, Y.; Huan, C.; Bui, H.H.; Kuo, M.S.; Park, T.S.; Cao, G.; Jiang, X.C. Impact of sphingomyelin synthase 1 deficiency on sphingolipid metabolism and atherosclerosis in mice. Arterioscler. Thromb. Vasc. Boil. 2012, 32, 1577–1584. 24. Lands, W.E. Metabolism of glycerolipids. 2. The enzymatic acylation of lysolecithin. J. Boil. Chem. 1960, 235, 2233–2237. 25. Lands, W.E.; Merkl, I. Metabolism of glycerolipids. III. Reactivity of various acyl esters of coenzyme a with α'-acylglycerophosphorylcholine, and positional specificities in lecithin synthesis. J. Boil. Chem. 1963, 238, 898–904. 26. Merrill, A.H., Jr. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev. 2011, 111, 6387–6422. 27. Bligh, E.G.; Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911–917. 28. Shah, V.; Castro-Perez, J.M.; McLaren, D.G.; Herath, K.B.; Previs, S.F.; Roddy, T.P. Enhanced data-independent analysis of lipids using ion mobility-tofmse to unravel quantitative and qualitative information in human plasma. Rapid Commun. Mass Spectrom. 2013, 27, 2195–2200. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
Supplementary Information Table S1. Plaque cholesteryl esters. Metabolite CE 18:2 CE 18:3 CE 22:4 CE 20:3 CE 24:2 CE 20:1 CE 18:1 CE 24:1 CE 22:2 CE 22:1 CE 22:5 CE 24:4 CE 16:1 CE 26:2 CE 18:0 CE 24:5 CE 24:3 CE 26:1 CE 26:3 CE 26:4 CE 20:2
Formula C45H79NO2 C45H77NO2 C49H83NO2 C47H81NO2 C51H91NO2 C47H85NO2 C45H81NO2 C51H93NO2 C49H87NO2 C49H89NO2 C49H81NO2 C51H87NO2 C43H77NO2 C53H95NO2 C45H83NO2 C51H85NO2 C51H89NO2 C53H97NO2 C53H93NO2 C53H91NO2 C47H83NO2
m/z Found 666.6204 664.6039 718.6522 692.6372 750.713 696.6668 668.6354 752.7309 722.6819 724.6968 716.6349 746.6808 640.6038 778.7448 670.6501 744.666 748.6974 780.7601 776.7288 774.7115 694.6499
mDa 1.5 0.7 2 2.7 0.2 1 0.9 2.5 0.4 −0.3 0.4 −0.7 0.6 0.7 −0.1 0.2 0.3 0.4 0.4 −1.3 −0.3
PPM 2.3 1 2.8 3.9 0.3 1.4 1.3 3.3 0.6 −0.5 0.5 −0.9 0.9 0.9 −0.1 0.2 0.4 0.5 0.5 −1.7 −0.4
Time 9.17 8.94 9.28 9.27 9.96 9.68 9.4 10.15 9.7 9.94 9.12 9.53 9.13 10.17 9.57 9.33 9.78 10.36 9.99 9.79 9.37
Area % 33.34 15.13 12.45 10.35 7.12 4.39 3.76 3.48 2.65 1.50 0.97 0.89 0.82 0.77 0.54 0.52 0.44 0.36 0.14 0.14 0.10
Table S2. Plaque triaclyglycerols. Metabolite TG 52:4 TG 54:6 TG 52:3 TG 54:5 TG 52:5 TG 52:2 TG 54:7 TG 54:4 TG 50:2 TG 50:1 TG 50:3 TG 54:3 TG 50:4 TG 54:2 TG 52:6 TG 48:0
Formula C55H101NO6 C57H101NO6 C55H103NO6 C57H103NO6 C55H99NO6 C55H105NO6 C57H99NO6 C57H105NO6 C53H101NO6 C53H103NO6 C53H99NO6 C57H107NO6 C53H97NO6 C57H109NO6 C55H97NO6 C51H101NO6
m/z Found 872.7699 896.7709 874.7882 898.7861 870.7543 876.8045 894.7551 900.8044 848.7718 850.7883 846.7545 902.8206 844.7394 904.8304 868.7403 824.7731
mDa −0.8 0.2 1.9 −0.2 −0.7 2.5 0.1 2.4 1.1 2 −0.5 3 0 −2.9 0.9 2.4
PPM −0.9 0.2 2.1 −0.3 −0.9 2.9 0.1 2.7 1.3 2.3 −0.6 3.3 0 −3.2 1 2.9
Time 8.56 8.38 8.8 8.62 8.35 9.03 8.14 8.84 8.76 8.96 8.52 9.06 8.31 9.26 8.1 8.95
Area % 10.17 8.89 7.40 7.32 7.23 6.13 5.82 5.67 5.45 4.97 4.38 3.99 3.06 2.89 2.50 2.32
S2 Table S2. Cont. Metabolite TG 48:2 TG 48:1 TG 48:3 TG 54:8 TG 50:5 TG 46:0 TG 56:6 TG 56:7 TG 46:1 TG 46:2 TG 56:5 TG 56:8 TG 48:4 TG 56:4 TG 56:3 TG 52:7
Formula C51H97NO6 C51H99NO6 C51H95NO6 C57H97NO6 C53H95NO6 C49H97NO6 C59H105NO6 C59H103NO6 C49H95NO6 C49H93NO6 C59H107NO6 C59H101NO6 C51H93NO6 C59H109NO6 C59H111NO6 C55H95NO6
m/z Found 820.738 822.7538 818.7233 892.7384 842.7247 796.7384 924.8013 922.7852 794.7227 792.7072 926.8188 920.77 816.7067 928.8317 930.8468 866.7227
mDa −1.4 −1.2 −0.4 −1 1 −1 −0.7 −1.1 −1 −0.9 1.2 −0.7 −1.4 −1.6 −2.1 −1
PPM −1.7 −1.5 −0.5 −1.1 1.1 −1.2 −0.7 −1.2 −1.3 −1.1 1.3 −0.8 −1.7 −1.7 −2.3 −1.2
Time 8.5 8.73 8.26 7.91 8.07 8.72 8.69 8.49 8.48 8.24 8.89 8.28 8.02 9.09 9.28 7.87
Area % 2.28 1.64 1.34 1.13 1.05 0.47 0.46 0.40 0.39 0.38 0.30 0.26 0.25 0.19 0.18 0.15
Table S3. Plaque phosphatidylcholines. Metabolite PC 34:2 PC 36:2 PC 34:1 PC 36:3 PC 36:4 PC 36:4 PC 38:4 PC 36:1 PC 32:1 PC 34:3 PC 38:6 PC 34:3 PC 32:2 PC 38:4 PC 38:5 PC 38:5 PC 38:4 PC 36:5 PC 38:2 PC 38:3 PC 36:5 PC 38:3 PC 36:5 PC 40:4 PC 34:4
Formula C42H80NO8P C44H84NO8P C42H82NO8P C44H82NO8P C44H80NO8P C44H80NO8P C46H84NO8P C44H86NO8P C40H78NO8P C42H78NO8P C46H80NO8P C42H78NO8P C40H76NO8P C46H84NO8P C46H82NO8P C46H82NO8P C46H84NO8P C44H78NO8P C46H88NO8P C46H86NO8P C44H78NO8P C46H86NO8P C44H78NO8P C48H88NO8P C42H76NO8P
m/z Found 758.5709 786.6002 760.5865 784.586 782.5703 782.5684 810.6002 788.6161 732.5553 756.5541 806.5701 756.5545 730.5395 810.5999 808.5854 808.5854 810.6024 780.555 814.6329 812.6171 780.5558 812.6154 780.5543 838.6312 754.5413
mDa 0.9 −1.1 0.9 0.4 0.3 −1.6 −1.1 −0.8 1 −0.2 0.1 0.2 0.8 −1.4 −0.2 −0.2 1.1 0.7 0.3 0.2 1.5 −1.5 0 −1.4 2.6
PPM 1.2 −1.3 1.2 0.5 0.4 −2 −1.3 −1 1.4 −0.3 0.2 0.3 1.2 −1.7 −0.3 −0.3 1.4 0.9 0.4 0.2 1.9 −1.9 0 −1.6 3.5
Time 4.83 5.41 5.28 4.91 4.74 4.44 5.35 5.83 4.68 4.39 4.34 4.31 4.2 4.98 4.77 4.97 5.15 4.34 5.9 5.56 4.01 5.45 4.21 5.72 4.13
Area % 21.34 16.69 12.96 12.09 10.89 5.22 3.74 3.53 3.35 2.21 1.80 1.36 0.77 0.64 0.60 0.57 0.38 0.36 0.27 0.25 0.23 0.23 0.18 0.13 0.11
S3 Table S4. Plaque sphingomyelins. Metabolite SM 16:0 SM 24:0 SM 24:1 SM 16:1 SM 22:0 SM 24:1 SM 24:2 SM 20:0 SM 22:1 SM 18:1 SM 14:0 SM 20:1 SM 26:0
Formula C39H79N2O6P C47H95N2O6P C47H93N2O6P C39H77N2O6P C45H91N2O6P C47H93N2O6P C47H91N2O6P C43H87N2O6P C45H89N2O6P C41H81N2O6P C37H75N2O6P C43H85N2O6P C49H99N2O6P
m/z Found 703.5763 815.699 813.683 701.5619 787.6685 813.6852 811.6678 759.6374 785.653 729.5923 675.5459 757.6223 843.7312
mDa 0.9 −1.6 −1.9 2.2 −0.8 0.3 −1.5 −0.6 −0.6 1.3 1.8 0 −0.7
PPM 1.3 −1.9 −2.4 3.1 −1 0.3 −1.8 −0.8 −0.8 1.7 2.7 0 −0.8
Time 4.59 6.8 6.28 4.05 6.34 6.42 5.88 5.78 5.92 4.69 3.94 5.34 7.24
Area % 30.27 18.49 12.05 8.66 8.55 7.68 5.46 4.47 2.49 0.68 0.67 0.28 0.14
Table S5. Plaque diacylglycerols. Metabolite DG 46:11 DG 44:10 DG 38:4 DG 46:10 DG 36:2 DG 34:2 DG 36:3 DG 36:4 DG 34:1 DG 36:1
Formula C49H77NO5 C47H75NO5 C41H75NO5 C49H79NO5 C39H75NO5 C37H71NO5 C39H73NO5 C39H71NO5 C37H73NO5 C39H77NO5
m/z Found 760.5865 734.573 662.5737 762.601 638.5737 610.5426 636.5576 634.5419 612.5568 640.5883
mDa −1.5 0.7 1.4 −2.6 1.4 1.6 0.9 0.9 0.1 0.3
PPM −1.9 0.9 2.1 −3.4 2.2 2.6 1.5 1.4 0.2 0.5
Time 5.28 5.19 6.52 5.75 6.62 6.12 6.17 6.01 6.5 6.97
Area % 51.32 28.13 8.04 5.83 2.66 1.95 0.63 0.46 0.41 0.31
Table S6. Plaque PE-plas. Metabolite PE_plas 38:4 PE_plas 40:4 PE_plas 38:5 PE_plas 36:1 PE_plas 36:4 PE_plas 40:5 PE_plas 38:4 PE_plas 40:6 PE_plas 40:5 PE_plas 34:1 PE_plas 36:2 PE_plas 38:3 PE_plas 32:4
Formula C43H78NO7P C45H82NO7P C43H76NO7P C41H80NO7P C41H74NO7P C45H80NO7P C43H78NO7P C45H78NO7P C45H80NO7P C39H76NO7P C41H78NO7P C43H80NO7P C37H66NO7P
m/z Found 752.5596 780.5913 750.5443 730.5765 724.5288 778.5758 752.5593 776.5585 778.575 702.5452 728.5615 754.5743 668.4722
mDa 0.2 0.6 0.6 1.5 0.7 0.8 −0.1 −0.9 0 1.5 2.1 −0.7 6.7
PPM 0.3 0.8 0.7 2 1 1 −0.1 −1.2 −0.1 2.1 2.9 −1 10
Time 5.82 6.17 5.31 6.28 5.23 5.99 5.65 5.66 5.84 5.75 5.91 6.08 7.05
Area % 49.86 13.92 7.30 6.56 6.48 4.08 2.89 2.54 2.07 1.97 1.45 0.63 0.16
S4 Table S7. Plaque PC−plas. Metabolite PC-plas 36:4 PC-plas 34:0 PC-plas 38:4 PC-plas 36:3 PC-plas 38:4 PC-plas 38:5 PC-plas 38:3 PC-plas 32:0 PC-plas 34:1 PC-plas 36:1 PC-plas 34:4 PC-plas 36:2 PC-plas 36:1 PC-plas 40:3
Formula C44H80NO7P C42H84NO7P C46H84NO7P C44H82NO7P C46H84NO7P C46H82NO7P C46H86NO7P C40H80NO7P C42H82NO7P C44H86NO7P C42H76NO7P C44H84NO7P C44H86NO7P C48H90NO7P
m/z Found 766.5742 746.6058 794.6055 768.5907 794.6052 792.5909 796.6212 718.576 744.5905 772.6226 738.5445 770.6043 772.6212 824.6522
mDa −0.8 −0.5 −0.8 0 −1.1 0.2 −0.8 1 −0.2 0.6 0.8 −2 −0.8 −1.1
PPM −1.1 −0.7 −1.1 0 −1.4 0.3 −1 1.3 −0.3 0.8 1 −2.7 −1 −1.3
Time 5.03 5.64 5.61 5.11 5.15 5.07 5.7 5.5 5.19 5.7 5.54 5.69 6.11 6.22
Area % 33.06 21.89 13.61 10.66 6.24 4.65 2.62 2.53 1.65 1.52 0.56 0.38 0.36 0.12
Table S8. Plaque fatty acids. Metabolite FA 20:3 FA 18:0 FA 18:1 FA 22:3 FA 20:4 FA 22:4 FA 20:2 FA 20:1 FA 20:0 FA 22:2 FA 20:5 FA 22:5
Formula C20H32O2 C18H34O2 C18H32O2 C22H36O2 C20H30O2 C22H34O2 C20H34O2 C20H36O2 C20H38O2 C22H38O2 C20H28O2 C22H32O2
m/z Found 303.2341 281.2485 279.2339 331.2642 301.2178 329.2487 305.2492 307.2643 309.2802 333.2789 299.2022 327.2331
mDa 1.7 0.4 1.5 0.5 1 0.6 1.1 0.6 0.8 −0.5 1.1 0.7
PPM 5.5 1.5 5.2 1.4 3.4 1.9 3.7 1.8 2.6 −1.4 3.6 2
Time 2.05 2.62 2.14 2.52 2.13 2.12 2.33 2.76 3.32 3.02 1.66 1.88
Area % 45.41 22.95 12.11 5.96 5.72 2.88 2.06 0.76 0.78 0.64 0.49 0.12
Table S9. Plaque phosphatidylserines. Metabolite PS 36:2 PS 38:4 PS 36:1 PS 40:5 PS 38:1 PS 38:0 PS 40:1 PS 40:3 PS 46:3 PS 44:3
Formula C42H78NO10P C44H78NO10P C42H80NO10P C46H80NO10P C44H84NO10P C44H86NO10P C46H88NO10P C46H84NO10P C52H96NO10P C50H92NO10P
m/z Found 786.5281 810.5272 788.5422 836.5426 816.5726 818.5909 844.6029 840.5715 924.6756 896.645
mDa −0.4 −1.3 −2 −1.6 −2.9 −0.2 −3.9 −4 6.2 6.9
PPM −0.6 −1.6 −2.5 −1.9 −3.5 −0.3 −4.6 −4.7 6.7 7.7
Time 4.91 4.91 5.36 4.91 5.14 5.56 5.7 5.05 6.97 6.52
Area % 77.41 9.14 5.84 4.66 1.25 0.87 0.32 0.22 0.18 0.11
S5 Table S10. Plaque lyso-phosphatidylcholines. Metabolite LPC 18:0 LPC 16:0 LPC 18:1 LPC 18:2
Formula C26H54NO7P C24H50NO7P C26H52NO7P C26H50NO7P
m/z Found 524.3723 496.3405 522.3562 520.3405
mDa 0.7 0.2 0.3 0.2
PPM 1.4 0.4 0.5 0.4
Time 1.94 1.49 1.56 1.31
Area % 53.57 38.20 4.68 3.56
Time 7.46 7.03 7.05 6.58 7.12 5.5 6.64 6.09 6.67 6.16 6.25 7.84 3.06
Area % 30.80 20.79 12.31 8.22 6.58 6.55 6.51 3.65 2.65 1.17 0.26 0.25 0.24
Time 4.03 4.48 4.97 3.27 3.66 4.99 4.19 4.97 4.91 3.6 4.38 3.82 4.32 3.94 3.47 3.04 3.46 5.05
Area % 19.62 19.41 11.26 10.80 7.12 6.51 3.58 3.48 3.23 2.39 2.21 2.19 1.53 1.47 1.09 0.60 0.56 0.44
Table S11. Plaque ceramides. Metabolite Cer 24:0 Cer 24:1 Cer 22:0 Cer 20:0 Cer 24:1 Cer 16:0 Cer 24:2 Cer 18:0 Cer 22:1 Cer 20:1 Cer 24:3 Cer 26:0 Cer 16:4
Formula C43H85NO5 C43H83NO5 C41H81NO5 C39H77NO5 C43H83NO5 C35H69NO5 C43H81NO5 C37H73NO5 C41H79NO5 C39H75NO5 C43H79NO5 C45H89NO5 C35H61NO5
m/z Found 694.6354 692.6192 666.6036 638.5743 692.6201 582.5099 690.6045 610.5396 664.5893 636.5552 688.5886 722.6662 574.4483
mDa 0.4 −0.1 −0.1 1.9 0.8 0.1 0.8 −1.5 1.3 −1.5 0.6 −0.1 1.1
PPM 0.6 −0.2 −0.1 3 1.1 0.2 1.2 −2.4 1.9 −2.4 0.8 −0.1 2
Table S12. Plaque phosphatidylglycerols. Metabolite PG 36:3 PG 36:2 PG 36:1 PG 36:4 PG 36:3 PG 38:2 PG 36:2 PG 36:2 PG 36:2 PG 36:4 PG 34:1 PG 36:3 PG 36:2 PG 34:2 PG 34:2 PG 34:3 PG 36:4 PG 38:1
Formula C42H77O10P C42H79O10P C42H81O10P C42H75O10P C42H77O10P C44H83O10P C42H79O10P C42H79O10P C42H79O10P C42H75O10P C40H77O10P C42H77O10P C42H79O10P C40H75O10P C40H75O10P C40H73O10P C42H75O10P C44H85O10P
m/z Found 771.5178 773.5335 775.5472 769.5003 771.5144 801.5627 773.5325 773.5319 773.5331 769.5 747.5172 771.5154 773.5317 745.5021 745.5005 743.4861 769.5016 803.576
mDa 0.2 0.2 −1.7 −1.7 −3.2 −1.9 −0.8 −1.4 −0.2 −2 −0.4 −2.2 −1.6 0.1 −1.5 −0.2 −0.4 −4.2
PPM 0.2 0.3 −2.2 −2.2 −4.2 −2.4 −1 −1.8 −0.2 −2.6 −0.6 −2.9 −2 0.2 −2 −0.3 −0.5 −5.3
S6 Table S12. Cont. Metabolite PG 36:4 PG 34:1 PG 34:3 PG 38:4 PG 38:6 PG 34:2 PG 34:2 PG 34:2 PG 36:3 PG 36:5 PG 38:4
Formula C42H75O10P C40H77O10P C40H73O10P C44H79O10P C44H75O10P C40H75O10P C40H75O10P C40H75O10P C42H77O10P C42H73O10P C44H79O10P
m/z Found 769.5003 747.5153 743.4866 797.5336 793.5034 745.4987 745.5016 745.4984 771.5158 767.4822 797.5334
mDa −1.7 −2.3 0.3 0.3 1.4 −3.3 −0.4 −3.6 −1.8 −4.1 0.1
PPM −2.2 −3.1 0.4 0.4 1.8 −4.4 −0.5 −4.8 −2.4 −5.4 0.1
Time 3.06 4.21 3.44 4.07 3.05 3.58 3.63 3.69 4.2 3.02 4.21
Area % 0.40 0.37 0.30 0.25 0.22 0.19 0.18 0.17 0.15 0.14 0.14
Table S13. Plaque phosphatidylinositols. Metabolite PI 38:4 PI 38:3 PI 36:1 PI 36:2 PI 38:5 PI 36:4 PI 34:1 PI 36:3 PI 34:2
Formula C47H83O13P C47H85O13P C45H85O13P C45H83O13P C47H81O13P C45H79O13P C43H81O13P C45H81O13P C43H79O13P
m/z Found 885.5511 887.5631 863.5639 861.5517 883.5317 857.5182 835.5361 859.532 833.5151
mDa 1.8 −1.9 −1.1 2.4 −2 0.2 2.4 −1.7 −2.9
PPM 2 −2.1 −1.3 2.7 −2.2 0.2 2.9 −2 −3.5
Time 4.72 4.95 4.95 4.79 4.2 4.13 4.65 4.28 4.19
Area % 55.41 32.95 4.96 1.77 1.40 1.18 0.89 0.75 0.71
Table S14. Plaque phosphatidylethanolamines. Metabolite PE 36:4 PE 38:4 PE 38:5 PE 36:2 PE 36:1 PE 34:2 PE 36:3 PE 34:1 PE 40:5 PE 42:1 PE 38:2
Formula C41H74NO8P C43H78NO8P C43H76NO8P C41H78NO8P C41H80NO8P C39H74NO8P C41H76NO8P C39H76NO8P C45H80NO8P C47H92NO8P C43H82NO8P
m/z Found 738.5074 766.5383 764.5226 742.5384 744.5533 714.5077 740.5206 716.5231 792.5518 828.647 770.5706
mDa 0 −0.4 −0.5 −0.3 −1.1 0.3 −2.5 0 −2.6 −1.3 0.6
PPM 0 −0.5 −0.6 −0.4 −1.4 0.4 −3.3 0.1 −3.2 −1.5 0.8
Time 4.95 5.53 4.99 5.61 6.01 4.99 5.1 5.49 5.73 7.34 6.05
Area % 36.80 24.49 18.22 10.57 3.85 3.13 1.95 0.54 0.22 0.12 0.10
Copyright of International Journal of Molecular Sciences is the property of MDPI Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
International Journal of
Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article
Lipidome of Atherosclerotic Plaques from Hypercholesterolemic Rabbits Lazar A. Bojic, David G. McLaren, Vinit Shah, Stephen F. Previs, Douglas G. Johns and Jose M. Castro-Perez * Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA; E-Mails: [email protected] (L.A.B.); [email protected] (D.G.M.); [email protected] (V.S.); [email protected] (S.F.P.); [email protected] (D.G.J.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-908-740-5033. External Editor: Mario Ollero Received: 23 October 2014; in revised form: 9 December 2014 / Accepted: 10 December 2014 / Published: 15 December 2014
Abstract: The cellular, macromolecular and neutral lipid composition of the atherosclerotic plaque has been extensively characterized. However, a comprehensive lipidomic analysis of the major lipid classes within atherosclerotic lesions has not been reported. The objective of this study was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression. Male New Zealand White rabbits were administered regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Our lipidomic analyses of plaques isolated from rabbits fed the HC diet, using ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry, detected most of the major lipid classes including: Cholesteryl esters, triacylglycerols, phosphatidylcholines, sphingomyelins, diacylglycerols, fatty acids, phosphatidylserines, lysophosphatidylcholines, ceramides, phosphatidylglycerols, phosphatidylinositols and phosphatidylethanolamines. Given that cholesteryl esters, triacylglycerols and phosphatidylcholines comprise greater than 75% of total plasma lipids, we directed particular attention towards the qualitative and quantitative assessment of the fatty acid composition of these lipids. We additionally found that sphingomyelins were relatively abundant lipid class within lesions, and compared the abundance
Int. J. Mol. Sci. 2014, 15
23284
of sphingomyelins to their precursor phosphatidylcholines. The studies presented here are the first approach to a comprehensive characterization of the atherosclerotic plaque lipidome. Keywords: lipidomics; atherosclerosis; plaque; lipids; mass spectrometry
1. Introduction For the past century, cardiovascular disease (CVD) has been the leading cause of death in the industrialized world and is projected to soon achieve this status worldwide [1,2]. At the core of most cardiovascular events is atherosclerosis, a chronic inflammatory condition of the macrovasculature initiated by the subendothelial retention of apolipoprotein B (apoB)-containing lipoproteins [3]. The trapping of lipoproteins by proteoglycans within the arterial intima increases the propensity for various lipoprotein modifications (oxidation, hydrolysis, aggregation) which trigger maladaptive immune responses that potentiate lesion development [3]. Eventually, atherosclerotic lesions reach an unstable state that is prone to rupture, ultimately resulting in acute cardiovascular events [3,4]. Monogenic disorders in which plasma low-density lipoprotein cholesterol (LDL-C) levels are substantially elevated (such as familial hypercholesterolemia) result in significantly increased cardiovascular event rates, and a number of randomized controlled trials of LDL-C lowering interventions, namely statins, consistently demonstrate reductions in CVD risk [5]. As a result, this evidence places beyond a reasonable doubt that LDL-C is a causative biomarker of atherogenesis. Given this breadth of existing data surrounding the link between plasma cholesterol and atherosclerotic cardiovascular disease [5], a logical assumption would be that cholesterol predominates as the lipid species within atheromatas. However, two recent studies have suggested that in a hypercholesterolemic rabbit model of atherosclerosis, triacylglycerols (TGs) actually represent a major constituent of atherosclerotic plaques [6,7]. Despite these studies, a detailed lipidomic analysis of rabbit plaque composition has not yet been reported. Such information may highlight novel therapeutic strategies to induce the regression of established atherosclerotic lesions, which current LDL-lowering therapies have only modestly achieved [8]. The goal of this study was to construct a detailed framework of the lipids that comprise atherosclerotic lesions that could help us to identify novel treatment strategies that promote lesion regression. Here we report a comprehensive lipidomic analysis of atherosclerotic plaques that develop as a consequence of hypercholesterolemia. We took advantage of ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry to gain insight into lesion composition. We quantified relative amounts of lipid subclasses based on acyl chain length and degree of acyl chain saturation within major lipid fractions detected in aortic plaques. 2. Results Male New Zealand White rabbits were administered either regular chow (RC) or regular chow supplemented with 0.5% cholesterol (HC) for 12 weeks to induce hypercholesterolemia and atherosclerosis. Rabbits fed the HC diet developed hypercholesterolemia compared to rabbits fed the RC diet (43.65 ± 3.67 versus 0.93 ± 0.15 mmol/L) without an elevation in plasma TGs
Int. J. Mol. Sci. 2014, 15
23285
(0.65 ± 0.06 versus 0.53 ± 0.07 mmol/L) These data are consistent with recently published studies from our laboratory [9]. In order to assess plaque lipid content, we took advantage of high resolution time of flight (ToF) mass spectrometry which can delineate analytes with a mass accuracy of less than 5 parts per million (Tables S1–S14). Thus, we were able to accurately measure the relative quantities of lipids within the atherosclerotic plaque. We isolated lipids from the full-length of the aorta beginning with the aortic arch through the descending aorta to the iliac bifurcation, and subjected the plaque to LC-MS/MS on ToF. Our lipidomic analyses detected most of the major lipid classes including: Cholesteryl esters (CEs), TGs, phosphatidylcholines (PCs), sphingomyelins (SMs), diacylglycerols (DGs), fatty acids (FAs), phosphatidylserines (PSs), lysophosphatidylcholines (LPCs), ceramides (Cers), phosphatidylglycerols (PGs), phosphatidylinositols (PIs) and phosphatidylethanolamines (PEs). Herein, we present a relative quantitation of the fatty acyl composition of these lipid fractions, with particular attention to CEs, TGs, and PCs, the three plasma lipid classes that account for greater than 75% of total plasma lipid [10]. We additionally focused on the lipidomics of plaque SMs, as we found this lipid class to be relatively abundant in lesions. The remaining lipidomic analyses of the lipid classes mentioned above have been cataloged in the supplemental material (Tables S5–S14). Figure 1. Fatty acid (FA) biosynthetic pathways and rabbit diet fatty acyl composition. (A) Schematic representation of three major FA biosynthetic pathways: Nonessential FAs (grey), ω-3 FAs (yellow), ω-6 FAs (lavender). Enzymes are depicted in light green; and (B) FA composition of rabbit diets. Regular chow (RC) and high cholesterol (HC).
(A)
Int. J. Mol. Sci. 2014, 15
23286 Figure 1. Cont.
(B) Fatty acids identified in plasma lipids are often categorized as: (i) Nonessential FAs, namely saturated FAs (SFAs) and mono- or polyunsaturated FAs (MUFAs and PUFAs); (ii) Essential FAs that are metabolites from either the ω-3 pathway (linolenic acid derivatives) or the ω-6 pathway (linoleic acid derivatives). Nonessential FAs can originate from the diet or from de novo lipogenesis (DNL), whereas essential FAs can only originate from the metabolism of essential ω-3 or ω-6 FAs obtained from diet (Figure 1A and [9]). We have recently shown that in cholesterol-fed rabbits, over 75% of the FAs within plasma CEs are SFAs and MUFAs, suggesting a strong dietary influence on the plasma lipidome [9]. Given the dietary composition of the diet in this study (Figure 1B), the CE 18:2 and 18:3 species that predominate as the CE species within rabbit plaque (Figure 2A, Table S1) are most likely CE 18:2n6 and CE 18:2n3, both of which are derived from diet. Together with CE 20:1, 18:1, 24:1 and 22:1, over 60% of lesion CEs can be attributed to dietary or nonessential FA contributions (Figure 2A, Table S1). Similar to plasma [9], ω-6 FAs are the next major constituents of plaque CEs, with CE 22:4, 20:3 and 22:2 accounting for another 25% of fatty acyl CEs within rabbit lesions (Figure 2A, Table S1). Rabbits administered the HC diet had identical FA composition of plasma TGs compared to rabbits administered the RC diet, with nearly 80% of FAs within TGs being derived from nonessential FAs [9]. Here, we find that the most abundant TGs in plaque are comprised of FAs obtained from the diet, accounting for over 80% of TGs in plaque (Figure 2B, Table S2). Akin to our findings in the plasma, metabolites of the ω-3 and ω-6 pathways are likely only minor contributors of TG deposition to the plaque (Figure 2B, Table S2). In our previous report, 55% of FAs in PCs came from nonessential FAs, 40% came from the ω-6 pathway and the remaining 5% came from the ω-3 pathway [9]. Interestingly, 91% of the FAs in plaque PCs stemmed from dietary nonessential FAs and ~8% stemmed from metabolites of the ω-6 pathway (Figure 2C, Table S3), suggesting that the dietary FAs in circulating PCs are highly prone to deposition within atherosclerotic plaque PCs.
Int. J. Mol. Sci. 2014, 15
23287
Figure 2. Fatty acyl composition of plaque lipids: Targeted analysis of lipids was conducted on full-length aortae isolated from rabbits fed the HC diet by UPLC/TOF-MS. (A) Plaque cholesteryl esters (CE); (B) Plaque triacylglycerols (TGs); (C) Plaque phosphatidylcholines (PCs); and (D) Plaque sphingomyelins (SMs). All data is presented as the percent of the given lipid species of the total lipid class.
In relative terms, CEs, TGs and PCs constitute over 65% of the lipid classes detectable in rabbit plaque. However, another relatively abundant lipid class detected in plaque, which is not appreciably detected in plasma, is the SMs [9]. As shown in Figure 3, SMs account for roughly 20% of plaque lipids. Within this lipid class, SM 16:0 is unequivocally the most abundant SM, most likely originating from PC 34:2 (Figure 2D, Table S4). SM 16:0, together with the next five most abundant SMs (SM 24:0, SM 24:1, SM 16:1, SM 22:0 and an SM 24:1 variant) account for about 85% of detectable plaque SMs (Figure 2D, Table S4).
Int. J. Mol. Sci. 2014, 15
23288
Figure 3. Summary of aortic lipid class distribution: Targeted analysis of lipids was conducted on full-length aortae isolated from rabbits fed the HC diet by UPLC/TOF-MS. Data is expressed as the percent of the lipid class of the total lipid content in the aorta, calculated as the total peak area of each lipid class, divided by the total peak area of all lipid classes. TG: Triacylglycerol; SM: Sphingomyelin; PC: Phosphatidylcholine; DG: Diacylglycerol; FA: Fatty acid; PE-plas: Phosphatidylthanolamine; PC-plas: Phosphatidyl choline; CE: Cholesteryl ester; Cer: Ceramide; PS: Phosphatidylserine; LPC: Lyso-phosphatidic acid; PE: Phosphatidylethanolamine; PG: Phosphatidylglycerol; and PI: Phosphatidylinositol.
3. Discussion Whole body lipid homeostasis is regulated through the exogenous uptake and the endogenous synthesis of fatty acids and cholesterol, as well as the trafficking of these lipids to the appropriate anatomical depots. The perturbation of any number of mechanisms in these pathways can lead to the development of dyslipidemia, thereby increasing the propensity for lipid delivery and retention within the artery wall. Although the macromolecular components and major neutral lipid species of atherosclerotic plaques have been extensively defined [3,4,11], the lipidome of atheromatas has not been reported. Our goal was to produce a detailed framework of the lipids that comprise the atherosclerotic lesion of a widely used pre-clinical model of plaque progression, with particular attention to the most abundant lipid classes found in plasma. Lipoprotein lipase (LPL) is the primary enzyme responsible for the liberation of FAs from lipoprotein particles, whereas hepatic lipase (HL) is the key enzyme responsible for the rapid removal of the resultant lipoprotein remnant particles from the circulation [12]. It is well established that rabbits are HL deficient, which causes extreme cholesterol accumulation within chylomicron remnant and β very low-density lipoprotein (β-VLDL) particles as a result of lowered metabolic clearance rate, when rabbits are challenged with a HC diet [12]. Although the balance between LPL and HL activities regulates lipoprotein clearance, the lipid composition of lipoprotein particles is primarily owing to the lecithin:cholesterol acyltransferase (LCAT) and acyl-coenzyme A:cholesterol acyltransferase (ACAT) enzymes, which are responsible for the conversion of FC to CE. Lipoprotein-bound LCAT cleaves sn2 position FAs (preferentially PUFAs) of PCs and transfers them to the 3-β-hydroxyl group
Int. J. Mol. Sci. 2014, 15
23289
of FC, thereby generating CE [13]. ACATs use MUFAs as their preferential substrate in the addition of exogenous dietary FAs or endogenous FAs generated through DNL, to FC [14]. The fact that we found predominantly dietary FAs (namely SFAs and MUFAs) within CEs of rabbit atherosclerotic lesions, suggests that ACAT is the major enzyme for the biosynthesis of CEs deposited in plaque. Moreover, our findings are consistent with the verity that ACAT-derived CE is the major atherogenic lipid in plasma [15], whereas LCAT-derived CE is the major atheroprotective lipid in blood, as it is mainly carried within high-density lipoprotein (HDL) particles [13]. Interestingly, our previous study demonstrated a higher dietary influence on plasma CE than on the plaque CE we observed in this study [9]. This discrepancy could potentially be due to macrophage metabolism of CE-derived FAs to other FA subspecies within the site of the lesion [16], warranting further lipidomic analyses of models of atherogenesis. The data presented in this study are consistent with two recent reports which have demonstrated that TGs constitute a relatively abundant lipid species within rabbit atherosclerotic plaques [6,7]. Although challenging these animals with a HC diet does not elevate their plasma TG levels [9], TG accumulation within the atherosclerotic lesion has long been suggested to occur as a consequence of chylomicron and β-VLDL remnant retention in the artery wall [17], as well as DNL in the vascular intima from precursors such as acetate, glucose, and long-chain free fatty acids [18–20]. Our results are perhaps more consistent with the former, as we found that over 80% of the FAs within plaque TGs were nonessential FAs. Thus, our data suggest that lipoprotein-derived TGs are the major driver of plaque TG deposition. While in situ lipogenesis may certainly contribute at least part of the remaining portion of lesion TG, further studies would be required to delineate the exact contributions of lipoprotein versus lipogenic TG sources. Phospholipids are major precursors to a host of signaling lipids, including eicosanoids and SMs [21], both of which are known to be involved in proinflammatory responses in atherogenesis [22,23]. In turn, their FA composition within atherosclerotic plaques is of particular interest. According to the Lands cycle, the ratio of saturated to unsaturated FAs within PCs in particular, is tightly regulated, resulting in a saturated FA occupying the sn1 position and an unsaturated FA occupying the sn2 position [24,25]. The result, at least in plasma, is an approximately equal distribution of FAs from nonessential and ω-6/ω-3 pathways in PCs [9]. In contrast, the distribution of plaque PCs is heavily slated towards the nonessential FA class, suggesting that FAs from other lipid classes within the lesion may influence plaque PC composition. For example, the generation of sphingomyelin results from the transfer of phosphorylcholine from PC to ceramide, which liberates diacylglycerols as a byproduct [26]. Given our finding that sphingomyelins and diacylglycerols are relatively abundant lipid classes within the lesion (Figure 3), the interplay between PCs, SMs and DGs may have influenced the observed distribution of FAs in plaque PCs. Furthermore, the relatively equal abundance of PCs and SMs (~20% of plaque, each) further supports the relationship between these lipid classes within the plaque. Therefore, the complex interactions between lipid classes in plaque warrants further study, as biomarkers of atherosclerosis progression, and perhaps changes in lipid profiles observed during plaque regression, may point towards biomarkers which can be used to validate therapeutics that modulate the disease.
Int. J. Mol. Sci. 2014, 15
23290
4. Materials and Methods 4.1. Animals and Diets Male New Zealand White rabbits (n = 20) were obtained from Covance (Princeton, NJ, USA) and were individually housed in a temperature- and humidity-controlled environment with a 12 h light/dark cycle. Animals were fed purified chow (Purina Mills LabDiet 5326, LLC, St. Louis, MO, USA) with 0.5% added cholesterol ad libitum for 12 weeks and had free access to water. At the end of the study, rabbits were euthanized with 1 mL/kg of Beuthanasia-D solution (Merck, Whitehouse Station, NJ, USA) via the marginal ear vein. Plasma lipid levels were determined on EDTA-plasma as described previously [9]. Full-length aortae were excised from each animal beginning with the aortic root through the aortic arch and the descending aorta to the iliac bifurcation. Aortae were dissected free of fat and connective tissue, flash-frozen in foil packets using liquid nitrogen and stored at −80 °C until future analyses. All animal experiments, procedures and protocols, were reviewed and approved by the Merck Research Laboratories’ Institutional Animals Care and Use Committee. 4.2. Lipid Extraction and Reagents All organic solvents used were of chromatographic grade or better. Leucine enkephalin (Sigma Aldrich, St. Louis, MO, USA) solution was used for lock mass correction. Lipids were extracted from full-length aortae as per a modified Bligh and Dyer method as described previously [27,28]. During the homogenization and extraction process, samples were treated with anti-oxidants (0.5 mg of 2,6-di-tert-butyl-4-methylphenol). Extracts were evaporated to dryness and re-constituted in isopropanol/ acetonitrile/water (65%:30%:5% (v/v/v)). 4.3. Analytical Instrumentation for Relative Quantitation of Lipid Fractions Rabbit plaque lipid extracts were analyzed using an Acquity UPLC system coupled to a Synapt G2-HDMS mass spectrometer system (Waters Corp., Milford, MA, USA). For analyses, 5 µL of extract was injected. Chromatographic separation was achieved using a mobile phase A solution of 60% acetonitrile/40% water with 10 mM ammonium formate solution, and a mobile phase B solution of 90% isopropanol/10% acetonitrile with 10 mM ammonium formate. An HSS T3 C18 reverse-phase column was utilized (1.7 µm, 2.1 mm × 100 mm; Waters Corp.) coupled to the following chromatographic conditions: A linear gradient from 40% B to 100% B in 0 to 10 min followed by a 2 min hold at 100% B. The mass spectral data was acquired in electrospray positive and negative ion modes using nitrogen as the desolvation gas with a flow rate of 700 L/h. The source and desolvation temperatures were maintained at 120 and 450 °C, respectively. The capillary voltage, source cone voltage, and extraction cone voltage were maintained at +/−3 kV, +/−30 V, and 4 V, respectively. Mass spectra were acquired over the m/z range of 50 to 1200 at a resolution of 25,000 FWHM (full width half height mass resolution).
Int. J. Mol. Sci. 2014, 15
23291
5. Conclusions The observations presented in this study demonstrate the ability to obtain specific diet-induced lipid phenotypes from directly within the atherosclerotic plaque. However, these measurements do not provide any specific insights into the pathophysiology of the disease. For example, it is possible that in situ DNL and/or the metabolism of CE-, TG-, PC- and SM-derived FAs gives rise to signaling lipids such as oxylipin eicosanoids; known mediators of onset and progression of atherosclerosis. Although this example is speculative based on the present study, the data presented here provide the first complete characterization of the atherosclerotic plaque lipidome of a widely used in vivo model of the disease. This detailed framework of the lipids within the vessel wall provides a powerful lipid profile to enable future studies to expand on the lipidomic characterization of atherosclerotic lesions. Supplementary Materials Supplementary tables can be found at http://www.mdpi.com/1422-0067/15/12/23283/s1. Acknowledgments We thank the Merck Research Laboratories’ Postdoctoral Fellowship Program for financial support. Lazar A. Bojic is supported by a research fellowship provided by the MRL Postdoctoral fellowship program. Author Contributions Lazar A. Bojic conducted data analyses and drafted, edited and finalized the paper; David G. McLaren conducted analytical experiments and contributed to editing the paper; Vinit Shah conducted analytical experiments and contributed to editing the paper; Stephen F. Previs assisted in study design and execution, and contributed to editing the paper; Douglas G. Johns assisted in study design and execution, and contributed to editing the paper; and Jose M. Castro-Perez contributed to study design and execution, conducted analytical experiments, conducted data analyses, and contributed to editing the paper. Conflicts of Interest The authors declare no conflict of interest. References 1.
2. 3. 4.
Go, A.S.; Mozaffarian, D.; Roger, V.L.; Benjamin, E.J.; Berry, J.D.; Borden, W.B.; Bravata, D.M.; Dai, S.; Ford, E.S.; Fox, C.S.; et al. Heart disease and stroke statistics—2013 update: A report from the American Heart Association. Circulation 2013, 127, e6–e245. Libby, P. The forgotten majority: Unfinished business in cardiovascular risk reduction. J. Am. Coll. Cardiol. 2005, 46, 1225–1228. Moore, K.J.; Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011, 145, 341–355. Libby, P.; Ridker, P.M.; Hansson, G.K. Progress and challenges in translating the biology of atherosclerosis. Nature 2011, 473, 317–325.
Int. J. Mol. Sci. 2014, 15 5. 6.
7.
8. 9.
10.
11. 12. 13.
14. 15.
16. 17. 18. 19. 20. 21.
23292
Laufs, U.; Weintraub, W.S.; Packard, C.J. Beyond statins: What to expect from add-on lipid regulating therapy? Eur. Heart J. 2013, 34, 2660–2665. Lattermann, A.; Matthaus, C.; Bergner, N.; Beleites, C.; Romeike, B.F.; Krafft, C.; Brehm, B.R.; Popp, J. Characterization of atherosclerotic plaque depositions by raman and ftir imaging. J. Biophotonics 2013, 6, 110–121. Matthaus, C.; Dochow, S.; Bergner, G.; Lattermann, A.; Romeike, B.F.; Marple, E.T.; Krafft, C.; Dietzek, B.; Brehm, B.R.; Popp, J. In vivo characterization of atherosclerotic plaque depositions by Raman-probe spectroscopy and in vitro coherent anti-stokes Raman scattering microscopic imaging on a rabbit model. Anal. Chem. 2012, 84, 7845–7851. Williams, K.J.; Feig, J.E.; Fisher, E.A. Rapid regression of atherosclerosis: Insights from the clinical and experimental literature. Nat. Clin. Pract. Cardiovasc. Med. 2008, 5, 91–102. Yin, W.; Carballo-Jane, E.; McLaren, D.G.; Mendoza, V.H.; Gagen, K.; Geoghagen, N.S.; McNamara, L.A.; Gorski, J.N.; Eiermann, G.J.; Petrov, A.; et al. Plasma lipid profiling across species for the identification of optimal animal models of human dyslipidemia. J. Lipid Res. 2012, 53, 51–65. Quehenberger, O.; Armando, A.M.; Brown, A.H.; Milne, S.B.; Myers, D.S.; Merrill, A.H.; Bandyopadhyay, S.; Jones, K.N.; Kelly, S.; Shaner, R.L.; et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 2010, 51, 3299–3305. Libby, P.; Ridker, P.M.; Hansson, G.K. Inflammation in atherosclerosis: From pathophysiology to practice. J. Am. Coll. Cardiol. 2009, 54, 2129–2138. Chang, S.; Borensztajn, J. Hepatic lipase function and the accumulation of β-very-low-density lipoproteins in the plasma of cholesterol-fed rabbits. Biochem. J. 1993, 293, 745–750. Rousset, X.; Vaisman, B.; Amar, M.; Sethi, A.A.; Remaley, A.T. Lecithin: Cholesterol acyltransferase—From biochemistry to role in cardiovascular disease. Curr. Opin. Endocrinol. Diabetes Obes. 2009, 16, 163–171. Chang, T.Y.; Chang, C.C.; Lin, S.; Yu, C.; Li, B.L.; Miyazaki, A. Roles of acyl-coenzyme a: Cholesterol acyltransferase-1 and -2. Curr. Opin. Lipidol. 2001, 12, 289–296. Lee, R.G.; Kelley, K.L.; Sawyer, J.K.; Farese, R.V., Jr.; Parks, J.S.; Rudel, L.L. Plasma cholesteryl esters provided by lecithin:cholesterol acyltransferase and acyl-coenzyme a:cholesterol acyltransferase 2 have opposite atherosclerotic potential. Circ. Res. 2004, 95, 998–1004. Norris, P.C.; Dennis, E.A. A lipidomic perspective on inflammatory macrophage eicosanoid signaling. Adv. Biol. Regul. 2014, 54, 99–110. Vost, A. Uptake and metabolism of circulating chylomicron triglyceride by rabbit aorta. J. Lipid Res. 1972, 13, 695–704. Howard, C.F., Jr. De novo synthesis and elongation of fatty acids by subcellar fractions of monkey aorta. J. Lipid Res. 1968, 9, 254–261. Stein, Y.; Stein, O. Incorporation of fatty acids into lipids of aortic slices of rabbits, dogs, rats and baboons. J. Atheroscler. Res. 1962, 2, 400–412. Vost, A. Lipid accretion in the perfused rabbit aorta. J. Atheroscler. Res. 1969, 9, 221–238. Zhou, L.; Nilsson, A. Sources of eicosanoid precursor fatty acid pools in tissues. J. Lipid Res. 2001, 42, 1521–1542.
Int. J. Mol. Sci. 2014, 15
23293
22. Buczynski, M.W.; Dumlao, D.S.; Dennis, E.A. Thematic review series: Proteomics. An integrated omics analysis of eicosanoid biology. J. Lipid Res. 2009, 50, 1015–1038. 23. Li, Z.; Fan, Y.; Liu, J.; Li, Y.; Huan, C.; Bui, H.H.; Kuo, M.S.; Park, T.S.; Cao, G.; Jiang, X.C. Impact of sphingomyelin synthase 1 deficiency on sphingolipid metabolism and atherosclerosis in mice. Arterioscler. Thromb. Vasc. Boil. 2012, 32, 1577–1584. 24. Lands, W.E. Metabolism of glycerolipids. 2. The enzymatic acylation of lysolecithin. J. Boil. Chem. 1960, 235, 2233–2237. 25. Lands, W.E.; Merkl, I. Metabolism of glycerolipids. III. Reactivity of various acyl esters of coenzyme a with α'-acylglycerophosphorylcholine, and positional specificities in lecithin synthesis. J. Boil. Chem. 1963, 238, 898–904. 26. Merrill, A.H., Jr. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev. 2011, 111, 6387–6422. 27. Bligh, E.G.; Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911–917. 28. Shah, V.; Castro-Perez, J.M.; McLaren, D.G.; Herath, K.B.; Previs, S.F.; Roddy, T.P. Enhanced data-independent analysis of lipids using ion mobility-tofmse to unravel quantitative and qualitative information in human plasma. Rapid Commun. Mass Spectrom. 2013, 27, 2195–2200. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
Supplementary Information Table S1. Plaque cholesteryl esters. Metabolite CE 18:2 CE 18:3 CE 22:4 CE 20:3 CE 24:2 CE 20:1 CE 18:1 CE 24:1 CE 22:2 CE 22:1 CE 22:5 CE 24:4 CE 16:1 CE 26:2 CE 18:0 CE 24:5 CE 24:3 CE 26:1 CE 26:3 CE 26:4 CE 20:2
Formula C45H79NO2 C45H77NO2 C49H83NO2 C47H81NO2 C51H91NO2 C47H85NO2 C45H81NO2 C51H93NO2 C49H87NO2 C49H89NO2 C49H81NO2 C51H87NO2 C43H77NO2 C53H95NO2 C45H83NO2 C51H85NO2 C51H89NO2 C53H97NO2 C53H93NO2 C53H91NO2 C47H83NO2
m/z Found 666.6204 664.6039 718.6522 692.6372 750.713 696.6668 668.6354 752.7309 722.6819 724.6968 716.6349 746.6808 640.6038 778.7448 670.6501 744.666 748.6974 780.7601 776.7288 774.7115 694.6499
mDa 1.5 0.7 2 2.7 0.2 1 0.9 2.5 0.4 −0.3 0.4 −0.7 0.6 0.7 −0.1 0.2 0.3 0.4 0.4 −1.3 −0.3
PPM 2.3 1 2.8 3.9 0.3 1.4 1.3 3.3 0.6 −0.5 0.5 −0.9 0.9 0.9 −0.1 0.2 0.4 0.5 0.5 −1.7 −0.4
Time 9.17 8.94 9.28 9.27 9.96 9.68 9.4 10.15 9.7 9.94 9.12 9.53 9.13 10.17 9.57 9.33 9.78 10.36 9.99 9.79 9.37
Area % 33.34 15.13 12.45 10.35 7.12 4.39 3.76 3.48 2.65 1.50 0.97 0.89 0.82 0.77 0.54 0.52 0.44 0.36 0.14 0.14 0.10
Table S2. Plaque triaclyglycerols. Metabolite TG 52:4 TG 54:6 TG 52:3 TG 54:5 TG 52:5 TG 52:2 TG 54:7 TG 54:4 TG 50:2 TG 50:1 TG 50:3 TG 54:3 TG 50:4 TG 54:2 TG 52:6 TG 48:0
Formula C55H101NO6 C57H101NO6 C55H103NO6 C57H103NO6 C55H99NO6 C55H105NO6 C57H99NO6 C57H105NO6 C53H101NO6 C53H103NO6 C53H99NO6 C57H107NO6 C53H97NO6 C57H109NO6 C55H97NO6 C51H101NO6
m/z Found 872.7699 896.7709 874.7882 898.7861 870.7543 876.8045 894.7551 900.8044 848.7718 850.7883 846.7545 902.8206 844.7394 904.8304 868.7403 824.7731
mDa −0.8 0.2 1.9 −0.2 −0.7 2.5 0.1 2.4 1.1 2 −0.5 3 0 −2.9 0.9 2.4
PPM −0.9 0.2 2.1 −0.3 −0.9 2.9 0.1 2.7 1.3 2.3 −0.6 3.3 0 −3.2 1 2.9
Time 8.56 8.38 8.8 8.62 8.35 9.03 8.14 8.84 8.76 8.96 8.52 9.06 8.31 9.26 8.1 8.95
Area % 10.17 8.89 7.40 7.32 7.23 6.13 5.82 5.67 5.45 4.97 4.38 3.99 3.06 2.89 2.50 2.32
S2 Table S2. Cont. Metabolite TG 48:2 TG 48:1 TG 48:3 TG 54:8 TG 50:5 TG 46:0 TG 56:6 TG 56:7 TG 46:1 TG 46:2 TG 56:5 TG 56:8 TG 48:4 TG 56:4 TG 56:3 TG 52:7
Formula C51H97NO6 C51H99NO6 C51H95NO6 C57H97NO6 C53H95NO6 C49H97NO6 C59H105NO6 C59H103NO6 C49H95NO6 C49H93NO6 C59H107NO6 C59H101NO6 C51H93NO6 C59H109NO6 C59H111NO6 C55H95NO6
m/z Found 820.738 822.7538 818.7233 892.7384 842.7247 796.7384 924.8013 922.7852 794.7227 792.7072 926.8188 920.77 816.7067 928.8317 930.8468 866.7227
mDa −1.4 −1.2 −0.4 −1 1 −1 −0.7 −1.1 −1 −0.9 1.2 −0.7 −1.4 −1.6 −2.1 −1
PPM −1.7 −1.5 −0.5 −1.1 1.1 −1.2 −0.7 −1.2 −1.3 −1.1 1.3 −0.8 −1.7 −1.7 −2.3 −1.2
Time 8.5 8.73 8.26 7.91 8.07 8.72 8.69 8.49 8.48 8.24 8.89 8.28 8.02 9.09 9.28 7.87
Area % 2.28 1.64 1.34 1.13 1.05 0.47 0.46 0.40 0.39 0.38 0.30 0.26 0.25 0.19 0.18 0.15
Table S3. Plaque phosphatidylcholines. Metabolite PC 34:2 PC 36:2 PC 34:1 PC 36:3 PC 36:4 PC 36:4 PC 38:4 PC 36:1 PC 32:1 PC 34:3 PC 38:6 PC 34:3 PC 32:2 PC 38:4 PC 38:5 PC 38:5 PC 38:4 PC 36:5 PC 38:2 PC 38:3 PC 36:5 PC 38:3 PC 36:5 PC 40:4 PC 34:4
Formula C42H80NO8P C44H84NO8P C42H82NO8P C44H82NO8P C44H80NO8P C44H80NO8P C46H84NO8P C44H86NO8P C40H78NO8P C42H78NO8P C46H80NO8P C42H78NO8P C40H76NO8P C46H84NO8P C46H82NO8P C46H82NO8P C46H84NO8P C44H78NO8P C46H88NO8P C46H86NO8P C44H78NO8P C46H86NO8P C44H78NO8P C48H88NO8P C42H76NO8P
m/z Found 758.5709 786.6002 760.5865 784.586 782.5703 782.5684 810.6002 788.6161 732.5553 756.5541 806.5701 756.5545 730.5395 810.5999 808.5854 808.5854 810.6024 780.555 814.6329 812.6171 780.5558 812.6154 780.5543 838.6312 754.5413
mDa 0.9 −1.1 0.9 0.4 0.3 −1.6 −1.1 −0.8 1 −0.2 0.1 0.2 0.8 −1.4 −0.2 −0.2 1.1 0.7 0.3 0.2 1.5 −1.5 0 −1.4 2.6
PPM 1.2 −1.3 1.2 0.5 0.4 −2 −1.3 −1 1.4 −0.3 0.2 0.3 1.2 −1.7 −0.3 −0.3 1.4 0.9 0.4 0.2 1.9 −1.9 0 −1.6 3.5
Time 4.83 5.41 5.28 4.91 4.74 4.44 5.35 5.83 4.68 4.39 4.34 4.31 4.2 4.98 4.77 4.97 5.15 4.34 5.9 5.56 4.01 5.45 4.21 5.72 4.13
Area % 21.34 16.69 12.96 12.09 10.89 5.22 3.74 3.53 3.35 2.21 1.80 1.36 0.77 0.64 0.60 0.57 0.38 0.36 0.27 0.25 0.23 0.23 0.18 0.13 0.11
S3 Table S4. Plaque sphingomyelins. Metabolite SM 16:0 SM 24:0 SM 24:1 SM 16:1 SM 22:0 SM 24:1 SM 24:2 SM 20:0 SM 22:1 SM 18:1 SM 14:0 SM 20:1 SM 26:0
Formula C39H79N2O6P C47H95N2O6P C47H93N2O6P C39H77N2O6P C45H91N2O6P C47H93N2O6P C47H91N2O6P C43H87N2O6P C45H89N2O6P C41H81N2O6P C37H75N2O6P C43H85N2O6P C49H99N2O6P
m/z Found 703.5763 815.699 813.683 701.5619 787.6685 813.6852 811.6678 759.6374 785.653 729.5923 675.5459 757.6223 843.7312
mDa 0.9 −1.6 −1.9 2.2 −0.8 0.3 −1.5 −0.6 −0.6 1.3 1.8 0 −0.7
PPM 1.3 −1.9 −2.4 3.1 −1 0.3 −1.8 −0.8 −0.8 1.7 2.7 0 −0.8
Time 4.59 6.8 6.28 4.05 6.34 6.42 5.88 5.78 5.92 4.69 3.94 5.34 7.24
Area % 30.27 18.49 12.05 8.66 8.55 7.68 5.46 4.47 2.49 0.68 0.67 0.28 0.14
Table S5. Plaque diacylglycerols. Metabolite DG 46:11 DG 44:10 DG 38:4 DG 46:10 DG 36:2 DG 34:2 DG 36:3 DG 36:4 DG 34:1 DG 36:1
Formula C49H77NO5 C47H75NO5 C41H75NO5 C49H79NO5 C39H75NO5 C37H71NO5 C39H73NO5 C39H71NO5 C37H73NO5 C39H77NO5
m/z Found 760.5865 734.573 662.5737 762.601 638.5737 610.5426 636.5576 634.5419 612.5568 640.5883
mDa −1.5 0.7 1.4 −2.6 1.4 1.6 0.9 0.9 0.1 0.3
PPM −1.9 0.9 2.1 −3.4 2.2 2.6 1.5 1.4 0.2 0.5
Time 5.28 5.19 6.52 5.75 6.62 6.12 6.17 6.01 6.5 6.97
Area % 51.32 28.13 8.04 5.83 2.66 1.95 0.63 0.46 0.41 0.31
Table S6. Plaque PE-plas. Metabolite PE_plas 38:4 PE_plas 40:4 PE_plas 38:5 PE_plas 36:1 PE_plas 36:4 PE_plas 40:5 PE_plas 38:4 PE_plas 40:6 PE_plas 40:5 PE_plas 34:1 PE_plas 36:2 PE_plas 38:3 PE_plas 32:4
Formula C43H78NO7P C45H82NO7P C43H76NO7P C41H80NO7P C41H74NO7P C45H80NO7P C43H78NO7P C45H78NO7P C45H80NO7P C39H76NO7P C41H78NO7P C43H80NO7P C37H66NO7P
m/z Found 752.5596 780.5913 750.5443 730.5765 724.5288 778.5758 752.5593 776.5585 778.575 702.5452 728.5615 754.5743 668.4722
mDa 0.2 0.6 0.6 1.5 0.7 0.8 −0.1 −0.9 0 1.5 2.1 −0.7 6.7
PPM 0.3 0.8 0.7 2 1 1 −0.1 −1.2 −0.1 2.1 2.9 −1 10
Time 5.82 6.17 5.31 6.28 5.23 5.99 5.65 5.66 5.84 5.75 5.91 6.08 7.05
Area % 49.86 13.92 7.30 6.56 6.48 4.08 2.89 2.54 2.07 1.97 1.45 0.63 0.16
S4 Table S7. Plaque PC−plas. Metabolite PC-plas 36:4 PC-plas 34:0 PC-plas 38:4 PC-plas 36:3 PC-plas 38:4 PC-plas 38:5 PC-plas 38:3 PC-plas 32:0 PC-plas 34:1 PC-plas 36:1 PC-plas 34:4 PC-plas 36:2 PC-plas 36:1 PC-plas 40:3
Formula C44H80NO7P C42H84NO7P C46H84NO7P C44H82NO7P C46H84NO7P C46H82NO7P C46H86NO7P C40H80NO7P C42H82NO7P C44H86NO7P C42H76NO7P C44H84NO7P C44H86NO7P C48H90NO7P
m/z Found 766.5742 746.6058 794.6055 768.5907 794.6052 792.5909 796.6212 718.576 744.5905 772.6226 738.5445 770.6043 772.6212 824.6522
mDa −0.8 −0.5 −0.8 0 −1.1 0.2 −0.8 1 −0.2 0.6 0.8 −2 −0.8 −1.1
PPM −1.1 −0.7 −1.1 0 −1.4 0.3 −1 1.3 −0.3 0.8 1 −2.7 −1 −1.3
Time 5.03 5.64 5.61 5.11 5.15 5.07 5.7 5.5 5.19 5.7 5.54 5.69 6.11 6.22
Area % 33.06 21.89 13.61 10.66 6.24 4.65 2.62 2.53 1.65 1.52 0.56 0.38 0.36 0.12
Table S8. Plaque fatty acids. Metabolite FA 20:3 FA 18:0 FA 18:1 FA 22:3 FA 20:4 FA 22:4 FA 20:2 FA 20:1 FA 20:0 FA 22:2 FA 20:5 FA 22:5
Formula C20H32O2 C18H34O2 C18H32O2 C22H36O2 C20H30O2 C22H34O2 C20H34O2 C20H36O2 C20H38O2 C22H38O2 C20H28O2 C22H32O2
m/z Found 303.2341 281.2485 279.2339 331.2642 301.2178 329.2487 305.2492 307.2643 309.2802 333.2789 299.2022 327.2331
mDa 1.7 0.4 1.5 0.5 1 0.6 1.1 0.6 0.8 −0.5 1.1 0.7
PPM 5.5 1.5 5.2 1.4 3.4 1.9 3.7 1.8 2.6 −1.4 3.6 2
Time 2.05 2.62 2.14 2.52 2.13 2.12 2.33 2.76 3.32 3.02 1.66 1.88
Area % 45.41 22.95 12.11 5.96 5.72 2.88 2.06 0.76 0.78 0.64 0.49 0.12
Table S9. Plaque phosphatidylserines. Metabolite PS 36:2 PS 38:4 PS 36:1 PS 40:5 PS 38:1 PS 38:0 PS 40:1 PS 40:3 PS 46:3 PS 44:3
Formula C42H78NO10P C44H78NO10P C42H80NO10P C46H80NO10P C44H84NO10P C44H86NO10P C46H88NO10P C46H84NO10P C52H96NO10P C50H92NO10P
m/z Found 786.5281 810.5272 788.5422 836.5426 816.5726 818.5909 844.6029 840.5715 924.6756 896.645
mDa −0.4 −1.3 −2 −1.6 −2.9 −0.2 −3.9 −4 6.2 6.9
PPM −0.6 −1.6 −2.5 −1.9 −3.5 −0.3 −4.6 −4.7 6.7 7.7
Time 4.91 4.91 5.36 4.91 5.14 5.56 5.7 5.05 6.97 6.52
Area % 77.41 9.14 5.84 4.66 1.25 0.87 0.32 0.22 0.18 0.11
S5 Table S10. Plaque lyso-phosphatidylcholines. Metabolite LPC 18:0 LPC 16:0 LPC 18:1 LPC 18:2
Formula C26H54NO7P C24H50NO7P C26H52NO7P C26H50NO7P
m/z Found 524.3723 496.3405 522.3562 520.3405
mDa 0.7 0.2 0.3 0.2
PPM 1.4 0.4 0.5 0.4
Time 1.94 1.49 1.56 1.31
Area % 53.57 38.20 4.68 3.56
Time 7.46 7.03 7.05 6.58 7.12 5.5 6.64 6.09 6.67 6.16 6.25 7.84 3.06
Area % 30.80 20.79 12.31 8.22 6.58 6.55 6.51 3.65 2.65 1.17 0.26 0.25 0.24
Time 4.03 4.48 4.97 3.27 3.66 4.99 4.19 4.97 4.91 3.6 4.38 3.82 4.32 3.94 3.47 3.04 3.46 5.05
Area % 19.62 19.41 11.26 10.80 7.12 6.51 3.58 3.48 3.23 2.39 2.21 2.19 1.53 1.47 1.09 0.60 0.56 0.44
Table S11. Plaque ceramides. Metabolite Cer 24:0 Cer 24:1 Cer 22:0 Cer 20:0 Cer 24:1 Cer 16:0 Cer 24:2 Cer 18:0 Cer 22:1 Cer 20:1 Cer 24:3 Cer 26:0 Cer 16:4
Formula C43H85NO5 C43H83NO5 C41H81NO5 C39H77NO5 C43H83NO5 C35H69NO5 C43H81NO5 C37H73NO5 C41H79NO5 C39H75NO5 C43H79NO5 C45H89NO5 C35H61NO5
m/z Found 694.6354 692.6192 666.6036 638.5743 692.6201 582.5099 690.6045 610.5396 664.5893 636.5552 688.5886 722.6662 574.4483
mDa 0.4 −0.1 −0.1 1.9 0.8 0.1 0.8 −1.5 1.3 −1.5 0.6 −0.1 1.1
PPM 0.6 −0.2 −0.1 3 1.1 0.2 1.2 −2.4 1.9 −2.4 0.8 −0.1 2
Table S12. Plaque phosphatidylglycerols. Metabolite PG 36:3 PG 36:2 PG 36:1 PG 36:4 PG 36:3 PG 38:2 PG 36:2 PG 36:2 PG 36:2 PG 36:4 PG 34:1 PG 36:3 PG 36:2 PG 34:2 PG 34:2 PG 34:3 PG 36:4 PG 38:1
Formula C42H77O10P C42H79O10P C42H81O10P C42H75O10P C42H77O10P C44H83O10P C42H79O10P C42H79O10P C42H79O10P C42H75O10P C40H77O10P C42H77O10P C42H79O10P C40H75O10P C40H75O10P C40H73O10P C42H75O10P C44H85O10P
m/z Found 771.5178 773.5335 775.5472 769.5003 771.5144 801.5627 773.5325 773.5319 773.5331 769.5 747.5172 771.5154 773.5317 745.5021 745.5005 743.4861 769.5016 803.576
mDa 0.2 0.2 −1.7 −1.7 −3.2 −1.9 −0.8 −1.4 −0.2 −2 −0.4 −2.2 −1.6 0.1 −1.5 −0.2 −0.4 −4.2
PPM 0.2 0.3 −2.2 −2.2 −4.2 −2.4 −1 −1.8 −0.2 −2.6 −0.6 −2.9 −2 0.2 −2 −0.3 −0.5 −5.3
S6 Table S12. Cont. Metabolite PG 36:4 PG 34:1 PG 34:3 PG 38:4 PG 38:6 PG 34:2 PG 34:2 PG 34:2 PG 36:3 PG 36:5 PG 38:4
Formula C42H75O10P C40H77O10P C40H73O10P C44H79O10P C44H75O10P C40H75O10P C40H75O10P C40H75O10P C42H77O10P C42H73O10P C44H79O10P
m/z Found 769.5003 747.5153 743.4866 797.5336 793.5034 745.4987 745.5016 745.4984 771.5158 767.4822 797.5334
mDa −1.7 −2.3 0.3 0.3 1.4 −3.3 −0.4 −3.6 −1.8 −4.1 0.1
PPM −2.2 −3.1 0.4 0.4 1.8 −4.4 −0.5 −4.8 −2.4 −5.4 0.1
Time 3.06 4.21 3.44 4.07 3.05 3.58 3.63 3.69 4.2 3.02 4.21
Area % 0.40 0.37 0.30 0.25 0.22 0.19 0.18 0.17 0.15 0.14 0.14
Table S13. Plaque phosphatidylinositols. Metabolite PI 38:4 PI 38:3 PI 36:1 PI 36:2 PI 38:5 PI 36:4 PI 34:1 PI 36:3 PI 34:2
Formula C47H83O13P C47H85O13P C45H85O13P C45H83O13P C47H81O13P C45H79O13P C43H81O13P C45H81O13P C43H79O13P
m/z Found 885.5511 887.5631 863.5639 861.5517 883.5317 857.5182 835.5361 859.532 833.5151
mDa 1.8 −1.9 −1.1 2.4 −2 0.2 2.4 −1.7 −2.9
PPM 2 −2.1 −1.3 2.7 −2.2 0.2 2.9 −2 −3.5
Time 4.72 4.95 4.95 4.79 4.2 4.13 4.65 4.28 4.19
Area % 55.41 32.95 4.96 1.77 1.40 1.18 0.89 0.75 0.71
Table S14. Plaque phosphatidylethanolamines. Metabolite PE 36:4 PE 38:4 PE 38:5 PE 36:2 PE 36:1 PE 34:2 PE 36:3 PE 34:1 PE 40:5 PE 42:1 PE 38:2
Formula C41H74NO8P C43H78NO8P C43H76NO8P C41H78NO8P C41H80NO8P C39H74NO8P C41H76NO8P C39H76NO8P C45H80NO8P C47H92NO8P C43H82NO8P
m/z Found 738.5074 766.5383 764.5226 742.5384 744.5533 714.5077 740.5206 716.5231 792.5518 828.647 770.5706
mDa 0 −0.4 −0.5 −0.3 −1.1 0.3 −2.5 0 −2.6 −1.3 0.6
PPM 0 −0.5 −0.6 −0.4 −1.4 0.4 −3.3 0.1 −3.2 −1.5 0.8
Time 4.95 5.53 4.99 5.61 6.01 4.99 5.1 5.49 5.73 7.34 6.05
Area % 36.80 24.49 18.22 10.57 3.85 3.13 1.95 0.54 0.22 0.12 0.10
Copyright of International Journal of Molecular Sciences is the property of MDPI Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
