Quantitative determination of esterified eicosanoids and related oxygenated metabolites after base hydrolysis
2018; Elsevier BV; Volume: 59; Issue: 12 Linguagem: Inglês
10.1194/jlr.d089516
ISSN1539-7262
AutoresOswald Quehenberger, Signe Dahlberg-Wright, Jiang Jiang, Aaron M. Armando, Edward A. Dennis,
Tópico(s)Diet, Metabolism, and Disease
ResumoEicosanoids and related metabolites (oxylipins) possess potent signaling properties, elicit numerous important physiologic responses, and serve as biomarkers of disease. In addition to their presence in free form, a considerable portion of these bioactive lipids is esterified to complex lipids in cell membranes and plasma lipoproteins. We developed a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance LC coupled with mass spectrometric analysis. Detailed evaluation of the data revealed that several oxylipins are susceptible to alkaline-induced degradation. Nevertheless, of the 136 metabolites we examined, 56 were reproducibly recovered after alkaline hydrolysis. We classified those metabolites that were resistant to alkaline-induced degradation and applied this methodology to quantify metabolite levels in a macrophage cell model and in plasma of healthy subjects. After alkaline hydrolysis of lipids, 34 metabolites could be detected and quantified in resting and activated macrophages, and 38 metabolites were recovered from human plasma at levels that were substantially greater than in free form. By carefully selecting internal standards and taking the observed experimental limitations into account, we established a robust method that can be reliably employed for the measurement of esterified oxylipins in biological samples. Eicosanoids and related metabolites (oxylipins) possess potent signaling properties, elicit numerous important physiologic responses, and serve as biomarkers of disease. In addition to their presence in free form, a considerable portion of these bioactive lipids is esterified to complex lipids in cell membranes and plasma lipoproteins. We developed a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance LC coupled with mass spectrometric analysis. Detailed evaluation of the data revealed that several oxylipins are susceptible to alkaline-induced degradation. Nevertheless, of the 136 metabolites we examined, 56 were reproducibly recovered after alkaline hydrolysis. We classified those metabolites that were resistant to alkaline-induced degradation and applied this methodology to quantify metabolite levels in a macrophage cell model and in plasma of healthy subjects. After alkaline hydrolysis of lipids, 34 metabolites could be detected and quantified in resting and activated macrophages, and 38 metabolites were recovered from human plasma at levels that were substantially greater than in free form. By carefully selecting internal standards and taking the observed experimental limitations into account, we established a robust method that can be reliably employed for the measurement of esterified oxylipins in biological samples. Eicosanoids and related metabolites, sometimes referred to as oxylipins, are a group of structurally diverse metabolites that derive from the oxidation of PUFAs, including arachidonic acid, linoleic acid, α and γ linolenic acid, dihomo γ linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. They are locally acting bioactive signaling lipids that regulate a diverse set of homeostatic and inflammatory processes (1.Funk C.D. Prostaglandins and leukotrienes: advances in eicosanoid biology.Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3030) Google Scholar, 2.Astarita G. Kendall A.C. Dennis E.A. Nicolaou A. Targeted lipidomic strategies for oxygenated metabolites of polyunsaturated fatty acids.Biochim. Biophys. Acta. 2015; 1851: 456-468Crossref PubMed Scopus (104) Google Scholar). Given the important regulatory functions in numerous physiological and pathophysiological states, the accurate measurement of eicosanoids and other oxylipins is of great clinical interest and lipidomics is now widely used to screen effectively for potential disease biomarkers (3.Quehenberger O. Dennis E.A. The human plasma lipidome.N. Engl. J. Med. 2011; 365: 1812-1823Crossref PubMed Scopus (302) Google Scholar). The biosynthesis of eicosanoids and oxylipins involves the action of multiple enzymes organized into a complex and intertwined lipid-anabolic network (4.Dennis E.A. Norris P.C. Eicosanoid storm in infection and inflammation.Nat. Rev. Immunol. 2015; 15: 511-523Crossref PubMed Scopus (845) Google Scholar). Generally, the enzymatic formation of eicosanoids requires free fatty acids as substrates; thus, the pathway is initiated by the hydrolysis of phospholipids (PLs) by phospholipase A2 upon physiological stimuli (5.Dennis E.A. Cao J. Hsu Y.H. Magrioti V. Kokotos G. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.Chem. Rev. 2011; 111: 6130-6185Crossref PubMed Scopus (768) Google Scholar). The hydrolyzed PUFAs are then processed by three enzyme systems: cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 enzymes. Each of these enzyme systems produces unique collections of oxygenated metabolites that function either as end-products or as intermediates for a cascade of downstream enzymes. The resulting eicosanoids exhibit diverse biological activities, half-lives, and utilities in regulating many physiologic processes in health and disease, including the immune response, inflammation, and homeostasis (6.Smith W.L. Urade Y. Jakobsson P.J. Enzymes of the cyclooxygenase pathways of prostanoid biosynthesis.Chem. Rev. 2011; 111: 5821-5865Crossref PubMed Scopus (350) Google Scholar, 7.Morisseau C. Hammock B.D. Impact of soluble epoxide hydrolase and epoxyeicosanoids on human health.Annu. Rev. Pharmacol. Toxicol. 2013; 53: 37-58Crossref PubMed Scopus (361) Google Scholar, 8.Lone A.M. Tasken K. Proinflammatory and immunoregulatory roles of eicosanoids in T cells.Front. Immunol. 2013; 4: 130Crossref PubMed Scopus (90) Google Scholar, 9.Serhan C.N. Pro-resolving lipid mediators are leads for resolution physiology.Nature. 2014; 510: 92-101Crossref PubMed Scopus (1835) Google Scholar, 10.Kuhn H. Banthiya S. van Leyen K. 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Two potential mechanisms for the formation of eicosanoid-containing PLs have been proposed: i) direct oxidation of PUFAs on the intact PLs; and ii) reacylation of preformed free oxylipins into lysoPLs. COXs require free fatty acid as substrate and show little activity toward PUFAs in intact PLs (19.Lands W.E. Samuelsson B. Phospholipid precursors of prostaglandins.Biochim. Biophys. Acta. 1968; 164: 426-429Crossref PubMed Scopus (232) Google Scholar). A number of subsequent studies support the concept that prostaglandins (PGs) are first formed enzymatically and then incorporated into PLs by the sequential actions of long-chain acyl-CoA synthases and lysoPL acyltransferases (20.Aldrovandi M. Hammond V.J. Podmore H. Hornshaw M. Clark S.R. Marnett L.J. Slatter D.A. Murphy R.C. Collins P.W. O'Donnell V.B. Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation.J. 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Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate.J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar, 24.O'Donnell V.B. Murphy R.C. New families of bioactive oxidized phospholipids generated by immune cells: identification and signaling actions.Blood. 2012; 120: 1985-1992Crossref PubMed Scopus (66) Google Scholar). Similarly, the endocannabinoid 2-arachidonylglycerol is a substrate for COX-2 and is metabolized to PGH2 glycerol ester as effectively as free arachidonic acid (25.Kozak K.R. Rowlinson S.W. Marnett L.J. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2.J. Biol. Chem. 2000; 275: 33744-33749Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The final products derived from this direct PL oxygenation pathway include esterified PGs as well as 11-HETE and 15-HETE. PUFAs contained in PLs can also be oxidized by nonenzymatic reactions. Free radical peroxidation reactions observed under conditions of oxidative stress can freely proceed on intact PLs resulting in the formation of isoprostanes (26.Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts L.J. Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids.Proc. Natl. Acad. Sci. USA. 1992; 89: 10721-10725Crossref PubMed Scopus (666) Google Scholar). Previously, we and others applied LC-MS/MS protocols to test whether plasma levels of oxylipins can be used as biomarkers to differentiate the progressive form of nonalcoholic fatty liver disease, termed nonalcoholic steatohepatitis, from the milder form termed nonalcoholic fatty liver. In that study, we identified a panel of nonesterified oxylipins that when used together is able to discriminate nonalcoholic steatohepatitis from nonalcoholic fatty liver with a high degree of certainty (27.Loomba R. Quehenberger O. Armando A. Dennis E.A. Polyunsaturated fatty acid metabolites as novel lipidomic biomarkers for noninvasive diagnosis of nonalcoholic steatohepatitis.J. Lipid Res. 2015; 56: 185-192Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Another study used an approach that included an alkaline hydrolysis step with the aim of measuring the sum total of free and esterified oxylipins (28.Feldstein A.E. Lopez R. Tamimi T.A. Yerian L. Chung Y.M. Berk M. Zhang R. McIntyre T.M. Hazen S.L. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J. Lipid Res. 2010; 51: 3046-3054Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Of the markers monitored, products derived from free radical-mediated oxidation of linoleic acid were reported to be significantly elevated in nonalcoholic steatohepatitis. These results differed significantly from our findings, but can in part be explained by the difference in the experimental approach, as we measured the free oxylipins present in plasma, not those appearing after alkaline hydrolysis (27.Loomba R. Quehenberger O. Armando A. Dennis E.A. Polyunsaturated fatty acid metabolites as novel lipidomic biomarkers for noninvasive diagnosis of nonalcoholic steatohepatitis.J. Lipid Res. 2015; 56: 185-192Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In order to quantitatively capture the sum total of esterified and free oxylipins, all plasma samples need to be hydrolyzed, which requires strong alkaline conditions to quantitatively release the oxidized PUFAs before analysis. However, neither any specific experimental conditions nor systematic testing of the effect of strong bases on eicosanoid stability were reported in the later study (28.Feldstein A.E. Lopez R. Tamimi T.A. Yerian L. Chung Y.M. Berk M. Zhang R. McIntyre T.M. Hazen S.L. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J. Lipid Res. 2010; 51: 3046-3054Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In contrast, in the present study, we have specifically determined the stability of the oxylipins under the hydrolysis conditions employed, and compiled a list of metabolites that can be reproducibly measured in biological samples. From previous studies in our and other laboratories, we know that eicosanoids and specifically PGs are sensitive to alkaline-induced degradation. The objective of the current study was to develop precise conditions to minimize degradation of lipid metabolites during alkaline treatment and to identify specific eicosanoids and related oxidized PUFAs that are released intact from esterified lipids and which can be quantitatively included in searches for potential biomarkers. All solvents were ultra-performance LC (UPLC) grade and were purchased from Fisher Scientific (Waltham, MA). All primary standards (PSTDs) for standard curves and deuterated internal standards (ISTDs) were purchased from Cayman Chemicals (Ann Arbor, MI) or Enzo Life Sciences (Farmingdale, NY). Strata-X polymeric reversed phase columns were purchased from Phenomenex (Torrance, CA). Human plasma was purchased from Gemini Bio Products (West Sacramento, CA). RAW 264.7 cells (ATCC) were used in all cell experiments. Briefly, 4x1E5 cells were plated into each well of a 6-well plate and cultured overnight in 2 ml of DMEM containing 10% FBS (Atlanta Biologicals, Flowery Branch, GA) and were grown overnight at 37°C and 5% CO2. For the experiment, the culture medium was exchanged with 2 ml of DMEM without FBS, and the cells were primed with Kdo2-lipid A (Avanti Polar Lipids, Alabaster, AL) at 100 ng/ml for 4 h, then stimulated with ATP (5 mM) for an additional 20 h. At the end of the incubation period, DMEM was removed and cells were harvested into 1 ml of PBS, counted, homogenized, and both cell homogenate and medium fractions were frozen. For analysis, we used 500 μl of the cell homogenates. For the extraction of free eicosanoids, 50 μl of plasma, 500 μl of cell homogenates, or the PSTD collection consisting of 136 individual standards were spiked with 100 μl of the ISTD mix (1 ng of each of 26 deuterated standards in ethanol) and diluted with Dulbecco's PBS to give a 10% total ethanol concentration (29.Wang Y. Armando A.M. Quehenberger O. Yan C. Dennis E.A. Comprehensive ultra-performance liquid chromatographic separation and mass spectrometric analysis of eicosanoid metabolites in human samples.J. Chromatogr. A. 2014; 1359: 60-69Crossref PubMed Scopus (124) Google Scholar). Eicosanoids were then isolated by solid phase extraction (SPE) using Strata-X polymeric reversed phase columns. The columns were activated with consecutive washes of 3 ml of 100% methanol and 3 ml of water. The samples were loaded and washed with 3 ml of 10% methanol. Eicosanoids were then eluted with 1 ml of 100% methanol, dried under vacuum, and dissolved in 50 μl of buffer A consisting of water/acetonitrile/acetic acid (60/40/0.02, v/v/v). Samples were immediately analyzed using UPLC-MS/MS. A complete list of all PSTDs used for standard curves and deuterated standards and their assignments for normalization is provided in supplemental Table S1. To extract total eicosanoids, 50 μl of plasma, 500 μl of cell homogenates, or 50 μl of the PSTDs mix were spiked with ISTDs (in 100 μl of ethanol) and added to a mixture consisting of 100 μg of butylated hydroxytoluene (in 100 μl ethanol), 250 μl methanol, 50 μl KOH (4 M), and water to a final volume of 1 ml (Fig. 1). The mixture was kept for 30 min at 37°C to hydrolyze the esterified eicosanoids. Following hydrolysis, 3.5 ml of glycine-HCl buffer (0.1 mM, pH 4) were added. The free eicosanoids were then isolated by SPE and analyzed according to the protocol for the free eicosanoids, as described above. An unadulterated mix of pure PSTDs and ISTDs that was not subjected to hydrolysis conditions or SPE served as a control to estimate recoveries. Separation was performed on an Acquity UPLC system (Waters, Milford, MA), equipped with a RP C18 BEH shield column (2.1 × 100 nm; 1.7 μm; Waters). For the separation of eicosanoids, a binary buffer system was used consisting of buffer A (described above) and buffer B composed of acetonitrile/2-propanol (50/50, v/v). At a flow rate of 0.5 ml/min, buffer A was held at 100% for 1 min followed by a gradient over 3 min to 55% buffer B, then further increased over 1.5 min to 100% buffer B and kept at this level for 0.5 min. The starting conditions were reconstituted in 1 min. The column was kept at 40°C and the sample manager at 4°C. The samples (10 μl) were injected via partial loop injection using needle overfill mode. To minimize carryover, needle washes were carried out between samples. Data were collected on an AB/Sciex 6500 QTRAP hybrid triple quadrupole mass spectrometer (Sciex, Framingham, MA) using negative electrospray and scheduled multiple reaction monitoring (MRM) mode. The source settings were as follows: curtain gas (CUR = 20 psi), nebulizer gas (GS1 = 30 psi), turbo heater gas (GS2 = 20 psi), electrospray voltage (TEM = −4,500 V), source temperature (500°C), and collision gas (CAD = medium). Eicosanoids were quantified by the stable isotope dilution method. Briefly, identical amounts of ISTDs were added to each sample and to all the PSTDs. Nine point standard curves were generated for each of the 136 PSTDs, ranging from 0.03 ng to 10 ng. To calculate the amount of eicosanoids in a sample, ratios of peak areas between endogenous eicosanoids and matching deuterated internal eicosanoids were calculated. Ratios were converted to absolute amounts by linear regression analysis of standard curves. Currently, we quantify most eicosanoids at low femtomole levels. To determine recovery values of the PSTDs under alkaline hydrolysis conditions, MS peak areas were compared before and after the addition of base. All measurements were performed in triplicate or five replicate measurements and the data are reported as averaged values. The coefficient of variance (CV) determines the precision of this quantitation method. The PL measurements were carried out by LC-MS using a multiplex approach as previously described (30.Baker P.R. Armando A.M. Campbell J.L. Quehenberger O. Dennis E.A. Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.J. Lipid Res. 2014; 55: 2432-2442Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Briefly, 50 μl of human plasma were hydrolyzed as described above and extracted according to Bligh and Dyer (31.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). As a control, nonhydrolyzed plasma was included. The organic solvent was removed and the lipids were reconstituted in buffer A (isopropanol/hexane/water = 59/40/1, v/v/v, with 10 mM ammonium acetate) and analyzed on a Waters Acquity UPLC-Sciex 6500 QTrap mass spectrometer system. The lipids were separated on a silica column (2.1 × 150 mm; 3 μm; Phenomenex) using a binary solvent gradient from 100% A to 100% B (isopropanol/hexane/water = 50/40/10, v/v/v, with 10 mM ammonium acetate) over 16 min. PL molecular species (432 isobaric species in total) were identified by MS using precursor ion and neutral loss scans in positive and negative modes. For the PL class analysis, the sum total of individual molecular species within each class was used. Eicosanoids are important lipid metabolites that are involved in a number of physiological processes at the cellular level. As with fatty acids, they can exist either in the free form or esterified to complex lipids, such as PLs. The objective of the current study was to develop precise conditions that allow the quantitative measurement of both free and esterified oxylipins. To achieve this, we first examined the recovery of free eicosanoids during the prepurification step prior to LC-MS analysis using a defined set of quantitation standards consisting of 161 authentic metabolites that were mixed at precisely measured concentrations. Included in the standard cocktail were also 26 deuterated analogs that can be used to offset any potential losses during workup. The standard mix was then divided into two aliquots and analyzed by LC-MS with or without SPE prepurification. Figure 2 shows the recovery of a subset of eicosanoids that were selected based on their metabolic pathway. In order to assess the degree of any potential losses during sample preparation and SPE purification, we plotted the raw MS data after SPE without normalization as percent recovery compared with the raw MS data obtained with standards set that did not undergo SPE purification. As can be seen, the recovery of these lipid metabolites in their free nonesterified form and undergoing our standard purification procedure was largely quantitative, ranging between 90% and 100%, even without normalization to ISTDs (Fig. 2). Any potential losses were minimal and could easily be offset with the application of our routine normalization procedure using deuterated eicosanoid analogs as ISTDs. A complete list of the recoveries for all metabolites as well as ISTD assignments, pertinent technical information, and instrument settings are provided in the supplemental Table S1. A number of studies reported that a portion of eicosanoids are naturally esterified and can be contained in membrane lipids in the form of esters. To profile quantitatively all eicosanoids incorporated into the various lipid fractions using an approach that preserves the intact molecule represents an enormous technical challenge. An alternative approach is to release the eicosanoids first by hydrolysis and then measure the metabolites in their free form. Several laboratories have applied alkaline hydrolysis for this purpose; however, the hydrolysis conditions varied considerably as they were often optimized for the analysis of certain subclasses of eicosanoids, including isoprostanes, fatty acid alcohols, ketones, and epoxides (26.Morrow J.D. Awad J.A. Boss H.J. Blair I.A. Roberts L.J. Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids.Proc. Natl. Acad. Sci. USA. 1992; 89: 10721-10725Crossref PubMed Scopus (666) Google Scholar, 32.Mallat Z. Nakamura T. Ohan J. Leseche G. Tedgui A. Maclouf J. Murphy R.C. The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis.J. Clin. Invest. 1999; 103: 421-427Crossref PubMed Scopus (163) Google Scholar, 33.Newman J.W. Kaysen G.A. Hammock B.D. Shearer G.C. Proteinuria increases oxylipid concentrations in VLDL and HDL but not LDL particles in the rat.J. 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Brennecke S.P. Mountford L.A. Brunt J.D. Turnbull A.C. Development and validation of a radioimmunoassay for prostaglandin E2 metabolite levels in plasma.J. Clin. Endocrinol. Metab. 1983; 57: 101-106Crossref PubMed Scopus (30) Google Scholar, 38.Younger E.W. Szabo R.M. The stability of prostaglandin E1 in dilute physiological solutions at 37 degrees C.Prostaglandins. 1986; 31: 923-927Crossref PubMed Scopus (21) Google Scholar), it is important to balance hydrolysis efficiency and structural preservation of the analytes. To achieve this, we explored mild alkaline conditions for their efficacies to hydrolyze oxylipins esterified to complex lipids, including PLs. We established the optimal base concentration at 0.2 M KOH and tested the hydrolysis efficiency at this concentration on human plasma at various temperatures and incubation times. The majority of base-stable metabolites that are generated by enzymes, including the fatty acid epoxides, are contained in PLs (22.Spector A.A. Fang X. Snyder G.D. Weintraub N.L. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function.Prog. Lipid Res. 2004; 43: 55-90Crossref PubMed Scopus (489) Google Scholar). Thus, we focused on the PL fraction to measure hydrolysis efficiency. The mass chromatograms for several PL classes taken before and after base hydrolysis indicated that the mildest condition, 0.2 N KOH at 37°C for 30 min, was sufficient to hydrolyze >95% of the plasma PLs (Fig. 3). There was some remaining sphingomyelin, which contains N-linked fatty acids that are more resistant to hydrolysis, even at 60°C. No lysoPLs were detectable post hydrolysis, which indicates that the PLs were not converted to the lyso moieties and the hydrolysis step effectively released all sn1 and sn2 fatty acids (Fig. 3B). We also subjected some selected oxylipin standards to the same conditions and observed that at 37°C and 30 min, the base-induced destruction of these metabolites was least, as exemplified by the recovery of intact 7-hydroxy-docosahexaenoic acid (Fig. 4). Like the nondeuterated metabolites, the degradation of the deuterated ISTDs increased similarly with increasing temperature and time. As a result, the sensitivity and precision of the analysis decreases proportionally. Our data show that the hydrolysis condition of 0.2 N KOH at 37°C for 30 min provides the optimal balance between hydrolysis efficiency and structural preservation of the analytes. Deviating from these conditions augments metabolite degradation and low abundance metabolites may fall below the lower limit of detection. Furthermore, reproducibility decreases with increasing degradation and, consequently, the precision of the analysis deteriorates.Fig. 4Recovery of 7-hydroxy-docosahexaenoic acid (7-HDoHE) subjected to alkaline conditions. The 7-HDoHE is shown as a representative example to test the stability of related hydroxylated PUFAs. Three conditions were tested: A, no saponification; B, 37°C for 30 min; C, 37°C for 60 min; and D, 60°C for 30 min. The mass spectral response for 7-HDoHE is shown in panel A. The average relative intensity of triplicate measurements is shown in panel B. The untreated control was set at 100%. As can be seen, there was a time- and temperature
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