Quantitative determination of esterified eicosanoids and related oxygenated metabolites after base hydrolysis

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 liquid chromatography 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.


INTRODUCTION
Eicosanoids and related metabolites, sometimes referred to as oxylipins, are a group of structurally diverse metabolites that derive from the oxidation of polyunsaturated acids (PUFAs) including arachidonic acid (AA), linoleic acid, alpha and gamma linolenic acid, dihomo gamma linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. They are locally acting bioactive signaling lipids that regulate a diverse set of homeostatic and inflammatory processes (1,2). 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).
The biosynthesis of eicosanoids and oxylipins involves the action of multiple enzymes organized into a complex and intertwined lipid-anabolic network (4). 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 A 2 upon physiological stimuli (5). The hydrolyzed PUFAs are then processed by three enzyme systems: cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 enzymes (CYP450). 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)(7)(8)(9)(10).
Additionally, non-enzymatic processes can produce oxidized PUFA metabolites via free radical reactions giving rise to isoprostanes and other oxidized fatty acids (11).
Eicosanoids are either secreted and signal through G-protein-coupled receptors in an autocrine or paracrine fashion or act intracellularly via various peroxisome proliferator-activating receptors (4,12,13).
For optimal biological activity, these mediators need to be present in their free, non-esterified form.
However, a number of studies reported that a portion of eicosanoids are naturally esterified and can also be contained in cell membrane lipids, including PLs, in the form of esters (14)(15)(16). The role of esterified 4 eicosanoids is not clear but they may be signaling molecules in their own right or serve as a cellular reservoir for the rapid release upon cell stimulation (17,18).
Two potential mechanisms for the formation of eicosanoids-containing PLs have been proposed, i) direct oxidation of PUFAs on the intact PLs, and ii) re-acylation of preformed free oxylipins into lysoPLs.
Cyclooxygenases require free fatty acid as substrate and show little activity toward PUFAs in intact PLs (19). A number of subsequent studies support the concept that prostaglandins are first formed enzymatically and then incorporated into PLs by the sequential actions of long-chain acyl-CoA synthases and lysophospholipid acyltransferases (20,21). Additionally, preformed fatty acid epoxides, including the regioisomers of epoxyeicosatrienoic acid (EET), are effectively incorporated primarily into the phospholipid fraction of cellular lipids, presumably via CoA-dependent mechanisms (22).
The final products derived from this direct PL oxygenation pathway include esterified prostaglandins (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).
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 non-esterified oxylipins that when used together is able to discriminate nonalcoholic steatohepatitis from nonalcoholic fatty liver with a high degree of certainty (27). 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). 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 5 experimental approach as we measured the free oxylipins present in plasma, not those appearing after alkaline hydrolysis (27). 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). 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.

Reagents
All solvents are 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, Michigan) 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).

Free Eicosanoids
For the extraction of free eicosanoids, 50 l of plasma, or 500 ul of cell homogenates or the PSTDs collection consisting of 136 individual standards were spiked with 100 ul of the ISTDs mix (1 ng of each of 26 deuterated standard in ethanol) and diluted with Dulbecco's PBS to give a 10% total ethanol concentration (29). 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.

7
Eicosanoids were then eluted with 1 ml of 100% methanol, dried under vacuum, and dissolved in 50 l 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, deuterated standards and their assignments for normalization is provided in the Supplemental Table 1.

Total Eicosanoids
To extract total eicosanoids, 50 ul of plasma, or 500 ul of cell homogenates, or 50 ul of the PSTDs mix were spiked with ISTDs (in 100 ul ethanol) and added to a mixture consisting of 100 g butylated hydroxytoluene (in 100 ul ethanol), 250 ul methanol, 50 ul KOH (4M) and water to a final volume of 1 ml ( Figure 1). The mixture was kept for 30 minutes at 37°C to hydrolyze the esterified eicosanoids.
Following hydrolysis, 3.5 ml of glycine-HCl buffer (0.1mM, pH=4) was added. The free eicosanoids were then isolated by SPE and analyzed according to the protocol for the free eicosanoid, as described above. An unadulterated mix of pure primary standards and internal standards that was not subjected to hydrolysis conditions or solid phase extraction served as a control to estimate recoveries.

Chromatographic Separation
Separation was performed on an Acquity ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA), equipped with a RP C18 BEH shield column (2.1 x 100 nm; 1.7 m; 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 re-constituted 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.

Quantitation
Eicosanoids were quantified by the stable isotope dilution method. Briefly, identical amounts of ISTDs were added to each sample and to all the primary standards. 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.

Phospholipid Measurements
The phospholipid measurements were carried out by LC-MS using a multiplex approach as previously described (30). Briefly, 50 ul of human plasma were hydrolyzed as described above and extracted according to Bligh and Dyer (31). As a control, non-hydrolyzed 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 10mM ammonium acetate) and analyzed on a Waters Acquity UPLC-Sciex 6500 QTrap mass spectrometer system. The lipids were separated on a silica column (2.1mm x 150mm; 3m; Phenomenex) using a binary solvent gradient from 100% A to 100% B (isopropanol/hexane/water=50/40/10, v/v/v, with

Quantitation of eicosanoid related oxylipins in their free, non-esterified form
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 phospholipids.
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 2 aliquots and analyzed by LC-MS with or without SPE pre-purification. 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, non-esterified form and undergoing our standard purification procedure was largely quantitative, ranging between 90-100%, even without normalization to internal standards ( Figure 2). Any potential losses were minimal and could easily be offset with the application of our routine normalization procedure using deuterated eicosanoid analogs as internal standards. A complete list of the recoveries for all metabolites as well as internal standard assignments, pertinent technical information and instrument settings are provided in the Supplemental Table S1.

Optimization of hydrolysis conditions to preserve oxylipin structure
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 11 the analysis of certain sub-classes of eicosanoids including isoprostanes, fatty acid alcohols, ketones and epoxides (26,(32)(33)(34)(35). Considering that many oxylipins are unstable under extreme alkaline or acidic conditions (36)(37)(38), 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 phospholipids. 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 PL (22). Thus, we focused on the PL fraction to measure hydrolysis efficiency. The mass chromatograms for several phospholipid classes taken before and after base hydrolysis indicated that the mildest condition, 0.2 N KOH, 37C, 30 min was sufficient to hydrolyze >95% of the plasma phospholipids ( Figure 3). There was some remaining sphingomyelin, which contains N-linked fatty acids that are more resistant to hydrolysis, even at 60C. No lyso-PLs were detectable post hydrolysis, which indicates the PLs were not converted to the lyso moieties and the hydrolysis step effectively released all sn1 and sn2 fatty acids ( Figure 3B). We also subjected some selected oxylipin standards to the same conditions and observed that at 37C and 30 min, the baseinduced destruction of these metabolites was least, as exemplified by the recovery of intact 7-hydroxydocosahexaenoic acid (Figure 4). Like the non-deuterated 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, 37C, 30 min provide 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.

Analysis of eicosanoids after alkaline treatment
Next, we expanded our stability tests and examined our entire library of primary standards for their resistance to base-induced degradation ( Figure 5). As shown in Figure 6 and Supplemental Table S2, 12 many of the eicosanoids are susceptible to base induced degradation, as indicated by the changes in their mass spectral intensities. The data shown represent the average of five replicate measurements performed on a single day. In particular, prostaglandins and leukotrienes were virtually undetectable. The exceptions were PGF 2a , and LTB 4 , which were resistant to degradation (Supplemental Table S2). Additionally, fatty acids containing hydroxy, di-hydroxy or epoxy groups showed resistance to degradation to various degrees. Even though some degradation may have occurred during the saponification step, when we normalized the spectral data of the non-deuterated eicosanoids to their deuterated analogues, we were able to neutralize the loss for many of the eicosanoids. For example, the non-normalized 'raw' recovery of TXB 2 was about 78%. However, when we normalized the value to its deuterated analogue measured under identical conditions, the recovery was calculated to be 100%. Similarly, the normalized recoveries were quantitative for 5-HETE, 13-HODE and 9,10 EpOME, compared with the non-normalized recoveries of about 70-80% based on raw mass spectral peak areas ( Figure 6). As a class, the epoxides and diols are quite stable under basic conditions (33), and many of them were recovered quantitatively from the standard mix under alkaline hydrolysis conditions (Table 1). For some eicosanoids normalization to internal standards did not improve the overall recovery. This applies specifically to the ones with more complex structural elements including prostaglandins, and leukotrienes as wells as ketones (Supplemental Table S2).
Most naturally occurring PGs have a considerable potential for hydrolysis, dehydration, or isomerization, depending on their immediate environment (39). PGs contain multiple hydroxyl groups, keto groups and a rigid 5-member prostane ring. The resulting -hydroxy ketone system is unstable and readily undergoes dehydration under acidic or basic conditions to A-or B-type PGs (36,40). Alternatively, PGD and PGE can oxidize to the 9,11-diketones PGK1 and PGK2, which were generated and increased about 3 fold during alkaline hydrolysis. Furthermore, bicyclo PGE 2 , a base-catalyzed breakdown product of PGE 2 and 13,14-dihydro-15-keto PGE 2 (dhk PGE 2 ) was found to be substantially increased after alkaline hydrolysis (Supplemental Table 2). These breakdown or conversion products cannot be reliably measured after alkaline hydrolysis and should not be included in quantitative analyses of esterified eicosanoids.

Precision of the method
For the method to be applicable to biological samples, it has to be accurate and reliable. For this purpose, we compiled a panel of eicosanoids that a) were either resistant to base-induced degradation or suffered only minor destruction and b) were reproducibly recovered. Each metabolite in our library of standards was measured in five replicate measurements on three consecutive days with and without saponification, normalized to internal standards and averaged. The recovery after alkaline hydrolysis was calculated and the precision was expressed as the coefficient of variation.
For practical purpose, we used a cut-off point of 80-120% normalized recovery and a precision (CV) of 15% or less to assemble a list of eicosanoids that can be reproducibly measured (Table 1). Of the 136 eicosanoids in the standard mix we used for this purpose (Supplemental Table 2), 56 metabolites satisfied these criteria and were included in the list. The three-day average was close to 100% recovery for most of these metabolites. These data indicated that the assay is reproducible and with careful selection of internal standards for normalization and neutralization of potential losses, the method is useful for the analysis of selected esterified eicosanoids.

Application of the method to measure esterified eicosanoids in biological samples
To establish usefulness, we applied this protocol to the analysis of esterified eicosanoids in biological samples including RAW cells, a cell model of mouse macrophages (41,42), and human plasma (43). For the cell model, mouse RAW macrophages were activated with the Toll-like receptor agonist Kdo2-lipid A, and ATP, and eicosanoids were analyzed in their free and esterified form in both stimulated and unstimulated control cells. Considering the limitations outlined in Table 1 and using the algorithm for the identification of stable metabolites, we identified and quantified a number of eicosanoids that were present in their free and esterified form ( Table 2). In total, we detected 34 metabolites that met the criteria for inclusion. In the unstimulated control cells, the COX-derived metabolites 11-HETE and PGF 2a were present only in their free form. In contrast, metabolites formed by CYP450 and non-enzymatic pathways were mainly found in their esterified form. As can be seen, most eicosanoids increased during stimulation.
The eicosanoid ratio esterified vs free in the stimulated cells is a reflection of de-novo synthesis, 14 hydrolysis by phospholipase A 2 and release into the extracellular medium. Of course it is possible that some eicosanoids are tightly bound to receptors or carrier proteins even during SPE purification, but are released upon base hydrolysis and would therefore be included in the "esterified" measure. Additionally, it is well established that prostaglandins, including PGF 2a are formed and rapidly secreted during stimulation with Toll-like receptor agonists and ATP (41). Consequently, there may be a relative increase in the esterified portion due to secretion of metabolites in their free form. Of note, the data contain the cell associated free eicosanoids and do not include the secreted fraction.
We next examined esterified eicosanoids in human plasma. In all, we recovered 38 eicosanoids after base hydrolysis (Table 3). For comparison, we also measured these metabolites in their free form as previously reported (3,43). The dynamic range for the total eicosanoids span several orders of magnitude from about 5 to 44,500 pmol/ml and on average, about 70% of all eicosanoids were esterified. A potential matrix effect adds to the complexity of the measurement. The matrix effect was determined previously by spiking the internal standard mixture into a plasma sample (29). The results showed that the recovery of internal standards that are resistant to alkaline-induced degradation is greater than 80%, indicating that matrix effects and ion suppression are minimal. The plasma measurements were carried out on 50 ul of plasma of which the equivalent of 10 ul was injected into the UPLC/MS for an analysis. Thus, the lower limit of quantification for eicosanoid measurements in biological material are in the low femtomole range.

CONCLUSION
In this study, we assessed the suitability of base hydrolysis to quantify esterified eicosanoids in biological material. We showed that about 75% of all cell associated eicosanoids were esterified. Globally this percentage remained largely unchanged between control and Kdo2-lipid A/ATP-stimulated cells even though some selected metabolites increased either in their free or esterified form. In a previous report, we demonstrated the presence of free eicosanoids in human plasma (43) and found that the levels are correlated with various inflammatory and metabolic diseases including non-alcoholic fatty liver disease (27). In the current study, we expanded our method to include esterified eicosanoids in the analysis. Overall, we fine-tuned the alkaline hydrolysis conditions to minimize metabolite degradation, adjusted the pre-purification steps to enhance metabolite recovery, optimized the assignment of internal standards to compensate for potential losses and established an algorithm based on recovery and reproducibility to compile a table of metabolites that can be accurately measured. When taking these limitations into consideration, this method can now be successfully applied to accurately measure the sum total, i.e., free and esterified oxylipins in human plasma and a variety of biological samples.

Acknowledgments
This work was supported by National Institutes of Health Grants R01 GM20501, R01 DK105961, P30 DK063491 and NIGMS LIPID MAPS "Glue Grant" U54 GM069338. EAD is a co-founder, director, and consultant to LipoNexus, Inc., which has licensed IP from UCSD. The set of primary standards was supplemented with internal standards, subjected to alkaline hydrolysis conditions and analyzed by LC-MS. An identical set of untreated standards was analyzed in parallel and served as control. The analyses were performed in five replicates on each of three consecutive days.
Recoveries after alkaline treatment were calculated by comparing the mass spectral intensities with those of untreated standards and after normalization to internal standards. Shown is the list of metabolites that were recovered at 80-120% and with a CV of 15%.     A collection of primary oxylipin standards was analyzed with and without SPE purification. Recoveries were determined by comparing MS intensities obtained with standards after undergoing SPE purification with MS intensities of standards without SPE purification that were analyzed in parallel and served as controls. The open bars show non-normalized recoveries (Non-Norm), which were calculated from the raw MS peak areas without normalization to internal standards; the closed bars show the same data set but normalized to internal standard (Norm). All data are expressed as percent of untreated controls. The mean and SD of triplicate measurements are displayed. Shown is a representative subset of standards (See Supplemental Table S1 for the complete data set).     A set of pure oxylipin standards (136 metabolites) were subjected to alkaline hydrolysis conditions, purified by SPE and analyzed by LC-MS. Recoveries were determined by comparing MS intensities of the standards after alkaline treatment with MS intensities of the untreated standards that were analyzed in parallel and served as controls. The open bars show non-normalized recoveries (Non-Norm), which were calculated from the raw MS peak areas without normalization to internal standards; the closed bars show the same data set but normalized to internal standard (Norm). All data are expressed as percent of untreated controls. The mean and SD of five replicate measurements are displayed. Shown is a representative subset of standards (See Supplemental Table S2 for the complete data set).