biomedicines Article HPLC-MS/MS Oxylipin Analysis of Plasma from Amyotrophic Lateral Sclerosis Patients Mauricio Mastrogiovanni 1 , Andrés Trostchansky 1 , Hugo Naya 2,3 , Raúl Dominguez 4 , Carla Marco 4 , Mònica Povedano 4 , Rubèn López-Vales 5,6,7, * and Homero Rubbo 1, * 1 Departamento de Bioquímica, Facultad de Medicina and Centro de Investigaciones Biomédicas, Universidad de la República, Montevideo 11800, Uruguay; maurimastro@fmed.edu.uy (M.M.); trocha@fmed.edu.uy (A.T.) 2 Unidad de Bioinformática, Institut Pasteur-Montevideo, Montevideo 11400, Uruguay; naya@pasteur.edu.uy 3 Departamento de Producción Animal y Pasturas, Facultad de Agronomía, Universidad de la República, Montevideo 12900, Uruguay 4 Instituto de Investigación Biomédica de Bellvitge, 08907 Hospitalet del Llobregat, Spain; rauldominguez86@hotmail.com (R.D.); carlamarcocazcarra@gmail.com (C.M.); 30058mpp@gmail.com (M.P.) 5 Institute of Neurosciences, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 6 Department Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 7 Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, 28031 Madrid, Spain * Correspondence: ruben.lopez@uab.cat (R.L.-V.); hrubbo@fmed.edu.uy (H.R.); Tel.: +34-935811348 (R.L.-V.); +598-91238702 (H.R.) Abstract: Oxylipins play a critical role in regulating the onset and resolution phase of inflammation. Despite inflammation is a pathological hallmark in amyotrophic lateral sclerosis (ALS), the plasma  oxylipin profile of ALS patients has not been assessed yet. Herein, we develop an oxylipin profile-  targeted analysis of plasma from 74 ALS patients and controls. We found a significant decrease in Citation: Mastrogiovanni, M.; linoleic acid-derived oxylipins in ALS patients, including 9-hydroxy-octadecadienoic acid (9-HODE) Trostchansky, A.; Naya, H.; and 13-HODE. These derivatives have been reported as important regulators of inflammation on dif- Dominguez, R.; Marco, C.; Povedano, M.; López-Vales, R.; Rubbo, H. ferent cell systems. In addition, some 5-lipoxygenase metabolites, such as 5-hydroxy- eicosatetraenoic HPLC-MS/MS Oxylipin Analysis of acid also showed a significant decrease in ALS plasma samples. Isoprostanes of the F2α family were Plasma from Amyotrophic Lateral detected only in ALS patients but not in control samples, while the hydroxylated metabolite 11-HETE Sclerosis Patients. Biomedicines 2022, significantly decreased. Despite our effort to analyze specialized pro-resolving mediators, they were 10, 674. https://doi.org/10.3390/ not detected in plasma samples. However, we found the levels of 14-hydroxy-docosahexaenoic acid, biomedicines10030674 a marker pathway of the Maresin biosynthesis, were also reduced in ALS patients, suggesting a Academic Editor: Jun Lu defective activation in the resolution programs of inflammation in ALS. We further analyze oxylipin concentration levels in plasma from ALS patients to detect correlations between these metabolites Received: 27 January 2022 and some clinical parameters. Interestingly, we found that plasmatic levels of 13-HODE and 9-HODE Accepted: 16 February 2022 positively correlate with disease duration, expressed as days since onset. In summary, we developed Published: 15 March 2022 a method to analyze “(oxy)lipidomics” in ALS human plasma and found new profiles of metabolites Publisher’s Note: MDPI stays neutral and novel lipid derivatives with unknown biological activities as potential footprints of disease onset. with regard to jurisdictional claims in published maps and institutional affil- Keywords: amyotrophic lateral sclerosis; lipidomics; oxylipin; specialized pro-resolving mediators; iations. mass spectrometry Copyright: © 2022 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Amyotrophic lateral sclerosis (ALS) is a heterogeneous neurodegenerative disease distributed under the terms and characterized by the degeneration of both upper and lower motor neurons, leading to motor conditions of the Creative Commons and extra-motor symptoms [1]. In most patients, the cause of ALS is unknown, although Attribution (CC BY) license (https:// some individuals have a family antecedent, which is associated with mutations in genes creativecommons.org/licenses/by/ that have a wide range of roles. Some of the implicated genes are incompletely penetrant in 4.0/). familial ALS, and with rare exceptions, genotype does not necessarily predict phenotype [1]. Biomedicines 2022, 10, 674. https://doi.org/10.3390/biomedicines10030674 https://www.mdpi.com/journal/biomedicines Biomedicines 2022, 10, 674 2 of 13 As is the case with other neurodegenerative diseases, it is now recognized that ALS is a heterogeneous condition associated with more than one pathogenic mechanism and with different clinical characteristics [2]. Degeneration of motor neurons is accompanied by neuroinflammatory processes, with the proliferation of astroglia, microglia, and oligoden- droglia [3]. Neuroinflammation has been observed by imaging studies in patients with ALS, human post-mortem samples, and rodent models of ALS [4]. Moreover, biomarkers of neuroinflammation such as MCP-1, CHIT1, and YKL-40, are elevated in patients with ALS and have been shown to correlate with disease severity and predict disease progres- sion [5,6]. Growing evidence suggests that, in addition to microglia, several other innate immune cell subsets, including macrophages, monocytes, dendritic and natural killer cells, mastocytes, and neutrophils are implicated in the ALS pathogenesis [7]. Indeed, the rate of disease progression in ALS has been suggested to be related to the degree of systemic monocyte/macrophage activation [8]. A large body of evidence shows that systemic inflammatory response can alter central inflammation in degenerative processes. Pro-inflammatory responses are present in the periphery in addition to neuroinflammatory disease within the central nervous system (CNS) [9]. Levels of circulating cytokines are abnormal in ALS patients, with higher levels of TNF-α, IL-1 β, IL-2, IL-8, IL-12p70, IL-4, IL-5, IL-10, and IL-13, and lower levels of INF-γ when compared to healthy controls [10]. IL-6 was also strongly associated with C-reactive protein levels and was found to increase the expression toward end-stage disease in a longitudinal analysis [10]. As ALS is a clinical disease with a heterogeneous phenotypic manifestation and clinical course, diagnostic and prognostic biomarkers are needed for stratification [1]. Neurofilament light polypeptide and phosphorylated neurofilament heavy polypeptide in cerebrospinal fluid (CSF) or blood is probably the most promising biofluid marker of ALS. Its levels correlate with the rate of disease progression and are most strongly associated with the involvement of the upper motor neuron system [1,11]. However, integration into standard clinical practice represents major difficulties [1]. Several reports investigated other potential biomarkers. A large primary care cohort study identified novel metabolic markers including alterations in carbohydrate, lipid, and apolipoprotein profiles, that were associated with an increased risk of ALS in later life [11]. Regarding inflammation, a recent meta-analysis involving 25 studies on peripheral cytokines levels in ALS confirmed that TNF-α, IL-1 β, IL-6, IL-8, and VEGF measured in blood were significantly elevated in ALS cases compared with controls [12]. Although inflammation is not ALS-specific, exploration of inflammation biomarkers may improve the knowledge of ALS pathogenesis. Regarding lipid mediators of inflam- mation, some evidence on alterations of PGE2 levels in ALS patients has been previously reported [13]. Eicosanoid analysis of ALS patient samples showed a significant increase in PGE2 levels in cerebrospinal fluid and also in postmortem spinal cord and cortex sam- ples [14,15]. Recent reports in which the omics approaches have been applied found alterations in ALS patients’ lipid metabolism [16–19]. Henriques et al. [16] analyzed the HPLC profiles of fatty acids derived from total lipids in serum and clotted blood cells from ALS patients and controls and reported as a major conclusion that blood cell 16:1/16:0 ratio and palmitoleate levels correlate with disease progression. Blasco et al. [17] performed a lipidomics analysis of CSF from ALS patients searching for diagnostic and predictive footprints. They found predictive models for the variation of ALSFRS-r score from the lipidome baseline with 71% accuracy. Besides, significant prediction of clinical evolution correlated with sphingomyelins and triglycerides with long-chain fatty acids. A recent arti- cle by Area-Gomez et al. [18] reports a lipidomic analysis (complex lipids) of plasma from patients with ALS and primary lateral sclerosis (PLS, a more benign form of motor neuron disorders that only affects upper motor neurons). They found that common aspects of these pathologies suggest that PLS and ALS behave as a part of a continuum of motor neuron diseases from the lipidomics point of view. Another study from O’Reilly et al. [19] evalu- ated the association between pre-diagnostic plasma polyunsaturated fatty acids (PUFA) Biomedicines 2022, 10, 674 3 of 13 levels and ALS. They found that the majority of individual PUFAs were not associated with ALS, although in men linoleic acid (LA) was inversely correlated with the risk of ALS, and docosahexaenoic acid (DHA) was positively correlated to the risk of ALS. In women, they found that arachidonic acid (AA) was positively related to the risk. However, these findings warrant confirmation in further studies. Recently Fernandez-Eulate et al. [20] have reported a lipidomic analysis in sera from ALS patients and healthy volunteers, in which hydroxyl- derivatives from LA were measured. They claim one of the most comprehensive lipid profiling of ALS, which did not reveal any serum lipid signature discriminating patients with ALS from controls. To our knowledge, there is no lipidomic analysis containing a broad spectrum of oxylipins, applied to human samples from ALS patients. Thus, it is currently unknown whether the levels of oxylipins in the circulating plasma could reflect the ALS inflammatory component of the disease. In the present study, we performed a targeted liquid chromatography-mass spectrometry (HPLC-MS/MS) analysis to detect and quantify a full panel of oxylipins in plasma samples from 78 ALS and age-matched control healthy individuals. 2. Materials and Methods 2.1. Materials All lipids were purchased from Cayman Chemicals, and specific working mixes were prepared by dilution with HPLC grade methanol. Internal standard mix (IS mix) contains 500 pg/µL of each of the following deuterated standards: (d4) 6k PGF1α, (d4) TxB2, (d4) PGF2α, (d4) PGE2, (d4) PGD2, (d8) 15-HETE, (d8) 12-HETE, (d8) 5-HETE, (d4) 8-isoPGF2α, (d11) 5-isoPGF2α, (d5) RvD1, (d5) MaR1, (d4) RvE1, (d4) 13-HODE and (d4) 9-HODE. 2.2. Sample Preparation Blood samples were collected in Barcelona according to The Research Protocol for Biosamples approved by the Ethics Committee of the Bellvitge University Hospital. In- formed consent was obtained from all participants. Plasma samples were obtained between 3 and 66 months after the beginning of symptoms, and ALS was diagnosed according to the El Escorial criteria for ALS. The ALS-functional rate was calculated and validated by two independent neurologists. The progression rate was calculated (at baseline or last visit) as 48 minus the ALS Functional Rating Scale-Revised score, divided by the disease duration from onset of symptoms. Plasma was separated by centrifugation, immediately frozen and kept at −80 ◦ C. Oxylipin analysis was performed by the MS platform of CEINBIO in Montevideo, Uruguay. Therefore, plasma samples were shipped in dry ice to CEINBIO and stored at −80 ◦ C until analysis. Sample processing was performed as described previously [21]. Briefly, plasma sam- ples are allowed to thaw on ice, and 500 µL aliquots are spiked with 2.5 µL of IS mix and diluted in an equal volume of 20% MeOH prepared in acidified water (HCl, pH 3). Tubes are gently vortexed and centrifuged for 30 min at 14,000× g, 4 ◦ C. Clear supernatant is directly transferred to preconditioned SPE cartridges (Strata™-X, Polymeric sorbent 33 µ, 60 mg, 3 mL; Phenomenex). After the vacuum-assisted flow of the samples is achieved, cartridges are washed with 10% MeOH solution in acidified water (HCl, pH 3) and submit- ted to high vacuum (~11 In Hg) for no more than 5 min. Finally, elution of the oxylipins is performed with 1 mL MeOH, and tubes are transferred to SpeedVac to allow evaporation to dryness. Before HPLC-MS/MS analysis, samples are reconstituted in 50 µL of 50% MeOH in ultrapure water. 2.3. Oxylipin Analysis Analysis and quantitation of oxylipins in plasma were performed by HPLC-MS/MS employing an Infinity 1260 HPLC system (Agilent), coupled to a hybrid triple quadrupole/ linear ion trap mass spectrometer QTRAP4500 (ABSciex, Framingham, MA, USA). A complete list of analyzed oxylipins is shown in Table S1. Biomedicines 2022, 10, 674 4 of 13 Chromatographic separation was performed on a Luna 5µm C18(2) reversed-phase col- umn (100 × 2.0 mm, 100 A, Phenomenex) using a security guard cartridge (C18, 4 × 3.0 mm, Phenomenex) at 30 ◦ C. Solvent A consisted of 0.1% formic acid in ultrapure water and solvent B was 0.1% formic acid in acetonitrile. The flow rate was set to 300 µL/min and the gradient was as follows: 30% B from 0 to 0.1 min, linear increase to 95% B at 11 min, maintained at 95% B until 15 min, and back to 30% B in 0.1 min and maintained for 5 min. For MS detection, the QTRAP4500 was operated in the negative ion mode and set in multiple periods of MRM experiments. All standards were first diluted in methanol for parameter optimization and directly infused into the mass spectrometer. Three main experiments were performed for each compound; first, a mass spectra employing the quadrupole 1 (Q1 experiment) and an enhanced resolution spectra using the ion trap (ER experiment), for m/z definition and then a fragmentation experiment (Product Ion experiment) to determine the fragmentation pattern and define possible MRM transitions. In all cases parameters were ramped to define the values that allow the best signal, finding declustering potential (DP) and collision energy (CE), which are the parameters with more influence on the signal quality (see all fragmentation spectra in Figure S1). Finally, flow injection analysis was performed using a mixture of ultrapure water and methanol with 0.1% formic acid as mobile phase, and parameters were adjusted to define the final HPLC-MS/MS method. General parameters were set as follows: curtain gas (CUR): 50; collision gas (CAD): medium; ion spray voltage (IS): 4500 V; ion source temperature (TEM): 400 ◦ C; nebulizer gas (GS1): 40; drying gas (GS2): 30; entrance potential (EP): −10; and collision exit potential (CXP): −13. Probe position (TIS) was set at 0.300 cm on the vertical axis and at 0.500 cm on the horizontal axis, electrode protrusion was 1–1.5 mm. Each oxylipin was monitored with at least three MRM transitions (Table S2). Detection was carried out in four periods of MRM experiments: Period 1 from 0 to 8 min, Period 2 from 8 to 10.5 min, Period 3 from 10.5 to 13 min, and Period 4 from 13 min to end. This strategy allows increasing the time averaging for each data point (dwell time), thus decreasing the noise levels and increasing the signal-to-noise ratio. A full list of analytes contained in each period is shown in Table S2 along with the specific compound parameters. As a reference, Period 1 covers mostly triol fatty acids and COX-derived prostaglandins, Period 2 covers mostly diol fatty acids, Period 3 covers mostly monohydroxylated fatty acids, and Period 4 covers unmodified fatty acids. An exemplary chromatogram for all oxylipins is shown in Figure S2. Instrument control and data acquisition were performed with Analyst 1.6.2 (AB- Sciex, Framingham, MA, USA) software. PeakView 2.2 and MultiQuant 2.1 (ABSciex, Framingham, MA, USA) software were employed for data visualization and quantitation purposes, respectively. 2.4. Quantitation Quantitation was based on external calibration utilizing 15 deuterated internal stan- dards covering all the chromatographic regions. Both natural and isotope-labeled stan- dards for most oxylipins were assessed to construct calibration curves ranging from LOD to 500–2500 pg, plotting the peak area ratio (analyte/IS) against the mass ratio (pg, analyte/IS) (see Table S3 for calibration curves details). Those oxylipins for which standards were not available were quantified employing calibration curves of the most similar molecule as calibrant (Table S4). 2.5. General LOD and LOQ Determination The limit of detection (LOD) and lower limit of quantification (LOQ) for all ana- lyzed metabolites are shown in Table S5. Along with calibration curves, blank samples (50–70 samples) containing IS mix were submitted to HPLC-MS/MS analysis, and standard deviation of area ratio was obtained for each analyte. LOD and LOQ values were calculated as the concentration in which signal corresponds to the blank plus 3 or 5 times the standard deviation of the blank, respectively. To translate into plasmatic LOD, dilution factor and % Biomedicines 2022, 10, 674 5 of 13 recovery were used. The dilution factor is 10, as oxylipins derived from 500 µL of plasma are eventually dried and resuspended in 50 µL prior analysis. Percentage recovery for each analyte was assumed as the %recovery of the internal standard employed as reference. 2.6. Specilized Pro-Resolving Lipid Mediators LOD and LOQ To determine the limit of detection (LOD) and lower limit of quantification (LOQ), a standard mix containing all specialized pro-resolving mediators (SPM) standards available in our lab was prepared at a low concentration (lower values of calibration curves) and then diluted in half with methanol several times. Internal standard mix was added and then HPLC-MS/MS analysis was carried out. The Signal Finder algorithm (Multiquant 2.1, ABSciex) was employed for peak area integration of the most intense transition for each SPM. Then, LOD and LOQ were defined as the mass (pg) at which the signal-to-ratio (S/N) was equal or greater than 3 or 5, respectively (see Figure S3 and Table S6 for complete data). 2.7. Statistical Analysis For individual oxylipin analysis, a non-parametric Mann Whitney´s test was used for single comparisons between Control and ALS groups, performed in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Tukey plot of plasmatic concentration of selected oxylipins and correlation plots were performed with the same software. Other statistical analyses were performed in the statistical package R [22]. In an attempt to identify combinations of oxylipins concentrations that could discriminate be- tween groups of patients we performed several oxylipins multivariate exploratory analyses, including hierarchical and non-hierarchical (k-means) clustering, metric and non-metric multidimensional scaling, discriminant analysis, and orthogonal partial least squares dis- criminant analysis (OPLS-DA). Non-parametric analysis of variance (Kruskal–Wallis test) was used to detect sig- nificant differences in continuous variables according to patient groups. Correlations between continuous variables were assessed using the Pearson correlation test after loga- rithmic scaling (in particular for oxylipins) with posterior Benjamini-Hochberg correction for multiple testing. 3. Results 3.1. Oxylipin Analysis Plasma samples of ALS patients, as well as healthy volunteers, were obtained from the Biobank HUB-ICO-IDIBELL (PT17/0015/0024) and the Motor Neuron Unit of Bellvitge University Hospital. Control samples were obtained from patients without any type of neurological disorder. The demographic and clinical characteristics of donors are shown in Table 1. Frozen plasma samples were analyzed by HPLC-MS/MS to detect and quantify free oxylipins. Analyzed oxylipins include LA, AA, eicosapentaenoic acid (EPA), and DHA metabolites, derived from enzymatic and non-enzymatic pathways. A full list of analyzed metabolites is shown in Table S1. Table 1. Demographic and clinical characteristics of ALS patients and control volunteers. ALS (n = 78) Control (n = 9) Gender Female 31 7 Male 47 2 BMI a 25.2 (+/− 3.8) 23.2 (+/− 1.8) b Site at onset Spinal 42 - Bulbar 28 - Other 8 - Biomedicines 2022, 10, 674 6 of 13 Table 1. Cont. ALS (n = 78) Control (n = 9) Age at onset (years) a 62.2 (+/− 11.8) - sample collection (years) a 63.8 (+/− 11.1) 47.3 (+/− 15.8) Time since onset (months) a 23.1 (+/− 30.9) - ALSFRS-r slope at last visit 1.35 (+/− 1.61) - (n = 65) SP 28 - NP 17 - FP 24 - a Values are given as mean ± standard deviation. b Data obtained for n = 5 control volunteers. 3.2. Individual Oxylipin Analysis Plasmatic concentration levels for all detected oxylipins are shown in Table 2 and a comparison analysis between the Control group and ALS patients was performed (Mann– Whitney test). As shown in Table 2 and Figure 1A,B, LA-derived metabolites show a significant decrease in ALS patients compared to control samples. Particularly, lipoxyge- nase derivatives show a decrease by 50%, while CYP450 derivatives show an even more considerable reduction (~3–9 fold). Regarding the AA pathway, it should first be noted that there is no significant difference in the plasmatic concentration of the precursor (Figure 1B). However, some 5-lipoxygenase metabolites (Figure 1A,B) such as 5-HETE and 5-oxoETE show a significant decrease in ALS patients, while for others such as LTB4 the reduction does not reach statistical significance (p-value = 0.07). Similarly, 12-hydroxyeicosatetraenoic acid (12-HETE) also indicates a decrease in ALS patients although the significance is not achieved. Regarding the non-enzymatic pathways, isoprostanes of the F2α family were detected but only in 17 ALS patients. Interestingly, the hydroxylated metabolite, 11-HETE was detected in all samples, showing a significant decrease in ALS patients. As a result of this analysis, a schematic representation of major altered oxylipin pathways in plasma from ALS patients is shown in Figure 1C. Table 2. Plasma oxylipin concentrations in Control and ALS samples assessed by HPLC-MS/MS. Control (n = 9) ALS (n = 74) Metabolites p-Value b Median (pg/mL) Percentile a Median (pg/mL) Percentile LA (18:2n-6) metabolome 12,13-DiHOME 1084 (969.3; 1381) 389 (201; 666) 0.0002 9,10-DiHOME 126 (73.1; 163) 19.4 (1.20; 60.0) <0.0001 13-HODE 7865 (5375; 11,132) 4049 (3044; 5773) 0.0001 9-HODE 6332 (4769; 10,706) 3881 (2775; 5951) 0.0073 AA (20:4n-6) metabolome AA 811,395 (457,556; 1,046,666) 588,400 (429,801; 825,059) 0.1601 TxB2 440 (46.9; 614) 125 (46.6; 412) 0.2800 PGE2 0 (0; 7.73) 0 (0; 0) 0.6111 PGD2 0 (0; 0) 0 (0; 0) 0.9029 isoPGF2 0 (0; 0) 0 (0; 443) 0.1935 5-HETE 636 (469; 685) 350 (261; 505) 0.0044 8-HETE 124 (83.0; 137) 84.7 (58.7; 117) 0.0531 11-HETE 191 (109; 224) 117 (87.9; 167) 0.0459 12-HETE 3144 (2673; 4431) 1616 (771; 4125) 0.0550 15-HETE 247 (181; 271) 187 (139; 247) 0.1312 5-oxoETE 3.29 (1.61; 3.52) 0 (0; 3.22) 0.0089 Biomedicines 2022, 10, 674 7 of 13 Table 2. Cont. Control (n = 9) ALS (n = 74) Metabolites p-Value b Median (pg/mL) Percentile a Median (pg/mL) Percentile 12-oxoETE 36.4 (0; 59.8) 0 (0; 42.3) 0.4171 15-oxoETE 67.9 (0; 119) 37.0 (27.7; 63.5) 0.5207 14,15-DiHETrE 379 (321; 446) 320 (269; 383) 0.1064 LTB4 33.2 (11.9; 45.5) 14.9 (0; 32.1) 0.0741 EPA (20:5n-3) metabolome EPA 198,372 (121,928; 361,172) 208323 (146,448; 292,417) 0.9713 12-HEPE 117 (90.1; 169) 92.7 (0; 157) 0.1816 DHA (22:6n-3) metabolome DHA 329,956 (189,149; 560,821) 376709 (261,062; 529,711) 0.8010 4-HDoHE 260 (158; 297) 196 (147; 283) 0.4142 7-HDoHE 75.6 (0; 111) 69.9 (0; 102) 0.8521 10-HDoHE 27.2 (20.0; 33.9) 24.3 (18.8; 31.1) 0.5217 11-HDoHE 30.0 (19.9; 39.2) 23.9 (16.9; 30.1) 0.1384 13-HDoHE 87.4 (45.3; 125) 56.1 (33.9; 81.3) 0.0675 14-HDoHE 117 (86.5; 222) 78.5 (41.1; 127) 0.0381 16-HDoHE 66.9 (37.5; 102) 56.1 (43.4; 84.9) 0.5114 20-HDoHE 31.2 (10.7; 44.0) 23.5 (16.1; 35.9) 0.6488 19,20-DiHDPA 160 (149; 201) 157 (136; 193) 0.4929 a 25% percentile and 75% percentile are shown; b derived from the Mann–Whitney test. Metabolites in italics are significantly different between groups according to the Mann–Whitney test. There are no significant differences in plasmatic-free fatty acids levels regarding omega 3 fatty acids, EPA and DHA. Detected omega 3 fatty acid derivatives consisted mainly in the monohydroxylated fatty acids and no significant differences were detected between ALS and Control groups for any of them (Table 2). Despite our effort to detect SPM, these metabolites were not detected in ALS nor control plasma samples. Then, to evaluate the complete method for SPM detection in human plasma, we performed a supplementation experiment of SPM to fresh human plasma (which was previously analyzed and SPM levels were < LOD). An SPM mix containing LXA4, LXB4, RvE1, RvD1, RvD2, RvD3, RvD5, MaR1, and PD1 was spiked at different concentration levels (ranging from 0.1 to 10 nM, specific values shown in Figure S4). Then, both regular sample processing and HPLC-MS/MS analysis were performed. Detection of spiked SPM was verified for all compounds, even in the lower concentrations tested (0.1–0.5 nM, Figure S4), thus verifying the method’s ability to detect SPM in the low nM range. Despite SPM were undetected in plasma samples, we observed that the levels of 14-hydroxy-docosaexaenoic acid (14-HDoHE), one of the most abundant hydroxy-DHA derivatives detected in plasma and a key activation marker of the Maresin biosynthesis pathway, were significantly reduced in ALS patients, further suggesting ALS patients fail to switch on the production of lipids that are responsible to resolve inflammation, at least in the periphery. Biomedicines 2022, 10, x FOR PEER REVIEW 8 of 13 Biomedicines 2022, 10, 674 8 of 13 A B Linoleic acid derivatives 5 12,13-DiHOME 9,10-DiHOME 3.0×10 3 * 4.0×10 2 * 9,10-DiHOME 13-HODE 4 3.0×10 2 -log10 (p-value) 2.0×10 3 12,13-DiHOME 2.0×10 2 3 1.0×10 3 1.0×10 2 5-HETE 9-HODE 2 0 Ctrl ALS 0 Ctrl ALS 14-HDoHE 1 13-HODE 9-HODE 2.0×10 4 2.5×10 4 * * 0 2.0×10 4 1.5×10 4 -3 -2 -1 0 1 1.5×10 4 1.0×10 4 log2 (Fold Change) 1.0×10 4 5.0×10 3 5.0×10 3 0 0 Ctrl ALS Ctrl ALS C Arachidonic acid derivatives AA 5-HETE 2.0×10 6 1.5×10 3 9-HODE ↓ 9,10-EpOME ↓ * LOX CYP450 1.5×10 6 Linoleic acid 1.0×10 3 13-HODE ↓ 12,13-EpOME ↓ 1.0×10 6 5.0×10 2 5.0×10 5 0 0 5-LOX non-enz Ctrl ALS Ctrl ALS 5-HETE ↓ Arachidoinc acid 11-HETE ↓ 5-oxoETE LTB4 3.0×10 2 * 2.0×10 2 2.0×10 2 5-oxoETE ↓ 5.0×10 0 1.0×10 2 5.0×10 1 0 0 Ctrl ALS Ctrl ALS Figure1.1.Individual Figure Individual oxylipin analysis. oxylipin (A) (A) analysis. Volcano plot representation Volcano indicating plot representation the −log indicating 10 (p-value) the −log10 (p- and value) and2 the log2 (fold change) of the oxylipin concentration in plasma for ALS vs. Controlshown the log (fold change) of the oxylipin concentration in plasma for ALS vs. Control group group inshown Table in1. Table Dots 1. in Dots blackinrepresent p < 0.05. black represent p <(B) 0.05.Tukey plot for (B) Tukey plotplasma concentration for plasma (pg/mL) concentration of (pg/mL) of selected selected oxylipins oxylipins in Control in Control (Ctrl) (Ctrl) and and ALSALS patients patients (ALS). (ALS). UpperUpper panels, panels, LA-derived LA-derived metabo- metabolites lites detected were were detected and a significant and a significant decreasedecrease is shown is for shown ALSfor ALS samples. plasma plasma samples. Lowerarachidonic Lower panels, panels, ara- chidonic acid, acid, and 5-lipoxygenase-derived and 5-lipoxygenase-derived metabolitesmetabolites plasma concentration. plasma concentration. (C) Scheme (C)representing Scheme represent- major ing major altered oxylipin pathways in plasma of ALS patients. Significantly altered metabolites are altered oxylipin pathways in plasma of ALS patients. Significantly altered metabolites are derived derived from LOX and CYP450 pathways. * p < 0.05, Mann–Whitney test. from LOX and CYP450 pathways. * p < 0.05, Mann–Whitney test. There are Analysis 3.3. Multivariate no significant differences in plasmatic-free fatty acids levels regarding omega 3 fatty acids, Considering EPA andoxylipins, the detected DHA. Detected omega multivariate we performed 3 fatty acid derivatives consisted analysis (metric and mainly in the monohydroxylated fatty acids and no significant differences non-metric multidimensional scaling, hierarchical clustering, k-means clustering, were detected and between ALS discriminant and Control analysis) groups in search for any of between of differences them (Table 2). the Control group and ALS patients. Despite our effort to detect SPM, these metabolites However, it was impossible to discriminate between patients and were notcontrols detected in ALS (data nor con- not shown). trol plasma samples. Then, to evaluate the complete method for SPM We also performed comparative tests between ALS subgroups, regarding phenotype (bulbar detection in human plasma, or we performed spinal onset), a supplementation progression rate, gender, andexperiment other clinicalof parameters SPM to fresh human (data plasma not shown), (which where nowas previously significant analyzed differences anddetected. were SPM levels were < LOD). An SPM mix containing LXA4, LXB4, RvE1, RvD1, RvD2, RvD3, RvD5, MaR1, and PD1 was spiked at different 3.4. Correlation Analysis concentration levels (ranging from 0.1 to 10 nM, specific values shown in Figure S4). Then, bothWeregular sample further analyzedprocessing and HPLC-MS/MS oxylipins concentration analysis were performed. levels detected Detection in ALS patient sam-of spiked ples, SPM was to assess the verified for all correlation compounds, between even in metabolites andtheclinical lower concentrations tested (0.1– parameters. Logarithmic 0.5 nM, Figure S4), transformation thus verifyingvalues of concentration the method’s ability toand was performed detect theSPM in thecorrelation Pearson low nM range. test Despite SPM evaluated were undetected the correlation between inoxylipin plasma concentration samples, we observed that theage, and phenotype, levels of 14-hy- progression droxy-docosaexaenoic rate, and time since disease acidonset (14-HDoHE), one since (elapsed time of theonset mosttoabundant hydroxy-DHA blood sample extraction)deriv- with Benjamini-Hochberg correction for multiple testing. As shown in Figure 2, plasmatic levels We further analyzed oxylipins concentration levels detected in ALS patient samples, to assess the correlation between metabolites and clinical parameters. Logarithmic trans- formation of concentration values was performed and the Pearson correlation test evalu- ated the correlation between oxylipin concentration and phenotype, age, progression rate, Biomedicines 2022, 10, 674 and time since disease onset (elapsed time since onset to blood sample extraction)9with of 13 Benjamini-Hochberg correction for multiple testing. As shown in Figure 2, plasmatic lev- els of LA-derived 13-HODE and 9-HODE, positively correlate with disease duration, ex- pressed of as days LA-derived since onset 13-HODE (logarithmic and 9-HODE, transformation). positively Nodisease correlate with correlation was found duration, be- expressed tween the concentration values of other oxylipins and the above-mentioned clinical as days since onset (logarithmic transformation). No correlation was found between the pa- rameters. concentration values of other oxylipins and the above-mentioned clinical parameters. Figure 2. Correlation Figure 2. Correlation plot plot between between LALA metabolites metabolites and and disease disease duration duration in in ALS ALS patients. patients. Pearson Pearson correlation tests tests and p-valueadjustment andp-value adjustment(Benjamini-Hochberg) (Benjamini-Hochberg) were performed forfor were performed all detected all detected ox- oxylipins ylipins andand clinicalparameters. clinical parameters.AApositive positivecorrelation correlationwas wasfound foundfor for 13-HODE 13-HODE and 9-HODE with time since since onset onset (logarithmic (logarithmic transformation). transformation). 4. 4. Discussion Discussion A misbalance in pro- and anti-inflammatory and pro-resolving lipid mediators could A misbalance in pro- and anti-inflammatory and pro-resolving lipid mediators could play a critical role in neuroinflammation associated with ALS disease. Despite some evi- play a critical role in neuroinflammation associated with ALS disease. Despite some evi- dence on alterations in AA-derived metabolites that have been reported, to our knowledge, dence on alterations in AA-derived metabolites that have been reported, to our no “(oxy)lipidomics” approach in ALS human samples has been performed. Herein, we de- knowledge, no “(oxy)lipidomics” approach in ALS human samples has been performed. veloped an oxylipin profile analysis of plasma samples of ALS patients and healthy volunteers. Herein, we developed an oxylipin profile analysis of plasma samples of ALS patients and Previous reports on eicosanoid analysis in ALS patient samples showed a significant healthy volunteers. increase in PGE2 plasma levels [13], and postmortem spinal cord and cortex tissue [14,15]. Previous reports on eicosanoid analysis in ALS patient samples showed a significant This was not observed in the samples analyzed here, emphasizing the relevance of disease increase in PGE2 plasma levels [13], and postmortem spinal cord and cortex tissue [14,15]. onset conditions. Samples in previous work were from postmortem tissues or advanced This was not observed in the samples analyzed here, emphasizing the relevance of disease ALS patients whereas our analysis included patients with only 1–3 years of disease onset. In addition, the reported determinations of PGE2 were obtained by immunoassays while here we performed a LC-MS analysis having greater specificity and sensitivity. Regarding SPMs, as demonstrated for other neurodegenerative diseases, e.g., Alzheim- er´s disease [23] and multiple sclerosis [24,25], lack of activation of resolution of inflam- mation programs is represented as a decrease in SPM levels which leads to sustained inflammation. We recently showed that the production of the main lipid mediators that activate resolution programs of inflammation is deficient in serum and active brain lesions of multiple sclerosis patients, as well as in the spinal cord of experimental autoimmune encephalomyelitis mice [25]. Furthermore, these observations were accompanied by an increase in AA-derived pro-inflammatory lipid mediators, such as LTB4, PGE2, PGF2 and TXB2, emphasizing an imbalance between pro-inflammatory and pro-resolving lipid medi- ators [25]. Thus, we hypothesized that a similar scenario could be responsible for the per- sistent inflammatory component in ALS. We developed an HPLC-MS/MS method [21,25] based on previous reports [26–30] to study plasma oxylipin profiles in ALS patients. As described in the Material and Methods section, this method comprises sample processing by solid-phase extraction and reversed-phase liquid chromatography before mass spectrom- etry detection. However, no SPMs were detected either in Control or ALS groups despite our effort. We then analyzed plasma samples spiked with a mixture of SPM and detection was performed for all SPMs, even in pg/mL concentration values (Figure S4). Values of SPMs concentration in plasma from healthy individuals range from 1 to 2000 pg/mL in some reports [31,32] while others have reported levels