www.nature.com/scientificreports Corrected: Author Correction OPEN Identifcation of specialized pro- resolving mediator clusters from healthy adults after intravenous Received: 6 July 2018 Accepted: 25 November 2018 low-dose endotoxin and omega-3 Published online: 21 December 2018 supplementation: a methodological validation Paul C. Norris1, Ann C. Skulas-Ray2, Ian Riley1, Chesney K. Richter2, Penny M. Kris-Etherton3, Gordon L. Jensen4, Charles N. Serhan 1 & Krishna Rao Maddipati 5 Specialized pro-resolving mediator(s) (SPMs) are produced from the endogenous ω-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and accelerate resolution of acute infammation. We identifed specifc clusters of SPM in human plasma and serum using LC-MS/MS based lipid mediator (LM) metabololipidomics in two separate laboratories for inter- laboratory validation. The human plasma cluster consisted of resolvin (Rv)E1, RvD1, lipoxin (LX)B4, 18-HEPE, and 17-HDHA, and the human serum cluster consisted of RvE1, RvD1, AT-LXA4, 18-HEPE, and 17-HDHA. Human plasma and serum SPM clusters were increased after ω-3 supplementation (triglyceride dietary supplements or prescription ethyl esters) and low dose intravenous lipopolysaccharide (LPS) challenge. These results were corroborated by parallel determinations with the same coded samples in a second, separate laboratory using essentially identical metabololipidomic operational parameters. In these healthy subjects, two ω-3 supplementation protocols (Study A and Study B) temporally increased the SPM cluster throughout the endotoxin-challenge time course. Study A and Study B were randomized and Study B also had a crossover design with placebo and endotoxin challenge. Endotoxin challenge temporally regulated lipid mediator production in human serum, where pro-infammatory eicosanoid (prostaglandins and thromboxane) concentrations peaked by 8 hours post-endotoxin and SPMs such as resolvins and lipoxins initially decreased by 2 h and were then elevated at 24 hours. In healthy adults given ω-3 supplementation, the plasma concentration of the SPM cluster (RvE1, RvD1, LXB4, 18-HEPE, and 17-HDHA) peaked at two hours post endotoxin challenge. These results from two separate laboratories with the same samples provide evidence for temporal production of specifc pro-resolving mediators with ω-3 supplementation that together support the role of SPM in vivo in infammation-resolution in humans. Infammation arises from responses to tissue injury and microbial stimuli to prevent the spread of infection1. Failure to resolve excessive infammation is a central component of many chronic diseases1–4, including ather- osclerosis and rheumatoid arthritis, thus contributing to the burden on public health. Nutritional intervention 1Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine and Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, 02115, USA. 2Department of Nutritional Sciences, University of Arizona, Tucson, AZ, 85721, USA. 3Department of Nutritional Sciences, Pennsylvania State University, University Park, PA, 16802, USA. 4Larner College of Medicine, University of Vermont, Burlington, VT, 05405, USA. 5Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan, USA. Paul C. Norris and Ann C. Skulas-Ray contributed equally. Correspondence and requests for materials should be addressed to C.N.S. (email: [email protected]) or K.R.M. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:18050 | DOI:10.1038/s41598-018-36679-4 1 www.nature.com/scientificreports/ Figure 1. Study Design and Sampling Schematic. (A) Design of 5-month parallel arm supplementation study. (B) Design of 8-week supplementation crossover study. (C) Timing of blood sampling during the endotoxin (lipopolysaccharide, LPS) challenge testing visit; all time points were the same for both studies except for 120 hrs (measured in Study A) and 168 hrs (measured in Study B). Participants were required to fast for 12 hours prior to endotoxin administration. studies suggest that intake of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and other ω-3 pol- yunsaturated fatty acids (PUFA) may provide tissue protection4. Te ω-3 PUFA are precursors for specialized pro-resolving mediators (SPM) that include resolvins, protectins and maresins that are produced in the reso- lution phase of acute infammation. By defnition, each SPM pathway stimulates resolution of infammation and infections by limiting the exposure to pathogens and collateral damage from tissue-destructive neutrophils. SPMs enhance innate host defense responses that include macrophage phagocytosis of apoptotic neutrophils and microbes3. Tese proresolving mechanisms include limiting neutrophil infltration and stimulation of mac- rophage mediated uptake of apoptotic neutrophils, cellular debris and microbes. Each action is stimulated at pico to nanomolar ranges of SPM, requiring stereospecifc biosynthesis (reviewed in ref.3). Identifcation and profl- ing of SPMs has recently been operationalized with liquid chromatography-mass spectrometry (LC-MS) based approaches by multiple laboratories5–7. Tis has enabled elucidation of specifc functional SPM clusters in several human tissues and fuids, including blood5–7, placenta8, and emotional tears9. In the present report, we demonstrate alignment between two independent laboratories for the identif- cation and quantifcation of lipid mediators (LM) and SPM via metabololipidomic profling of subjects who received intravenous low-dose endotoxin (lipopolysaccharide; LPS) and ω-3 PUFA supplementation in two studies (Fig. 1 and Table 1). In Study A, healthy volunteers were randomized to 900–1800 mg/d EPA and DHA (as the triglyceride form of a fsh oil dietary supplement) or a soybean oil placebo for fve months followed by intravenous low-dose endotoxin (lipopolysaccharide, LPS), which is a model of acute, systemic infammatory challenge10. In Study B, which utilized a randomized crossover design, healthy men were given 3400 mg/d EPA or DHA and an olive oil placebo, each for 8 weeks prior to low-dose intravenous endotoxin challenge, with an 8-week washout (16 weeks separating each LPS testing visit). Using the above-specifed metabololipidomic profling approach with serum and plasma, we identifed a cluster of potent bioactive3–5 pro-resolving media- tors consisting of resolvin E1 (5S, 12R, 18R-trihydroxy-eicosa-6Z, 8E, 10E, 14Z, 16E-pentaenoic acid; RvE1), resolvin D1 (7S, 8R, 17S-trihydroxy-docosa-4Z, 9E, 11E, 13Z, 15E, 19Z-hexaenoic acid; RvD1), aspirin-triggered [R-epimer] (AT)-lipoxin A4 (5S, 6R, 15S-trihydroxy-eicosa-7E, 9E, 11Z, 13E-tetraenoic acid; LXA4), 18-hydroxy-eicosapentaenoic acid (HEPE), and 17-hydroxy-docosahexaenoic acid (HDHA) and elucidated their temporal regulation during infammatory challenge and ω-3 PUFA supplementation. Results LM-SPM were profled from serum in Study A and plasma in Study B using LC-MS/MS (Fig. 2, Supplementary Table 1, Supplementary Fig. 1). Te duration and dose of ω-3 supplementation implemented in the two studies was selected based on results from earlier studies where supplementation reduced triglycerides and increased incorporation of both EPA and DHA into erythrocytes (i.e., ω-3 index11,12). Additional criteria for the individual studies were as follows: In Study A, supplementation was designed to refect a range of dietarily achievable doses of EPA and DHA <2 grams/day, and the duration of supplementation was longer (~5 months) to permit incor- poration of EPA and DHA into erythrocyte membranes11. In Study B, we implemented a crossover design with higher dosing of 3.4 g/d EPA and DHA and shorter duration (8–12 weeks) of supplementation (Fig. 1 illustration). SCIENTIFIC REPORTS | (2018) 8:18050 | DOI:10.1038/s41598-018-36679-4 2 www.nature.com/scientificreports/ Study A (n = 6)b Study B (n = 3)c Placebo (n = 3; 1M, 2F) EPA and DHA (n = 3; 2M, 1F) Placebo EPA and DHA Age (y) 24 ± 2 (21–28) 25 ± 0.3 (24–25) 29 ± 1.7 (29–32) 29 ± 1.7 (29–32) Body mass index (kg/m2) 26.6 ± 0.7 (25.6–28.0) 23.8 ± 2.0 (21.4–27.7) 25.2 ± 2.1 (22.5–29.3) 25.9 ± 2.3 (24.5–30.4) Systolic blood pressure 121 ± 4 (114–126) 118 ± 3 (114–124) 120 ± 3 (115–124) 119 ± 4 (115–127) Diastolic blood pressure 77 ± 2 (74–80) 69 ± 6 (58–76) 77 ± 4 (70–85) 72 ± 2 (68–76) TC (mg/dL) 178 ± 20 (157–218) 120 ± 10 (104–137) 136 ± 15 (111–163) 139 ± 14 (111–157) LDL-C (mg/dL) 112 ± 15 (90–140) 58 ± 8 (52–67) 72 ± 12 (50–93) 77 ± 14 (52–102) HDL-C (mg/dL) 46 ± 10 (27–56) 53 ± 6 (41–59) 43 ± 4 (41–50) 49 ± 6 (47–60) TC:HDL-C 4.2 ± 0.9 (2.9–5.8) 2.3 ± 0.1 (2–2.5) 3.3 ± 0.6 (2.7–4.4) 3.0 ± 0.5 (2.4–4.0) TG (mg/dL) 97 ± 15 (68–113) 45 ± 4 (41–53) 109 ± 31 (60–166) 66 ± 7 (57–80) CRP (mg/L) 1.6 ± 0.7 (0.7–3.0) 0.3 ± 0.1 (0.2–0.6) 0.3 ± 0.1 (0.2–0.4 0.2 ± 0.1 (0.2–0.3) Peak CRP (mg/L) 20.4 ± 1.1 (18.4–22.2) 17 ± 2.1 (14.1–21) 16.3 ± 2.2 (12–19) 14.2 ± 3.2 (9.6–20.2) TNF-α (pg/mL) 1.6 ± 0.1 (1.3–1.7) 1.5 ± 0.1 (1.4–1.7) 1.0 ± 0.2 (0.7–1.3) 0.8 ± 0.1 (0.7–1.0) IL-6 (pg/mL) 1.7 ± 0.6 (0.6–2.3) 1.0 ± 0.2 (0.8–1.3) 0.9 ± 0.2 (0.5–1.3) 0.8 ± 0.1 (0.6–1.1) Erythrocyte fatty acid content (% of total fatty acids) Linoleic acid (LA; 18:2n6) 13.6 ± 1.04 (12.5–15.6) 12.5 ± 0.52 (11.6–13.4) 12.6 ± 0.29 (12.1–13.0) 12.1 ± 0.61 (11.3–13.3) Arachidonic acid (AA; 20:4n6) 15.7 ± 0.60 (14.9–16.9) 13.8 ± 0.66 (12.8–15.0) 16.5 ± 0.97 (14.7–18.0) 14.4 ± 0.34 (13.9–15.0) Eicosapentaenoic acid (EPA; 20:5n3) 0.44 ± 0.05 (0.39–0.55) 2.47 ± 0.71 (1.32–3.77) 0.31 ± 0.06 (0.28–0.37) 2.2 ± 0.09 (2.0–2.3) Docosahexaenoic acid (DHA; 22:6n3) 3.81 ± 0.82 (2.67–5.39) 7.40 ± 0.80 (5.90–8.66) 3.80 ± 0.58 (3.20–4.94) 6.66 ± 0.16 (6.36–6.89) Omega-3 index (EPA + DHA) 4.26 ± 0.87 (3.06–5.95) 9.88 ± 1.50 (7.23–12.4) 4.11 ± 0.60 (3.49–5.31) 8.85 ± 0.14 (8.58–9.06) Table 1.