Identification of specialized pro-resolving mediator clusters from healthy adults after intravenous low-dose endotoxin and omega-3 supplementation: a methodological validation

Abstract

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 inflammation. We identified specific 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-inflammatory 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 specific pro-resolving mediators with ω-3 supplementation that together support the role of SPM in vivo in inflammation-resolution in humans.

Conflict of interest statement

The authors declare no competing interests.

Figures

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.

Figure 2

LM-SPM metabolomic protocol. Schematic of LM extraction, LC-MS/MS data acquisition and analysis implemented by two laboratories independently on same samples.

Figure 3

SPM identification with MS/MS fragmentation analysis in human serum. LC-MS/MS fragmentation and identification of SPMs and pathway markers based on presence of >6 diagnostic ions, denoted in the insets. Spectra are representatives from one of six healthy donors, and confirmation was carried out by both labs.

Figure 4

Pro-inflammatory leukotriene and prostaglandin identification with MS/MS fragmentation analysis in human serum. LC-MS/MS fragmentation and identification of prostaglandins, leukotrienes, and pathway markers based on presence of >6 diagnostic ions, denoted in the insets. Spectra are representatives from one of six healthy donors, and confirmation was carried out by both labs.

Figure 5

EPA and DHA supplementation increases SPM production in human serum after LPS challenge. Principal Component Analysis (PCA) for human LPS-challenged serum, with and without EPA + DHA supplementation. (A) 3-dimensional score plot of blood sampling time points; green circles are representative of ω-3 FA supplementation study group (mean of three donors), while blue circles are representative of placebo study group (mean of three donors). Gray ellipse denotes 95% confidence interval. (B) 3-dimensional loading plot of LM-SPMs identified in human serum upon LPS challenge; green circles are those mediators associated with the study group supplemented with ω-3 FA, while the blue circles are those mediators associated with the placebo study group. (C) Total SPM cluster and total prostaglandins, thromboxane, and LTB4 in human serum 120 h post LPS injection. Results are means ± SEM of 3 healthy subjects; **P < 0.01 for the ω-3 FA group vs. placebo group.

Figure 6

Time course of prostaglandins and thromboxane in human serum with LPS challenge. (A) Thromboxane concentrations during 120 h LPS time course in serum. (B) Prostaglandin and thromboxane corresponding pathway fold-change in ω-3 FA group vs placebo group at 0 h; circle size represents ω-3 FA group LM quantities (pg/mL) at 0 h. (C) C-reactive protein (CRP) concentrations during 120 h LPS time course in serum. Results are means ± SEM of 3 healthy subjects.

Figure 7

Time course of SPM in human serum with LPS challenge. (A) Total SPM cluster amounts (top) and 17-HDHA (bottom) during 120 h LPS time course in serum. (B) Corresponding DHA-derived pathway changes (top) and AA-derived SPM and leukotriene pathway changes between ω-3 FA group vs. placebo group at 0 h; circle size represents ω-3 FA group LM quantities in pg/mL at 0 h. Results are means ± SEM of 3 healthy subjects.

Figure 8

SPM production is increased in human plasma via ω-3 PUFA supplementation and immunologic challenge. (A) Total SPM cluster concentrations in human plasma with ω-3 PUFA supplementation or placebo 1–168 h post intravenous LPS injection; *P < 0.05, **P < 0.01 for ω-3 PUFA supplement group vs. placebo group with one-tailed ratio t-test. (B) Comparison of total SPM cluster concentrations in human plasma at 2 h between separate laboratories. Results are means ± SEM of 3 healthy subjects; *P < 0.05; ns = not statistically different using paired one-tailed ratio t-test.