Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS

Abstract

Objectives Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma. Methods Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion. Results We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2). Conclusions Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.

E-series resolvin metabolome, biosynthesis and critical role of stereochemistry of specialized pro-resolving mediators (SPMs) in inflammation-resolution: Preparing SPMs for long COVID-19, human clinical trials, and targeted precision nutrition

“…It is appropriate to emphasize that these early fundamental studies are vital to understanding the pathogenesis of COVID-19 infections and the potential role of lipid mediators that could give useful new directions for treatments of COVID-19, long COVID and in lasting immunity. These new findings ( Table 1 ) and many other human studies for SPMs from international experts [ [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] ] would not have been possible without the i) complete stereochemical assignments of each of the potent bioactive SPMs produced by human and mouse leukocytes, ii) their biosynthesis from n-3 essential fatty acids (EPA and DHA) and iii) total organic synthesis with rigorous matching studies with endogenous SPMs along with our dedicated and talented organic chemistry collaborators summarized herein. In this relatively new SPM field, the Serhan lab has collaborated for many years with Professor Nicos Petasis and colleagues as part of the total synthesis core of our NIH-supported Program Project grant P01GM095467, Professor Trond Hansen and team in Oslo, Norway [ 56 ], Professor Bernd Spur and Anna Rodriguez [ [57] , [58] , [59] ], and the organic synthesis group at Cayman Chemical; for examples, see the custom synthesis of benzo-RvD1 analog mimetic [ 60 ] and the new RvE4 [ 61 ].…”

“…Importantly, the many other elegant human studies on SPMs would not have been possible without reliance on the complete stereochemically defined synthetic SPMs and their internal standards for LC-MS-MS-based analyses with adult tissues [ [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] ] and adolescent human peripheral blood samples [ 38 ] as well as their scale-up for commercial availability [ 62 ]. A recent clinical trial reported that local topical treatment of gingival inflammation with a proresolving lipoxin/resolvin stable analog is safe and effective as well as activated endogenous production of SPMs present in peripheral blood [ 63 ].…”

“…The structure of resolvin D5 was established and confirmed with results from total organic synthesis [ 148 , 149 ], leukocyte biosynthesis, and from enzymatic studies [ 150 ] with 15-lipoxygenase-2. RvD5 is increased in human blood with n-3 supplementation [ 38 ] and is present in human skin in lesions of psoriasis [ 151 ]. With skin cells, RvD5 reduces expression of IL-24 and S100A12 with human keratinocytes [ 151 ] and remains elevated one year after discharge in ICU patients with abdominal septic shock [ 152 ], suggesting the need for long-term follow-up of ICU patients.…”

“… 184 ]. The biosynthesis of SPM in humans requires substrate EPA and DHA in adequate amounts and locations in vivo to enable SPM biosynthesis in vivo in humans; for examples, see [ [38] , [39] , [40] , 50 , [185] , [186] , [187] ], to yield precise chemical structures of the SPMs with protective function for the host [ 188 ]. These early results of our studies underscore the importance of precision nutrition in the optimal progression of host defense and resolution of the acute inflammatory response in humans and model organisms [ 189 ].…”

Formation, Signaling and Occurrence of Specialized Pro-Resolving Lipid Mediators—What is the Evidence so far?

“…Unlike other methods they used a base modified LC solvent, thus enhancing the ionization process in negative ESI mode. This alternative analytical technique is most probably the reason why their method is 100-fold more sensitive ( Hartling et al, 2021 ).…”