Metabolomics-lipidomics of eicosanoids and docosanoids generated by phagocytes

Rong Yang, Nan Chiang, Sungwhan F Oh, Charles N Serhan, Rong Yang, Nan Chiang, Sungwhan F Oh, Charles N Serhan

Abstract

Lipid mediators derived from essential fatty acids, such as arachidonic acid, play important roles in physiologic and pathophysiologic processes. Prostaglandins, thromboxane, and leukotrienes are well-known eicosanoids that play critical roles in hemodynamics and inflammation. New families of mediators were recently uncovered that constitute a new genus stimulating resolution of acute inflammation, and are organ-protective. These include the resolvins (E-series and D-series), protectins (neuroprotectin D1/protectin D1), and maresins biosynthesized from omega-3 essential fatty acids. Phagocytes play major roles in tissue homeostasis and have a high capacity to produce these mediators, which depend on their tissue and state of activation. It is important to select appropriate methods for identifying target mediators and pathway biomarkers. In this unit, we review state-of-the-art approaches to identify and profile eicosanoid and docosanoid pathways, including specialized pro-resolving mediators resolvins, protectins, and maresins, in relation to their biosynthesis and inactivation by neutrophils and macrophages.

Figures

Figure 1
Figure 1
Overview of A) eicosanoid and docosanoid biosynthesis and B) lipidomic analysis procedures. COX: cyclooxygenase; LOX: lipoxygenase; P-450: cytochrome P-450.
Figure 1
Figure 1
Overview of A) eicosanoid and docosanoid biosynthesis and B) lipidomic analysis procedures. COX: cyclooxygenase; LOX: lipoxygenase; P-450: cytochrome P-450.
Figure 2
Figure 2
HPLC substrate isolation and GC/MS quality control. A) UV chromatogram (235nm) of HPLC isolation of commercial DHA; B) GC/MS Total Ion Chromatogram (TIC) and spectrum of methylated isolated DHA. See text for details.
Figure 3
Figure 3
Mediator lipidomics flow chart for lipidomic matching/identification, profiling, structure elucidation, and defining novel lipid mediator and its biosynthesis.
Figure 4
Figure 4
Lipid mediator identification/matching criteria.
Figure 5
Figure 5
Deuterium labeled compounds for mediator lipidomic profiling.
Figure 6
Figure 6
LC-UV-MS-MS-based lipidomics: authentic and synthetic eicosanoids and docosanoids. LM lipidomics was conducted with an ABI Qtrap using MRM IDA EPI method (See text for details). A) Total ion chromatogram of 12 authentic and synthetic LM; Chromatogram of precursor/product ion pairs m/z 349 > 195 for RvE1 and m/z 375 > 141 for RvD1. B) Demonstration of online UV spectra for RvE1 and RvD1. MS-MS spectra of C) RvD1, and D) RvE1.
Figure 7
Figure 7
Macrophages generate lipoxin A4 during phagocytosis of apoptotic PMN. A) Lipoxin A4 production (red circle, quantitated with ELISA) peaked at 15 min during macrophage phagocytosis (green diamond). In contrast, inflammatory LTB4 production was not increased during this process as shown inset. Quantitation of LXA4 with ELISA was confirmed by LC-MS-MS. B) Tandem mass spectrum of LXA4.
Figure 8
Figure 8
Macrophages generate RvE1 during phagocytosis of apoptotic PMN: impact of aspirin and EPA. A) Macrophages, when treated with aspirin prior to coincubation with apoptotic PMN, generated RvE1. This level increased in presence of substrate EPA. B) MS-MS spectra of the authentic synthetic RvE1 and biogenic RvE1 from murine macrophages.
Figure 9
Figure 9
Transcellular biosynthesis: aspirin-dependent biosynthesis regulation of thromboxane, 15-epi-Lipoxin A4 and resolvin E1 (see text for details).
Figure 10
Figure 10
E series resolvin pathway metabolome. Profiling of E series resolvin biosynthesis and inactivation in human and murine with LC-UV-MS-MS. Chromatogram of precursor/product ion pairs m/z 317 > 259 for 18-HEPE, m/z 333 > 115 for RvE2, and m/z 349 > 195 for RvE1. See text for more details.
Figure 11
Figure 11
Tandem mass spectrum of A) 18-HEPE, B) RvE2, C) RvE1, and D) 20-carboxy-RvE1 with online UV spectrum.
Figure 12
Figure 12
RvD1 pathway metabolome. Profiling of RvD1 biosynthesis and further metabolism with LC-UV-MS-MS. Chromatogram of precursor/product ion pairs m/z 343 > 245 for 17-HDAH, m/z 359 > 153 for PD1, and m/z 375 > 141 for RvD1. See text for more details.
Figure 13
Figure 13
Tandem mass spectra of A) 17-HDHA and B) PD1. Refer to Figure 6 for RvD1 tandem mass spectrum.
Figure 14
Figure 14
LC-UV-MS-MS based chiral analysis of racemic 4-HDHA, 7-HDHA, and 14-HDHA mixture. Chiralities are determined by marching retention time with optical pure compounds. Total Ion Chromatogram (TIC) of racemic 4-HDHA, 7-HDHA, and 14-HDHA mixture is shown in brown color. Chromatogram of precursor/product ion pair m/z 343>101 for racemic 4-HDHA is shown in blue, m/z 343 >141 for 7-HDHA in orange, and m/z 343 > 205 for 14-HDHA in green.
Figure 15
Figure 15
LC-UV-MS-MS based chiral analysis shows incubation of porcine 12-LOX with DHA generated product with > 98% S-configuration. The incubation was reduced with NaBH4 before analysis. The assignment was confirmed by co-eluting (retention time), online UV and diagnostic ions of ms/ms spectrum with authentic synthetic standards. The spectrum of 14-HDHA is shown on the right.
Figure 16
Figure 16
GC/MS based LM lipidomics. A)Overview of GC/MS procedure. B) GC-MS C-value determination.
Figure 17
Figure 17
Examples of database search of RvD1 spectrum on Qtrap 3200 with Analyst 1.41.

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir