Targeted chiral analysis of bioactive arachidonic Acid metabolites using liquid-chromatography-mass spectrometry

Abstract

A complex structurally diverse series of eicosanoids arises from the metabolism of arachidonic acid. The metabolic profile is further complicated by the enantioselectivity of eicosanoid formation and the variety of regioisomers that arise. In order to investigate the metabolism of arachidonic acid in vitro or in vivo, targeted methods are advantageous in order to distinguish between the complex isomeric mixtures that can arise by different metabolic pathways. Over the last several years this targeted approach has become more popular, although there are still relatively few examples where chiral targeted approaches have been employed to directly analyze complex enantiomeric mixtures. To efficiently conduct targeted eicosanoid analyses, LC separations are coupled with collision induced dissociation (CID) and tandem mass spectrometry (MS/MS). Product ion profiles are often diagnostic for particular regioisomers. The highest sensitivity that can be achieved involves the use of selected reaction monitoring/mass spectrometry (SRM/MS); whereas the highest specificity is obtained with an SRM transitions between an intense parent ion, which contains the intact molecule (M) and a structurally significant product ion. This review article provides an overview of arachidonic acid metabolism and targeted chiral methods that have been utilized for the analysis of the structurally diverse eicosanoids that arise.

Figure 1

Pathways of arachidonic acid metabolism. Abbreviations: COX, cyclooxygenase; CYP, cytochrome P540; EET, epoxyeicosatrienoic acid; EH, epoxide hydrolase; FLAP, 5-lipoxygenase activating protein; GGT, γ-glutamyltranspeptidase; GSH, glutathione; GST, glutathione-S-transferase; H, hydrolase; HEDH, hydroxyeicosanoid dehydrogenase; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HX, hepoxillin; LOX, lipoxygenase; LT, leukotriene; LX, lipoxins; PG, prostaglandin; PGDH, prostaglandin dehydrogenase; POX, peroxidase; S, synthase; TX thromboxane; UGT, UDP-glucuronosyltransferases.

Figure 2

Chiral separation of HETEs (top panel) and hydroxylated metabolites of EPA (middle panel) and DHA (lower panel). Reprinted with permission from Ref. [93].

Figure 3

Formation and action of COX-2-derived eicosanoids in epithelial cell models. arachidonic acid is released from membrane phospholipids by calcium-dependent cytosolic phospholipase A₂ (cPLA₂). The released arachidonic acid undergoes COX-2-mediated metabolism to PGs or forms the lipid hydroperoxides, 15(S)-hydroperoxyeicosatetraenoic acid (HPETE), 15(R)-HPETE and 11(R)-HPETE, which are reduced to the corresponding HETEs. PGD₂ and PGE₂ are inactivated by 15-PGDH-mediated conversion to their 15-oxo metabolites. Both 15-oxo-PGD₂ and 15-oxo-PGE₂ are converted to 13,14-dihydro-5-oxo-PG metabolites. Intact PGD₂ secreted by the epithelial cells can undergo albumin-mediated dehydration to 15d-PGJ₂. PGE₂ secreted from the epithelial cells by the ABCC4 transporter is pro-proliferative for tumor cells. Reuptake of PGE₂ by OATP2A1 into the epithelial cells leads to further 15-PGDH-mediated inactivation. In contrast to PGE₂ and PGD₂, 15(S)-HETE and 11(R)-HETE are activated by 15-PGDH-mediated oxidation to 15-oxo-ETE and 11-oxo-ETE, respectively. The oxo-ETEs are further conjugated to form OEGs. Secreted 15- and 11-oxo-ETE that escape further metabolism can then inhibit endothelial cell proliferation. Therefore, down-regulation of 15-PGDH and OATP2A1 would result in increased PGE₂-mediated tumor and endothelial cell proliferation. Reprinted with permission from Ref [110].

Figure 4

Targeted chiral lipidomics analysis of COX-2-derived eicosanoids from LoVo cells. LoVo cells were lysed; eicosanoids were extracted, derivatized with PFB bromide, and analyzed by LC-ECAPCI/SRM/MS. LoVo cell lysates were pretreated with 50 μM CAY10397 to inhibit 15-PGDH to be able to detect the 11-, 15-HETEs and PGE₂. Representative chromatograms are shown for (top to bottom) (a) 11(R)-HETE-PFB (m/z 319 → 167), (b) [²H₈]-15(S)-HETE-PFB internal standard (m/z 327 → 226), (c) 11-oxo-ETE-PFB (m/z 317 → 165) and 15-oxo-ETE-PFB (m/z 317 → 165), (d) [¹³C₂₀]-15-oxo-ETE-PFB internal standard (m/z 337 → 120), (e) PGE₂-PFB (m/z 351 → 271), (f) [²H₄]-PGE₂-PFB (m/z 355 → 275), (g) 13,14-dihydro-15-oxo-PGE₂-PFB (m/z 351 → 235), (h) [²H₄]-13,14-dihydro-15-oxo-PGE₂-PFB (m/z 355 → 239). Reprinted with permission from Ref. [110].

Figure 5

5-LOX-mediated formation of arachidonic acid metabolites and dGuo-adducts. HPNE, 4-hydroperoxy-2(E)-nonenal; DOOE, dioxo-6-octenoic acid. Reprinted with permission from Ref. [108].

Figure 6

Amount of lipid peroxidation metabolites from CESS cells. A, 5-HETEs. B, LTB4. C, PGE2, PGD2, and PGF2α. D, 13-HODEs. NT, no treatment; CA, treated with 1.0 μm A23187; CA+VC, treated with 1.0 μm A23187 and 1.0 mm vitamin C; CA+MK, treated with 1.0 μm A23187 and 1.0 μm MK886; CA+ASP, treated with 1.0 μm A23187 and 200.0 μm aspirin. Analyses were performed by stable isotope dilution LC-ECAPCI/SRM/MS of PFB derivatives. Determinations were conducted in triplicate (means ± S.D.). Reprinted with permission from Ref. [108].

Figure 7

LC-SRM/MS analysis and quantitation of 15-LOX-derived eicosanoids from R15L cells and RMock cells treated with arachidonic acid. A, representative chromatograms of 15-LOX-derived lipid metabolites released by R15L cells after 5-min treatment with 10 μM arachidonic acid. SRM chromatograms are shown for 15-oxo-ETE-PFB (m/z 317 → 273) (a), [²H₆]5-oxo-ETE-PFB internal standard (m/z 323 → 279) (b), 15-(R,S)-HETE-PFB (m/z 319 → 219) (c), and [²H₈]15-(S)-HETE-PFB internal standard (m/z 327 → 226) (d). B, concentration-time graph of 15-HETE (R- and S-form) released by R15L or RMock cells treated with 10 μM arachidonic acid for 24 h. C, concentration-time graph of 15-oxo-ETE released by R15L or RMock cells treated with 10 μM arachidonic acid for 24 h. Cell supernatants were collected at each time point. Lipid metabolites in the cell supernatants were extracted and derivatized with PFB. Determinations were conducted in triplicate (means ± S.E.M.) by stable isotope dilution chiral LC-ECAPCI/SRM/MS analyses of PFB derivatives. Reprinted with permission from Ref [108].

Figure 8

Biosynthesis of epoxyeicosatrienoic acids (EETs) by CYP isoforms. Reprinted with permission from Ref. [138].

Figure 9

Enantioselective biosynthesis of EETs by CYP family 2 isoforms: (A) hCYP2C19 and (B) hCYP2D6. Reprinted with permission from Ref. [138].

Figure 10

Analysis of epoxyeicosatrienoic acids by chiral liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry using a [¹³C]‐analog internal standards. Reprinted with permission from Ref. [138].