Anti-Inflammatory Actions of Neuroprotectin D1/Protectin D1 and Its Natural Stereoisomers: Assignments of Dihydroxy-Containing Docosatrienes

Abstract

Protectin D1, neuroprotectin D1 when generated by neural cells, is a member of a new family of bioactive products generated from docosahexaenoic acid. The complete stereochemistry of protectin D1 (10,17S-docosatriene), namely, chirality of the carbon-10 alcohol and geometry of the conjugated triene, required for bioactivity remained to be assigned. To this end, protectin D1/neuroprotectin D1 (PD1) generated by human neutrophils during murine peritonitis and by neural tissues was separated from natural isomers and subjected to liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Comparisons with six 10,17-dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed that PD1 from human cells carrying potent bioactivity is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Additional isomers identified included trace amounts of Δ15-trans-PD1 (isomer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer IV), and a double dioxygenation product 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer I), present in exudates. 18O2 labeling showed that 10S,17S-diHDHA (isomer I) carried 18O in the carbon-10 position alcohol, indicating sequential lipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at 10 nM attenuated (∼50%) human neutrophil transmigration, whereas Δ15-trans-PD1 was essentially inactive. PD1 was a potent regulator of polymorphonuclear leukocyte (PMN) infiltration (∼40% at 1 ng/mouse) in peritonitis. The rank order at 1- to 10-ng dose was PD1 ≈ PD1 methyl ester ≫ Δ15-trans-PD1 > 10S,17S-diHDHA (isomer I). 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (isomer VI) proved ≥ PD1 in blocking PMN infiltration, but was not a major product of leukocytes. PD1 also reduced PMN infiltration after initiation (2 h) of inflammation and was additive with resolvin E1. These results indicate that PD1 is a potent stereoselective anti-inflammatory molecule.

Biogenic Synthesis, Purification, and Chemical Characterization of Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6)

“…Overall, many of the products characterized upon using DPAn-6 or DPAn-3 as substrates were similar to those obtained from DHA, as reported earlier (1,3,5). As expected, the 15-lipoxygenase products were primarily oxygenated at the C-17-position, ultimately producing 17-HDHA, 17-HDPAn-6, and 17-HDPAn-3 from the respective fatty acids.…”

“…The number of peaks, integration, and chemical shifts in both the one-dimensional 1 H and the one-dimensional 13 C spectra were consistent with the molecular formula C 22 H 34 O 3 and were consistent with spectra predicted by Chemdraw and ACDlabs software. The two-dimensional 1 H, 1 H COSY unambiguously showed that hydroxylation was at the 17-position. The protons belonging to C (17) -H-OH centered at 4.12 ppm produced cross-peaks to C (16) -H and C (18) -H but not with protons belonging to C (13) or C (14) .…”

“…Fig. 5 shows the one-dimensional 1 H, one-dimensional 13 C, and two-dimensional 1 H, 1 H COSY spectra of 17-HDPAn-6. The number of peaks, integration, and chemical shifts in both the one-dimensional 1 H and the one-dimensional 13 C spectra were consistent with the molecular formula C 22 H 34 O 3 and were consistent with spectra predicted by Chemdraw and ACDlabs software.…”

Transgenic mice rich in endogenous omega-3 fatty acids are protected from colitis

“…Newly identified potent n-3 fatty acid-derived mediators such as the resolvins, including RvE1 and resolvin D3 (RvD3), and protectins, i.e., neuroprotectin D1 (NPD1)͞protectin D1 (PD1), are antiinflammatory (20,21). We assessed both n-6 and n-3 PUFA-derived mediators from colons using liquid chromatography-UV-tandem MS mediator informatics to determine whether the difference in DSS-induced colitis observed between WT and fat-1 mice was associated with these pathways.…”

“…The newly identified n-3-derived resolvins and protectins are potent antiinflammatory mediators in various settings, including trinitrobenzene sulfonate colitis and periodontitis (20,21,29,30). These lipid mediators have been shown to decrease formation of inflammatory cytokines such as TNF␣, IL-6, and others (31,32).…”