Abstract
A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.
Highlights
A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P) DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z, 13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase, and potato tuber lipoxygenase in various combinations
10,17(S)-dihydroxydocosahexaenoic acid with postulated 4Z,7Z,11E,13E,15Z,19Z geometry of the double bonds, which was termed neuroprotectin D1 and 10,17(S)-docosatriene in previous publications [2,3,4,5, 8], is of special interest in that it was shown that this compound appears to be produced by a variety of mammalian cells
Molecular masses of the DHA oxidation products were determined on an LCQ Deca XP Max MSn mass spectrometer with an electrospray ionization (ESI) ion source operating in either negative (M 2 H1, free fatty acids, and/or M 1 Cl2 adducts) or positive (M 1 H1 and/or M 1 Na1 adducts) mode
Summary
A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P) DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z, 13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. 10,17(S)-dihydroxydocosahexaenoic acid with postulated 4Z,7Z,11E,13E,15Z,19Z geometry of the double bonds, which was termed neuroprotectin D1 and 10,17(S)-docosatriene in previous publications [2,3,4,5, 8], is of special interest in that it was shown that this compound appears to be produced by a variety of mammalian cells. Added exogenously, it had potent antiapoptotic activity, effectively protected the cells during oxidative stress [3], and increased the healing rate of damaged mouse cornea by a mechanism that differed from its antiinflammatory activity [9].
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