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Seven Z-octadecenoic acids having the double bond located in positions 6Z to 13Z were photooxidized. The resulting hydroperoxy-E-octadecenoic acids [HpOME(E)] were resolved by chiral phase-HPLC-MS, and the absolute configurations of the enantiomers were determined by gas chromatographic analysis of diastereoisomeric derivatives. The MS/MS/MS spectra showed characteristic fragments, which were influenced by the distance between the hydroperoxide and carboxyl groups. These fatty acids were then investigated as substrates of cyclooxygenase-1 (COX-1), manganese lipoxygenase (MnLOX), and the (8R)-dioxygenase (8R-DOX) activities of two linoleate diol synthases (LDS) and 10R-DOX. COX-1 and MnLOX abstracted hydrogen at C-11 of (12Z)-18:1 and C-12 of (13Z)-18:1. (11Z)-18:1 was subject to hydrogen abstraction at C-10 by MnLOX and at both allylic positions by COX-1. Both allylic hydrogens of (8Z)-18:1 were also abstracted by 8R-DOX activities of LDS and 10R-DOX, but only the allylic hydrogens close to the carboxyl groups of (11Z)-18:1 and (12Z)-18:1. 8R-DOX also oxidized monoenoic C14-C20 fatty acids with double bonds at the (9Z) position, suggesting that the length of the omega end has little influence on positioning for oxygenation. We conclude that COX-1 and MnLOX can readily abstract allylic hydrogens of octadecenoic fatty acids from C-10 to C-12 and 8R-DOX from C-7 and C-12.

Abstract: Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O2. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n−6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n−6 and n−9 fatty acids and by analysis of D-KIE. COX-1 oxidized C20 and C18 fatty acids in the following order of rates: 20:2n−6>20:1n−6>20:3n−9>20:1n−9 and 18:3n−3≥18:2n−6>18:1n−6. 18:2n−6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n−6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n−6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-2H]18:2n−6 with similar D-KIE (~53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n−6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-2H2]- and [11S-2H]18:2n−6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n−6 by COX-1 and 18:1n−9 by 8R-DOX were both accompanied by large D-KIE (>20). [Copyright &y& Elsevier]

In the mature retina, the components of the macular pigment, lutein (L), R,R-zeaxanthin (RRZ), R,S-zeaxanthin (RSZ, meso-zeaxanthin) are most concentrated in the central macula. L and RRZ are of dietary origin but RSZ is produced in situ from L. The relative proportions of L and Z isomers vary across the retina with eccentricity in the adult retina. Early reports have shown that during development, the proportions of L and Z isomers undergo changes as the total pigment levels increase. The methods described here demonstrate the unique utility of chiral phase HPLC to measure the amounts of L, RRZ, and RSZ, discriminating between the two zeaxanthin stereoisomers. In three concentric retinal sections of macaque retinas chiral phase HPLC has been employed to document the developmental changes in the distribution of each L, RSZ, and RRZ during the period just prior to full term gestation through 19 months after birth. The net rate of accumulation of carotenoids within the central retina during the first 20 months is quasi-linear and fit by a linear regression. During development, the rate of transport of L (0.12 (±0.033)ngmm -2 mo -1 (SE)) into the central 2mm of the retina is double that of RRZ (0.062 (±0.02)ngmm -2 mo -1 (SE)). The rate of accumulation of RSZ (0.06 (±0.01)ngmm -2 mo -1 (SE)) is comparable to that of RRZ. In the peripheral retina, the rates of accumulation of L and RRZ are not correlated with increasing age, whereas accumulation of RSZ does correlate with age. The changing proportions of L to Z isomers in the central retina during development are explained by the rates for carotenoid accumulation within the central retina. At birth, the macular pigment in the central retina is dominated by L and RRZ, 0.35±0.11 and 0.21±0.054ngmm -2 . In the central retina, RSZ was rarely detected in the youngest tissues analyzed. It can be estimated to represent 6% of the total macular pigment (0.033±0.11ngmm -2 ) at birth based on extrapolation from measurements in the peripheral retina and the ratio of L/(RRZ+RSZ) is ≈1.5. At maturity, the concentrations for L, RRZ, and RSZ in the central macaque retina are estimated to be 1.7, 1.8 and 1.08ngmm -2 , with L/(RRZ+RSZ) being 0.6., (Copyright © 2022 Elsevier Inc. All rights reserved.)