Bis-allylic hydroxylation of polyunsaturated fatty acids by hepatic monooxygenases and its relation to the enzymatic and nonenzymatic formation of conjugated hydroxy fatty acids.

[14C]Linoleic acid was incubated with phenobarbital-induced rat liver microsomes and formation of cis-trans-conjugated hydroxy fatty acids was investigated. 13-Hydroxy-9Z,11E-octadecadienoic acid (13-HODE), 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), and three novel metabolites were identified, viz. 11-hydroxy-9Z,12Z-octadecadienoic acid (11-HODE), 8-HODE, and 14-HODE. 11-HODE (59% R), the main product, was unstable and converted to 9(R, S)-HODE and 13(R, S)-HODE in acidic media. All metabolites contained oxygen from O2. Experiments under oxygen-18 gas showed that 13-HODE and 9-HODE contained equal or less amounts of oxygen-18 than the other metabolites. In the former case, 9-HODE and 13-HODE were formed with stereo-selectivity (80-82% R). [11S-2H]Linoleic acid was metabolized to 13R-HODE with loss of deuterium (24% 2H) and to 9R-HODE with deuterium retention (95% 2H), while [11R-2H]linoleic acid was metabolized to 13R-HODE that largely retained the label (71% 2H) and to 9R-HODE that lost most of the label (22% 2H). These data indicated that P450 catalyzed abstraction of the pro-R hydrogen at C11, double bond migration and suprafacial oxygen insertion at C9 to give 9R-HODE, while abstraction of the pro-S hydrogen at C11, followed by double bond migration and oxygen insertion, yielded 13R-HODE. Hepatic microsomes of the cynomolgus monkey metabolized 18:2n-6 as above and 20:4n-6 to 13-hydroxyeicosatetraenoic acid, likely formed in analogy with 11-HODE. In summary, one mechanism in the biosynthesis of cis-trans-conjugated hydroxy fatty acids by P450 involves suprafacial hydrogen abstraction and oxygen insertion. In addition, hydrolysis of the unstable bis-allylic hydroxy metabolites may contribute to the formation of conjugated hydroxy fatty acids.