LOX-Mediated Reactions

In addition to LOX-mediated reactions, AA-derived HETEs and LA-derived HODEs can also be biosynthesized by CYP pathways (Bylund et al. 1998). HODEs which have been determined as CYP products include 9-, 13-, and 18-HODE (Bylund et al. 1998), while some work on rodents and primates found the additional products 8-HODE, 11-HODE (acid-labile converts into 9/13-HODE), and 14-HODE (Oliw et al. 1993). 8-HODE, 11-HODE, and 14-HODE were all formed via an allylic hydroxylation, whereas the 9/J-HODE and 131 -HODE were stereoselectively biosynthesized via hydroxylation with double bond migration (Oliw et al. 1993). AA-derived HETEs that can be generated by CYP include 7-, 10-, and 13-HETE (acid-labile) and 5,6-diHETrE. CYP enzymes can also catalyze the formation of terminal and subterminal hydroxylated species such as 16-, 18-, 19-, 20-HETE, all of which have been shown to exhibit vasodilatory properties (Carroll et al. 1996). 19-HETE has been reported to affect vascular tone and ion transport. 20-HETE is a CYP4 family AA oxidation product, which has been found to be a potent vasoconstrictor but it also has been identified in some studies as having a pro-carcinogenic role contributing to cell proliferation and tumor growth (Guo et al. 2008 Alexanian et al. 2009 Liu et al. 2010). 

When the substrate (e.g., linoleic acid) also acts as a cosubstrate (i.e., LH = XH), the cooxidation will also lead to hydroperoxides among other oxygenated products. However, these hydroperoxides lack the stereospecificity of the direct enzyme products (i.e., a mixture of 9R-, 9S-, 13R-, and 13S-LOOH would be produced). This together with the isomerization reactions described earlier, could explain the lack of product specificity observed in certain cases. Weber and Grosch (1976) suggest that the extent of cooxidation (and loss of stereospeciflcity) depends on the lifetime of the radical intermediates and the relative efficiency of the LOX-mediated radical reduction. 

One of numerous examples of LOX-catalyzed cooxidation reactions is the oxidation and demethylation of amino derivatives of aromatic compounds. Oxidation of such compounds as 4-aminobiphenyl, a component of tobacco smoke, phenothiazine tranquillizers, and others is supposed to be the origin of their damaging effects including reproductive toxicity. Thus, LOX-catalyzed cooxidation of phenothiazine derivatives with hydrogen peroxide resulted in the formation of cation radicals [40]. Soybean LOX and human term placenta LOX catalyzed the free radical-mediated cooxidation of 4-aminobiphenyl to toxic intermediates [41]. It has been suggested that demethylation of aminopyrine by soybean LOX is mediated by the cation radicals and neutral radicals [42]. Similarly, soybean and human term placenta LOXs catalyzed N-demethylation of phenothiazines [43] and derivatives of A,A-dimethylaniline [44] and the formation of glutathione conjugate from ethacrynic acid and p-aminophenol [45,46],... 

For the COX-1 and COX-2 assay, the diluted solution of the test compound is incubated with COX-1 or COX-2 according to the standard method and then the enzyme reaction is initiated by the addition of [l- C]-arachidonic acid. From the incubation mediate, prostaglandin E2 and prostaglandin D2 (PED2) are extracted to measure radioactivity. Indomethacin is used as a positive control. For the 5-LOX assay, the inhibitory activity of the test compound is evaluated. The test compound is incubated with 5-LOX (46pg of protein) at 24°C for lOmin, and then the enzyme reaction is initiated by the addition of [l- C]-arachidonic acid (50nCi). After 5 min, 4-M formic acid is added to terminate the enzyme reaction. Indomethacin is used as a positive control. 

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