We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects. Cell incubation with AA (0.3-150 μM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry. Maximal amounts of 12-HETE were 126 ± 21 pmol/106 cells (± SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 μM AA. Two epoxyeicosatrienoic acids derived from AA were also identified. Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects. Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that >99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets. Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions. The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.
|Number of pages||8|
|Journal||Journal of Clinical Investigation|
|State||Published - 1989|