Oxidative metabolism of a fatty acid amide hydrolase-regulated lipid, arachidonoyltaurine

Abstract

A novel class of lipids, N-acyltaurines, was recently discovered in fatty acid amide hydrolase knockout mice. In some peripheral tissues, such as liver and kidney, N-acyltaurines with long, polyunsaturated acyl chains are most prevalent. Polyunsaturated fatty acids are converted to a variety of signaling molecules by cyclooxygenases (COXs) and lipoxygenases (LOXs). The ability of COXs and LOXs to oxygenate arachidonoyltaurine was evaluated to gain insight into the potential metabolic fate of N-acyltaurines. Although arachidonoyltaurine was a poor substrate for COXs, mammalian 12 S- and 15 S-LOXs oxygenated arachidonoyltaurine with similar or better efficiency than arachidonic acid. Products of arachidonoyltaurine oxygenation were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The positional specificity of single oxygenation was retained for 15 S-LOXs. However, platelet-type 12 S-LOX produced 12- and 15-hydroxyeicosatetraenoyltaurines (HETE-Ts). Furthermore, LOXs generated dihydroxyeicosatetraenoyltaurines (diHETE-Ts). Metabolism of arachidonoyltaurine by murine resident peritoneal macrophages (RPMs) was also profiled. Arachidonoyltaurine was rapidly taken up and converted primarily to 12-HETE-T. Over prolonged incubations, RPMs also generated small amounts of diHETE-T. Oxidative metabolism of polyunsaturated N-acyltaurines may represent a pathway for the generation or termination of novel signaling molecules.

Figure 1

Structures of arachidonoyl derivatives.

Figure 2

Concentration-dependence of arachidonoyltaurine metabolism by pl12-LOX. Maximal rate of metabolism of arachidonoyltaurine (0.78 to 50 μM) by pl12-LOX (closed circles) was determined by monitoring change in absorbance at 236 nm. Due to substrate inhibition, data did not fit hyperbolic Michaelis-Menten equation; v_max/K_M was estimated from linear regression (dashed line) at low substrate concentrations. Substrate inhibition was observed at high concentrations of arachidonoyltaurine, but could be partially alleviated by co-incubation with 13-HpODE (open square). All data points represent the average ± SEM of at least two separate trials.

Figure 3

MS and CID of arachidonoyltaurine in negative ion mode. A, Full scan MS of arachidonoyltaurine standard. B, CID of m/z 410. The structure of arachidonoyltaurine and proposed fragmentation is also shown.

Figure 4

UV chromatographic profiles of in vitro arachidonoyltaurine oxidation by LOXs. Substrate was incubated with each indicated enzyme for 15 minutes. Hydroperoxides were reduced in situ by addition of sodium dithionite, and products extracted and analyzed by LC-UV-MS. Chromatograms at 236 nm showed similar distribution of products as observed in the selected ion chromatograms at m/z 426.

Figure 5

MS and CID of monoxygenated products of arachidonoyltaurine metabolism in vitro. A, Full scan MS of product at 5.4 min from incubation of arachidonoyltaurine with r15-LOX-1. Products at 5.4 min and 5.6 min from enzymatic incubations afforded similar full scan MS. B, CID of product at 5.4 min. C, CID of product at 5.6 min. The proposed structure and fragmentation are also shown; ions in italics were also observed for arachidonoyltaurine (refer to Fig. 3).

Figure 6

Selected ion chromatograms of dioxygenated products (m/z 442) of in vitro arachidonoyltaurine oxidation by LOXs. Substrate was incubated with each indicated enzyme for 15 minutes. Hydroperoxides were reduced in situ by addition of sodium dithionite, and products extracted and analyzed by LC-UV-MS. The extracted chromatograms for m/z 442 are shown, and the absolute ion intensity is shown at the left of each panel. Absolute ion intensities for HETE-T were on the order of 3-8×10⁷ for each enzyme.

Figure 7

Chromatographic profiles of RPMs incubated with arachidonoyltaurine. RPMs were incubated with arachidonoyltaurine for either 30 s (A) or 15 min (B). UV chromatogram at 236 nm and selected mass ranges for arachidonoyltaurine and its mono- and di-oxygenated products are shown. The absolute ion intensity is noted for each mass range.

Figure 8

Proposed pathway of arachidonoyltaurine metabolism in RPMs. Arachidonoyltaurine is rapidly converted to 12-HETE-T by a 12/15-LOX. Following prolonged incubation, a diHETE-T is observed. DiHETE-T may arise from secondary oxidation of 12-H(p)ETE-T.