DOCOSAHEXAENOIC ACID CAUSES ACCUMULATION OF FREE ARACHIDONIC-ACID IN RAT PINEAL-GLAND AND HIPPOCAMPUS TO FORM HEPOXILINS FROM BOTH SUBSTRATES

Hepoxilins (Hx) are biologically active metabolites of arachidonic aci d (AA) formed regioselectively from 12(S)-HPETE by 'hepoxilin synthase '. Hx modulate synaptic neurotransmission in hippocampal CA1 neurons, and inhibit norepinephrine release in hippocampal slices. During the c ourse of our studies we investigated whether docosahexaenoic acid (DHA ) was a substrate for hepoxilin formation. We used two tissues, the pi neal gland and hippocampal slices. Tissues were incubated alone or wit h AA (20 mu g/ml) or DHA (20 mu g/ml). After 60 min at 37 degrees C, s amples were acid-extracted to convert Hx into their stable trioxilin ( TrX) form and analyzed as the Me-TMSi derivatives by EI-GC/MS to deter mine the structures of the DHA metabolites, and as PFB-TMSi derivative s by GC/MS in the NICI mode using SIM to simultaneously quantify TrX p roducts of the 3-series (derived from AA) monitored at m/z 569, while those of the 5-series (derived from DHA) were monitored at m/z 593, Re sults show good conversion of both substrate fatty acids by the rat pi neal gland and hippocampal slices, into the 3-series (21.3 +/- 5.8 and 12.5 +/- 2.2 ng/mu g protein, respectively) and 5-series TrX (12.3 +/ - 2.7 and 2.9 +/- 0.4 ng/mu g protein, respectively), Surprisingly tho ugh, experiments with DHA, in both tissues, also showed formation of T rX derived from endogenous AA (3-series) (10.4 +/- 8.3 and 3.1 +/- 2.1 ng/mu g protein, respectively). These experiments demonstrate previou sly unreported actions of DHA causing the accumulation of AA, which is converted into hepoxilins. In order to prove that AA is accumulated d uring DHA stimulation of the tissue? we carried out separate experimen ts with hippocampal slices in which the neutral lipids and phospholipi ds were labeled with [C-14]AA. DHA caused a time-dependent appearance of free [C-14]AA which was released mostly from the TG pool. Measureme nt of the AA/DHA ratio in the TG pool by GC/MS further indicated that DHA is incorporated into the TG at the expense of AA. These results de monstrate that DHA competes with AA for acylation into the metabolical ly active TG fraction, and both fatty acids are converted into hepoxil ins of the corresponding series.