Figure 2.

Identification and biosynthesis of NADA in brain. (a) Identical mass spectra of NADA and material in bovine striatal extract. Standard and extract were analyzed in negative-ion, product-ion scanning mode by using a triple-quadrupole mass spectrometer. (b) Purified extract of bovine striatum and synthetic NADA were treated with acetic anhydride to form N-arachidonoyl-3,4-diacetoxyphenylethylamine (di-acetylNADA). Multiple-reaction monitoring on a triple-quadrupole mass spectrometer revealed coeluting peaks with molecular/fragment ions 524/196 atomic mass units. (c) Quadrupole time-of-flight MS analysis of material in the brain extract in positive-ion mode yielded a mass estimate of 440.3189, which is within 4.6 ppm of the mass of NADA. Exact masses of fragment ions permitted reconstruction of NADA as shown in d. (e) Distribution of NADA (pmol/g wet weight tissue, means ± SEM, n = 4–12) in central and peripheral nervous system. hippoc., hippocampus; cerebell., cerebellum; thal., thalamus, mid., midbrain. (f) Biosynthesis of [³H]NADA from [³H]arachidonic acid (50 μM) and tyrosine (50 μM) in rat brain homogenates. Lipids from different incubates (boiled homogenate, enzymatically active homogenate, and homogenate in the presence of the catechol-O-methyltransferase inhibitor OR-486 at 30 μM (a concentration that blocks the conversion of tyrosine into dopamine) were extracted and purified by thin layer chromatography. A peak sensitive to OR-486 with the same R_f as synthetic NADA was scraped from the plate and reanalyzed under different eluting conditions (Inset). Similar results (except for the effect of OR-486) were obtained when incubating [³H]arachidonic acid (50 μM) and dopamine (50 μM). The data are representative of three experiments.