Synaptamide biosynthesis monitored by stable isotope labeling and high resolution mass spectrometry

Authors

• KR KEVALA
• M LAGARDE
• AA SPECTOR
• HY KIM

Abstract

Synaptamide is an N-acylethanolamide derived endogenously from docosahexaenoic acid (DHA), and shows potent neurogenic, synaptogenic and anti-inflammatory activity. It is structurally similar to the endocannabinoid N-arachidonylethanolamine (anandamide) which is biosynthesized via N-acylphosphatidylethanolamine (NAPE), specifically N-arachidonylphosphatidylethanolamine (NArPE). Structural similarity suggests that synaptamide may also use N-docosahexaenoylphosphatidylethanolamine (NDoPE) as an intermediate for its biosynthesis. This putative precursor of synaptamide has been found in rat brains. NArPE is believed to be biosynthesized via transfer of the arachidonyl group in the sn-1 position of phosphatidylcholine (PC) to phosphatidylethanolamine (PE). In this study, we quantitatively examined the time course of both NDoPE and synaptamide biosynthesis from an equimolar (2 micromolar) mixture of uniformly 13C labeled DHA (U-13C-DHA) and either the biomimetic compound 1-acetyl, 2-DHA-PC (AcDoPC) or 1-DHA, 2-lyso-PC (lyso-DHA-PC) in mouse neuroblastoma cell culture by high-resolution mass spectrometry. Cells were found to produce significantly larger amounts of both NDoPE and synaptamide from free DHA compared to that produced from either AcDoPC or lyso-DHA-PC after 1, 4, and 8 hours of incubation. The results suggest that NDoPE may be an intermediate for synaptamide production. They also show that, under the conditions studied, free DHA is a better substrate for NDoPE production than the putative sn-1 DHA-PC type precursor compounds.

Scientific Focus Area: Molecular Biology and Biochemistry

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