In previous work, methods for analysis of NAEs by GC-MS [Venables et al., 2005
] and phospholipids by direct infusion electrospray ionization mass spectrometry [Welti et al., 2007
] have been developed. Here, in order to comprehensively analyze lipids in the NAE biosynthetic pathway, we developed a method for direct infusion electrospray ionization mass spectrometry analysis of NAPEs. In solvent containing ammonium acetate, NAPEs form positively charged ammonium adducts [M + NH4
by electrospray ionization. By collision induced dissociation, the ammonium adducts of NAPEs can be fragmented into a neutral head group fragment and a charged diradylglycerol (i.e. diacylglycerol or alk(en)yl,acylglycerol) fragment (). Scanning for neutral loss of an ammoniated N
-fatty amide head group in a triple quadrupole mass spectrometer produces a spectrum revealing the NAPE molecular species that contain the fragment, in other words, a spectrum of the NAPE class (). Sequential scanning for neutral loss of ammoniated N
-fatty amide head groups corresponding to each common fatty acid provides a method to detect the molecular species of NAPE in all N
-acyl classes ().
Collision induced fragmentation of the ammonium adduct of N-16:0 PE.
Figure 3 Neutral loss scans targeting analysis of NAPE species with varying N-acyl groups. From top to bottom panels, the scans target N-16:0 PE (NL 396.3), N-18:2 PE (NL 420.3), N-18:1 PE (NL 422.3), N-18:0 PE (NL 424.3), N-20:4 PE (NL 444.3), N-22:6 PE (NL 468.3), (more ...)
To determine whether neutral loss scanning provides an appropriate method for quantification, the linearity of the mass spectral response of the neutral loss scans was investigated (). The signals for two pure NAPEs were determined in relation to the signal from a known quantity of an internal standard, N-17:0 di16:0 PE. The response was determined to be linear, but both N-16:0 di16:0 PE and N-20:4 di18:1PE produced somewhat less signal per mole than N-17:0 di16:0 PE. This suggests that different NAPE molecular species vary somewhat in their ability to ionize and/or to undergo fragmentation under particular fixed mass spectral conditions. On the other hand, a spike-in experiment in which pure NAPE species were added to a biological mixture of lipids showed that their mass spectral signals, normalized to the N-17:0 di16:0 PE internal standard, were approximately proportional to the amount of each NAPE species added (). Finally, varying amounts of the biological sample while holding the level of internal standard constant resulted in normalized mass spectral responses proportional to the amount of sample added (). Taken together, the data in through 6 show that, although response factors for individual NAPE molecular species vary (), the NAPE analysis provides a reliable means to compare levels of NAPE species among samples.
Figure 4 Linearity of the mass spectral response. Varying amounts of N-16:0 di16:0 PE and N-20:4 di18:1 PE were combined with 200 pmol of internal standard N-17:0 di16:0 PE. Neutral loss signals for each NAPE molecular species are presented in relation to the (more ...)
Figure 5 The ability of ESI-MS/MS lipid profiling to detect small changes in NAPE molecular species. The lipid species in roughly 10 mg FW of mouse brain were analyzed and, to demonstrate the ability of the lipid profiling methodology to detect changes, small, (more ...)
Figure 6 Variation in mass spectral response with variation in amount of biological sample. Varying amounts of mouse brain extract were combined with 200 pmol of internal standard N-17:0 di16:0 PE. The amounts of mouse brain extract corresponded to approximately (more ...)
Endocannabinoid metabolism is influenced by both phospholipase D (PLD)-mediated hydrolysis of NAPE and FAAH-mediated breakdown of NAEs (). We examined the effect of FAAH disruption on the content and composition of NAE and various phospholipids, including NAPE and PE, in brain and heart tissue of wild type and FAAH −/− mice. Total levels are summed from the individual molecular species. The data show that lipid content was generally higher in brain than in the heart tissue (). Compared to brain, heart phospholipids had little PS or PA. Both total NAE and PE content were significantly elevated in brain tissue of FAAH −/− mice compared to the wild type controls. On the other hand, in heart tissue there was a significant increase only in PE, PI, and lysoPC content, while NAE content was the same between FAAH −/− and control mice.
Figure 7 Endogenous phospholipids and ethanolamine-containing NAE metabolites. Total lipid extracts of brain and heart tissue of wild type (WT, black bars) and FAAH −/− (KO, hatched bars) mice were analyzed for phospholipid, including PE and NAPE (more ...)
To examine the relationship between NAE composition and the NAPE precursor pool, NAPEs were quantified according to N
-acyl head group and these NAPE classes were compared to the principal NAE types. Absolute quantities of NAPE and NAE and their composition were quite different between brain and heart tissue of mice ( and ). The significantly higher level of NAE in the brain tissue of FAAH −/−
mice, as compared to wild type mice, was attributable mostly to 16:0 NAE (); the level of N
-16:0 PE was also significantly higher in the brain tissue of FAAH −/−
mice, as compared to wild type mice. The concentration of 18:0 NAE species was higher in brain tissue of FAAH −/−
mice as well. In contrast to predominant 16:0 NAEs and N
-16:0 PEs in brain, heart tissue did not reveal a prevalent NAPE class or NAE type. Murine hearts showed no differences between FAAH −/−
and wild type animals in NAE types or NAPE classes. This suggests that FAAH disruption has a considerably greater effect on steady-state levels of endocannabinoid pathway metabolites in brain tissue than in heart tissue. Major differences in steady-state anandamide levels in brain extracts between FAAH −/− mice and wildtype littermates were not evident in our studies, which was inconsistent with previous reports quantifying 15-fold higher anandamide levels in brain tissues of FAAH−/− mice compared to FAAH +/+ mice (Clement et al., 2003 Cravatt and Lichtman, 2004
). Differences may be due to organ preparation (euthanasia rather than decapitation) and/or tissue extraction procedures since anandamide levels seem to be particularly sensitive to preparation methods (Muccioloi and Stella, 2008). Nonetheless, the principal saturated NAE types (NAE18:0 and NAE16:0) quantified in our samples showed a significant elevation in brain tissues of knockout mice as expected ().
Figure 8 Brain NAE species (dark gray bars) and NAPE classes (hatched bars) in wild type (WT) and FAAH −/− (KO) mice. N = 4 or 5 ± SD. “X” indicates species that were not determined. Significance (P < 0.05) was determined (more ...)
Figure 9 Heart NAE species (dark gray bars) and NAPE classes (hatched bars) in wild type (WT) and FAAH −/− (KO) mice. N = 4 or 5 ± SD. “X” indicates species that were not determined. Significance (P < 0.05) was determined (more ...)
Although useful for visualizing potential NAE precursors, grouping of NAPE species by common N-acyl chains ( and ) does not reveal the remarkable complexity of NAPEs. Therefore, a detailed molecular species profile of NAPE was generated for brain and heart tissue of wild type and FAAH −/− mice (Figs. and ). In brain tissue of mice, the most abundant molecular species with 16:0 at the N-position contained PE 36:2, 36:1, 38:6, 38:4, and 40:6, where the species are indicated by total acyl carbons:total carbon-carbon double bonds in the combined acyl chains in the 1 and 2 positions on the glycerol (). Many N-16:0-containing species were significantly elevated in knockout mice compared to wild type, suggesting that the elevation of the N-16:0 PE molecular species in FAAH −/− mice affected those molecular species already prevalent in wild type mice. N-18:2-containing NAPE molecular species were much less abundant overall; only 5 molecular species were quantified at more than 0.1 nmol/g FW and none of these were elevated in FAAH −/− brain tissue (). N-18:1 and N-18:0 molecular species were somewhat more abundant and distributed among diradyl species in a manner similar to N-16:0 PEs; several molecular species were significantly higher in the FAAH −/− knockout tissues (). The anandamide-containing (N-20:4) NAPE pool was relatively minor in terms of overall abundance, and this subgroup showed no differences between wild type and FAAH −/− in terms of quantity or composition. Similarly the N-22:6 PE molecular species were not very abundant, and the N-22:5 PE class was very minor in brain tissue, with only a few molecular species identified in this subgroup. There were no significant differences in N-22:6 and N-22:5 PE species between wild type and FAAH −/− mice.
Figure 10 Detailed profile of NAPE molecular species from brain tissue of wild type (WT) and FAAH −/− (KO) mice. Designations for diradyl molecular species are: numbers only represent diacyl species as total acyl carbons: total carbon-carbon double (more ...)
Figure 11 Detailed profile of NAPE molecular species from heart tissue of wild type (WT) and FAAH −/− (KO) mice. Designations for diradyl molecular species are: numbers only represent diacyl species as total acyl carbons: total carbon-carbon double (more ...)
Similar to brain tissue, murine heart tissue showed complexity in the molecular species composition of NAPE (). Some differences were immediately evident between heart and brain NAPE molecular species. First, the content of all NAPE molecular species in heart tissue was two- to four-fold less than in the brain tissue, and, as suggested from the NAPE class data in , there was no difference in molecular species content of any NAPE type in heart tissue of FAAH −/− mice compared to wild type ().
Comparisons of PC and PE compositions of brain and heart ( and ) show that the diradyl compositions of these two major phospholipid classes are quite different from each other. In brain, 40:6 PE was the most abundant diradyl species for N-acylation with 36:1, 38:6, and 38:4 species also being prominent (). In heart, 40:6 and 38:6 were most prominent with 38:4 and 36:4 also abundant (). These NAPE compositions resemble the PE compositions ( and ), supporting the notion that NAPE is derived from PE ().
Figure 12 PC and PE species from brain tissue of wild type (WT) and FAAH −/− (KO) mice. Designations for diradyl molecular species are: numbers only represent diacyl species as total acyl carbons: total carbon-carbon double bonds; e before a number (more ...)
Figure 13 PC and PE species from heart tissue of wild type (WT) and FAAH −/− (KO) mice. Designations for diradyl molecular species are: numbers only represent diacyl species as total acyl carbons: total carbon-carbon double bonds; e before a number (more ...)