|Changes in N-acylethanolamine Pathway Related Metabolites
in a Rat Model of Cerebral Ischemia/Reperfusion
|Aruna Kilaru1,¶,#, Pamela Tamura2#, Puja Garg3,4#, Giorgis Isaac2,§, David Baxter1, R. Scott Duncan3,4, Ruth Welti2, Peter Koulen1,3,4, Kent D.Chapman1 and Barney J. Venables1*
|1University of North Texas, Center for Plant Lipid Research, Department of Biological Sciences, Denton, TX 76203
|2Kansas State University, Kansas Lipidomics Research Center, Division of Biology, Manhattan, KS 66506
|3University of Missouri – Kansas City, School of Medicine, Vision Research Center, Kansas City, MO 64108
|4University of Missouri – Kansas City, School of Medicine, Departments of Basic Medical Science and Ophthalmology, Kansas City, MO 64108
|¶current address: Michigan State University, Department of Plant Biology, East Lansing, MI 48824
|§current address: Pacific Northwest National Laboratory, PO Box 999, MSIN: K8-98, Richland, WA 99352
||Dr. Barney J. Venables
University of North Texas, Center for Plant Lipid Research, Department of Biological Sciences, Denton
TX 76203,1155 Union Circle #305220
|Received October 08, 2010; Accepted November 16, 2010; Published November
|Citation: Kilaru A, Tamura P, Garg P, Isaac G, Baxter D, et al. (2011) Changes
in N-acylethanolamine Pathway Related Metabolites in a Rat Model of Cerebral
Ischemia/Reperfusion. J Glycom Lipidom 1:101. doi:10.4172/2153-0637.1000101
|Copyright: © 2011 Kilaru A, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
|In mammals, the endocannabinoid signaling pathway provides protective cellular responses to ischemia. Previous
work demonstrated increases in long-chain N-acylethanolamines (NAE) in ischemia and suggested a protective role for
NAE. Here, a targeted lipidomics approach was used to study comprehensive changes in the molecular composition
and quantity of NAE metabolites in a rat model of controlled brain ischemia. Changes of NAE, its precursors,
N-acylphosphatidylethanolamines (NAPE), major and minor phospholipids and free fatty acids (FFA) were quantified
in response to ischemia. The effect of intraperitoneal injection of N-palmitoylethanolamine (NAE 16:0) prior to ischemia
on NAE metabolite and phospholipid profiles was measured. While ischemia, in general, resulted in elevated levels of
N-acyl 16:0 and18:0 NAE, NAPE and FFA species, pretreatment with NAE 16:0 reduced infarct volume, neurological
behavioral deficits in rats and FFA content in ischemic tissues. Pretreatment with NAE 16:0 did not affect the profiles of
other NAE metabolites. These studies demonstrate the utility of a targeted lipidomics approach to measure complex and
concomitant metabolic changes in response to ischemia. They suggest that the neuroprotective effects of exogenous
NAE 16:0 and the reduction in inflammatory damage may be mediated by factors other than gross changes in brain NAE
levels, such as modulation of transcriptional responses.
|Free fatty acid; N-acylphosphatidylethanolamine;
Lipid signaling; N -palmitoylethanolamine; Phospholipids; Mass
|BSTFA: N,O-Bis(trimethylsilyl)trifluoroacetamide;ePC: alk(en)yl, acylglycerophosphocholine ; ePE: alk(en)yl,acyl
glycerophosphoethanolamine; ESI: Electrospray Ionization; FAAH:
Fatty Acid Amide Hydrolase; FAME: Fatty Acid Methyl Ester;
FW: Fresh Weight; ICA: Internal Carotid Artery; I/R: Ischemia
Reperfusion; MCAO: Middle Cerebral Artery Occlusion; NAE: N-acylethanolamine; NAPE: N-acyl PE; NAT: N-acyltransferase;
PA: diacyl glycerophosphate (Phosphatidic Acid); PC: diradyl
glycerophosphocholine (Phosphatidylcholine); PE: diradyl
glycerophosphoethanolamine (Phosphatidylethanoline); PI: diacyl
glycerophosphoinositol (Phosphatidylinositol); PLD: Phospholipase
D; PPAR: Peroxisome Proliferator-activated Receptor; PS: diacyl
glycerophosphoserine (Phosphatidylserine); SM: Sphingomyelin;
SPE: Solid Phase Extraction; TMS: Trimethylsilyl; TTC:
2,3,5-triphenyltetrazolium Chloride; X:Y designates total fatty acid
carbons: carbon-carbon double bonds or total acyl carbons: total
carbon-carbon double bonds
|Endocannabinoids are trace lipid signaling molecules that are
endogenous ligands of cannabinoid receptors. Several of these
ligands are important lipid mediators that regulate a wide range of
biological processes in vertebrates , invertebrates  and plants
[3,4]. Specifically, the endocannabinoid system plays an important
role in overall biochemical response to ischemia, which results in
nutrient deprivation and accumulation of injurious metabolites
that challenge physiological homeostasis in mammals [see reviews
[1,5,6]]. Previous studies of experimentally induced ischemia in rodent brains both support and in some cases contradict [7,8]
the view that endocannabinoid responses are neuroprotective
[reviewed by ]. Increased free radical production, over-stimulation
of glutamate receptors and elevated intracellular calcium levels
resulting from ischemic damage are thought to be counteracted by
endocannabinoid responses .
|Anandamide, the first endocannabinoid discovered , is an
arachidonic acid-derived member of the N-acylethanolamines (NAE).
NAE are derived from N-acylated phosphatidylethanolamines (PE),
which include molecular species with a variety of saturated and
unsaturated N-acyl groups, as well as a heterogeneous mix of chains in
the diradyl glycerol component [NAPE; reviewed in ]. Anandamide
formation is thought to originate with the transfer of arachidonic
acid from the sn-1 position of precursor phospholipids, such as
phosphatidylcholine (PC), to the amine of PE by N-acyltransferases
[NAT; [12,13]]. Free NAE are produced from their NAPE precursors by
NAPE-phospholipase D (NAPE-PLD) mediated hydrolysis. Free NAE are
rapidly eliminated as signaling sources, likely by transporter-aided cellular reuptake followed by hydrolysis of the NAE; predominantly
fatty acid amide hydrolase (FAAH) terminates NAE signaling, yielding
free fatty acids (FFA) and ethanolamine [14-16].
|Recognition and characterization of the N-acylationphosphodiesterase
pathway, i.e. the endocannabinoid signaling
pathway, originated from the observation of sharp increase in
concentrations of relatively trace lipids, NAPE and NAE, in ischemic
tissues [17-20]. It is now recognized that the role of NAE signaling in
response to ischemia is complex; the behavior of NAPE accumulation
can differ significantly from NAE release and can vary among acyl
groups of varying chain lengths and degrees of saturation . Also,
several studies have demonstrated that the damaging effects of
ischemia can be reduced by exogenous treatment with NAE [21-24].
Among the various NAE species, NAE 16:0 has received particular
attention as having potential neuroprotective effects against ischemic
damage [25-29]. NAE 16:0 is among the most abundant of NAE
molecular species in a wide range of plants and animals [26,30,31]
and it is among the most highly up-regulated in response to a variety
of stimuli, including hypoxia and ischemia .
|A comprehensive study of lipid metabolism is required to
adequately describe the dynamics of interactions among exogenous
addition, storage, release and degradation of various molecular
species that may be of importance in modulation of response to
ischemic stress. Targeted lipidomics approaches focusing on effects
of altered NAE metabolism in mice  and the role of arachidonic
acid  in signaling have recently been conducted. Here we
adopted a lipidomics strategy to measure changes in the molecular
composition and quantity of NAE metabolites and describe lipidomic
changes associated with ischemia in a transient middle cerebral artery
occlusion (MCAO), with and without the exogenous administration
of NAE 16:0. Our analyses include detailed measurement of the
concentrations of individual acyl groups comprising the NAE family
and their NAPE and PE precursors and other [phosphatidylcholine
(PC), phosphatidylserine (PS), sphingomyelin (SM), alk(en)yl,acyl PE
(ether-linked PE or ePE), phosphatidylinositol (PI), phosphatidic acid
(PA), lysoPE, lysoPC and alk(en)yl,acyl PC (ePC)] phospholipid classes.
Finally, we also analyzed the quantity and composition of FFA, since
they are important biochemical indicators of metabolic pathways in
various pathological conditions, such as membrane disintegration
via mechanisms such as deacylation, peroxidation, or free radical
reaction [33, 34] and are products of NAE hydrolysis.
|Materials and Methods
|All NAE standards were purchased from Cayman Chemicals (Ann
Arbor, MI, USA). Fatty acid methyl ester (FAME) standards were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Solvents (HPLC
grade) and N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) were
purchased from Fisher Scientific (Pittsburgh, PA, USA). Other reagents
were of the highest purity commercially available.
|Rats were randomly divided into four experimental groups: (1)
sham-operated group (S) that received mock MCAO surgery and
treatment with vehicle (ethanol), (2) sham-operated group that
received mock MCAO surgery and treatment with 10 mg/kg NAE
16:0 intraperitoneally, (3) ischemia-reperfusion group (I or I/R) that
experienced 90 min of MCAO followed by 24 h of reperfusion and
treatment with vehicle (ethanol) and (4) I/R group that received 10 mg/kg NAE 16:0 intraperitoneally, concomitantly with MCAO surgery.
Brain tissue from ipsilateral (I) and contralateral (C) hemispheres of
each of these groups was analyzed separately, yielding lipidomics
results for the eight tissue sources summarized in (Table 1).
|Rat MCA transient occlusion – a model of brain ischemia
|Male Sprague Dawley rats (Harlan Laboratories, Inc., Indianapolis,
IN, USA) weighing 300-325 g were fed ad libitum and kept in a 22-
25°C temperature-controlled room on a 12 h light/dark cycle. All
animal-related care and procedures conformed with the Public Health
Service Policy on Humane Care and Use of Laboratory animals and
were reviewed and approved by the Institutional Animal Care and Use
Committee at the University of North Texas Health Science Center.
|Brain ischemia reperfusion injury was modeled using transient
MCAO as described by us previously [23,35,36]. In brief, rats were
anesthetized with a ketamine/xylazine cocktail (60 and 10 mg/kg body
weight, respectively) and body temperature was maintained at 37 ±
0.5°C throughout surgery. After exposing the left common carotid,
the left external carotid and the left internal carotid artery (ICA), a
monofilament nylon suture (3-0; Ethilon; Ethicon Inc., Sommerville,
NJ, USA) was introduced through a puncture into the lumen of the
ICA. The suture was advanced until adequate resistance was felt to
accomplish MCA occlusion. After ninety minutes of MCAO, the suture
was withdrawn, followed by 24 h of reperfusion. Sham surgeries
were identical except no suture was inserted. After recovery, animals
were kept with free access to water and food. Rats were sacrificed by
pentobarbital overdose at 24 h after ischemia, death was assured by
pneumothorax and the rats were decapitated. Brains were removed
and immediately (within 2 min) flash frozen in liquid nitrogen and
stored at -80°C until extraction.
|Measurement of cerebral infarct volume
|In order to determine cerebral infarct volume, the brains of
identically treated animals were dissected after decapitation.
Brains were placed in ice-cold saline for 5 min after removal. Seven
coronal slices were then sectioned from each brain at a thickness
of 2 mm and sections were incubated for 15 min at 37°C in a 2%
2,3,5-triphenyltetrazolium chloride (TTC) solution. In the stained
sections, infarct area appeared pale-colored and viable areas were
colored pink/red. The infarction volume was calculated according
to Swanson et al. . In brief, the infarct area in each section was
calculated by subtracting the non-infarcted areas of the ipsilateral
side from the total corresponding area of the contralateral side that
was not affected by the I/R injury. Infarction areas on each section
were summed and multiplied by section thickness to compute the
total infarction volume, which was expressed as a percentage of total
brain volume. Computation and analysis for infarct volume in each
brain slice were performed using PCI version 5.3.1 high performance
imaging software (Compix Inc., Cranberry Township, PA, USA).
|Evaluation for neurological deficits, expressed as neurological
deficit scores, was performed on all animals just before euthanasia
and scored as described [23,38] using the following criteria: 0, no
observable deficit (normal); 1, failure to extend left forepaw on lifting
the whole body by tail (mild); 2, circling towards the contralateral
side (moderate); 3, leaning to the contralateral side at rest or no
spontaneous motor activity (severe).
|Rat brain tissues were homogenized in 2 ml hot 2-propanol
(70°C) and 1 ml of chloroform. Samples were vortexed and incubated
on ice for 30 min prior to overnight extraction at 4°C. Monophasic
lipid extracts were partitioned with 2 ml of 1 M KCl. The lower
organic phase was washed three additional times with 1 M KCl
and subsequently dried to completion under nitrogen. The total
lipid content was estimated gravimetrically and the samples were
dissolved in chloroform and stored under nitrogen at -20°C until
|Phospholipid analysis and quantification
|An automated electrospray ionization (ESI)-tandem mass
spectrometry approach was used and sample preparation and
data acquisition were carried out as described previously ,
with modifications. Briefly, an aliquot of 2 µl of brain lipid extract,
equivalent to 0.6-0.8 mg tissue fresh weight (FW), was analyzed.
Precise amounts of internal lipid standards for PC, lysoPC, PE, lysoPE,
PA, PS and PI, as well as solvent, were added.
|Mass spectra were acquired on a triple quadrupole MS system
(API 4000, Applied Biosystems, Foster City, CA, USA). Sequential
precursor and neutral loss scans of the extracts produced a series of
spectra, with each spectrum revealing a set of lipid species, PC, PE,
PA, PS, or PI, containing a common head group fragment. SM was
determined from the same mass spectrum as PC (precursors of m/z
184 in positive mode) [39, 40].
|The data were processed as described . Peaks corresponding
to the target lipids in these spectra were identified, quantified
in comparison to the internal standards in the same lipid class
and corrected for instrument/sample background interference
and isotopic overlap. SM quantification was by comparison with
PC internal standards, using an experimentally determined molar
response factor of 0.39 for SM (in comparison with PC). Data were
reported as nmoles of each detected lipid metabolite/g tissue FW.
|NAPE analysis and quantification
|NAPE analysis and quantification were carried out as described
previously , with modifications. N-17:0 di16:0 PE was synthesized
 and employed as an internal standard to quantify the NAPE
species in the sample extracts. The same rat lipid brain extracts were
used for both phospholipid and NAPE analysis. NAPE standard (1.1
nmoles) and solvent were added to an aliquot of extract equivalent
to 6-10 mg tissue FW.
|Mass spectra were acquired by automated ESI-tandem mass
spectrometry on a triple quadrupole MS system (API 4000 QTrap,
Applied Biosystems). Sequential neutral loss scans produced a
series of spectra, with each spectrum revealing a set of lipid species
containing a common ammoniated N-fatty amide head group
fragment. N-16:0, N-17:0, N-18:2, N-18:1, N-18:0, N-20:4, N-22:6 and
N-22:5 species were detected.
|The data were processed as described . Peaks corresponding
to the target lipids in each N-acyl class (each spectrum) were
identified, quantified relative to the N-17:0 di16:0 PE internal standard
and corrected for instrument/sample background interference and
isotopic overlap. Due to the interfering presence in the N-20:4 scan
of peaks with m/z inconsistent with NAPEs in the m/z range of N-20:4-
36:4, N-20:4-36:2, N-20:4-38:6, N-20:4-38:5, N-20:4-40:7, N-20:4-
40:5 and N-20:4-40:4 NAPEs, these compounds were not measured. Data were reported as mass spectral signal normalized to N-17:0
di16:0 PE/g tissue FW; the amount of signal produced by 1 nmol
of N-17:0 di16:0 PE is 1. Data were evaluated for possible outliers
using the Q-test  on NAPE lipid class totals; one sham-operated
contralateral (SC) rat brain replicate (out of 4) was determined to be
an outlier, most likely due to sample mishandling and was removed
from calculations. No response correction factors were employed for
potentially different mass spectral responses of various N-acyl chains
. NAPE lipidomic data, thus, allow comparison of samples, but
may not represent the relative amounts of various molecular species.
|NAE extraction and quantification
|NAE extraction was conducted as described previously .
Each brain hemisphere was removed and homogenized in ice-cold
chloroform using a glass tissue grinder. The homogenate was
sonicated (60 W) for 2 min to disrupt cells. Approximately 500 mg
of tissue was then added to 10 ml of ice-cold chloroform containing
deuterated NAE standards (D4-NAE 16:0 and D4-NAE 20:4, 50
ng each). Lipids were extracted by Folch extraction (chloroform/
methanol/water, 4:2:1) by the addition of 5 ml cold methanol and 2.5
ml cold PBS buffer and sonication (60 W) in an ice-cold water bath for
10 min, followed by centrifugation. The organic phase was collected
for further purification by solid phase extraction (SPE).
|Silica SPE cartridges (100 mg, 1.5 ml; Alltech, Deerfield, IL, USA)
were conditioned with 2 ml methanol and 4 ml chloroform and
subsequent to loading the sample, the column was washed with
2 ml chloroform and NAE were eluted with 2 ml of ethyl acetate/
acetone (1:1). The eluate was collected, evaporated under nitrogen
and derivatized with 50 µl BSTFA and 25 µl dichloromethane for 30
min at 55°C. After derivatization, the samples were again evaporated
under nitrogen and reconstituted in 50 µl hexane.
|NAE were identified via selective ion monitoring and quantified
against the internal deuterated standards (saturated species against
deuterated NAE 16:0 and unsaturated species against deuterated NAE
20:4) as trimethylsilyl (TMS)-ether derivatives by GC-MS (Model 6890
GC coupled with a 5973 mass selective detector; Agilent, Wilmington,
DE, USA), as described previously . NAE concentration was
calculated based on FW.
|Total lipid extracts were combined with 50 µg of heptadecanoic
acid (17:0), dried under nitrogen and derivatized with 400 µl methanol
and 6.5 µl 2 M trimethylsilyldiazomethane . The reaction mixture
was shaken vigorously for 30 min at room temperature, the reaction
was terminated with the addition of 0.2 µl concentrated acetic
acid and the solvent was evaporated under nitrogen. FAME were
solubilized in 10 µl acetonitrile and 2 µl were analyzed by GC (Agilent
6890) using flame ionization detection and a capillary DB-23 column
(30 m x 0.25 mm; 0.25 µm film thickness; J&W Scientific, Agilent).
Helium was used as a carrier gas at a flow rate of 1 ml/min and the
temperature gradient was as follows: 150°C for 1 min, 150 – 200°C
at 8°C /min, 200 – 250°C at 25°C /min and 250°C for 6 min. FAME
quantification was based on FAME 17:0 as the internal standard.
|In all of the lipid analyses experiments, N = 4 (for NAE
quantification, N = 6) and the data were expressed as means ±
SD. Statistical differences between contra- and ipsilateral, vehicle
and treatment and ischemia and sham tissues were determined by
comparing the means, using the two-tailed, unpaired Student’s t-test and significance (P < 0.05) was indicated by ‘a’, ‘b’ and ‘c’ respectively.
Histochemistry and neurological assessment data were plotted as
the means ± SE. The determination of statistical significance (P
< 0.05) was performed with ANOVA and post-hoc Newman Keuls
and Bonferroni multiple comparison tests and GraphPad Prism 4
|The major metabolites in the endocannabinoid pathway include
PE, NAPE, NAE and FFA. In order to understand ischemia-induced
changes in NAE metabolite content and composition, we used
a targeted lipidomics approach and conducted comprehensive
analyses of total lipid extracts from the contralateral and ipsilateral
brain tissues of rats. Ischemia was confirmed in all of the animals
that received reperfusion injury, by neurological evaluation (Figure
1A) and histochemistry (Figure 1B). Sham-operated rats that had
been treated with vehicle or NAE 16:0 showed no ischemic lesions
or neurological deficits (data not shown; reported in ). The range
of neurological deficit scores was between 2 and 3 in vehicle-treated
ischemic rats, corresponding to a medium to severe neurological
deficit phenotype (Figure 1A). These animals showed extensive
infarction in the cortical and subcortical areas, as determined by vital
staining with TTC in coronal brain slices (Figure 1B). The neurological
deficit score of I/R NAE 16:0-treated rats improved significantly to a
range of 0-1 (Figure 1A) and the data were confirmed by significant
reduction of ipsilateral infarct volume (Figure 1B).
||Figure 1: Assessment of neurological deficit scores and histochemistry confirm
that NAE 16:0 protects against ischemic brain damage following MCAO I/R.
(A) MCAO-mediated I/R caused moderate to severe neurological deficits, as
measured with the standardized neurological deficit score, and treatment with
NAE 16:0 reduced neurological deficits significantly (phenotype: none to mild).
(B) The size of the ipsilateral ischemic lesion volume (infarct volume) was
significantly reduced by NAE 16:0 treatment. Data are shown as mean ± SE. aP
< 0.05, compared with I/R vehicle-treated group as determined using one-way
ANOVA with Newman Keuls multiple comparison post-hoc test.
|Lipids extracted from the contralateral and ipsilateral brain
regions of the I/R and sham-operated rats which had been treated
with vehicle or NAE 16:0 (Table 1) were analyzed for changes in NAE
metabolites. Levels of total NAE and their precursor ethanolamine containing
lipid classes, NAPE and PE and FFA, were determined by
summing the analyzed individual molecular species concentrations within each lipid class (Figure 2). The total amount of PE in contra
and ipsilateral tissues showed no significant change associated
with ischemia or treatment. In contrast, ischemia with or without
treatment resulted in an approximate 3-fold increase in total NAPE
and an order of magnitude increase in total NAE, only in the ipsilateral
brain tissues (II vs. IC groups, Figure 2), compared to shams (II vs. SI
groups, Figure 2). Prior administration of NAE 16:0, which resulted
in reduced neurological deficit score and infarct volume (Figure 1),
had no effect on endogenous PE, NAPE, or NAE content of ischemic
tissues but resulted in a two-fold decline in total FFA content in the
ipsilateral tissue (II-NAE 16:0 vs. II-Vehicle, Figure 2) and an increase
in sham-operated contralateral tissue (SC-NAE 16:0 vs. SC-Vehicle, Figure 2). The total FFA content was two-fold higher in ischemic
tissues that did not receive NAE 16:0 treatment, compared to sham-operated tissues (IC- and II-Vehicle groups compared with SC- and
SI-Vehicle groups, respectively, Figure 2).
||Figure 2: Ischemia/reperfusion- and sham-operation-induced changes in total
PE, NAPE, NAE, and FFA content in contralateral (IC and SC) and ispsilateral
(II and SI) brain tissues of rats treated with vehicle or exogenous NAE 16:0.
Data are shown as mean ± SD. Statistical significance (P < 0.05; N = 4 (for
NAE samples N = 6)) between contra- and ipsilateral tissues is indicated by ‘a’,
and vehicle and NAE 16:0 treatment by ‘b’ and ischemia and sham tissues by
‘c’ as determined by unpaired Student’s t-test. NAPE values indicate relative
normalized mass spectral signal per g of sample FW, where 1 unit of signal
indicates the amount of signal that is observed for 1 nmol of the internal
standard, N-17:0 di16:0 PE; all other lipid classes are expressed as nmol/g FW.
||Table 1: Identification of experimental groups.
|The changes in acyl-chain composition of NAPE, NAE and FFA, in
response to ischemia, were analyzed (Figure 3). All three metabolite
groups were dominated by the 16:0, 18:0 and 18:1 chains. Arachidonic
acid (20:4) represented a larger fraction of FFA than of the N-acyl chains
in NAPE or NAE. The dominant acyl groups, 16:0, 18:0 and 18:1, were
also the molecular species that primarily contributed to the higher levels in total NAPE and NAE content in ischemic ipsilateral and not
contralateral preparations as compared to shams (II vs IC groups and
II vs. SI groups in Figure 2 and Figure 3). In sham-operated tissues,
the content of various molecular species of NAPE and NAE was low
and similar to that of ischemic contralateral tissues (IC vs. SC groups, Figure 3). However, similar to ischemic groups, the major NAPE and
NAE species in shams were higher in the ipsilateral tissues compared
with contralateral (SI vs. SC groups, Figure 3). The abundance of FFA
16:0 and 18:0 also was, in general, low in sham-operated tissues of
vehicle-treated rats; however, the levels increased by two- to threefold
in rats that were treated with NAE 16:0 (SC- and SI- NAE 16:0
vs. SC- and SI-Vehicle, Figure 3). Ischemia induced increases in FFA
16:0 and 18:0 content in both contra- and ipsilateral tissues, but
these increases tended to be reduced in rats that were pre-treated
with NAE 16:0 (II-NAE 16:0 vs II-Vehicle, Figure 3). Ischemia-related
changes observed in the polyunsaturated species, N-linked 18:2, 20:4
and 22:6 of NAPE and NAE and 18:2 and 20:4 of FFA, were not as
dramatic as in the saturated and mono-unsaturated species and their
contribution to total accumulation of metabolite was small.
||Figure 3: Total lipid extracts from ischemia/reperfusion or sham-operated
contralateral (IC and SC) and ipsilateral (II and SI) brain tissues of rats that were
treated with vehicle or exogenous NAE 16:0 were analyzed for the composition
of NAPE, NAE, and FFA species, differentiated by their acyl chains. Data are
shown as mean ± SD. Statistical significance (P < 0.05; N = 4 (for NAE samples
N = 6)) between contra- and ipsilateral tissues is indicated by ‘a’, and vehicle and
NAE 16:0 treatment by ‘b’, and ischemia and sham tissues by ‘c’ as determined
by unpaired Student’s t-test. NAPE values indicate relative normalized mass
spectral signal per g of sample FW, where 1 unit of signal indicates the amount
of signal that is observed for 1 nmol of the internal standard, N-17:0 di16:0
PE; NAE and FFA lipid classes are expressed as nmol/g FW. Acyl chain
designations are indicated by x:y, or carbon chain length:number of double
|In order to further characterize the ischemia-induced changes in
NAPE, NAPE sharing a common N-acyl group were characterized in
terms of the total acyl carbons and carbon-carbon double bonds in
the two chains linked to glycerol. Mass spectral signals for NAPE were
assigned to molecular species based on the mass of the intact ion
and an N-acyl-containing fragment (major contributing NAPE, Figure
4; minor contributing NAPE, Figure 5). The distribution patterns of
individual NAPE species with 16:0, 18:0 and 18:1 at the N-position
(Figure 4) and those with 18:2, 20:4 and 22:6 at the N-position (Figure
5) were all similar and were dominated by diacyl species 40:6, 38:4,
38:6 and 36:1. Once again, as seen in Figure 3, ischemia-related
increases in the dominant NAPE species are evident in the ipsilateral
tissue (II vs. IC groups, Figure 4 and Figure 5). Molecular species
with 1-alk(en)yl, acyl linkages were present only at low amounts in
the NAPE profiles (data not shown). There was no apparent effect of
exogenous NAE 16:0 treatment on the NAPE profiles.
||Figure 4: Comprehensive analysis of ischemia/reperfusion-induced levels
of NAPE molecular species with acyl chains N-16:0, N-18:0 and N-18:1, in
contralateral (IC) and ipsilateral (II) brain tissue of groups treated with vehicle
or exogenous NAE 16:0. Data are shown as mean ± SD and N = 4. NAPE
molecular species labels indicate the diacyl component (total acyl carbons: total
carbon-carbon double bonds) of each detected NAPE species. NAPE species
mass spectral signals were normalized against the signal, assigned to be 1 unit,
observed for 1 nmol of the internal standard, N-17:0 di16:0 PE.
||Figure 5: Comprehensive analysis of ischemia/reperfusion-induced levels
of NAPE molecular species with acyl chains N-18:2, N-20:4, and N-22:6, in
contralateral (IC) and ipsilateral (II) brain tissue of groups treated with vehicle
or exogenous NAE 16:0. Data are shown as mean ± SD and N = 4. NAPE
molecular species labels indicate the diacyl component (total acyl carbons: total
carbon-carbon double bonds) of each detected NAPE species. “ND” indicates
molecular species that were not determined (see Experimental Procedures).
NAPE species mass spectral signals were normalized against the signal,
assigned to be 1 unit, observed for 1 nmol of the internal standard, N-17:0
|Finally, major and minor phospholipid class concentrations were
compared among the various treatment groups (Figure 6). Estimates
of the total phospholipid content of each treatment group are
presented in (Table 2). Ischemia, with or without pretreatment with
NAE 16:0, had no effect on any of the major and minor phospholipids
examined, in both contra- and ipsilateral tissues (IC- and II-Vehicle/
NAE 16:0 vs. SC- and SI-Vehicle/NAE 16:0 groups, Figure 6). Absence
of an ischemia/reperfusion-induced change in phospholipid patterns
may have been due to sampling too early to detect the change or to
ischemic damage too small to be reflected in statistically significant
changes in phospholipid pattern. Further examination of specific
molecular species that contributed to PE indicated a profile similar
to that of NAPE molecular species distribution with 40:6, 38:4, 38:6,
40:4 and 36:1 being the predominant diacyl species, which did not
change substantially in response to ischemia (Figure 7). A complete
presentation of results for all phospholipid molecular species is
provided as Supplemental Data (Table 3).
||Figure 6: Quantification of major and minor classes of phospholipids in
ischemic contralateral and ipsilateral tissues (IC and II) that were treated
with vehicle or exogenous NAE 16:0, compared with sham-operated tissues
(SC and SI). Data are shown as mean ± SD and N = 4. Abbreviations:
phosphatidylethanolamine (PE), alk(en)yl,acyl glycerophosphoethanolamine
(ePE), phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine
(PS), phosphatidylinositol (PI), lysophosphatidylethanolamine (lysoPE), alk(en)
yl,acyl glycerophosphocholine (ePC), lysophosphatidylcholine (lysoPC), and
phosphatidic acid (PA).
||Figure 7: Effect of ischemia/reperfusion injury on levels of PE molecular
species in contralateral (IC) and ipsilateral (II) brain tissue of groups treated with
vehicle or exogenous NAE 16:0, compared with corresponding sham-operated
tissues. Data are shown as mean ± SD and N = 4. PE molecular species labels
indicate the diacyl component (total acyl carbons: total carbon-carbon double
bonds) of each detected species.
||Table 2: Total phospholipid content in the rat brain tissues of experimental groups. Total phospholipid content in the rat brain tissues of experimental groups.
|In a comprehensive review of available information on the
involvement of endocannabinoids in response to cerebral ischemia,
Hillard pointed out the potential importance of increased flux of
NAE through the biosynthetic pathway and the fact that relatively
modest changes reported for anandamide, NAE 20:4, are dwarfed
by consistent and much larger increases in saturated and monounsaturated members of the NAE family, particularly NAE 16:0 .
Our results support this view and offer new insight into the ischemia related
responses of the dominant molecular species of NAE and their
|As shown previously, MCAO I/R produced a robust ipsilateral
lesion as measured with histochemistry and was accompanied by a
significant increase in the neurological deficit scores (; Figure 1).
Also paralleling previous studies, NAE 16:0 protected against ischemic
brain damage both structurally and functionally by significantly
reducing the neurological deficit scores and infarct volume (; Figure 1). Our results also confirm the accumulation of both NAE and NAPE in cerebral ischemia [20,29,44-46], with the relative increase
in NAE greatly exceeding that of the NAPE (; Figure 2). A clear
preferential accumulation of species containing saturated and monounsaturated
N-acyl groups in both NAE and NAPE was observed
(Figure 3) and the molecular species profile of accumulating NAE
closely resembled that of the accumulating NAPE (Figure 4 and Figure
5). This supports the view that PLD hydrolysis of NAPE is non-specific
and that the preferential accumulation of saturated and monounsaturated
NAE is controlled by the availability of NAPE precursors.
NAPE formation via transfer of acyl groups by N-acyltransferases to PE
has been suggested to be both the rate-limiting step of NAE formation
as well as the source of substrate specificity which is responsible for
acyl patterns in both NAE and NAPE [9,12,13].
|We saw no evidence of the accumulation of anandamide (NAE
20:4) or any of its NAPE precursors after 24 hours of ischemia
reperfusion injury (Figure 3 and Figure 5). Other studies have reported increased anandamide concentrations occurring rapidly,
30 min to 5 hours after MCAO [47-49]. This accumulation has been
attributed to evidence that ischemia is accompanied by increased
NAPE levels, increased conversion of NAPE to NAE and decreased
FAAH expression [9,47,48]. These previous studies focused only on
occlusion effects and did not include reperfusion in the experimental
design. There is evidence that the bulk of anandamide accumulation
occurs during reperfusion in both mice  and rats . In contrast,
although our study measured lipid concentrations after 24 hours of
reperfusion after MCAO, we did not observe I/R-related accumulation
of anandamide. Degn et al.  observed the same result in a study
conducted with mice using a time frame similar to ours (24 h postischemia),
in which neither increases in anandamide or changes in
the enzymatic potential for its accumulation were observed.
|Despite various discrepancies noted above, it is clear that NAE
accumulation in ischemia, as well as after ischemia reperfusion injury,
is dominated by saturated and mono-unsaturated species that closely
resemble profiles of the precursor NAPE species. N-acyltransferases
may be responsible for the observed preferential accumulation of
saturated and mono-unsaturated N-acyl groups in NAPE, or perhaps
the pool of acyl groups available for transfer is enriched in these
species. On the other hand, the lack of specificity of PLD may account
for the fact that N-acyl groups of NAE resemble those of NAPE.
Relatively high rates of NAPE hydrolysis compared to NAE catabolism
may be responsible for the observed accumulation of NAE .
|One of the goals of our study was to decipher the implications
of administering NAE 16:0 as an ischemia protectant . Curiously,
neither NAPE nor NAE content was affected by NAE 16:0 treatment; however, the decline noted in the levels of saturated and unsaturated
FFA suggests that the protective role of NAE 16:0 is perhaps
independent of gross changes in NAE content (Figure 3). Typically,
an ischemic energy crisis accelerates calcium influx into the cytosol,
which activates phospholipases and thus the liberation of FFA .
On the other hand, accumulation of NAE in ischemic tissues may
represent a component of cellular defense mechanisms against the
detrimental changes brought about by ischemia and may act as an
inhibitor of calcium uptake and electron transport, which has been
shown to be concentration dependent . Perhaps the 10-fold
increase in NAE content that we reported in response to ischemia
was not sufficient to attenuate the increase in FFA. However, it is
reasonable to speculate that providing exogenous NAE 16:0, although
it did not affect NAE levels at 24 h post-ischemia, may have earlier
contributed to the inhibition of FFA formation and thereby offered
protection to the tissues by preventing production of reactive oxygen
species, destruction of the antioxidant system and inflammation
during ischemia, responses associated with increase in FFA content,
specifically polyunsaturated FFA [52,53]. The functional protective
effects of NAE 16:0 did not extend significantly into the perfusion
period (although structural protection was seen with administration
2 h after occlusion) in an earlier study . A possible mechanism
for this protective effect lies in peroxisome proliferator-activated
receptor (PPAR)-mediated anti-inflammatory responses .
PPAR-alpha is considered a molecular target of NAE 16:0 and 18:1
[55,56] and its activation has been shown to be critical in cerebral
infarct volume reduction produced by exogenous administration of
NAE 18:1 in mice . Certainly many other possible mechanistic
explanations exist for neuroprotection, including other pathways
involving oxidative signals and this will be resolved only with further
|In conclusion, our targeted, but unbiased lipidomics approach has
provided insights and alternatives into understanding the complexity
of biochemical events that occur in ischemia and the role of NAE as
|We would like to thank Mary R. Roth for expert technical assistance. A joint
seed grant from the UNT- UNTHSC helped initiate these studies. This work was
funded in part by a grant from the U.S. Department of Energy, Office of Science
(BES, DE-FG02-05ER15647) to KDC as well as partial support by grants EY014227
from NIH/NEI, RR022570 from NIH/NCRR and AG010485, AG022550 and
AG027956 from NIH/NIA and by The Garvey Texas Foundation and the Felix and
Carmen Sabates Missouri Endowed Chair in Vision Research (P.K.). Instrument
acquisition and method development at the Kansas Lipidomics Research Center
were supported by NSF grants MCB 0455318 and DBI 0521587, K-INBRE (NIH
Grant P20 RR16475 from the INBRE program of the National Center for Research
Resources) and NSF EPSCoR grant EPS-0236913 with matching support from the
State of Kansas through Kansas Technology Enterprise Corporation and Kansas
State University (R.W).
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