728
Degradation of Phospholipid Molecular Species
During Experimental Cerebral Ischemia in Rats
Yasunobu Goto, MD, Shinichiro Okamoto, MD, PhD, Yasuhiro Yonekawa, MD, PhD,
Waro Taki, MD, PhD, Haruhiko Kikuchi, MD, PhD,
Hajime Handa, MD, PhD, and Makoto Kito, PhD
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Previous investigators have shown that free fatty acids that accumulate during ischemia are an
indicator of evolution in ischemic brain damage. Our study describes the temporal relations between
free fatty acid accumulation and degradation of phospholipid molecular species after cerebral
ischemia. Using the four-vessel occlusion model of adult Wistar rats, we analyzed quantitatively the
cerebral phospholipid molecular species of diacyi phosphatidylcholine and diacyl phosphatidylethanolamine and released free fatty acids during ischemia. Total diacyl phosphatidylcholine molecular
species decreased gradually but did not show any significant difference even at 60 minutes. By contrast,
total diacyl phosphatidylethanolamine abruptly decreased after 5 minutes and continued to decrease
significantly thereafter. Polyunsaturated molecular species showed a higher ratio of degradation than
saturated and monounsaturated molecular species of either phosphatidylcholine or phosphatidylethanolamine. Total free fatty acid accumulated according to the time elapsed, and statistical
significance was obtained after 10 minutes. Free arachidonic and docosahexaenoic acids were
attributed to these significant accumulations at 10,15, and 30 minutes. At 60 minutes, individual free
fatty acids increased nonspecifically. Free fatty acids, which are hydrolyzed from phospholipid classes,
are known to be further metabolized to bioactive substances such as prostaglandins and leukotrienes.
Rapid degradation of phospholipid molecular species, especially of diacyl polyunsaturated molecular
species, could be an important finding to membrane perturbation. Effective prevention of these
changes might enhance tolerance to ischemic brain damage. (Stroke 1988; 19:728-735)
B
rain tissue contains a large amount of lipids,
composed mostly of phospholipids, as the
major structural component of biological
membranes. The integrity of the membranes is essential to brain function. Various adverse conditions, such
as ischemia,1"10 hypoxia," hypogrycemia,12 and seizures,1 induce decomposition of membrane phospholipids and release of free fatty acids (FFAs). FFA
accumulation is said to be the only biochemical
correlate of the evolution of ischemic brain damage.13
Released FFAs are known to impair mitochondrial
function.1415 Arachidonic acid metabolites and prostaglandins may induce alterations in the blood-brain
barrier16 and disturbances of neuronal function.17 Many
pathways have been suggested for the release of FFAs
from membrane phospholipids; the exact mechanisms
responsible for the initiation and continuation of this
increase are not yet clear. In addition, individual
phospholipids (which have different concentrations
and compositions of fatty acids) that may be involved
in the release mechanism have not yet been identified.18
Membrane phospholipids are also implicated in synaptic transmission.19
From the Department of Neurosurgery, Faculty of Medicine (Y.G.,
S.O., W.T., H.K.) and the Research Institute for Food Science
(M.K.), Kyoto University, Kyoto, the Hamamatsu Rosai Hospital,
Hamamatsu (H.H.), and the Department of Neurosurgery, National
Cardiovascular Center (Y.Y.), Suita, Japan.
Supported by Japanese Ministry of Education Grant B 61480309.
Address for reprints: Yasunobu Goto, MD, Department of
Neurosurgery, Faculty of Medicine, Kyoto University, Shogoinkawara-cho 54, Sakyo-ku, Kyoto 606, Japan.
Received March 20, 1987; accepted December 11, 1987.
Amount and composition of the FFAs released
cannot give us accurate information on the degradation
of phospholipids. Therefore, we describe a model for
simultaneous comprehensive measurement of the
cerebral contents of both phospholipid molecular
species and FFAs by using the model of cerebral
ischemia in rats, first, to explore the changes in
phospholipid molecular species and FFAs during
ischemia and second, to find out whether preferential
cleavage of phospholipid molecular species, especially
polyunsaturated molecular species, occurs during the
early or late ischemic period.
Materials and Methods
Induction of Ischemia and Methods of Obtaining
Brain Specimens
Male Wistar rats (350-450 g) of an SPF strain were
acclimatized to the laboratory, fed a normal rat diet,
and given free access to food and water until the time
of the study. Anesthesia was induced with 60 mg/kg i.p.
pentobarbital. Cerebral ischemia was produced by the
procedure described by Pulsinelli and Brierley20 after
minor modification.21 Rats were paralyzed with 0.5
mg/kg i.v. d-tubocurarine and ventilated through a
tracheostomy by a small-animal ventilator. A femoral
artery and vein were cannulated for monitoring blood
pressure and sampl ing blood Paco2, Pao2, and pH. Both
common carotid arteries were isolated from the vagus
nerve and jugular vein and encircled with fine nylon
threads that were then passed through Silastic tubes
(occlusive devices). Then the rats were turned to the
prone position. The alar foramina on each side were
Goto et al
exposed, and both vertebral arteries were electrocauterized; then, within 1 minute, the occlusive devices
were tightened, causing severe hemispheric ischemia in
all rats. In a preliminary experiment, regional cerebral
blood flow in the parietal lobe was lowered to a level
undetectable by the hydrogen clearance method
(n = 6). The duration of occlusion was 5, 10, 15, 30,
or 60 minutes. Control rats received sham operations.
After each occlusion period, the brain was frozen in
situ by pouring liquid N2 into a paper funnel22 fixed to
the carvarium. The frozen brain was cut into two or
three slices under intermittent irrigation with liquid N2,
and the whole brain was chiseled out in a cryostat at
- 30° C and stored in liquid N2 until analysis.
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Quantitative Analysis of Free Fatty Acids
After the addition of an internal standard, pentadecanoic acid (15:0), the brain was weighed and
homogenized for 30 seconds in 7 ml 5% trichloroacetic
acid and 30 ml chloroform:methanol (1:2 vol: vol)
in an ice bath using a Porytron homogenizer (Lucerne,
Switzerland). Lipids were extracted three times at 37°
C for 30 minutes according to the method of Kates.23
Then, 30 ml chloroform and 30 ml 0.01N HC1 were
added and centrifuged. The neutral lipids were separated on a Sep-pak silica cartridge (Milford). The
neutral lipids were eluted with 20 ml chloroform and
the phospholipids with 20 ml methanol. After drying
under a N2 stream, the neutral lipids were concentrated
and applied to a thin-layer chromatography (TLC) plate
with a solvent of hexane: ethyl ether: acetic acid
(50:50:1 vol:vol:vol). The amount of FFA was
estimated by reference to FFA standards that were run
parallel on each TLC plate and scraped off. FFAs were
methylated with BF3MeOH according to the method of
Morrison and Smith24 and determined by gas-liquid
chromatography (Shimadzu GC-9A, Kyoto, Japan) on
a column packed with 10% Silar IOC on 100-120 mesh
Chromosorb W (Kyoto, Japan), with a N2 flow rate of
40 ml/min and a temperature of from 160° to 240° C.
FFAs were identified by comparison of their retention
times with those of standards run under identical
conditions. Palmitic (16:0), stearic (18:0), oleic
^18:1), arachidonic (20:4), and docosahexaenoic
(22:6) acids were quantified in comparison with
known amounts of internal standards and expressed as
micTOgrams per gram wet weight. The sum of these five
values was considered to represent total nonesterified
fatty acids.
Quantitative Analysis of Phospholipid
Molecular Species
The phospholipids were separated by the methods
described above, weighed, and resuspended in chloroform. After the addition of distearoyl-phosphatidylcholine (18:0-18:0 PC) and distearoyl-phosphatidylethanolamine (18:0-18:0 PE) as internal standards, 2 mg phospholipid containing 10 p.g butylated
hydroxytoluene was applied and developed with a
solvent of chloroform: methanol: acetic acid: water
(180:150:30:10 vol:vol:vol:vol). Phosphatidyl-
Degradation of Phosphollpld Daring Ischemia
729
choline (PC) and phosphatidylethanolamine (PE)
were extracted three times from the TLC plates with
chloroform:methanol (2:1 vol:vol). After drying
under a N2 stream, molecular species in PC and PE
were identified and quantitatively analyzed according to the method of Kito and coworkers.25-26 PC and
PE were hydroryzed to diacylglycerols (DGs) by
phospholipase C. Thereafter, DGs and dinitrobenzoylchloride were incubated in dry pyridine at 60° C
for 10 minutes. The combined extract was dried
under a N2 stream to remove the pyridine. The
residue was dissolved in 2.0 ml «-hexane, and this
solution was washed. High-performance liquid chromatography (HPLC) was carried out on an Hitachi
655-15 liquid chromatograph (Tokyo, Japan). The
separated molecular species were determined at 254 nm
with an Hitachi 638-41 variable-wave UV monitor.
An Ultrasphere ODS column (5 jim, 4.6 mm i.d.
x250 mm, supplied by Altex Scientific, Berkeley,
California) was used for separation with a solvent of acetonitrile: 2-propanol (80:20 vol: vol). The
flow rate was 1.0 ml/min and the column temperature
was 25° C.
Statistical Analysis
Means ±SEM were determined. The results were
analyzed by one-way analysis of variance. A value of
p<0.05 was considered to reflect significant differences throughout this study.
Results
Physiologic Variables
Physiologic variables are given in Table 1. Systemic
arterial blood pressure and pH remained stable throughout the experiment. Four-vessel occlusion led PacOj
and Pao2 to successive increases according to the time
elapsed, but analysis of variance failed to show any
significant differences.
Separation of Phospholipid Molecular Species
In control rats, PC and PE accounted for 41.9 ± 3.5%
(n = 5) and 30.2 ±3.1% (n = 5) of the total phospholipids, respectively. Each PC and PE was further
separated by their acyl group composition, that is,
molecular species, using HPLC with a solvent of
acetonitrile: 2-propanol (80:20 vol: vol) as shown in
Figure 1, in which the nine major consistent peaks were
detected. The area of each peak was proportional to the
amount of that molecular species and was not affected
by differences in the structure of the molecular
species.25 Hence, the 18:0 (stearoyl)-18:0 species
was added as an internal standard to determine the absolute amount of each fraction. The relative composition of each molecular species was different for PC
and PE, as shown in Table 2. Saturated and monounsaturated molecular species (18: l[oleoyl]-18:1,
16:0[palmitoyl]-18:l, 16:0-16:0, 18:0-18:1, and
18:0-16:0) comprised 81.3% of the total diacyl
PC molecular species. In contrast, diacyl molecular
species of PE mainly consisted of poryunsaturated
molecular species (16:0-22:6[docosahexaenoyl],
730
Stroke
Vol 19, No 6, June 1988
TABLE 1. Physiologic Variables Before and After Ischemia in Male Wistar Rats
Ischemia (min)
Systemic arterial
pressure (mm Hg)
Blood gas analysis
PH
Pacoj (mm Hg)
PaO2 (mm Hg)
Controls
5
10
15
30
60
100.0±5.0
106.0±9.5
102.0±10.5
98.0±8.5
90.0±8.2
95.0±9.5
7.4±0.1
30.6 + 6.7
138.9+17.3
7.2±0.2
7.2±0.3
37.9 ±9.9
139.4±19.1
7.3 + 0.2
35.8±10.1
136.5±15.4
41.1±10.6
140.6±17.0
7.3±0.2
42.8 ±9.0
142.4±19.9
7.3±0.2
43.1 ±10.5
145.6±18.6
Values are mean±SEM, « = 5.
16:0-20:4[arachidonoyl], 18:0-22:6, and 18:0-20:4),
which comprised 67.0% of the total diacyl molecular
species of PE. More detailed data on the time-course
amount of each molecular species of PC and PE are given
in Tables 3 and 4, respectively.
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Changes in Phospholipid Molecular Species
During Ischemia
Figure 2 shows the time course of changes in total
PC and PE (sum of the nine major diacyl molecular
species). Total PC gradually decreased during the
experiment. However, the polyunsaturated molecular
species (especially 16:0-22:6 and 18:0-22:6) were
degraded rapidly after the initiation of ischemia (Table
3). Polyunsaturated molecular species of diacyl PC
decreased by 42% and saturated and monounsaturated
molecular species decreased by 32% within 60 minutes.
Total PE (sum of the nine major diacyl molecular
species) decreased abruptly within 5 minutes
(p<0.05), and then decreased gradually (Figure 2).
The rate and ratio of the decrement were more marked
than for PC. Polyunsaturated molecular species of PE
decreased by 66% and saturated and monounsaturated
molecular species decreased by 54% within 60 minutes
(Table 4). Thus, although the rate of degradation and
time course of decrement were different for PC and PE,
polyunsaturated molecular species of both diacyl PC
and PE were more liable to be degraded than the others
in the early ischemic period.
Free Fatty Acids
The changes in the amount of total FFAs after
four-vessel occlusion are shown in Figure 3. The
increase in total FFA was significant even after 10
minutes. The amount of total FFA increased progressively as the period of ischemia was prolonged and
reached as much as 2.4 times the control value after 60
minutes of ischemia.
The changes in the amounts of individual FFAs after
various periods of ischemia are given in Figure 4 and
Table 5. Although the saturated and monounsaturated
FFAs (16:0,18:0, and 18:1) were more prominent than
the polyunsaturated FFAs (20:4 and 22:6), in the FFA
brain pool of control rats significant change occurred only
in the polyunsaturated FFAs in the early stages of
ischemia. The pronounced increase of polyunsaturated
PC
PE
10-
20-
30-
40-
80-
60-
r
J
FIGURE 1. Separation of major molecular species of diacyl phosphatidylcholine (PC) and phosphatidylethaiwlamine (PE) by
high-performance liquid chromatography. a, 16 :0-22 : 6; b, 16 :0-20 -' 4; c, 18 :0-22 :6; d, 18 •' 0-20 •' 4; e, 18 :1-18 :1; f,
16 : 0-18 :1; g, 16 :0-16 : 0; h, 18 :0-18 •' 1; i, 18 : 0-16 •' 0; and j , 18 :0-18 :0 as internal standard.
Goto et al
TABLE 2. Relative Composition of Diacyl Molecular Species
of Phosphatidykholine and Phosphatldylethanolamine hi Control Rat Brain
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Molecular
Phosphatidylspecies
choline
Polyunsaturated
16:0-22:6
4.38±0.67
16:0-20:4
5.68±1.01
18:0-22:6
3.82±0.48
18:0-20:4
4.81 ±0.46
Total
18.78±1.97
Saturated and monounsaturated
18:1-18:1
3.01 ±0.22
16:0-18:1
39.35+1.20
16.59±1.84
16:0-16:0
18:0-18:1
16.44 + 0.57
5.92±0.68
18:0-16:0
Total
81.31 ± 1.92
Phosphatidylethanolaminc
9.94 + 0.58
3.53±0.19
31.32±2.74
22.17±1.04
66.96±2.78
6.70±1.01
6.54+1.31
4.55 ±0.30
12.95±1.31
2.31 ± 1 . 1 2
33.03±2.78
Values are mean±SD%, n = 5 in each group. Polyunsaturated,
16:0-22:6+16:0-20:4+18:0-22:6+18:0-20:4; saturated and
monounsaturated, 18:1-18:1 + 16:0-18:1 + 16:0-16:0+18:018:1 + 18:0-16:0.
FFAs was observed for as long as 30 minutes of
ischemia, and then the increase in every kind of FFA
became significant after 60 minutes of ischemia.
Discussion
Quantification of Molecular Species
Biological membranes are composed basically of
lipid bilayers in which various phospholipid classes and
their molecular species control membrane fluidity and
functions. HPLC with detection at 205 nm has been
used to separate molecular species. However, it is
Degradation of Phospholipid During Ischemia
731
difficult to quantify the eluted molecular species using
this wavelength because of the variable absorption
properties of the individual molecular species. The new
method we applied in our study, HPLC with detection
at 254 nm, is simple and sensitive for the microdetection of changes in membrane phospholipid molecular species.2526
Changes in Phospholipid Molecular Species
During Ischemia
Changes in phospholipids during ischemia and
recirculation have been reported from many laboratories. However, there is controversy about the results,
even if only the effects of ischemia itself are concerned.
Rehncrona et al8 observed no difference between the
size or composition of phospholipid pools in complete
and severe incomplete ischemia in rats. They suggested
that free radical damage and peroxidative degradation
of membrane lipids were not important to the accumulation of FFAs and were important rather to the
activation of phospholipase A2 during ischemia. Hattori et al 67 also reported no significant decrease and no
changes in the composition of phospholipids and no
significant changes in both deacylating and reacylating
enzyme activities in the rat decapitation model. They
considered that FFA release was attributed to the rapid
depletion of high-energy phosphate compounds. By
contrast, DeMedio et al4 observed decreases in the total
phospholipid, PC and PE, content in gerbil cortex after
10 minutes of ischemia. Yoshida et al10 observed a 4%
decrease in PC (not significant) and a 16% decrease in
PE content of gerbil brain after 30 minutes of ischemia.
Enseleit et al5 reported small changes in the total
cerebral phospholipid, PC, phosphatidylserine (PS),
and PE content, which might depend on the different
TABLE 3. Diacyl Molecular Species of Phosphatidylchollne in Rat Brain During Various Periods of Ischemia
Ischemia (min)
Molecular species
Controls
Polyunsaturated
8.46±0.70
16:0-22:6
16:0-20:4
10.99±1.06
7.44±0.65
18:0-22:6
18:0-20:4
9.37±0.80
36.26 ±2.77
Total
% of control
100
Saturated and monounsaturated
18:l-18'l
5.99±0.71
16:0-18:1
77.89 ±8.60
16:0-16:0
33.03±4.31
18.0-18:1
32.53 ±3.50
18:0-16:0
14.77 ±1.50
Total
164.21 ±18.05
% of control
100
200.47 ±20.53
Total
5
10
15
30
60
6.06±0.61*
9.03±0.91
5.86±0.57
7.02±0.42
27.97±1.54
77.1
5.72+1.15*
7.82±1.94
4.75±0.86*
7.00±1.68
25.29 ±5.49*
69.7
5.44±1.12*
7.57+1.05
4.57 ±0.72*
6.80±0.85*
24.38 ±3.70*
67.2
4.88±0.25*
7.57± 1.22
4.51 ±0.84*
5.97±0.33*
22.93 ±0.96*
63.1
4.67 ±1.02*
6.83 ±1.06
3.84±0.82*
5.77±0.91*
21.11±3.57*
58.1
4.64±0.53
68.36±4.72
32.81 ±2.28
28.25 ±2.45
13.59±2.15
147.65 ±11.75
90.0
175.62 ±12.98
4.59±1.00
68.10± 13.78
32.61 ±6.02
27.75±5.15
13.00±2.51
146.05±28.27
89.0
171.34±32.54
4.58 ±1.00
66.07 ±8.46
32.41 ±3.79
27.36±4.25
12.67±2.38
143.09± 19.37
87.1
167.47±22.91
4.43±0.41
57.83±3.48
31.71 ±7.30
24.44±1.81
10.62 ±1.65
129.03 ±11.65
78.6
151.96± 12.32
4.42±0.52
52.41 ±6.54
24.97 ±2.89
22.09 ±2.60
8.22±0.79
112.11 ± 13.06
68.3
133.22± 16.40
Values are mean±SEM nmol/mg phospholipid, n = 5 in each group. Polyunsaturated, 16:0-22:6+16:0-20:4+18:0-22:6 +
18-0-20-4; saturated and monounsaturated, 18:1-18:1 + 16:0-18:1 + 16:0-16 0+18:0-18:1 + 18:0-16:0.
*p<0.05 compared with controls.
732
Stroke
TABLE 4.
Vol 19, No 6, June 1988
Diacyl Molecular Species of Pbosphatidylethanolainine in Rat Brain During Various Periods of Ischemia
Ischemia (min)
Molecular species
Controls
5
10
15
30
60
10.41 ±1.04
3.70±0.35
32.77±3.36
23.26±2.4O
70.14±6.86
4.56 ±1.04*
4.51 ±1.38*
1.50±0.17*
12.88±2.54*
9.59±1.64*
4.13±0.71*
1.49±0.18*
12.88±1.31*
9.59 ±1.44*
3.54±0.41*
1.38±0.38*
10.51 ±1.97*
8.44±1.01*
28.48±5.60*
40.5
28.09±3.40*
40.1
3.98 ±0.39*
1.40±0.11*
12.01 ±0.67*
9.49±0.84*
26.98 ±1.64*
Polyunsaturated
16:0-22:6
16:0-20:4
18:0-22:6
18:0-20:4
Total
% of control
Saturated and
100
1.58±0.18*
13.02± 1.78*
10.11 ±1.59*
29.27±4.75*
41.8
38.5
23.87±2.99*
34.1
monounsaiurated
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
3.56 + 0.41*
3.63 ±0.70*
3.52±0.76*
3.46 ±1.04*
3.56±0.59*
6.99±0.79
4.29 ±0.76*
3.86±0.35*
4.13 ±0.29*
3.50±1.00*
6.73 ±0.72
4.27 ±0.89*
1.89±0.21*
2.55±0.38*
1.87 + 0.26*
1.67 ±0.24*
2.55±0.53*
4.76 ±0.47
6.3O±0.58*
6.64 ±0.79*
6.97 ±1.30*
6.19±1.82*
6.72±0.95*
13.48±1.23
2.32±0.85
1.76±0.30
1.66±0.36
2.20 ±0.59
0.78±0.13
2.38±0.59
17.98±1.81*
19.76±3.68*
19.3O±2.51*
17.21 ±1.83*
34.34±2.86
15.60±4.10*
52.5
57.7
56.3
50.1
45.5
100
49.03 ±8.42*
47.78 ±7.24*
46.07 ±5.07*
44.09±3.09*
39.47 ±6.14*
104.48±9.18
Total
Values are mean ± SEM nmol/mg phospholipid, n = 5 in each group. Polyunsaturated, 16:0-22:6+16:0-20:4+18:0-22:6 +
18:0-20:4; saturated and monounsaturated, 18:1-18:1 + 16:0-18:1 + 16:0-16 0+18:0-18:1 + 18:0-16:0.
*p<0.05 compared with controls.
18:1-18:1
16:0-18:1
16:0-16:0
18:0-18:1
18:0-16:0
Total
% of control
600
0>
200
500
o
E
c
m
<e
o
m
400f
a.
t 300
5 100
3
o
3
O
"5
200
100
0 5 1015
30
60
Tlme(mln)
FIGURE 2. Changes in major molecular species of diacyl
phosphatidylcholine (PC, •) and phosphatidylethanolamine
(PE, o) during various periods of cerebral ischemia in rats.
Values are mean ± SEM. *p < 0.05 compared with control. Note
that PC showed gradual decrease and PE showed abrupt
decrease, n = 5 in each group.
0 5 1015
30
60
Tlme(mln)
FIGURE 3. Total free fatty acid (FFA) concentrations during
various periods of cerebral ischemia in rats. Values are
mean ± SEM. *p<0.05 compared with control. Note that amount
of total FFAs released accumulated according to time elapsed.
Goto et al Degradation of Phospholipid During Ischemia
FFA
733
FFA
2OOr
200r
16:0
100
18:0
100
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
5
10 15
30
60
min
FFA
5
10
15
30
60
mln
FFA
200r
200
18:1
FIGURE 4. Individual free fatty acid
(FFA) concentrations after cerebral ischemia in rats. 16 •' 0, palmitic acid; 18 •' 0,
stearic acid; 18 • 1, oleic acid; 20 • 4,
arachidonic acid; 22 •' 6, docosahexaenoic
acid. Values are mean±SEM. *p<0.05
compared with control. Note that 22 •' 6 and
20 •' 4 increased significantly in early period; thereafter 16 : 0, 18 • 0, and 18 :1
were released more prominently
20:4
100
100
22:6
10 15
30
60
mln
5
10
Free
fatty acid
60
min
Our results show a gradual decrease in total diacyl
PC and an abrupt decrease in total diacyl PE, which are
in agreement with previously published reports. 410 The
cause of the abrupt decrease in diacyl PE molecular
species is not clear. Since most of the PC is located on
rates of lipid metabolism of gerbil brains subjected to
various periods of ischemia. These discrepancies are
probably due to a combination of factors such as
differences in sampling procedures, analytic methodology, animal model, and duration of the ischemic insult.
TABLE 5.
30
15
Free Fatty Acid Concentrations in Rat Brain During Various Periods of Ischemia
Ischemia (min)
Controls
Polyunsaturated
20:4
40.72±6.19
22:6
26.46 ±0.91
Saturated and monounsaturated
16:0
59.66±3.18
18:0
65.02±2.74
18:1
73.91 ±3.51
Total
265.77 ±15.22
5
10
15
30
60
64.40±7.53
31.00±9.82
89.08 ±18.98*
47.69±3.83*
95.17±32.61*
48.35 ±10.42*
99.76±9.68*
50.10±0.73*
108.61 ±8.37*
66.05 ±4.74*
70.94 ±10.24
86.08 ±15.72
81.92± 13.76
334.34±42.44
74.53 ±5.63
94.63±3.19
83.48±5.71
389.41 ±24.99*
74.52 ±8.18
94.53± 14.58
87.91 ±14.84
400.48 ±62.46*
78.15±0.46
104.10±9.19
88.38±4.56
420.49 ±21.88*
143.29± 12.24*
142.62 ±27.49*
183.55 ±7.47*
644.12 ±37.65*
Values are mean±SEM jtg/g brain, n = 5 in each group.
*p<0.05 compared with controls.
734
Stroke Vol 19, No 6, June 1988
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
the outer leaflet whereas PE is located mainly on the
inner leaflet of the plasma membrane, the intracellular
hydroh/tic enzymes could be more accessible to PE.27
In addition to the changes in the amounts of total PC
and PE, we did further analysis to clarify which
molecular species of PC and PE are involved in such
changes induced by cerebral ischemia. Rat brain PC
and PE were composed mainly of nine diacyl molecular
species, among which four diacyl molecular species
were poh/unsaturated and five were saturated or monounsaturated. The distribution of these molecular species for PC was different from that for PE, which may
be due to differences inherent in the phospholipid
classes. Our results reveal that, although ischemia
ultimately causes nonpreferential hydrolysis of phospholipid (PC and PE) molecular species during the later
period, poryunsaturated diacyl molecular species (of
both PC and PE) are more rapidly degraded than
saturated and monounsaturated molecular species during the early phase. Such preferential degradation of
poh/unsaturated diacyl molecular species in each phospholipid class may be involved in the rapid accumulation of poryunsaturated FFAs after ischemia. Similar
mechanisms may also explain, in part, the rapid
degradation of PE, which is more abundant in poh/unsaturated fatty acid residues than PC.
Free Fatty Acids
Cerebral ischemia results in marked time-dependent
enlargement of the FFA pool of the brain1"10 even after
30 seconds. DG also accumulates during ischemia.6-7-28-29 These phenomena might be attributed to the
imbalance between deacylation and reacylation. Our
results confirm that catabolism of phospholipids actually takes place and that it is the main cause of FFA
accumulation/-3-10
In the early ischemic period, poryunsaturated FFAs
such as 20:4 and 22:6 significantly increased. Such a
differential accumulation of poh/unsaturated FFAs has
been attributed to selective degradation of specific
phospholipid classes whose acyl groups are rich in
poh/unsaturated fatty acids. Our study also shows that
PE, which is rich in pofyunsaturated acyl groups, was
more rapidly degraded than PC. In addition, our results
imply that the preferential degradation of poh/unsaturated diacyl molecular species in each phospholipid
class is also a cause of the differential FFA accumulation. Since the absolute amounts of accumulated
FFAs are disproportionately high in poh/unsaturated
FFAs (20:4 and 22:6) between 10 and 30 minutes after
ischemia, phospholipase A2 is most likely involved in
the degradation during this period.30-31 Phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-diphosphate (PIP2) are known to be present
in large amounts and to disappear rapidly from the brain
post mortem. 3 " 3 Although we did not determine the
degradation of phosphoinositol lipids, the possibility
cannot be excluded that phosphatidylinositol, PIP, and
PIP2, which are composed mainly of 18:0-20:4
species, supply 20:4 and 18:0.
After 30 minutes of ischemia, 1 6 : 0 , 1 8 : 0 , and 18:1
accumulate much more than 20:4 and 2 2 : 6 .
This indicates that nonspecific cleavage of phospholipids occurs in the late ischemic period. In this case,
the participation of phospholipase A, and A2 cannot
be excluded. Different rates of FFA accumulation
between these early and late ischemic periods may be
due to different mechanisms, as indicated by other
workers.13;29>34
Relatively later, ischemia may cause profound perturbation of biomembranes by activating irreversible
processes such as massive calcium influx or degradation of h/sosomes.
Clinical Significance
The potential significance of the changes in phospholipid molecular species and FFAs in terms of brain
or neuronal function is obscure, but FFA accumulation
may correlate with the duration of ischemia even after
prolonged periods and may reflect the evolution of
ischemic brain injury.13 Reductions in high-energy
phosphates, increases in lactate and individual FFAs,
uncoupling of mitochondrial oxidative phosphorylation, and reduced oxygen utilization are also thought
to be correlated with irreversible brain injury. Therefore, the prevention of rapid degradation of poh/unsaturated molecular species, especially in diacyl PE,
might be clinically relevant in the treatment of cerebral
ischemia.
In conclusion, 1) in the early ischemic period the
selective hydrolysis of diacyl PE takes place, 2) the
selective hydrolysis of phospholipid (diacyl PC and
PE) molecular species possessing a poh/unsaturated
acyl group also takes place in the early ischemic period,
which strongly implies the involvement of phospholipase A2 during this period, and 3) in the later period,
additional nonspecific catalytic enzymes may be involved in the release and accumulation of FFAs.
References
1. Bazan NG Jr: Effects of ischemia and electroconvulsive shock
on free fatty acid pool in the brain. Biochim Biophys Acta
1970-,218:l-10
2. Bazan NGJr.de Bazan HEP, Kennedy WG, Joel CD: Regional
distribution and rate of production of free fatty acids in rat
brain. J Neurochem 1971,18:1387-1393
3. Bhakoo KK, Crockard HA, Lascelles PT: Regional studies of
changes in brain fatty acids following experimental ischemia
andreperfusioninthegerbil./^«uroc/w!ml984;43:1025-1031
4. De Medio GE, Brunetti M, Dorman RV, Droz B, Horrocks LA,
Porcellati G, Souyri F, Trovarelli G: Phospholipid metabolism
during central and peripheral damage and recovery in nervous
tissue. Birth Defects 1983;19:175-187
5. Enseleit WH, Domer FR, Jarrot DM, Baricos WH: Cerebral
phospholipid content and Na+,K+-ATPase activity during
ischemia and postischemic reperfusion in the Mongolian
gerbil. J Neurochem 1984;43:320-327
6. Hattori T, Sakai N, Yamada H, Kameyama Y, Nozawa Y: Brain
lipid metabolism during early period of global ischemia. No To
Shinkei 1985;37:377-383
7. Hattori T, Nishimura Y, Sakai N, Yamada H, Kameyama Y,
Nozawa Y: Effects of pentobarbital on lipid metabolism during
global ischemia. No To Shinkei 1986;38:585-591
8. Rehncrona S, Westerberg E, Akesson B, Siesjtf BKJ Brain
cortical fatty acids and phospholipids during and following
complete and severe incomplete ischemia. / Neurochem
1982;38:84-93
Goto et al
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
9. Yasuda H, Kishiro K, Izumi N, Nakanishi M: Biphasic
liberation of arachidonic and stearic acids during cerebral
ischemia. J Neurochem 1985;45:168-172
10. Yoshida S, Inoh S, Asano T, Sano K, Kubota M, Shimazaki
H, Ueta N: Effects of transient ischemia on free fatty acids and
phospholipids in the gerbil brain. / Neurosurg 1980;53:
323-331
11. Gardiner M, Nilsson B, Rehncrona S, Siesjo BK: Free fatty
acids in the rat brain in moderate and severe hypoxia. /
Neurochem 1981;36:150O-1505
12. Wielock T, Harris RJ, Symon L, Siesja BK; Influence of severe
hypogh/cemia on brain extracellular calcium and potassium
activities, energy, and phospholipid metabolism. J Neurochem
1984;43:160-168
13. Shiu GK, Nemoto EM: Barbiturate attenuation of brain free
fatty acid liberation during global ischemia. J Neurochem
1981;37:1448-1456
14. Lazarewicz JW, Strosznajder J, Gromek A: Effects of ischemia
and exogenous fatty acids on the energy metabolism in brain
mitochondria. Bull Acad Pol Sci 1972^0:599-606
15. Wojtczak L: Effect of long chain fatty acids and acyl-CoA on
mitochondria! permeability, transport, and energy-coupling
processes. / Bioenerg Biomembr 1976;8:293-311
16. Bhakoo KK, Crockard HA, Lascelles PC, Avery SF: Prostaglandin synthesis and oedema formation during reperfusion
following experimental brain ischemia in the gerbil. Stroke
1984;15:891-895
17. Wolfe LS, Coceani F: The role of prostaglandins in the central
nervous system. Annu Rev Physiol 1979;41:669-684
18. Marion J, Wolfe LS: Origin of the arachidonic acid released
post-mortem in rat forebrain. Biochim Biophys Acta
1979;574:25-32
19. Hokin-Neaverson M: Metabolism and role of phosphatidylinositol in acetylcholine stimulated membrane function, in
Bazan NG, Brenner RR, Guisto NM (eds): Function and
Biosynthesis ofUpids (Advances in Experimental Medicine and
Biohgy Series: Vol 83). New York, Plenum Publishing Corp,
1976, pp 429-446
20. Pulsinelli WA, Brierley JB: A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke
1979;10:267-272
21. Todd NV, Picozzi P, CrockardHA, Ross Russell R: Reperfusion
after cerebral ischemia: Influence of duration of ischemia.
Stroke 1986;17:460-466
22. Ponten U, Ratcheson RA, Salford LG, Siesjfl BK: Optimal
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Degradation of Phospholipid During Ischemia
735
freezing conditions for cerebral metabolites in rats. J Neurochem 1973;21:1127-1138
Kates M: Technique of lipidology, in Work TS, Work E (eds):
Amsterdam, North-Holland 1972, pp 347-350
Morrison WR, Smith LM: Preparation of fatty acid methyl
esters and dimethylacetals from lipids with boron fluoridemethanol. / Lipid Res 1964^:600-608
Kito M, Takamura H, Narita H, Urade R: A sensitive method
for quantitative analysis of phospholipid molecular species by
high performance liquid chromatography. J Biochem (Tokyo)
1985;98:327-331
Takamura H, Narita H, Urade R, Kito M: Quantitative analysis
of polyenoic phospholipid molecular species by high performance liquid chromatography. Lipids 1986;21:356-361
Crews FT, Camacho A, Philips I, Willink ECT, Calderini G,
Hirata F, Axelrod J, McGivney A, Siraganian R: Effects of
membrane fluidity on mast cell and nerve cell function, in
Horrocks L, Ansell GB, Porcellati G (eds): Phospholipids in
the Nervous System: Metabolism, Vbl 1. New York, Raven
Press Publishers, 1982, pp 21-35
Abe K, Kogure K: Accurate evaluation of 1,2-diacyIglycerol
in gerbil forebrain using HPLC and in situ freezing technique.
J Neurochem 1986;47:577-582
Aveldano MI, Bazan NG: Rapid production of diacjigrycerols
enriched in arachidonate and stearate during early brain
ischemia. / Neurochem 1975;25:919-920
Edgar AD, Strosznajder J, Horrocks LA: Activation of
ethanolamine phospholipase A2 in brain during ischemia. J
Neurochem 1982;39:1111-1116
Sun GY, Foudin L: On the status of rysolecithin in rat cerebral
cortex during ischemia. / Neurochem 1984;43:1081-1086
Nishihara M, Keenan RW: Inositol phospholipid levels of rat
forebrain obtained by freeze-blowing method. Biochem Biophys Acta 1985;835:415^U8
Dceda M, Yoshida S, Busto R, Santiso M, Ginsberg MD:
Poryphosphoinositides as a probable source of brain free fatty
acids accumulated at the onset of ischemia. / Neurochem
1986;47:123-132
Yasuda H, Kishore K, Izumi N, Nakanishi M: Biphasic
liberation of arachidonic and stearic acids during cerebral
ischemia. J Neurochem 1985;45:168-172
KEY WORDS • cerebral ischemia • fatty acids, nonesterified
• phospholipids • rats
Degradation of phospholipid molecular species during experimental cerebral ischemia in
rats.
Y Goto, S Okamoto, Y Yonekawa, W Taki, H Kikuchi, H Handa and M Kito
Stroke. 1988;19:728-735
doi: 10.1161/01.STR.19.6.728
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1988 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/19/6/728
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office.
Once the online version of the published article for which permission is being requested is located, click Request
Permissions in the middle column of the Web page under Services. Further information about this process is
available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Stroke is online at:
http://stroke.ahajournals.org//subscriptions/