316
CIRCULATION RESEARCH
myocytes: Isolation, morphology, and biochemical characteristics. J Mol Cardiol 11: 339-357
Williamson JR, Safer B, Rich T, Schaffer S, Kobayoshi K (1975)
Effects of acidosia on myocardial contractility and metabolism.
Acta Med Scand 587 (suppl): 95-111
Williamson Jft Schaffer SW, Ford C, Safer B (1976) Contribu-
VOL. 49, No. 2, AUGUST
1981
tion of tissue acidosis to ischemic injury in the perfused rat
heart. Circulation 53 (suppl I): 3-14
Zuurendonk PF, Tager JM (1974) Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver
cells Biochim Biophys. Acta 333: 393-399
Time Course of Changes in Porcine
Myocardial Phospholipid Levels during
Ischemia
A Reassessment of the Lysolipid Hypothesis
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NISAR A. SHAIKH AND EUGENE DOWNAR
SUMMARY This study was performed to determine the early and delayed metabolic effects of
myocardial ischemia on the major membrane phospholipids and to reassess the potential role of
lysophospholipids In the genesis of malignant dysrhythmias induced by ischemia. Samples taken from
in situ hearts before and at various intervals up to 40 minutes after abrupt ligation of LAD were
extracted by the classical Folch technique with modifications to avoid artlfactual lysophospholipid
production and losses. Following thin layer chromatography of lipid extracts, phospholipid fractions
were quantified by phosphorus estimation and lysophospholipids by a more sensitive method employing
gas liquid chromatography. The total phospholipid content with the exception of lysophospholipids
remained essentially constant throughout the early phases of acute ischemia, but fell by 6 and 14%
after 8 and 24 hours, respectively. At 8 minutes, lysophospholipid levels in ischemic myocardium were
significantly increased by 60% compared to pre-occlusion controls in tbe ischemic zone and by 25% in
post-occlusion controls. They changed little thereafter. The molecular species of lysophospholipids
remained unchanged throughout the period of ischemia gtudied. The mole fraction of other phospholipids as well as their fatty acyl and aldehyde profiles also were unchanged. Despite significant
elevations in lysophoapholiplds levels, their absolute quantities were very small (0.6% of total phospholipid P) and 15-fold smaller than that reported in vitro to simulate elcctrophysiological manifestation of ischemia. However, such small amounts in vivo, if produced in the microenvironment of certain
membrane-bound enrymes along with acidosis, hypoxia, and fatty acids, could be potentially deleterious
to cell functions. Ore Ren 48: 316-325,1981
MANY aspects of the metabolic changes associated
with myocardial ischemia have been studied intensively in the past (see Opie 1969; Jennings, 1976).
Despite early functional changes in membrane
properties that are responsible for the well known
electrophysiological manifestation of ischemia, little is known of myocardial phospholipid metabolism. The possibility that lysophospholipids contribute to ischemic damage in the heart was first
suggested by Hajdu et al (1957). Recently these
lipids have been reported to accumulate in ischemic
rabbit myocardium to an extent sufficient to proFrom th« Deportment of Medicine, University of Toronto, Toronto,
Ontario, Canada.
Supported by the Ontario Heart Foundation
Address for reprints: Dr. Nisar A Shaikh, Department of Medicine.
Medical Sciences Building, Room 7359, University of Toronto, Toronto,
Ontario, Canada M5S 1A8
Received October 1, 1980; accepted for publication February 2, 1981
duce electrophysiological changes similar to those
seen in ischemia (Sobel et al., 1978; Corr et al.,
1979). These studies led the authors to postulate a
possible involvement of lysophospholipid in the
genesis of malignant dysrhythmias induced by ischemia (Sobel et al., 1978). The pig, more than
other animals, is finding increasing favor as a model
for myocardial ischemia that is most relevant to the
human heart. The immediate and delayed effects of
abrupt coronary occlusion on the major phospholipid fractions, particularly the lysophospholipids,
were therefore investigated in the pig. Preliminary
results appeared elsewhere (Shaikh and Downar,
1980b).
The "lysolipid hypothesis" (Sobel and Corr,
1979) was reassessed by analyzing this lipid accurately and without artifactual contaminations. Lysophospholipids can be artifactually produced if
LYSOPHOSPHOLIPIDS IN ISCHEMIC MYOCARDIUM/S/iajM and Downar
tissue containing acid-labile plasmalogenic phospholipids are extracted with acidified solvents
(Shaikh and Downar, 1980a). In the present communication, lipids were extracted, along with internal standards, by a modification of the classical
Folch technique (Folch et al., 1957) to circumvent
the problems of artifactual intrapreparative lysophospholipid production, incomplete recoveries,
and losses of certain other phospholipids. A highly
reproducible assessment of lysophospholipid was
then made by gas liquid chromatography.
Methods
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All chemicals and solvents were reagent grade.
Petroleum ether (b.p. 30-60°C) was refluxed over
P2O5 and was triple distilled to obtain the 38-40°C
fraction. Methanol was redistilled.
Chromatographically pure phospholipids were
obtained from Serdary Research Laboratories. Cabbage phospholipase D, Naja Naja phospholipase A2,
1 - heptadecanoyl - sn - glycerol - 3 - phosphorylcholine
(H-LPC) and 2,3-diheptadecanoyl-sn-glycerol-lphosphorylcholine were purchased from Sigma
Chemical Co. Heptadecanoyl-glycerophosphorylcholine was purified from any phospholipid and free
fatty acid contaminants by TLC using chloroform:
methanol:acetic acid:water (C-M-AA-W, 100:45:
20:6.8, by volume) as a developing solvent (Shaikh
and Palmer, 1976). The lysophospholipid (lyso-PL)
was isolated from the silica gel with repeated extraction with C-M-AA-W (50:39:1:10, by volume)
and the extract washed with V3 volume of 4 M
NH4OH (Arvidson, 1968), dried in vacuo, and quantitated by phosphate analysis. Gas-liquid chromatographic analysis showed heptadecanoic acid as the
sole fatty acid constituent. Heptadecanoylglycerophosphorylethanolamine (H-LPE) was prepared
in the laboratory. Diheptadecanoylglycerophosphorylethanolamine was first prepared from diheptaglycerophosphorylcholine by transphosphatidylation catalyzed by phospholipase D in the presence of ethanolamine (Confurius and Zwaal, 1977).
This lipid then was purified on TLC, extracted from
silica gel, and treated with phospholipase A2
(Brockerhoff, 1965) to obtain H-LPE. The latter
compound was then recovered from the incubation
mixture, purified as described above, and quantified
by phosphate and fatty acid analyses.
Sample Preparation
Pigs (15-25 kg) were maintained on a standard
diet of pig Purina Chow for 2 days, then given
access to water only for 16 hours prior to each
experiment. Anesthesia was induced and maintained with iv sodium pentobarbital (30 mg/kg).
Positive pressure ventilation was maintained
through a cuffed endotracheal tube with a Harvard
respirator. Inspired air was supplemented with oxygen to maintain a normal arterial oxygen tension.
The chest was opened through a median sterno-
317
tomy and a segment of the left anterior descending
artery was prepared for ligation, proximal to its first
diagonal branch. In some pigs, full-thickness samples (weighing 100-150 mg) of the left ventricular
wall were obtained serially with a drill biopsy.
Bleeding from the biopsy site was immediately controlled with a cotton plug. lschemic myocardium
was always sampled in a predetermined pattern
from within the angle formed by the left anterior
descending artery and its first diagonal branch well
within the demarcation of the ischemic boundary.
In preliminary experiments, measurement of blood
flow throughout this region using radiolabeled microspheres (New England Nuclear) showed consistent reduction to less than 1% of control flow.
Non-ischemic myocardium was sampled from a
fixed site in the distribution of the obtuse branch of
the circumflex artery, well outside the demarcation
of the ischemic boundary. In these serial experiments, the non-ischemic control region was sampled
before coronary artery occlusion ("pre-control")
and again at the end of the ischemic period ("postcontrol"). In six pigs, no biopsy was taken until the
end of the ischemic period when the whole left
ventricle was rapidly excised and placed on ice. Two
full thickness samples (10-15 g) were then taken
with a circular biotome from the control (categorized as "post-control") and ischemic regions. The
ischemic sample encompassed all the sites used for
the serial-sample experiments. Over petri dishes
containing dry-ice, approximately 2-mm-thick slices
of endocardium, epicardium, and mid-ventricular
wall were rapidly sliced by hand. All the samples
were quickly rinsed in ice-cold saline, blotted dry,
and frozen in liquid N2 before being weighed in the
frozen state. The phospholipid composition data
were expressed on a wet weight basis since it has
been reported to give more constant values than
the dry weight (Rouser et al., 1968).
Lipid Extraction
Lipids were extracted by the method of Folch et
al. (1957) with some modification to eliminate phospholipid losses during purification of crude extracts
(Kuksis, 1977; Nelson, 1969) and to achieve virtually
complete recovery of all phospholipid fractions including lyso-PL (Shaikh and Downar, 1980a). This
technique also circumvents any hydrolysis of vinyl
ether-linked phospholipids (Plasmalogens) which
results when tissues containing plasmalogens are
extracted with acidified solvents. Approximately
100-150 mg of frozen myocardial tissue were placed
in 6-ml ice-cold C-M mixture (2:1, vol/vol) containing 0.005% BHT (antioxidant) and homogenized in
a Potter-Elvehjem homogenizer (kept in ice) as
soon as the tissue began to soften. Appropriate
amounts (0.75 ng P/mg tissue wet wt.) of H-LPC
and H-LPE were added as internal standards.
These standard lyso-PL were used to monitor the
recoveries of myocardial lyso-PL from extraction
318
CIRCULATION RESEARCH
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mixture and subsequently to calculate the absolute
amounts of corresponding lyso-PL after thin layer
chromatography (TLC)-gas liquid chromatography
(GLC). The homogenate was transferred to a 15-ml
screw cap centrifuge tube. The homogenizer was
rinsed twice with 3-ml portions of C-M mixture and
the washes were added to the first homogenate to
give a final 12-ml volume. After the homogenate
had been shaken in Buchler Evapo-Mix for 15 minutes at room temperature, a biphasic mixture then
was produced by the addition of 3 ml of 0.9%
aqueous NaCl solution. The tubes were shaken
briefly and the two phases were produced by centrifugation. By blowing air gently, a Pasteur pipette
was carefully inserted through the aqueous upper
phase and interfacial protein residue and placed at
the bottom of the tube. The lower lipid phase was
then withdrawn without any contamination of the
upper phase through this pipette by another Pasteur pipette and transferred to the round bottom
flask. The remaining upper phase and protein residue were successively re-extracted three time each
with 9-ml portions of "synthetic" lower phase (CM-0.58% NaCl, 86:14:1 by volume). The lipid lower
phase was removed after centrifugation as described above. A fourth and final extraction was
made with synthetic lower phase containing 1 N
HC1, the lower lipid phase was withdrawn and
neutralized with ammonia vapor. All extracts were
pooled in a round bottom flask and taken to near
dryness in vacuo. The lipid residue was quantitatively transferred to a 7-ml glass vial using repeated
extraction with C-M-W (75:25:2, by volume), dried
under a stream of N2, dissolved in 0.1 ml of moist
chloroform, and subjected to TLC on the same day
for lyso-PL isolation. The isolated lyso-PL were
then quantified by their fatty acid content using
GLC.
The recovery of phospholipids through use of the
above extraction scheme was ascertained by extracting the myocardial tissue in the presence of
•^P-labeled rat liver phospholipid fractions of
known radioactivity. For comparison, lipid extracts
were also made with C-M (2:1, vol/vol) mixture
and the extracts were not backwashed with aqueous
salt solutions. Instead, the non-lipid contaminants
were removed by partition chromatography over
Sephadex G-25 columns (Nelson, 1972). Ldpids were
also extracted through use of acidified butanol by
the method of Bjerve et al. (1974). After fractionation of lipid extracts by TLC, a measurement of the
radioactivity of phospholipid fraction directly on
silica gel scrapings was made by liquid scintillation
spectrometry (Shaikh and Palmer, 1977).
Thin Layer Chromatography of
Phospholipids
Since lyso-PL are relatively minor components
of lipid extracts, several two-dimensional TLC per
sample are required for one analysis. As a more
VOL. 49, No. 2, AUGUST 1981
practical alternative, lipid extracts were fractionated by a one-dimensional TLC technique. This
system has several advantages, namely (1) a larger
load of lipid can be applied in a shape of a band, (2)
three samples can be fractionated per plate, and (3)
complete fractionation of lysophospatidylcholine
(LPC) and lysophosphatidylethanolamine (LPE)
from other major phospholipids can be achieved in
a shorter time.
Lysophospholipids were fractionated on 0.3-mm
thick layers of silica gel H plates, which were air
dried and activated for an hour at 120°C before use.
Lipid extract was quantitatively transferred and
applied to TLC plates as an evenly spread 5-cm
long band with several washings of the lipid containing vial with C-M-W (75:25:2, by volume). Pig
liver LPC and LPE standards were also spotted as
references for mobility of lyso-PL on TLC. Chromatograms were run at 22-25°C in a paper-lined
tank containing C-M-AA-W (75:60:10:8, by volume) . Under these conditions, the sequence of phospholipid fractions on TLC were as follows: origin,
LPC, sphingomyelin (Sph), choline phosphoglycerides (CPG), LPE, phosphatidylserine (PS), phosphatidylinositol (PI), ethanolamine phosphoglyceride (EPG), and cardiolipin (CL). PI and PS were
often not well resolved, and CL fraction which
moved along with the solvent front probably also
contained phosphatidic acid (PA), phosphatidylglycerol (PG), and other neutral lipids. The purity of
lyso-PL was checked by additional chromatographic techniques as described previously (Shaikh
and Palmer, 1976). Phospholipid fractions were localized undeT UV after exposure to 0.05% 2',7'-dichlorofluorescein in M-W (3:1) mixture. Silica gel
areas corresponding to lyso-PL were scraped and
fatty acid methyl esters (FAME) were prepared on
the silica gel scrapings by the alkali-catalyzed transesterification method of Glass (1971). To the scrapings in screw-capped tubes, 2 ml of 0.1 N sodium
methoxide in dry methanol-benzene (6:4, vol/vol)
containing 0.01% phenolphthalein were added and
the tubes were mixed on a vortex mixer. After 30
minutes at room temperature, the tubes were transferred on ice and the reaction mixture was neutralized with 0.2 ml 1 N acetic acid in petroleum ether.
Fatty acid methyl esters then were extracted with
redistilled petroleum ether, washed with water,
passed through anhydrous sodium sulfate mini-columns, and analyzed by GLC.
For quantitative purposes, phospholipids other
than lyso-PL were fractionated by TLC on precoated silica gel HR plates (Analtech Inc.) as previously described (Shaikh and Palmer, 1976). Phospholipid fractions were localized on chromatograms
by brief exposure to iodine vapor or with the acidmolybdate reagent of Dittmer and Lester (1964)
and the phosphorus determined by the method of
Bartlett (1959) directly on the lipid-containing silica
gel (Shaikh and Palmer, 1976). Occasionally, phos-
LYSOPHOSPHOLIPIDS IN ISCHEMIC MYOCARDIUM/S/iawWi and Downar
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pholipid composition was confirmed by two-dimensional TLC (Rouser et al., 1970) on 0.5-mm thick
layers of silica gel H plates prepared with 3%
aqueous Mg-acetate. When the fatty acid profile of
individual phospholipids was required, the chromatogram was sprayed with dichlorofluorescein
and the lipid-containing silica gel was refluxed with
6% sulfuric acid in methanol for 3 hours at 80° C for
an acid-catalyzed transesterification (Christie,
1972). The tubes were cooled on ice and the reaction
mixture was brought to near neutrality with 4 M
ammonium hydroxide. Fatty acid methyl esters and
dimethyl acetals (DMA), corresponding to acyl and
alk-1-enyl analogues, respectively (Farquhar, 1962),
were extracted with petroleum ether as described
above and analyzed by GLC.
Plasmalogen content was determined after isolation of CPG and EPG on silica gel H plates with
C-M-W (65:25:5, by volume) as a developing solvent. These purified phospholipids were re-chromatographed following exposure to HC1 fumes on
the chromatographic plates to convert the alk-1enyl analogue to 1-lysophosphatidyl analogue of the
respective phospholipids (Paltauf, 1971). Alternatively, FAME and DMA of isolated phospholipids
were prepared as described above and analyzed by
GLC before and after TLC fractionations, using
benzene as the developing solvent. The percent
fraction of each component was then calculated.
Gas Liquid ChromatogTaphy
Fatty acid methyl esters and DMA were analyzed
by GLC (Kuksis, 1978) routinely performed on 6 ft.
x 1/8 in. glass columns packed with 10% EGSS-x
on 100-120 mesh Gas Chrom P (Applied Sciences
Laboratories). The columns were installed in a F
& M Scientific Model 402 Gas Chromatograph
equipped with flame ionization detector. The carrier gas was nitrogen at 40 ml/min. Isothermal runs
were made at 175°C with injector and detector
heaters at 200 and 25O°C, respectively. In several
experiments, fatty acid profiles were confirmed by
temperature programmed runs on 30-meter glass
capillary columns coated with SP-10O0 (Supelco)
through use of Hewlett Packard model 5880A gas
chromatograph equipped with a split mode injector,
flame ionization detector, and Level 4 microprocessor/integrator. The runs were made in the range of
120-210°C at a heating rate of 5°C/min with zero
grade helium as a carrier gas. The quantities of
individual fatty acids (nmol) were calculated by
comparing the peak areas with that of internal
standard after appropriate corrections for the differences in the recovery of fatty acids from the
column and in the sensitivity of the detector for
different fatty acids were made.
Statistics
Significance of the results were determined by
non-paired Student's {-test. P values of less than
0.05 were considered statistically significant.
Results
Recovery and Reproducibility of Phospholipid
Extraction and Analysis
Since lyso-PL levels in various tissues are very
low compared to levels of other phospholipids, it
was essential to adopt an extraction scheme for
cardiac tissue that would provide complete recoveries of lyso-PL without intrapreparative production of these compounds from plasmalogens. The
extraction scheme adopted in this communication
(modified Folch, see Methods) was tested for the
recoveries of endogenous lyso-PL and of exogenously added 32P-labeled lyso-PL of known radioactivity. Table 1 compares the recoveries obtained
with the modified Folch to that of other methods.
For reasons discussed elsewhere (Nelson, 1969;
Kuksis, 1977), the Folch extraction scheme using CM mixture followed by a time-consuming purification on Sephadex G-25 column (impractical for a
large number of analyses) was also used as a standard to evaluate other recoveries. The classical
Folch method, which involves purification of crude
lipid extracts in bipasic mixtures, resulted in 8790% recovery of endogenous lyso-PL and 89-94% of
exogenous radiolabeled lysocompounds. These recoveries could be improved to >98% if lipids were
extracted by the modified Folch. In this scheme,
TABLE 1 Recoveries of Lysophospholipids
LPC
Extra ctK>n
scheme
Folch/column
Classical Folch
Modified-Folch
Acid-butanol
No. of
analyse*
4
4
6
6
nmol
4.823 ± 0.052
4.341 ± 0.068
4.79«± 0.028
79.17 ± 11.65
319
LPE
dpm
7936 ±48
7063 ±68
7921 ± 2 3
7941 ±98
nmol
7.401 ±
6.439 ±
7.356 ±
122.59 ±
dpm
0.088
0.102
0.040
20.10
6831 ±61
6012 ± 72
6839 ±43
6824 ± 110
Data are expressed a* mean ± SD. Control myocardlal tissue was homogenized in 03% NaCl at 4'C and equal
portions of the homogenale representing 125 mg tissue wet weight were eitracted under each protocol. Lysophospholipids were then analyzed after TLC/GLC using internal standards (see Methods). Similar protocols were
repeated with added "P-labeled rat liver lygophospholipids (8021 and 7004 dpm/6 /ig P of LPC and LPE, respectively)
and, after fractionation of phospholipids by TLC, radioactivity w i s determined directly on silica jel scrapings of
appropriate fractions.
CIRCULATION RESEARCH
320
C
A. LPC
VOL. 49, No. 2, AUGUST
1981
LPE
I
I
MODIFIED FOLCH EXTRACTION
D. LPE
B. LPC
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ACID-BUTANOL EXTRACTION
v .
1 Comparison of the fatty acid profiles of lysophospholipid extracted with modified-Folch and acid-butanol
techniques. For details, see Methods. Exogenous palmitaldehyde was added to D. LPE before GLC to indicate its
position on the chromatogram.
FIGURE
the first three successive neutral solvent extractions
provided about 95% recovery of endogenous lysoPL and over 98% of exogenous labeled lyso-PL. The
final acidic extraction recovered virtually all of the
remainder. The recoveries of total phospholipids,
when compared to the Folch/column purification
scheme, were better than 98% for six analyses. Similar recoveries (98.9 ± 1.7% for three determinations) were also obtained for exogenously added
radiolabeled phospholipid fractions suggesting that,
under the experimental conditions used, intrapreparative hydrolyses of phospholipids did not occur.
The acid-butanol extraction scheme (Bjerve et al.,
1974) resulted in a 12- to 19-fold increase in lysoPL levels even though the entire procedure was
performed in a cold room on ice. The recoveries of
added radiolabeled lysocompounds remained unchanged. This suggested preferential hydrolysis of
phospholipids containing vinyl ether linkages. Gasliquid chromatographic analysis of fatty acyl profiles of lysocompounds and their precursor phospholipid (CPE and EPG) confirmed such hydrolysis. A substantial decrease in DMA content of CPG
and EPG was observed and was comparable to the
increase in unsaturated fatty acids in lysocompounds (see Fig. 1).
Aliquots of normal myocardium were extracted
in the presence of internal standards, and lyso-PL
were quantified by TLC/GLC. The reproducibility
of the entire method of analysis is presented in
Table 2. The maximum variation from the mean
was less than 1% for lyso-PL and 3% for total
phospholipids, and the difference between highest
and lowest levels was less than 1.5% and 5%, respectively. This highly reproducible method for the
quantitation of lyso-PL was adopted for all subsequent analyses.
Lysophospholipid Levels of Normal and
Ischemic Myocardium
Lyso-PL levels in normal and ischemic myocardium are presented in Table 3. Prior to occlusion,
levels of both LPC and LPE were very low (0.15%
of total phospholipids) and did not vary much
(<3%) among different hearts. Following 8 minutes
of coronary occlusion, the levels increased by about
60% compared to their own pre-control values or
the mean of the pre-controls. In a few instances,
321
LYSOPHOSPHOLIPmS IN ISCHEMIC MYOCARDIUM/Shaikh and Downar
TABLE 2 Reproducibility of Phosphohpid Extraction
and Analyses
Analysis
no.
LPC
(nmol)
LPE
(runol)
Total pho»phobpid
1.
2.
3.
4.
5.
6.
4.822
4.755
4.826
4.789
4.813
4.780
7.379
7.315
7.419
7.331
7.371
7.323
102.1
98.6
103.3
101.7
101.0
102.8
Mean
±SD
4.798
0.028
7.356
0.O4O
101.6
1.7
TABLE 4 Molecular Species of Lysophospholipids of
Normal and Ischemic Porcine Myocardium*
LPC (mole %)
Fatty acidif
LPE (mole %)
Normal
Iacheroic
Normal
Uchemic
1.35
30.85
0.40
36.57
15.82
1162
trace
3.39
1.24
29.20
0.70
34.02
16.47
14.31
trace
4.06
3.41
15.45
0.38
32.94
13.48
12.03
7.54
14.77
4.10
13.80
1.02
32.14
15.87
14.51
5.40
13.16
14:0
16:0
16:1
18:0
18:1
18:2
20:0
20:4
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Data expressed aa mean ± SD of (n) analyses- Aliquots of myocardial
homogcnatc (see legend Table 1) representing 125 mg tissue wet weight
were extracted along with internal standards by the modifled-Folch technique After fractionation of lipid eitracta by TLC, lyso-PL were analyzed
by GLC and other phospholipid fractions were qu»ntified by phosphate
analysis.
Result* are mean offivedeterminations.
• Samples were taken from normal (pre-control) and ischemic (40minute occlusion) myocardium. Values of iachemic myocardium are not
significantly different from the normal (P > 0.50). Both LPC »nd LPE
showed traces of 16 0 DMA, not recorded in table
f Carbon length • number of unsaturated bonds.
endocardium was isolated for analysis. The lyso-PL
levels in these cases did not appear to differ from
those observed in transmural samples.
In normal myocardium, the molecular species of
LPC is made up of 70% saturated and 30% unsaturated entities, whereas in LPE, the distribution is
about 50% each (Table 4). In the ischemic myocardium where the total content of lyso-PL increased,
the distribution of the individual molecular species
did not alter. This suggests that the increases in
lyso-PL levels were not restricted to certain molecular species but were evenly spread among all molecular entities.
Total phospholipid content of myocardium remained essentially constant throughout the first 40
minutes of ischemia (P > 0.50). In two pigs in which
coronary occlusion was maintained for 8 and 20
hours, the total phosphohpid content/g tissue wet
weight fell by 6 and 14%, respectively. The molar
proportions of individual phospholipidfractionsdid
not alter at any time with the exception of lysocompounds (Table 5). Plasmalogens constituted about
36 and 54% of CPG and EPG, respectively, and
remained unchanged in the ischemic myocardium
throughout the period of study.
Phosphohpid fractions of normal and ischemic
myocardium were also analyzed for their fatty acid
and aldehyde content (Table 6). The vinyl ether
linkages in plasmalogen when hydrolyzed produce
aldehydes which are quantified as DMA derivatives
by GLC. In CPG and EPG, aldehydes contributed
to about 18% and 26%, respectively, of the aliphatic
chains. These aldehydes are made up largely of
palmitaldehyde in the CPG fraction and of palmit-, stear-, and olealdehyde in the EPG fraction.
Both fatty acid and aldehyde profiles of the phosphohpid fractions did not appear to change with
the onset of ischemia, suggesting that a similar
metabolic specificity for all molecular species was
maintained during the ischemic insult.
TABLE 3
Discussion
In vivo concentrations of lyso-PL in porcine myocardium are very low. Artifactual lyso-PL can be
produced readily if tissue storage is prolonged even
at -20°C (Rouser et al., 1968) and by careless
Lysophospholipid Content of Porcine Myocardium
LPC
Time ligation
(min)
Normal zone
0 (Pre-control)
2-10 (Post-control)
Ischemic zone
2
4
6
12
20
40
runol/g wet wt.
LPE
%
change
nmol/g wet wt.
%
change
403 ± 1.3 (22)
51.0 ± 2.6 (19)
26.6
60.6 ± 3.9 (22)
75.5 ± 4.9 (19)
24.6
47.4 ± 2.9
49 0 ± 3.9
63.5 ± 5.2
50.6 ± 3 . 2
55.8 ± 3 . 6
55.2 ± 2 . 6
17.6
21.6
57.6
25.6
38.5
37.0
83.2 ± 10.0 (9)
98.1 ± 10.0 (6)
95.8 ± 8.4 (10)
97.4 ±8.4 (11)
103.5 ± 11.0 (7)
101.3 ± 10.0 (7)
37.3
61.9
58.1
60.7
70.8
67.2
(9)
(6)
(10)*
(11)
(7)*
(8)'
Data eiprested aj mean ± SD of (n) analyses. All values are significantly different (P < 0.001) than respective
pre-conLrot* (for LPC and LPE) and post-control (LPE).
* Significantly different from LPC post-control (P < 0.05)
322
CIRCULATION RESEARCH
VOL. 49, No. 2, AUGUST
1981
TABLE 5 Phospholipid Composition of Normal and Ischemw Myocardium
Normal zone
(0 min)
Time ligation
Ischemic zone
20-40 min)
(40 min)
(2-20 min)
(24 hr)
Vtf wet wt. (mean ± 3D of (n) observations)
Total phospholipids
825.3
±43.1
831.6
±52.6
818.3
±49.2
(5)
(5)
(5)
Phoapholipid fractions*
801.7
±62.8
(5)
715.6
±18.2
(3)
% of total lipid-P
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37.85
24.22
13.63
25.34
12.16
13.18
19.24
6.29
4.34
6.04
0.18
0.38
0.15
0.23
CPG
Diacyl
Alkenyl-acyl
EPG
Diacyl
Alkenyl-acyl
CL
PI
PS
Sph
PA
PG
LPC
LPE
37.10
23.68
13 42
25.68
12.26
13.42
18.73
7,12
4.12
6.18
.38.16
24.36
13.80
25.37
12.07
13.30
18.70
6.09
4.61
6.02
37.22
23.86
13.36
26.69
12.81
13.88
17.50
6.35
4.21
6.81
38.40
24.56
13.84
28.87
13.M
14.89
14.47
6.39
4.52
6 65
i<0.60
| <0.60
|<0.60
<0.60
0.19
0.28
0.21
0.34
0.22
0.40
0.25
0.45
Abbreviations used: CFG, choline phosphoglycendes; EPG, ethanolamine phosphoglycendes; CL, cardiolipin;
PI, phosphatidylinositol; PS, phosphatidylserine; Sph, sphingomyelin, PA, phoaphatidic acid; PG, phosphHtidylglyccrol. Total phosphohpid content was calculated by adding phosphate content of individual phospholipid fractions
with that of LPC and LPE amounts which were estimated by TLC/GLC as descnbed in Methods
* Mean of duplicate analyses of three independent samples. Data of the ischemic zone did not differ from that
of normal zone {P > 0 50) with the eiception of 24-hour samples which represents the mean of three independent
analyses of the same pig.
extraction and purification of lipids (Nelson, 1969).
Sub-cellular fractionation procedures also result in
the production of lyso-PL (Fleischer and Rouser,
1965; Rouser et al., 1972). These artifactual elevations result from hydrolysis of the precursor phospholipid by tissue phospholipases. Extraction of
tissues with mildly acidic solvents can also produce
lyso-PL by chemical hydrolysis of vinyl ether linkages (Snyder, 1969) present at position 1 of the
glycerol backbone of certain phospholipids (plasmalogens). On the other hand, failure to use acidic
solvents results in incomplete recoveries of certain
TABLE 6 Composition of Acyl and Alk-lenyl Moieties from Porcine Myocardial Phospholipids of Normal and
Ischemic Zones*
CPG
EPG
IscheComponentsf
PI
CL
Ische-
Normal
mic
Normal
mic
12.68
20.63
0.90
13.4S
19.37
0.84
0.61
1.59
6.66
1.93
17.10
27.39
0.56
10.76
3.15
0.84
0.89
8.59
18.12
3.80
5.01
17.52
0.77
9.52
2.84
0.86
0.71
10.04
18.67
3.84
4.42
16.03
0.67
Ische-
IscheNormal
mk
Normal
Sph
PS
mic
IscheNormal
mic
14:0
16:0DMA
16:0
16:1
17:0DMA
18:0DMA
18:0
18:1 DMA
18:1
18:2
18:3
20:0
20:2
20:4
22:0
1.05
1.37
6.80
1.98
17.22
27.38
0.74
4.70
6.50
25.36
27.14
1.60
7.39
1 55
9.78
1.60
1.55
45.11
16.29
71.13
tr.
16.98
68.11
10.57
13.30
3.38
tr.
3.62
0.58
1.17
1.24
17.66
18 42
9.73
45.13
44.28
44,46
14.37
14.0
10.21
11.31
19.05
14.28
18.21
14.02
tr.
tr.
tr.
tr
tr
21.41
1.23
2241
1.35
12.88
5.16
10.14
13.40
13.46
5 39
9fi4
11.57
tr
tr.
1 72
tr.
27.72
8 91
9.47
24:0
24.1
0.55
1.72
9.48
23:0
22:6
mic
tr.
tr.
tr.
25.64
IscheNormal
tr.
tr
tr.
" Simples were taken from normal and ischemic aones after 40 minutes of LAD occlusion and are mean of three analyses; DMA, dimethylaceuds
representing «lkenyl moieties, tr., trace Longer chain fatly acid, e g., 20:3, 20ft,22:5 in CPG, EPG, PI, and PS, were less than \% and are not recorded.
| Carbon length - number of uruaturated bonds
LYSOPHOSPHOLIPIDS IN ISCHEMIC MYOCARDIUM/Shaikh and Downar
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phospholipids (e.g., lyso-PL, PI, PS, CL, etc.). The
use of alkaline solvents (Rouser et al., 1963) also
produces deacylated products in small amounts.
Phospholipids, mainly acidic, can also be lost during
biphasic purification of lipid extracts (Table 1; Kuksis, 1977).
In the present study, myocardial samples were
quickly frozen in liquid nitrogen and then immediately subjected to lipid extraction without further
delay to avoid postmortem and intrapreparative
hydrolysis of phospholipids. Incomplete recoveries
and plasmalogenic hydrolysis were avoided by a
suitable adaptation of the classical Folch lipid extraction technique. Losses due to physical handling
of the lipid extracts (e.g., extraction from round
bottom flasks, transfer to TLC plates, transmethylation process, and extraction of FAME, etc.) were
corrected for by the use of appropriate internal
standards (see Methods). Consequently, a high degree of accuracy and reproducibility was achieved
in the analyses of total phospholipids and lyso-PL
in porcine myocardial tissue (Tables 2 and 3).
The total phospholipid content of porcine myocardium is in close agreement with previous reports
on ungulates (see White, 1973). However, LPC
levels of normal porcine myocardium reported in
this study are lower than those reported for different organs and heart muscle of other mammalian
species (0.15% vs. 0.4-1.6% of total lipid-P). LPE
content was not detectable in the heart muscle of
other mammals and often not analyzed in other
organs. Levels of LPC and LPE in the human heart
(1 hour 45 minutes postmortem) were 3.5 and 1.5%
of total lipid-P, respectively (Simon and Rouser,
1969), which is higher than reported here for porcine myocardium. All these studies were not directed principally toward the analyses of lyso-PL,
and their higher values might be attributable to the
intrapreparative/postmortem production of such
compounds. There has been no specific assessment
of lyso-PL in any myocardial tissue other than
rabbit myocardium (Sobel et al. 1978). That study
showed levels of lyso-PL manyfold (20-35X)
greater than those presented here for pig myocardium. This difference may, in part, be due to species
variation but is more likely due primarily to the
methodology used. The rabbit myocardium was
extracted with acidified butanol over a period of 2
hours. This would be expected to result in the
hydrolysis of acid-labile plasmalogens, thereby producing lysocompounds. The authors, however, attempted to refute this possibility with a strong
argument based on the fatty acid profiles of lysoPL, their structural analyses, and the chromatographic behavior of monoacylglycerols enzymatically produced in vitro from lyso-PL. Porcine myocardium, when extracted with acidified butanol for
10 minutes, produced an artifactual increment of
12- to 20-fold in lyso-PL levels that were about onehalf of that reported for rabbit myocardium. These
lyso-PL, upon fatty acid analysis, revealed a very
323
high content of unsaturated fatty acids which was
consistent with hydrolysis of plasmalogens (see Fig.
1). Furthermore, analyses of the precursor phospholipids (CPG, EPG) showed a reciprocal decrease in
the aldehyde content, confirming that vinyl ether
linkages had been hydrolyzed with acidified butanol. Thus, there is a strong possibility that the
much higher levels of lyso-PL reported in rabbit
myocardium were of intrapreparative origin.
Myocardial ischemia produces certain well
known metabolic effects such as accumulation of
K+, lactate, AMP, ionosine, and inorganic phosphate (see Opie, 1969; Jennings, 1976). Recently,
Sobel et al. (1978) reported that lyso-PL levels in
rabbit myocardium also increased during ischemia.
In porcine myocardium, a 60-70% increase in lysoPL levels occurred but, as already stated, the concentrations were very much lower. Lyso-PL concentrations in tissues are determined by the interplay
of phospholipid-metabolizing enzymes. Membranebound phospholipases can produce lyso-PL which,
in turn, can either be re-acylated or transacylated,
depending upon the energy state of the cell, to form
precursor PL or further degraded (Van den Bosch,
1974). Normally their concentration is maintained
very low, since lyso-PL in sufficient quantities are
potent detergents and could alter general properties
of the membrane such as fluidity and permeability
(see Weltzein, 1979). In in vivo studies, they have
been shown to be vasopressors (Takumura et al.,
1978) and have also been implicated in the genesis
of malignant arrhythmias (Corr et al., 1979). The
lyso-PL levels seen in ischemic porcine myocardium
represent <0.6% of total phospholipids and are
much lower than those shown to have deleterious
electrophysiological effects. Such concentrations, if
distributed evenly over the entire tissue membranes, are not likely to produce changes in membrane permeability. On the other hand, if these
accumulations are restricted to certain parts of the
membrane or subcellular organelles, they could be
sufficient to modify the microenvironment of certain enzymes so as to produce deleterious effects on
cell function. Lysophospholipids have been shown
to affect directly the activities of enzymes such as
sarcolemmal Na+-K+-ATPase and galactosyltransferase (Moorkerjea and Yung, 1974; Shier and Trotter, 1976; Owens et al., 1979). A number of other
enzymes have been shown to require phospholipids
for their activities (see Finean, 1973).
The small but significant elevation of lyso-PL in
non-ischemic porcine myocardium is surprising. It
may reflect a general activation of myocardial phospholipase activities due to indirect effects of ischemia, such as an increase in cardiac sympathetic
tone (Malliani et al., 1969). Although direct stimulation of phospholipases has yet to be described,
catecholamines produce an increase in intracellular
Ca2+ levels, and phospholipase activation has been
shown to be Ca2+-dependent. In ischemic myocardium, in addition to elevated Ca2+, other factors
CIRCULATION RESEARCH
324
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such as hypoxia, low levels of high energy phosphates, and bradykinin release may also lead to
activation of phospholipases. These factors, together with the lack of washout, result in a greater
accumulation of lyso-PL. The fact that lyso-PL
levels (LPC + LPE) did not continue to increase
beyond 8 minutes of ischemia suggests that, after
reaching a critical concentration, they are degraded
further by lysophospholipases and other enzymes.
The molecular species of lyso-PL (LPC, LPE) in
ischemic myocardium did not differ from that of
normal myocardium. The effect of ischemia therefore is probably mediated by an activation of phospholipases that is not species specific. It also indicates that the elevation in lyso-PL levels was not
likely due to entrapment of platelets and erythrocytes, since these would be expected to alter the
fatty acids profiles.
Ischemia of up to 40 minutes in duration did not
produce any change either in total phospholipid
content or in the individual phospholipid fractions.
When these were analyzed for fatty acyl and aldehyde content, no significant change was observed.
This suggests that, in general, the metabolic integrity of the membranes was maintained. The decreases in the total phospholipid content seen after
8 and 24 hours of ischemia may reflect the change
in tissue weight due to edema and/or a greater rate
of membrane phospholipid degradation, probably
due to leaky lysosomes (see Hoffstein et al., 1976).
Substantial decreases in some phospholipid fractions of subcellular organelles obtained from ischemic rat liver and canine myocardium favor the
latter view (Chien et al., 1978; Vasdev et al., 1979).
The observation that the mole fraction of individual
phospholipids of 24-hour ischemic myocardium did
not appear to alter indicates that the phospholipid
degradation was not selective.
In conclusion, although early ischemia produces
a significant elevation of lyso-PL in porcine myocardium, the absolute quantitites are very small
and alone cannot be responsible for the electrophysiological changes of ischemia. The functional
significance of these elevations in the presence of
other concomitant changes associated with ischemia remains to be determined.
Acknowledgments
We are indebted to Leon Lam for careful technical assistance.
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N A Shaikh and E Downar
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Circ Res. 1981;49:316-325
doi: 10.1161/01.RES.49.2.316
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