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The squalene content of atheromatous plaques A re-examination
Atherosclerosis, 22 (1975) 637-640
637
@IElsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communication
THE
SQUALENE
CONTENT
OF
ATHEROMATOUS
PLAQUES
A RE-EXAMINATION
R. W. LEWIS Department of Clinical Biochemistry, Otago University School of Medicine, Dunedin (New Zealand) (Received June 9th, 1975) (Accepted June lOth, 1975)
SUMMARY
Eight samples of human aorta wall and plaques contained between 0.35 and 1.3 ppm squalene, in contrast to the high concentrations of 550 ppm and 1000 ppm reported
previously
for atherosclerotic
pigeon aorta and human
atheromatous
plaques.
Key words : Anoxia - Atheromatous plaques - Squalene
INTRODUCTION
The squalene content of human atheromatous plaques and atherosclerotic pigeon aorta have been reported as reaching a maximum in the order of 0.1% of the wet weight (or 1000 ppm)r and approximately 500 ppm2, respectively. As normal human aorta contained 2 ppma and normal rabbit aorta 45 ppm relative to dry, delipidized tissue4, the values from atherosclerosis indicate an abnormal accumulation of squalene that might be associated with the pathology of the disease. For example, the high squalene content of atheromatous plaques has been considered as a possible indication of anoxia within the plaque because of the known requirement of oxygen for the conversion of squalene to sterolsa.
This work was supported
by the National Heart Foundation
of New Zealand.
638
SHORT COMMUNICATION
Since these results
carried
important
implications
and were given as approxi-
mations, analyses of aorta walls and plaques were undertaken to confirm and extend these findings using a method of proven accuracy and sensitivityspa. MATERIALS
AND
METHODS
Aortas were obtained
at necropsy
within 24 hours post mortem and were selected
to include a normal aorta, showing no evidence of plaque formation, and atherosclerotic aortas ranging from mildly diseased to severely diseased with ulcerated plaques and considerable calcification. If not dissected immediately, the aortas were stored at -20 “C. Adventitia was removed and samples were taken ofthe normal wall and of plaques with and without media. Lipids were extracted by the method of Folch et al.7.The extracts were dried over anhydrous sodium sulphate, evaporated under vacuum at 40°C weighed, and taken up in hexane. Solvents were redistilled and filter papers were eluted with chloroform before use. Squalene was assayed by a method previously described5,s which entailed addition of a known amount separation of the hydrocarbon
of n-tetracosane to the lipid as an internal standard, fraction by thin layer chromatography (TLC), and
analysis of this fraction by gas liquid chromatography (GLC). Squalene apparently occurs in all plant and animal tissues and usually in large enough amounts so that traces of alkanes in these tissues do not interfere with the analysis. However, because of the presence
of larger amounts
of alkanes
in aortic tissues, the method
was modified
as follows: The TLC solvent system was changed to petroleum ether (40”-60 “C)/ benzene 98/2 (v/v) to permit isolation of squalene (Rf0.5)free from alkanes, and the internal standard was added to the squalene after elution from the silica gel. In addition, the GLC on a 240 cm column of 1% SE-30 (60 ml Na/min) was done at two temperatures to broaden the standard peak for easier measurement: 220 “C until the n-tetracosane peak appeared, and 250 “C for the squalene peak. The amount of squalene could be calculated from the relationship of its peak area to that of the internal standard. Duplicate analyses were made by use of aliquots from the same lipid extract. RESULTS
The results are summarized in Table 1. It can be seen that the normal aorta and the wall from the diseased aorta gave values that were somewhat lower (less than 1 ppm) but generally agreeing with the 2 ppm and 2.6 ppm squalene previously reported for adult and child’s aortaa. In contrast, plaque tissue showed only slight increases in squalene content to a maximum of 1.3 ppm compared to the reported concentrations of up to approximately 1000 ppm. This latter value was supported by the report that the atherosclerotic pigeon aorta contained 500 ppm squalene2. The data in Table 1 show that there was no correlation between squalene content and lipid content, but there was some correlation with the severity of the disease.
SHORi
639
COMMUNICATION
TABLE SQUALENE
1 CONTENT
OF HUMAN
AORTA
AND
ATHEROMATOUS
Tissue
Normal aorta, Sample I Normal aorta, Sample 11 Moderately diseased aorta: wall plaque and wall plaque and wall Moderately diseased aorta: intima and plaque Moderately diseased aorta: intima and plaque Moderately diseased aorta: intima and plaque Heavily diseased aorta: intima and plaque Heavily diseased aorta, ulcerated and calcified : intima and plaque
Normal aorta gave the lowest concentrations a slight elevation and severe atherosclerosis
PLAQUES
Wet weight ig)
Lipid content Squalene ( 76) first analysis (PPM)
second analysis (PPm)
6.04
6.12
2.02 3.26
0.40 0.35
0.46 0.46
2.12 1.26 2.65
1.87 9.45 1.6
0.85 0.93 0.59
1.3 0.49
2.56
14.7
0.81
1.16
4.16
9.1
0.58
0.68
1.81
1.4
0.85
0.96
3.31
9.1
0.95
1.07
10.7
1.19
1.3
4.1
content
but moderate atherosclerosis produced had levels up to 3 times higher.
The method for assaying squalene has been tested on two previous occasions, giving recoveries of 85% and 86.7% at levels of 5 ppm and 53 ppm relative to lipid weight and variance of up to 10 ’ /, 596. Because of the marked disparity of the results from the data reported for plaques, the method was tested again on known concentrations of squalene in lipid and found to give the same average recoveries but a somewhat greater variance, probably because the internal standard gave narrower peaks with this GLC column,
This would increase
errors in measurement.
DISCUSSION
The question of cholesterol synthesis within the plaque has long been of concern in the study of atherosclerosis. Although the present study was directed toward confirming previous reports of high squalene concentrations, it offers some suggestive evidence on this question. Studies by Loud and Bucherg have shown that approximately lppm squalene would suffice for sterol synthesis in the rat liver over a period of lo-15 min. Since much of the cholesterol synthesized by the liver is for export in lipoproteins, a lower rate of synthesis would be expected in the aorta. The calculations of St Clair et al.2 are in agreement by showing that the pigeon aorta would synthesize a minimum of 0.5 ppm squalene in 4 hours. The average concentration of 0.42 ppm squalene found in the normal aorta in this study appears to be also at an
640
SHORT COMMUNICATION
appropriately
low level. The findings
of increased
sclerotic plaques (up to 1.3 ppm) is consistent for diseased aortasz. The reports
of larger
concentrations
amounts
of squalene
in athero-
with the greater sterol synthesis reported of squalene
(550 ppm and
1000 ppm) in
atherosclerotic pigeon aorta and human plaques are in obvious disagreement with the results of this study. Details of the methods by which squalene was assayed are not available findings
for appraisal
are not typical
in these reports, of atherosclerosis.
but at least it can be concluded Therefore,
which was considered as a possible explanation for squalenez, is not supported by the results of this study.
anoxia the large
within
that these the plaque,
concentrations
of
REFERENCES 1 BROOKS, C. J. W., HARLAND, W. A. AND STEEL, G., Squalene, 26-hydroxycholesterol and 7-ketocholesterol in human atheromatous plaques, Biochim. Biaphys. Acra, 125 (1966) 620. 2 ST. CLAIR, R. W., LOFLAND, H. B., PRICHARD, R. W. AND CLARKSON, T. B., Synthesis of squalene and sterols by isolated segments of human and pigeon arteries, Exp. Mol. Path., 8 (1968) 201. 3 GARBUZOV, A. G., PYATNITSKII AND PISKUNOV, A. K., Presence of squalene in human aorta. Fed. Proc., Trans. Suppl., 25 (1966) 463T. 4 STEFANOVICH, V. AND KAJIYAMA, G., Squalene and cholestanol in normal rabbit aorta, Atherosclerosis, 11 (1970) 401. 5 LEWIS, R. W., Squalene distribution in fish with normal and pathologically fatty livers, Inr. .f. Biochem., 2 (1971) 609. 6 LEWIS, R. W., The squalene content of plant tissues, Phytochem., 11 (1972) 417. 7 FOLCH, J., LEES, M. AND SLOANE STANLEY, G. H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 8 RAMSEY, R. B., AEXEL, R. T., JAIN, S. AND NICHOLAS, H. J., Distribution of alkanes in normal and atherosclerotic rabbit aortas, Atherosclerosis, 15 (1972) 301. 9 LOUD, A. V. AND BUCHER, N. L. B., The turnover of squalene in relation to the biosynthesis of cholesterol, J. Biol. Chem., 223 (1958) 37.
637
@IElsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communication
THE
SQUALENE
CONTENT
OF
ATHEROMATOUS
PLAQUES
A RE-EXAMINATION
R. W. LEWIS Department of Clinical Biochemistry, Otago University School of Medicine, Dunedin (New Zealand) (Received June 9th, 1975) (Accepted June lOth, 1975)
SUMMARY
Eight samples of human aorta wall and plaques contained between 0.35 and 1.3 ppm squalene, in contrast to the high concentrations of 550 ppm and 1000 ppm reported
previously
for atherosclerotic
pigeon aorta and human
atheromatous
plaques.
Key words : Anoxia - Atheromatous plaques - Squalene
INTRODUCTION
The squalene content of human atheromatous plaques and atherosclerotic pigeon aorta have been reported as reaching a maximum in the order of 0.1% of the wet weight (or 1000 ppm)r and approximately 500 ppm2, respectively. As normal human aorta contained 2 ppma and normal rabbit aorta 45 ppm relative to dry, delipidized tissue4, the values from atherosclerosis indicate an abnormal accumulation of squalene that might be associated with the pathology of the disease. For example, the high squalene content of atheromatous plaques has been considered as a possible indication of anoxia within the plaque because of the known requirement of oxygen for the conversion of squalene to sterolsa.
This work was supported
by the National Heart Foundation
of New Zealand.
638
SHORT COMMUNICATION
Since these results
carried
important
implications
and were given as approxi-
mations, analyses of aorta walls and plaques were undertaken to confirm and extend these findings using a method of proven accuracy and sensitivityspa. MATERIALS
AND
METHODS
Aortas were obtained
at necropsy
within 24 hours post mortem and were selected
to include a normal aorta, showing no evidence of plaque formation, and atherosclerotic aortas ranging from mildly diseased to severely diseased with ulcerated plaques and considerable calcification. If not dissected immediately, the aortas were stored at -20 “C. Adventitia was removed and samples were taken ofthe normal wall and of plaques with and without media. Lipids were extracted by the method of Folch et al.7.The extracts were dried over anhydrous sodium sulphate, evaporated under vacuum at 40°C weighed, and taken up in hexane. Solvents were redistilled and filter papers were eluted with chloroform before use. Squalene was assayed by a method previously described5,s which entailed addition of a known amount separation of the hydrocarbon
of n-tetracosane to the lipid as an internal standard, fraction by thin layer chromatography (TLC), and
analysis of this fraction by gas liquid chromatography (GLC). Squalene apparently occurs in all plant and animal tissues and usually in large enough amounts so that traces of alkanes in these tissues do not interfere with the analysis. However, because of the presence
of larger amounts
of alkanes
in aortic tissues, the method
was modified
as follows: The TLC solvent system was changed to petroleum ether (40”-60 “C)/ benzene 98/2 (v/v) to permit isolation of squalene (Rf0.5)free from alkanes, and the internal standard was added to the squalene after elution from the silica gel. In addition, the GLC on a 240 cm column of 1% SE-30 (60 ml Na/min) was done at two temperatures to broaden the standard peak for easier measurement: 220 “C until the n-tetracosane peak appeared, and 250 “C for the squalene peak. The amount of squalene could be calculated from the relationship of its peak area to that of the internal standard. Duplicate analyses were made by use of aliquots from the same lipid extract. RESULTS
The results are summarized in Table 1. It can be seen that the normal aorta and the wall from the diseased aorta gave values that were somewhat lower (less than 1 ppm) but generally agreeing with the 2 ppm and 2.6 ppm squalene previously reported for adult and child’s aortaa. In contrast, plaque tissue showed only slight increases in squalene content to a maximum of 1.3 ppm compared to the reported concentrations of up to approximately 1000 ppm. This latter value was supported by the report that the atherosclerotic pigeon aorta contained 500 ppm squalene2. The data in Table 1 show that there was no correlation between squalene content and lipid content, but there was some correlation with the severity of the disease.
SHORi
639
COMMUNICATION
TABLE SQUALENE
1 CONTENT
OF HUMAN
AORTA
AND
ATHEROMATOUS
Tissue
Normal aorta, Sample I Normal aorta, Sample 11 Moderately diseased aorta: wall plaque and wall plaque and wall Moderately diseased aorta: intima and plaque Moderately diseased aorta: intima and plaque Moderately diseased aorta: intima and plaque Heavily diseased aorta: intima and plaque Heavily diseased aorta, ulcerated and calcified : intima and plaque
Normal aorta gave the lowest concentrations a slight elevation and severe atherosclerosis
PLAQUES
Wet weight ig)
Lipid content Squalene ( 76) first analysis (PPM)
second analysis (PPm)
6.04
6.12
2.02 3.26
0.40 0.35
0.46 0.46
2.12 1.26 2.65
1.87 9.45 1.6
0.85 0.93 0.59
1.3 0.49
2.56
14.7
0.81
1.16
4.16
9.1
0.58
0.68
1.81
1.4
0.85
0.96
3.31
9.1
0.95
1.07
10.7
1.19
1.3
4.1
content
but moderate atherosclerosis produced had levels up to 3 times higher.
The method for assaying squalene has been tested on two previous occasions, giving recoveries of 85% and 86.7% at levels of 5 ppm and 53 ppm relative to lipid weight and variance of up to 10 ’ /, 596. Because of the marked disparity of the results from the data reported for plaques, the method was tested again on known concentrations of squalene in lipid and found to give the same average recoveries but a somewhat greater variance, probably because the internal standard gave narrower peaks with this GLC column,
This would increase
errors in measurement.
DISCUSSION
The question of cholesterol synthesis within the plaque has long been of concern in the study of atherosclerosis. Although the present study was directed toward confirming previous reports of high squalene concentrations, it offers some suggestive evidence on this question. Studies by Loud and Bucherg have shown that approximately lppm squalene would suffice for sterol synthesis in the rat liver over a period of lo-15 min. Since much of the cholesterol synthesized by the liver is for export in lipoproteins, a lower rate of synthesis would be expected in the aorta. The calculations of St Clair et al.2 are in agreement by showing that the pigeon aorta would synthesize a minimum of 0.5 ppm squalene in 4 hours. The average concentration of 0.42 ppm squalene found in the normal aorta in this study appears to be also at an
640
SHORT COMMUNICATION
appropriately
low level. The findings
of increased
sclerotic plaques (up to 1.3 ppm) is consistent for diseased aortasz. The reports
of larger
concentrations
amounts
of squalene
in athero-
with the greater sterol synthesis reported of squalene
(550 ppm and
1000 ppm) in
atherosclerotic pigeon aorta and human plaques are in obvious disagreement with the results of this study. Details of the methods by which squalene was assayed are not available findings
for appraisal
are not typical
in these reports, of atherosclerosis.
but at least it can be concluded Therefore,
which was considered as a possible explanation for squalenez, is not supported by the results of this study.
anoxia the large
within
that these the plaque,
concentrations
of
REFERENCES 1 BROOKS, C. J. W., HARLAND, W. A. AND STEEL, G., Squalene, 26-hydroxycholesterol and 7-ketocholesterol in human atheromatous plaques, Biochim. Biaphys. Acra, 125 (1966) 620. 2 ST. CLAIR, R. W., LOFLAND, H. B., PRICHARD, R. W. AND CLARKSON, T. B., Synthesis of squalene and sterols by isolated segments of human and pigeon arteries, Exp. Mol. Path., 8 (1968) 201. 3 GARBUZOV, A. G., PYATNITSKII AND PISKUNOV, A. K., Presence of squalene in human aorta. Fed. Proc., Trans. Suppl., 25 (1966) 463T. 4 STEFANOVICH, V. AND KAJIYAMA, G., Squalene and cholestanol in normal rabbit aorta, Atherosclerosis, 11 (1970) 401. 5 LEWIS, R. W., Squalene distribution in fish with normal and pathologically fatty livers, Inr. .f. Biochem., 2 (1971) 609. 6 LEWIS, R. W., The squalene content of plant tissues, Phytochem., 11 (1972) 417. 7 FOLCH, J., LEES, M. AND SLOANE STANLEY, G. H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 8 RAMSEY, R. B., AEXEL, R. T., JAIN, S. AND NICHOLAS, H. J., Distribution of alkanes in normal and atherosclerotic rabbit aortas, Atherosclerosis, 15 (1972) 301. 9 LOUD, A. V. AND BUCHER, N. L. B., The turnover of squalene in relation to the biosynthesis of cholesterol, J. Biol. Chem., 223 (1958) 37.