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Changes in lipid composition during incubation of pig aorta
SHORT COMMUNICATIONS
731
CHANGES IN L I P I D COMPOSITION DURING INCUBATION OF PIG AORTA
C. PRIES AND C. J. F. BOTTCHER Gaubius Institute of the University of Leyden (The Netherlands)
(Revised, received March 28th, 1968)
SUMMARY Experiments with pig aortae show that incubation for 6 hours in the presence of both oxygen and glucose does not effect materially the pattern of the aorta lipids. Without supply of oxygen and glucose the free fatty acids increase at the expense of the lecithins. In 18 hours' experiments without supply of oxygen and glucose about 30 % of the lecithins are hydrolysed, the free fatty acids increase considerably and indications are that under these conditions also the sphingolipids increase. If only glucose is added to the incubation medium hydrolysis of the lecithins is reduced to about half that value and no increase of the sphingomyelins is noted. The results of the investigation are discussed.
INTRODUCTION The accumulation of lipids in the arterial wall in relation to atherosclerotic disease has been studied thoroughly by many authors (see review by SCHETTLER1). The changes occurring in the lipid composition, the fatty acid patterns of the lipid fractions, and the phospholipid distribution have been described by BOTTCHER2,3, B~TTCHER AND VAN GENT4 and SMITH5,6. But the remarkable increase in stero! esters, the relative decrease in phospholipids (reflected especially in the lecithin fraction), the shift in the phospholipid distribution from lecithins to sphingomyelins, and other important findings have not yet been explained. In order to get more insight in the role of processes occurring in the arterial wall, it was thought worth-while to study the changes in lipid composition of an isolated healthy pig aorta incubated in an artificial medium in relation to the supply of oxygen and glucose, and the time of incubation. J. Atheroscler. Res., 1968, 8:731-734
732
SHORT COMMUNICATIONS
METHODS AND MATERIALS
Pig aortae were obtained immediately after the slaughter, transported on ice, and prepared in a room kept at 4~ After stripping the adventitia and about onethird of the media, the aorta was divided into two parts, longitudinally along the intercostal artery. One of these parts was extracted immediately, the other transferred to the sterilized incubation medium. All instruments, glass-ware, and solutions were also sterilized. Since sterilization of the aorta might effect its enzymatic activity this was omitted, thus accepting some danger of microbial infection. Extraction, isolation of the phospholipids and purification of the crude extract to remove non-lipid materials were according to BOTTCHER et al.7; isolation of free fatty acids by Amberlite IR 45 and titrimetric determination according to PRIES AND BOTTCHERS; determination of free and total sterols according to SPERRY AND WEBB9; estimation of triglycerides by a glycerol assay according to VAN HANDEL AND ZILVERSMIT10; and the analysis of the phospholipid fraction according to PRIES et al. 11. The basic isotonic salt solution for the incubation was prepared according to Ringer, modified for mammalian heart 12. RESULTS
In Table 1 the results of lipid analyses carried out before and after incubation are shown. It is seen that when incubating for 6 hours in the presence of both glucose and oxygen the lipid pattern did not change materially. Incubation for 6 hours without supply of glucose and oxygen gave rise to an increase of the free fatty acids and a decrease of the iecithins. In 18 hours' experiments this decrease amounted to about 30 % of the lecithins. On the other hand the sphingolipids appeared to be increased, if only glucose was present less lecithins were hydrolyzed and little if any increase of the sphingolipids was observed. DISCUSSION The results of 6 hours' experiments in the presence of glucose and oxygen indicate that under these conditions none of the systems involved in the lipid metabolism is running down. In the absence of oxygen and glucose the tissue is exhausted, coming to the fore in an accumulation of free fatty acids and a loss of lecithins. The presence of glucose counteracts the accumulation of free fatty acids. The synthesis of sphingolipids, which appears to occur under extreme conditions, could be tentatively interpreted as the last attempt of the cells to keep control of the situation; the breakdown of almost one-third of the lecithins can mean a serious danger to the biomembrane structure. If the results could be validly applied to the situation in vivo, it might be speculated that the shift in the ratio of lecithins to sphingomyelins observed in the atheroj. Atheroscler. Res., 1968, 8:731-734
SHORT COMMUNICATIONS
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734 sclerotic human
SHORT COMMUNICATIONS a r t e r i a l w a l l is c a u s e d b y i n s u f f i c i e n t s u p p l y of e n e r g y in p a r t i c u l a r
p a r t s of t h e t i s s u e .
REFERENCES 1 SCHETTLER, G., Arteriosklerose. A'tiologie, Pathologie, Klinik und Therapie, Georg Thieme,
Stuttgart, 1961, p. 94. 2 t36TTCHER, C. J. F., Phospholipids of atherosclerotic lesions in the h u m a n aorta. In: Proceedings of the Congress on Evolution of the Atherosclerotic Plaque, Chicago, 1963, University of Chicago Press, Chicago and London, 1964, p. 109. 3 BOTTCHER, C. J. F., Quelques aspects chimiques de l'athGro3clGrose et de la thrombose, Arch. Maladies Coeur Vaisseaux, 1965, Suppl. 3; Rev. A th~roscldr., 1965, 7: 3. 4 B('~TTCHER, C. J. F. AND C. M. VAN GENT, Changes in the composition of phospholipids and of phospholipid f a t t y acids associated with atherosclerosis in the h u m a n arterial wall, J. A theroscler. Res., 1961, 1: 36. 5 SMITH, E. B., Intimal and medial lipids in h u m a n aortas, Lancet, 1960, i: 799. 6 SMITH, E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, P a r t 1 (The lipids), J. Atheroscler. Res., 1965, 5: 224. 7 BOTTCHER, C. J. F., F. P. WOODFORD, E. BOELSMA-VAN HOUTE AND C. M. VAN GENT, Methods for the analysis of lipids extracted from h u m a n arteries and other tissues, Rec. Tray. Chim., 1959, 78: 794. 8 PRIES, C. AND C. J. F. BOTTCHER, Ultramicro titration of f a t t y acids is non-aqueous singlephase systems, Anal. Chim. A cta, 1964, 31 : 293. 9 SPERRY, W. M. AND M. WEBB, A revision of the Schoenheimer-Sperry method for cholesterol, J. Biol. Chem., 1950, 187: 97. I0 HANDEL, E. VAN AND D. B. ZILVERSMIT, Micromethod for direct determination of serum triglycerides, J. Lab. Clin. Chem., 1957, 50: 152. 11 PRIES, C., A. AUMONT AND C. J. F. BOTTCHER, Analysis of phospholipids, Biochim. Biophys. Acta, 1966, 125: 277. 12 Biochemists Handbook, b y C. LONG (Ed.), E. and F. N. Spon, London, 1961, p. 58.
J. Atheroscler. Res., 1968, 8 : 7 3 1 - 7 3 4
731
CHANGES IN L I P I D COMPOSITION DURING INCUBATION OF PIG AORTA
C. PRIES AND C. J. F. BOTTCHER Gaubius Institute of the University of Leyden (The Netherlands)
(Revised, received March 28th, 1968)
SUMMARY Experiments with pig aortae show that incubation for 6 hours in the presence of both oxygen and glucose does not effect materially the pattern of the aorta lipids. Without supply of oxygen and glucose the free fatty acids increase at the expense of the lecithins. In 18 hours' experiments without supply of oxygen and glucose about 30 % of the lecithins are hydrolysed, the free fatty acids increase considerably and indications are that under these conditions also the sphingolipids increase. If only glucose is added to the incubation medium hydrolysis of the lecithins is reduced to about half that value and no increase of the sphingomyelins is noted. The results of the investigation are discussed.
INTRODUCTION The accumulation of lipids in the arterial wall in relation to atherosclerotic disease has been studied thoroughly by many authors (see review by SCHETTLER1). The changes occurring in the lipid composition, the fatty acid patterns of the lipid fractions, and the phospholipid distribution have been described by BOTTCHER2,3, B~TTCHER AND VAN GENT4 and SMITH5,6. But the remarkable increase in stero! esters, the relative decrease in phospholipids (reflected especially in the lecithin fraction), the shift in the phospholipid distribution from lecithins to sphingomyelins, and other important findings have not yet been explained. In order to get more insight in the role of processes occurring in the arterial wall, it was thought worth-while to study the changes in lipid composition of an isolated healthy pig aorta incubated in an artificial medium in relation to the supply of oxygen and glucose, and the time of incubation. J. Atheroscler. Res., 1968, 8:731-734
732
SHORT COMMUNICATIONS
METHODS AND MATERIALS
Pig aortae were obtained immediately after the slaughter, transported on ice, and prepared in a room kept at 4~ After stripping the adventitia and about onethird of the media, the aorta was divided into two parts, longitudinally along the intercostal artery. One of these parts was extracted immediately, the other transferred to the sterilized incubation medium. All instruments, glass-ware, and solutions were also sterilized. Since sterilization of the aorta might effect its enzymatic activity this was omitted, thus accepting some danger of microbial infection. Extraction, isolation of the phospholipids and purification of the crude extract to remove non-lipid materials were according to BOTTCHER et al.7; isolation of free fatty acids by Amberlite IR 45 and titrimetric determination according to PRIES AND BOTTCHERS; determination of free and total sterols according to SPERRY AND WEBB9; estimation of triglycerides by a glycerol assay according to VAN HANDEL AND ZILVERSMIT10; and the analysis of the phospholipid fraction according to PRIES et al. 11. The basic isotonic salt solution for the incubation was prepared according to Ringer, modified for mammalian heart 12. RESULTS
In Table 1 the results of lipid analyses carried out before and after incubation are shown. It is seen that when incubating for 6 hours in the presence of both glucose and oxygen the lipid pattern did not change materially. Incubation for 6 hours without supply of glucose and oxygen gave rise to an increase of the free fatty acids and a decrease of the iecithins. In 18 hours' experiments this decrease amounted to about 30 % of the lecithins. On the other hand the sphingolipids appeared to be increased, if only glucose was present less lecithins were hydrolyzed and little if any increase of the sphingolipids was observed. DISCUSSION The results of 6 hours' experiments in the presence of glucose and oxygen indicate that under these conditions none of the systems involved in the lipid metabolism is running down. In the absence of oxygen and glucose the tissue is exhausted, coming to the fore in an accumulation of free fatty acids and a loss of lecithins. The presence of glucose counteracts the accumulation of free fatty acids. The synthesis of sphingolipids, which appears to occur under extreme conditions, could be tentatively interpreted as the last attempt of the cells to keep control of the situation; the breakdown of almost one-third of the lecithins can mean a serious danger to the biomembrane structure. If the results could be validly applied to the situation in vivo, it might be speculated that the shift in the ratio of lecithins to sphingomyelins observed in the atheroj. Atheroscler. Res., 1968, 8:731-734
SHORT COMMUNICATIONS
r
+
~
00 o~ tt~ ~0 it~
~o,., ~
~"' ,...,~~
0
0
09
733
tr~ ~1 0
l'~oO
44
,-~ 0
~0-~
+
~J
~o
~
~-~
i
~~ ~ J. Atheroscler. Res., 1968, 8 : 7 3 1 - 7 3 4
734 sclerotic human
SHORT COMMUNICATIONS a r t e r i a l w a l l is c a u s e d b y i n s u f f i c i e n t s u p p l y of e n e r g y in p a r t i c u l a r
p a r t s of t h e t i s s u e .
REFERENCES 1 SCHETTLER, G., Arteriosklerose. A'tiologie, Pathologie, Klinik und Therapie, Georg Thieme,
Stuttgart, 1961, p. 94. 2 t36TTCHER, C. J. F., Phospholipids of atherosclerotic lesions in the h u m a n aorta. In: Proceedings of the Congress on Evolution of the Atherosclerotic Plaque, Chicago, 1963, University of Chicago Press, Chicago and London, 1964, p. 109. 3 BOTTCHER, C. J. F., Quelques aspects chimiques de l'athGro3clGrose et de la thrombose, Arch. Maladies Coeur Vaisseaux, 1965, Suppl. 3; Rev. A th~roscldr., 1965, 7: 3. 4 B('~TTCHER, C. J. F. AND C. M. VAN GENT, Changes in the composition of phospholipids and of phospholipid f a t t y acids associated with atherosclerosis in the h u m a n arterial wall, J. A theroscler. Res., 1961, 1: 36. 5 SMITH, E. B., Intimal and medial lipids in h u m a n aortas, Lancet, 1960, i: 799. 6 SMITH, E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, P a r t 1 (The lipids), J. Atheroscler. Res., 1965, 5: 224. 7 BOTTCHER, C. J. F., F. P. WOODFORD, E. BOELSMA-VAN HOUTE AND C. M. VAN GENT, Methods for the analysis of lipids extracted from h u m a n arteries and other tissues, Rec. Tray. Chim., 1959, 78: 794. 8 PRIES, C. AND C. J. F. BOTTCHER, Ultramicro titration of f a t t y acids is non-aqueous singlephase systems, Anal. Chim. A cta, 1964, 31 : 293. 9 SPERRY, W. M. AND M. WEBB, A revision of the Schoenheimer-Sperry method for cholesterol, J. Biol. Chem., 1950, 187: 97. I0 HANDEL, E. VAN AND D. B. ZILVERSMIT, Micromethod for direct determination of serum triglycerides, J. Lab. Clin. Chem., 1957, 50: 152. 11 PRIES, C., A. AUMONT AND C. J. F. BOTTCHER, Analysis of phospholipids, Biochim. Biophys. Acta, 1966, 125: 277. 12 Biochemists Handbook, b y C. LONG (Ed.), E. and F. N. Spon, London, 1961, p. 58.
J. Atheroscler. Res., 1968, 8 : 7 3 1 - 7 3 4