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Changes in the composition of phospholipids and of phospholipid fatty acids associated with atherosclerosis in the human aortic wall
36
JOURNAL OF ATHEROSCLEROSIS RESEARCH
CHANGES IN T H E COMPOSITION OF P H O S P H O L I P I D S AND OF P H O S P H O L I P I D FATTY ACIDS ASSOCIATED W I T H A T H E R O S C L E R O S I S IN T H E HUMAN AORTIC WALL C. J. F. BOTTCHER AND C. M. VAN GENT
Department of Physical Chemistry, Leiden University (The Netherlands) (Received October 19, 1960)
INTRODUCTION
Several authors have produced evidence, some of it rather indirect, that in the phospholipids of the aorta the proportion of sphingomyelins increases with increasing atherosclerosis. WEINHOUSE AND HIRSCH1 showed that in later stages of the disease the ratio of ether-insoluble to ether-soluble pfiospholipids rises, STEELE AND KAYDEN2 found decreased lecithin-sphingomyelin ratios in atheromatous (compared with normal) coronary arteries and aorta, while SMITH3 showed that the sphingomyelins of the aortic media, which constitute about 45% of the phospholipids in apparently undiseased tissue, increase to 70% in highly atherosclerotic cases. Investigation of the fatty-acid composition of the total phospholipids extracted from intima-plus-media preparations of the human aortic wall in various stages of atherosclerosis (BOTTCHER et al. 4) led to another indication of an increase in sphingomyelin content. This evidence rested on the assumption that the different phospholipids (cephalins, lecithins, sphingomyelins, etc.) each possess - within certain limits - their characteristic fatty-acid compositions. Under abnormal conditions, for instance if a diet very rich in one particular f a t t y acid is given during a long period, this assumption would not be justified, but under normal conditions the supposition might be as true for phospholipid subfractions as it is for other lipid fractions, such as cholesterol esters and triglycerides 4. Moreover it receives support from the reported fatty-acid compositions of phospholipid fractions derived from other sources. Sphingomyelins, for example, are rich in saturated acids and contain unusually high proportions of C24-acids, while they are poor in oleic and arachidonic acids 5. The percentages of the former group of acids of the total phospholipid acids were indeed observed to increase and the percentages of the latter group to decrease with the degree of atherosclerosis 4. The present investigation was designed to check the observations of increasing sphingomyelin content in the course of atheroscterosis; to determine the characteristic J. Atheroscler. Res., 1 (1961) 36-46
P H O S P H O L I P I D FATTY ACIDS IN HUMAN AORTIC W A L L
37
fatty-acid patterns of as many fractions of aorta phospholipids as possible; and to investigate whether these fatty-acid patterns themselves undergo changes correlated with the degree of atherosclerosis. For these purposes we have employed in the first place chromatography on silicaimpregnated paper, with the developing solvents of MARINETTI, ERBLAND AND KOCHEN6, but using a new method of colouring the spots obtained (HooGHWINKEL AND NIEKERKT), in order to determine the composition of micro-quantities of phospholipid mixtures obtained from single aorta preparations. In addition, larger quantities of phospholipids, from pooled intima-plus media preparations of aortas at different stages of atherosclerosis, were fractionated by adsorption chromatography on silicic acid and the fractions purified when appropriate by differential hydrolysis, tn this way we obtained the fatty acids of seven phospholipid fractions (two different cephalin fractions, ethanolamine plasmalogens, lecithins, choline plasmalogens, sphingomyelins and lysolecithins). Their compositions were determined by gas-liquid chromatography on Apiezon " L " and on the polyester MAE (copolymer of maleic acid, adipic acid and ethylene glycol) as stationary phases. MATERIALS
Preparations of intima plus most of the media were made as previously described 4. Aortas were classified into four groups according to Buck AND ROSSITER8, the terminology of these authors being modified in accordance with the recommendations of the World Health Organisation 9, as follows: Stage 0 : no atherosclerotic lesions discernible at a magnification of 10. Stage I : fatty streaks and/or spots present. Stage II : fibrous plaques and/or atheromas present. Stage III: lesions as above, with additional complications, e.g. ulceration, necrosis, haemorrhage, thrombosis. The prepared material was freeze-dried and stored under methanol-chloroform (1 : 2, v/v) at -20~ until the extraction was carried out. METHODS
The extraction of lipids and preparation of phospholipids were as described previously 4. The major part of the lipids was extracted at room temperature by stirring with methanol-chloroform (1 : 2, v/v), and the remaining few per cent by 3 h Soxhlet extraction with the same solvent in the presence of an antioxidant (hydroquinone). After purification from non-lipid material on a column of powdered cellulose, the lipids other than phospholipids were separated by dialysis in petroleum ether (b.p. 40-60~ through a rubber membrane 10. In this non-polar solvent the phospholipids form micelles (consisting of about seventy molecules) which are too large to pass through the membrane. The concentration of the mixture in the dialysis bag must not exceed 50 mg/ml, otherwise cholesterol associates with the micelles and is not completely dialysed. J. Atherosder. Res., 1 (1961) 3 6 - 4 6
38
c.j.F.
BOTTCHER, C. M. VAN GENT
General precautions in the handling of the lipid factions to avoid oxidation and other degradation of unsaturated fratty acids were as described in a previous publication 11. Phospholipids from individual aorta preparations were subjected to chromatography on silica-impregnated paper; for column chromatography aorta preparations from each of the four stages were pooled to yield sufficient quantities of lipid material.
Chromatography of the phospholipids on silica-impregnated paper. This method for the quantitative estimation of phospholipid fractions was introduced by LEA, RHODES AND STOLL12 and developed by MARINETTI et al. 6. The impregnation of the paper (Schleicher and Schfill, 2043b Mgl) and elution of the spots with di-isobutylketoneacetic acid-water (8 : S : 1, v/v) followed the instructions of the latter authors. To each of six spots 5 cm from the lower edge of an impregnated sheet 15 cm wide by 25 cm high were applied Sttl of a 2% solution of the phospholipid mixture in chloroform. After 3 to 4 h of ascending development at room temperature the solvent front had reached a height of 15 cm; the chromatogram was removed from the chamber and dried in the air. Longer development is unnecessary since the separation is not improved and one increases the risk of hydrolysing the plasmalogens. The completed chromatograms were immersed for 16 h in a solution of 0.01 N HC1 containing 0.005% Ponceau Red (Supra Ponceau RS, Edicol) and 0.2% uranyl nitrate. This staining procedure 7 is simpler than that employing rhodamine 6G and subsequent determination of phosphorus. Red spots are obtained only for compounds having amphoteric character, so that any lipid contaminants other than phospholipids do not stain. The four spots (corresponding to cephalins, lecithins, sphingomyelins and lysolecithins) were outlined in pencil, the paper was dried in the air and the spots were accurately cut out and eluted with 3 ml of 0.6 N HC1/tert-butyl alcohol (prepared by mixing equal volumes of t-butanol and 1.2 N aqueous HC1). After 3 h extraction the extinctions at S10 m# were measured against the same solvent and expressed as percentages of the total of the extinctions for the four spots. Satisfactory independent standards are not available. Background colour, which is very slight, does not have to be determined because the presence of lipid on a spot completely prevents the weak reaction of the staining reagent with the fibres. Silicic-acid column chromatography. Mallinckrodt silicic acid, 100 mesh (13g) was made up as a slurry in methanol-chloroform (1 : 1, v/v) into a short, wide column (3 cm in diameter and 3.5 cm high). A ring of filter paper and 0.5 g of glass helices on top of the column prevented disturbance of the surface during additions. The adsorbent was activated by passing 200 ml of dry methanol-chloroform (1 : 1, v/v) through it, followed by 200 ml chloroform to remove the methanol. All solvents mentioned were subjected to a preliminary redistillation from Drierite. The phospholipid mixture (200-250 mg) was applied in a small volume of chloroform and subjected to linear gradient elution with increasing proportions of methanol in chloroform (0 to 80%). Fractions of 15 ml, 120 in number, were collected and evaporated to dryness in vacuo in weighed vessels, the weights determined and the J. Atheroscler. Res., 1 (1961) 36-46
PHOSPHOLIPID FATTY ACIDS IN HUMAN AORTIC WALL
39
fractions re-dissolved in chloroform for storage at low temperature under nitrogen. Fractions were combined according to their composition as judged by qualitative "thin-layer" chromatography on silica-coated glass plates (using the same developing solvent as for the paper chromatography described above). The fractions were as follows: (a) First "cephalin" fraction:contained at least 4 components, including most of the ethanolamine plasmalogens. (b) Second "cephalin" fraction: contained at least 3 components, with RE values different from those in the first cephalin fraction and including cerebrosides. (c) Lecithins plus most of the sphingomyelins, small amounts of inositides, possibly lyso-cephalins, and choline plasmalogens. (d) Lyso-lecithins plus a small percentage of the sphingomyelins, but no lecithins. Better separation of the lecithins and sphingomyelins can be achieved on longer columns, but at the expense of much longer running times. We considered it more satisfactory to obtain the fatty acids of the different types of phospholipids by subjecting the above-mentioned fractions to differential hydrolysis as described below. By this means the fatty acids of the plasmalogens, which are eluted together with the corresponding cephalins and lecithins in all known systems of column chromatography, are also separately obtained. The different sensitivities to saponification of glycerophosphatides (hydrolysed by mild alkali at 37~ plasmalogens (aldehydes split off by aqueous acid at room temperature) and sphingomyelins (fatty acids removed only by methanolysis) were discovered by SCHMIDT, BENOTTI, HERSHMANN AND THANNHAUSER 13, but methods for the quantitative recovery of the various products, especially water-insoluble products, have been newly developed for this investigation.
Differential hydrolysis. For fractions containing alkali-labile phospholipids, plasmalogens and alkali-stable phospholipids (i.e. sphingomyelins), the procedure is as follows: (1) Mild alkaline hydrolysis (N KOH at 37~ for 16 h) to saponify the alkalilabile compounds. (2) In the solution so obtained, 1 h acid saponification at room temperature to split the aldehydes from the plasmalogens, leaving the corresponding lyso-phospholipids. (3) Addition of water to this solution to retain the glycerophosphoric acid and other water-soluble fragments, and extraction of the rest (free fatty acids, aldehydes, lyso-compounds, and sphingomyelins) with chloroform. (4) Chromatography of the chloroform-extractable compounds over silicic acid to yield aldehydes plus fatty acids ("first fraction"), and lyso-compounds plus sphingomyelins ("second fraction"). (5) Extraction of fatty acids from the "first fraction" with aqueous alkali. (6) Mild alkaline hydrolysis of the "second fraction", acidification, chloroform extraction and separation of the fatty acids originating from plasmalogens from the sphingomyelins by silicic-acid chromatography. (7) Methanolysis of the sphingomyelins. In the two "cephalin" fractions (a and b), where no sphingomyelins occur, and in the lyso-lecithin fraction, where plasmalogens are virtually absent, appropriate steps J. Atheroscler. Res., 1 (1961) 36-46
40
C. J. F. BOTTCHER, C, M, VAN GENT
can be omitted. Plasmalogen f a t t y acids from fractions a and b were combined; sphingomyelins derived from fractions c and d were also combined. Since lysorcephalins are known to be eluted with the lecithins, i.e. in fraction c, it is impossible by this procedure to avoid contamination of lecithin fatty acids with lyso-cephalin acids, but this contamination is unlikely to be serious. The details concerning the various steps in the differential hydrolysis are: (1) "fhe phospholipid mixture, 5 to 100 mg, was warmed in a centrifuge tube of 50 to 100 ml capacity with 5 ml N methanolic K O H at 35 40~ for 16 h. (2) To the cooled solution were added 6 ml N aqueous HC1 and the mixture was kept at room temperature for 1 h. The addition of mercury salts, recommended by SCHMIDTet a l ) 4 for the complete removal of aldehydes from plasmalogens, has been avoided because of their possible oxidative effect on highly unsaturated acids. (3) Water (15 ml) and chIoroform (10 ml) were added and the contents of the tube vigorously shaken and centrifuged (3,0003,500 • g for 10 min). The chloroform layer was removed with a pipette into a second centrifuge tube. The saponification mixture was extracted twice more in the same manner, using chloroform containing 10% methanol. The combined chloroform extracts were washed free of acid by three extractions, carried out similarly, each with 10 ml of 20% methanol in water. The chloroform solution was dried over sodium sulphate and evaporated to small volume. (4) Silicie acid (13 g) was brought into a 3-cm diameter column as a slurry in 20% methanol in chloroform and activated with a further 100 ml of the same solvent. The chloroform solution was applied and the i a t t y acids and aldehydes were eluted with 20% methanol in chloroform (50 ml). Lyso-compounds (from plasmalogens) and sphingomyelins were eluted ("second fraction") with 150 ml methanol-chloroform-water (80 : 18 : 2, v/v). (5) To the 20% methanol-chloroform solution were added 30 ml 0.05 N aqueous KOH, the whole was shaken and centrifuged, and the f a t t y acids were recovered from the (upper) aqueous layer b y acidification and extraction with petroleum ether in the usual way. Aldehydes in the chloroform layer were stored for later examination. (6) The "second fraction" was evaporated to dryness and steps (1) (followed by acidification and immediate extraction), (3) and (4) were carried out. The first eluate from the silicicacid column, which this time contained only fatty acids (from plasmalogens), was inethylated. The second eluate contained only sphingomyelins. (7) Sphingomyelin mixtures obtained in this way from fraction c and fraction d of the original silicic-acid chromatography (see above) were combined before being subjected to methanolysis as described below. Methylation and methanolysis. Fatty-acid mixtures were methylated by refluxing them for 30 minutes with 0.05 N methanolic HC1. F a t t y acids of the sphingomyelins were obtained as their methyl esters by refluxing the sphingomyelin fraction for 3 h with 3 N methanolic HC1. Hydroquinone (l rag) was added to all refluxing solutions. The risk of oxidation can be still further guarded against by replacing the air condenser with a ground-glass stopper and gently heating the tubes for the specified times in a thermostaticaIIy controlled water-bath at 65-70~ To the cooled methanolic soluJ, Atheroscler. Res., 1 (1961) 36-46
PHOSPHOLIPID FATTY ACIDS IN HUMANAORTICWALL
41
tions 60% of distilled water was added and the methyl esters were extracted with petroleum ether. Gas chromatography. Although satisfactory analyses of many fatty-acid methyl ester mixtures can be made using solely a polar stationary phase, this is impossible for the fatty acids derived from phospholipids, since the acids above C20 present a complex picture which is hard to interpret without comparison with the pattern given by the same mixture when a non-polar phase is used. This results partly from the fact that a single peak on either phase may contain more than one ester. The combinations of such esters differ, however, depending on the phase employed. The comparison also enables one to decide whether late peaks eluted from the polar phase are long-chain esters or hydroxy-esters of shorter chain-length. We have some slender evidence that hydroxy-acids do occur, but to the extent of not more than 2 30/0. Apiezon L (10% on Celite 545, 50-80 mesh) was used as non-polar stationary phase at 203~ We have continued to find the most satisfactory polar stationary phase to be the co-polyester MAE (20% on Chromosorb, 80-100 mesh) previously described 4, 11; chromatography on this phase was carried out at 193~ The ester mixture was introduced in the liquid state (100-150#g) onto columns 5 mm by 120 cm, and eluted wifh argon at an inlet over-pressure of 60 cm of mercury (exit pressure atmospheric). Flow rates were 62 and 33 ml/min for Apiezon and MAE columns respectively. Under these conditions the retention time of lignoceric acid (C24) was 200 rain on Apiezon and 120 rain on the MAE colunm. Detection was by means of a fl-ray ionization detector. RESULTS
In Table I are shown the percentage compositions of the phospholipids at different stages of atherosclerosis obtained by chromatography on silica-impregnated paper. TABLE
I
PERCENTAGES OF MAJOR FRACTIONS IN TIlE PHOSPI-IOLIPIDS OF AORTA AT DIFFERENT STAGES OF ATHEROSCLEROSIS (Results o b t a i n e d b y c h r o m a t o g r a p h y on s i l i c a - i m p r e g n a t e d paper. F o r l i m i t a t i o n s of t h e m e t h o d see t e x t )
Number Stage
Average age
of Samples
Percentage Composition Cephalins
Lecithins
Sphingomyelins
Lysolecithins
0
7 (3-12)
5
17.9 (15.6-22.8)
42.7 (39.5-46.8)
34.8 (29.4-38.5)
4.6 (1.7- 7.7)
I
29 (14-55)
6
7.4 (3.5-13.4)
32.4 (27.2-37.9)
53.3 (47.6-56.5)
6.9 (4.1-10.0)
II
51 (25-82)
6
9.5 (6.7-12.8)
26.7 (13.4-36.3)
59.4 (47.2-75.2)
4.4 (2.3- 7.8)
III
61 (49-75)
5
9.2 (6.8 13.9)
23.4 (16.1-31.0)
62.5 (54.3-69.2)
4.9 (3.8- 6.1)
j . Atheroscler. Res., 1 (1961) 36-46
42
c.j.F.
BOTTCHER, C. M. VAN GENT
I t must be pointed out t h a t the m e t h o d of estimation described does not detect cerebrosides, inositides or phosphatidyl serines. Further investigations into these constituents are in progress, but they are known to be present in relatively small amounts. Ethanolamine plasmalogens and choline plasmalogens are included in the cephalin and lecithin percentages respectively. Quantitative data on the variation of plasmalogen content with degree of atherosclerosis will be published shortly. The main feature discernible in Table I is the increase in sphingomyelin percentage with degree of atherosclerosis, with corresponding decreases in lecithin and (to a lesser extent) in cephalin contents. The distinction between apparently undiseased tissue (stage 0) and stage I is particularly sharp. I t must be stressed t h a t decreases in percentage, where t h e y occur, are relative. As can be seen from our previous results (Table I of ref. 4), the average phospholipid content of the dry tissue increases b y a factor of 2.5 from stage 0 to I I I , so that the tissue percentages of lecithins and cephalins actually increase in the course of the disease. For reasons of space limitation it is impossible to show the detailed fatty-acid compositions of all seven of the fractions obtained at all four stages of atherosclerosis. I n a n y case the details would not be of great significance, as the results relate to single examples of each stage; the object of this investigation was to examine major TABLE II LECITHINS AND SPHINGOMYELINS OF AORTA AT DIFFERENT STAGES ATHEROSCLEROSIS. PERCENTAGE FATTY-ACID COMPOSITIONS*
Lecithins Acids +
Total phospholipids
Sphingomyelins
Stage II
III
o
I
II
III
Stage III
28 20 18 5
26 17 28 9
29 21 24 8
19 12 8 2
22 11 7 2
28 14 7 2
32 8 2 0
31 14 9 3
23 0
14 0
5 0
5 0
7 10
3 9
1 7
0 10
4 6
0 0
0 0
0 0
0 0
14 12
17 14
1I 11
13 19
7 11
37 19 44
46 22 32
47 36 17
54 29 17
60 24 16
65 25 lO
69 25 6
71 25 4
65 23
o
I
16 : 0 18 : 0 18:1 18:2
21 14 18 7
20:4 22:0 24 : 0 24 : 1
Saturated Monounsaturated Polyunsaturated
OF
Stage
12
* Percentages, determined to one decimal place, have been rounded off to the nearest whole number for simplicity of presentation. + The figure before the colon denotes chain-length; that after it, the number of double bonds. trends. The lecithin and sphingomyelin fractions, which together make up about 80% of the phospholipids at all stages of the disease, have, however, been selected to illustrate the m a r k e d difference between their fatty-acid characteristics. The percentages of the quantitatively most i m p o r t a n t individual acids are shown in Table II. J. Atherosder. Res., 1 (1961) 3 ~ 6
PHOSPHOLIPID
FATTY
ACIDS IN HUMAN
AORTIC
WALL
43
The high degree of s a t u r a t i o n a n d t h e high c o n t e n t of C22 a n d Ce4 acids in sphingomyelins is evident. T h e f a t t y - a c i d composition of the u n f r a c t i o n a t e d stage I I I phospholipids is shown in t h e last column. F o r c o m p a r i s o n of the differences between stages of atherosclerosis for t h e seven p h o s p h o l i p i d fractions, t h e f a t t y - a c i d p e r c e n t a g e s h a v e been s u m m e d i n t o t h r e e groups; s a t u r a t e d , m o n o u n s a t u r a t e d a n d p o l y u n s a t u r a t e d , a n d are shown in T a b l e I I I . W h i l e t h e changes w i t h increasing stage of atherosclerosis are n o t regular, certain TABLE I I I S A T U R A T E D , M O N O U N S A T U R A T E D AND P O L Y U N S A T U R A T E D ACIDS AS P E R C E N T A G E S OP T H E P A T T Y ACIDS OP P H O S P H O L I P I D S OP AORTA AT D I P P E R E N T STAGES OF A T H E R O S C L E R O S I S
Saturated
Phospholipid fraction Cephalins 1 Cephalins 2 Lecithins Sphingomyelins Lysolecithins Plasmalogens in the cephalin fractions Plasmalogens in the lecithin fraction
Monounsaturated
Stage II I I I
0
I
42 25 37 60 25
42 31 46 65 42
-52 47 69 43
44
69
Polyunsaturated
Stage II
III
0
I
18 15 22 25 22
-30 36 25 34
26 21 29 25 26
39 52 44 16 55
40 54 32 10 36
-18 17 6 23
30 37 17 4 14
20
33
36
25
11
22
8
30
36
36
27
0
I
44 42 54 71 60
19 23 19 24 20
45
56
31
34
37
Stage II I I I
t r e n d s are e v i d e n t w h i c h are c o m m o n to all fractious, n a m e l y increasing c o n t e n t s of s a t u r a t e d a n d decreasing a m o u n t s of p o l y u n s a t u r a t e d acids in the f a t t y - a c i d m i x t u r e . T h e decrease in p o l y u n s a t u r a t e d a c i d c o n t e n t in lecithins a n d s p h i n g o m y e l i n s is acc o u n t e d for largely b y t h e decrease in the p e r c e n t a g e of arachidonic acid (Table II), t h e differences in linoleic a c i d c o n t r i b u t i n g little if a n y t h i n g to t h e changes. T h e lysolecithin f a t t y acids closely resemble those from the lecithins (Table uI). This w o u l d n o t be t h e case if these c o m p o u n d s were p r e d o m i n a n t l y a- or p r e d o m i n a n t l y fl-lysolecithins, since it is k n o w n from studies of p h o s p h o l i p i d s from o t h e r sources is t h a t t h e former contain r e l a t i v e l y s a t u r a t e d , the l a t t e r r e l a t i v e l y u n s a t u r a t ed f a t t y acids. I n s e r u m phospholipids, for example, t h e lysolecithin f a t t y acids are m u c h m o r e s a t u r a t e d t h a n those from t h e lecithins is. Aortic lysolecithins are therefore p r o b a b l y a r a n d o m m i x t u r e of a - a n d fl-forms. T h e plasmalogens also show no over-riding preference for s a t u r a t e d or u n s a t u r a t e d acids. T h e results o b t a i n e d confirm t h e h y p o t h e s i s t h a t t h e o b s e r v e d changes in f a t t y - a c i d composition of t h e t o t a l p h o s p h o l i p i d s from i n t i m a - p l u s - m e d i a p r e p a r a t i o n s of the aortic wall are l a r g e l y d u e to a n increase in s p h i n g o m y e l i n content. I t is also r e v e a l e d t h a t t h e f a t t y - a c i d p a t t e r n of aortic sphingomyelins is similar to t h a t found in sphingomvelins from o t h e r sources, so far as these h a v e been r e p o r t e d , including those f o u n d
J. Atherosder. Res., 1 (1961) 36-46
44
C.J.F.
BOTTCHER, C. M. VAN GENT
in blood plasma. Furthermore, the results show that the compositions of the fatty acids from the phospholipid fractions isolated change with the degree of atherosclerosis. For the most part these changes take place in the same direction in the various fractions, namely towards a lower content of polyunsaturated and a higher content of saturated acids in the more diseased aortic wall. These observations are in accordance with our previous findings 4 on the fatty-acid composition of the unfractionated phospholipids, although the differences in the present examples are more pronounced. DISCUSSION
Certain criticisms of the chemical methods used in this first attempt at a detailed analysis of aorta phospholipids must be mentioned. No proof has been presented of the identity and purity of the fractions from which fatty acids have been isolated other than their behaviour on silicie acid columns and on differential hydrolysis. Control experiments with the purest available preparations of individual phospholipids (cephalins and lecithins) supported these identifications. Detailed analytical data will be published at a later date. It is clear already from our elution curves, the silicaimpregnated paper chromatography patterns and other evidence that the "cephalin" fractions are complex mixtures which include cerebrosides and possibly as yet undescribed phospholipids. On the other hand the combination of the procedures of chromatography and differential hydrolysis ensures that the fatty acids from lyso-lecithins and plasmalogens and from the two major fractions, lecithins and sphingomyelins, have been obtained reasonably free from one another. Evidence for the effective separation of lecithins and sphingomyelins is provided by a comparison of the fatty-acid compositions found for these phospholipids with those reported for the same phospholipids from other sources. The C22 and C24 acids, long known 17 to be a typical feature of the sphingomyelin (and cerebroside) acids, are completely absent from the lecithin analyses. Further, NELSON AND FREEMAN18 found for phospholipid fractions from serum lipids that "pahnitic is the most abundant fatty acid of lecithin and sphingomyelin, whereas the primary fatty acid of the phosphatidyl ethanolamine-serine (i.e. our "cephalin" fractions) appears to be stearic. The principal unsaturated fatty acids are palmitoleic, oleic, linoleic and arachidonic, with linolenic acid almost completely absent". All these statements are true of the corresponding aortic phospholipid fractions, which is remarkable in view of the probable fact that these phospholipids are independently synthesized. Within this framework of characteristic fatty-acid patterns for each phospholipid class there can occur, however, rather marked differences in the compositions according to the degree of atherosclerosis (Table III). The trends are the same in almost all the different fractions, and show decreasing percentages of polyunsaturated acids, especially arachidonic acid, with increasing degree of atherosclerosis. This is balanced by increasing percentages of saturated acids, especially palmitic acid, while the monounsaturated acid contents show no regular tendency. The largest quantitative change is the decrease of arachidonic acid percentage in J. Atheroscler. Res., 1 (1961) 3 6 - 4 6
PHOSPHOLIPID FATTY ACIDS IN HUMAN AORTIC WALL
45
the lecithins. We have the impression that in bIood phospholipids this percentage also shows a decrease with atherosclerosis. The results obtained to date can be fitted in to any of the existing theories concerning the source of the lipids in atherosclerotic vessels. If one accepts the theory that the lipids enter the wall by infiltration into the intima, the observations require that preferential deposition of relatively saturated phospholipids from the lumen takes place, or that there is indiscriminate deposition followed by preferential removal of relatively unsaturated phospholipids. Neither of these alternatives is intrinsically impossible, but the infiltration theory is not supported by the results of ZILVERSMIT AND MCCANDLESS 19, who demonstrated that virtually all the phospholipids in rabbit aorta are synthesized in situ. Similar experiments in man point in the same direction. Another hypothesis, based on the view held by many authors that essential fatty acid (EFA) deficiency of the whole organism leads to the onset of atherosclerosis, would be that such a shortage would lead to a decreased percentage of EFA in the phospholipids which have been synthesized - whether in the arterial wall or elsewhere during the development of the disease. Some light can be thrown on these questions by examining the changes of phospholipid and phospholipid fatty acid composition in the early stages of atherosclerosis in the intima and media separately, and investigations along these lines are proceeding. -
ACKNOWLEDGEMENTS
The expert assistance in some of the experimental work of Mrs. H. A. M. ROTHUISTER HAAR is gratefally acknowledged. Our thanks are due to Dr. C. Ch. TER HAAR ROMENY-WACHTER and Mr. L. BARNARD for the preparation of the aorta material and to the Laboratory of Pathological Anatomy, University Hospital, Leiden, for granting facilities for this work. The research was supported by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and the Netherlands Organization for Health Research (T.N.O.).
SUMMARY
The increasing percentage of sphingomyelins in the phospholipids of human aorta (intima plus media) with increasing degree of atherosclerosis has been confirmed. A method employing silicic-acid chromatography, differential hydrolysis, and gas-liquid chromatography is described which enables the fatty-acid composition of seven phospholipid fractions (two "cephalin" fractions, ethanolamine plasmalogens, lecithins, choline plasmalogens, sphingomyelins and lyso-lecithins) to be determined. It has been established by this means that the different fractions have characteristic fatty-acid compositions and that these compositions change lin the direction of increasing saturation with increasing degree of atherosclerosis. This is to be attributed mainly to a decline in the percentages of polyunsaturated acids, especially arachidonic acid. Possible reasons for these changes are discussed. J. Atheroscler. Res., 1 (1961) 36-46
46
c . J . F . BOTTCHER, C. M. VAN GENT RI~SUM]~
Les a u t e u r s o n t confirm6 que le p o u r c e n t a g e de sphingomy61ines d a n s les p h o s p h o lipides de l ' a o r t e h u m a i n e (intima plus media) a u g m e n t e lorsque le degr6 d ' a t h 6 r o s cl6rose augmente. Ils d6crivent une m 6 t h o d e c o m b i n a n t la c h r o m a t o g r a p h i e sur acide silicique et l ' h y d r o l y s e diff6rentielle qui p e r m e t de d 6 t e r m i n e r la composition en acides gras de sept fractions phospholipidiques (deux fractions de "c6phalines", 6thanolamineplasmalog&nes, 16cithines, choline-plasmalog~nes, sphingomy61ines et lysol6cithines). I1 a 6t6 6tabli p a r cette voie que les diff6rentes fractions ont des compositions caract6ristiques en acides gras et que ces compositions c h a n g e n t dans le sens d ' u n e s a t u r a t i o n a u g m e n t a n t e lorsque le degr6 d'athdroscl6rose augmente. I1 faut a t t f i b u e r ceci s u r t o u t ~ une d i m i n u t i o n des p o u r c e n t a g e s d'acides poly-nonsatur6s, p a r t i c u l i ~ r e m e n t de l ' a c i d e arachidonique. Les a u t e u r s d i s c u t e n t des raisons possibles de ces changements. ZUSAMMENFASSUNG Es w u r d e bestiitigt, dass bei z u n e h m e n d e r Atherosklerose der P r o z e n t s a t z der Sphing o m y e l i n e in den P h o s p h o l i p o i d e n der menschlichen A o r t a (intima plus media) z u n i m m t . L i n e aus S i l i c i u m o x y d c h r o m a t o g r a p h i e u n d Differentialhydrolyse bestehende M e t h o d e wurde beschrieben die es erm6glicht, die Fetts~iurenzusammensetzung von sieben P h o s p h o l i p o i d f r a k t i o n e n (zwei " C e p h a l i n " - F r a k t i o n e n , A t h a n o l a m i n p l a s malogene, Lezithine, Cholinplasmalogene, S p h i n g o m y e l i n e u n d Lysolezithine) zu b e s t i m m e n . So wurde festgestellt, dass die verschiedenen F r a k t i o n e n c h a r a k t e r i s t i s c h e F e t t s ~ u r e n z u s a m m e n s e t z u n g e n h a b e n u n d dass sich diese Z u s a m m e n s e t z u n g e n bei z u n e h m e n d e r Atherosklerose n a c h z u n e h m e n d e m S ~ t t i g u n g s g r a d hin ~ndern. Dies ist haupts/ichlich a u f eine A b n a h m e der m e h r f a c h unges~ittigten S~uren insbesondere der A r a c h i d o n s ~ u r e zuriickzufiihren. M6gliche Grfinde ftir diese A n d e r u n g e n werden er6rtert. REFERENCES 1 S. V~?EINHOUSEAND E. F. HIRSCH, A.M.A. Arch. Pathol., 29 (1940) 31. 2 j. M. STEELE AND H. J. KAYDEN, Trans. Assoc. Am. Phys., 68 (1955) 249. 3 E. B. SMITH, Lancet, i (1960) 799. 4 C. J. V. B6TTCHER, F. P. WOODFORD, C. CH. TER HAAR ROMENY-WACHTER, E. BOELSMA-VAN
HOUTE AND C. M. VAN GENT, Lancet, i (1960) 1378. 5 H. E. CARTER, in I. H. PAGE (ed.) Chemistry of Lipides as related to Atherosclerosis, Charles C. Thomas, Springfield, Ill., 1958, p. 85. 6 G. V. MARINETTI, J. ERBLAND AND J. NOCHEN, Fed. Proc., 16 (1957) 837. 7 G. HOOGHWINKELAND H. VAN NIEKERK, Proc. k. ned. Akad. Wetenschap., 63 B (1960) 469. 8 R. C. BUCK AND R. J. ROSSITER, A.M.A. Arch. Pathol., 51 (1951) 224. 9 World Health Organisation, Tech. Repts. Ser. No. 143 (1958) 4. 10 G. J. VANBEERS, H. DE IONGHAND J. BOLDINGH,in H. M. SINCLAIR(ed.), Essential Fatty Acids, Butterworths Sci. Publ., London, 1958, p. 43. 11 C. J . F . BOTTCHER, F. P. WOODFORD, E. BOELSMA-VAN HOUTE AND C. M. VAN GENT, Rec. trav. chim., 78 (1959) 794. 12 C. H. LEA, D. N. RHODES AND R. D. STOLL, Biochem. J., 60 (1955) 353. 13 G. SCHMIDT, J. BENOTTI, B. HERSHMANN AND S. THANNHAUSER, J. Biol. Chem., 166 (1946) 505. 14 G. SCHMIDT, B. OTTENSTEIN, W. A. SPENCER, K. NECK, R. BLIETZ, J. PAPAS, D. PORTER, M. L.
LEVIN AND S. J. THANNHAUSER, Amer. J. Dis. Child., 97 (1959) 691. 15 D. J. HANAHAN, H. BROCKERHOFF AND E. J. BARRON, J. Biol. Chem., 235 (1960) 1917. 16 E. GJONE, J. F. BERRY AND D. A. TURNER, Biochim. Biophys. Acta, 34 (1959) 288.
17 p. _A..LEVENE, J. Biol. Chem., 24 (1916) 69; E. KLENK, Z. physiol. Chem., 221 (1933) 259. 18 G. J. NELSON AND N. K. FREEMAN, J. Biol. Chem., 235 (1960) 578. 19 D. B. ZILVERSMIT AND E. L. MCCANDLESS, J. Lipid Res., 1 (1959) 118. J. Atheroscler. Res., 1 (1961) 36-46
JOURNAL OF ATHEROSCLEROSIS RESEARCH
CHANGES IN T H E COMPOSITION OF P H O S P H O L I P I D S AND OF P H O S P H O L I P I D FATTY ACIDS ASSOCIATED W I T H A T H E R O S C L E R O S I S IN T H E HUMAN AORTIC WALL C. J. F. BOTTCHER AND C. M. VAN GENT
Department of Physical Chemistry, Leiden University (The Netherlands) (Received October 19, 1960)
INTRODUCTION
Several authors have produced evidence, some of it rather indirect, that in the phospholipids of the aorta the proportion of sphingomyelins increases with increasing atherosclerosis. WEINHOUSE AND HIRSCH1 showed that in later stages of the disease the ratio of ether-insoluble to ether-soluble pfiospholipids rises, STEELE AND KAYDEN2 found decreased lecithin-sphingomyelin ratios in atheromatous (compared with normal) coronary arteries and aorta, while SMITH3 showed that the sphingomyelins of the aortic media, which constitute about 45% of the phospholipids in apparently undiseased tissue, increase to 70% in highly atherosclerotic cases. Investigation of the fatty-acid composition of the total phospholipids extracted from intima-plus-media preparations of the human aortic wall in various stages of atherosclerosis (BOTTCHER et al. 4) led to another indication of an increase in sphingomyelin content. This evidence rested on the assumption that the different phospholipids (cephalins, lecithins, sphingomyelins, etc.) each possess - within certain limits - their characteristic fatty-acid compositions. Under abnormal conditions, for instance if a diet very rich in one particular f a t t y acid is given during a long period, this assumption would not be justified, but under normal conditions the supposition might be as true for phospholipid subfractions as it is for other lipid fractions, such as cholesterol esters and triglycerides 4. Moreover it receives support from the reported fatty-acid compositions of phospholipid fractions derived from other sources. Sphingomyelins, for example, are rich in saturated acids and contain unusually high proportions of C24-acids, while they are poor in oleic and arachidonic acids 5. The percentages of the former group of acids of the total phospholipid acids were indeed observed to increase and the percentages of the latter group to decrease with the degree of atherosclerosis 4. The present investigation was designed to check the observations of increasing sphingomyelin content in the course of atheroscterosis; to determine the characteristic J. Atheroscler. Res., 1 (1961) 36-46
P H O S P H O L I P I D FATTY ACIDS IN HUMAN AORTIC W A L L
37
fatty-acid patterns of as many fractions of aorta phospholipids as possible; and to investigate whether these fatty-acid patterns themselves undergo changes correlated with the degree of atherosclerosis. For these purposes we have employed in the first place chromatography on silicaimpregnated paper, with the developing solvents of MARINETTI, ERBLAND AND KOCHEN6, but using a new method of colouring the spots obtained (HooGHWINKEL AND NIEKERKT), in order to determine the composition of micro-quantities of phospholipid mixtures obtained from single aorta preparations. In addition, larger quantities of phospholipids, from pooled intima-plus media preparations of aortas at different stages of atherosclerosis, were fractionated by adsorption chromatography on silicic acid and the fractions purified when appropriate by differential hydrolysis, tn this way we obtained the fatty acids of seven phospholipid fractions (two different cephalin fractions, ethanolamine plasmalogens, lecithins, choline plasmalogens, sphingomyelins and lysolecithins). Their compositions were determined by gas-liquid chromatography on Apiezon " L " and on the polyester MAE (copolymer of maleic acid, adipic acid and ethylene glycol) as stationary phases. MATERIALS
Preparations of intima plus most of the media were made as previously described 4. Aortas were classified into four groups according to Buck AND ROSSITER8, the terminology of these authors being modified in accordance with the recommendations of the World Health Organisation 9, as follows: Stage 0 : no atherosclerotic lesions discernible at a magnification of 10. Stage I : fatty streaks and/or spots present. Stage II : fibrous plaques and/or atheromas present. Stage III: lesions as above, with additional complications, e.g. ulceration, necrosis, haemorrhage, thrombosis. The prepared material was freeze-dried and stored under methanol-chloroform (1 : 2, v/v) at -20~ until the extraction was carried out. METHODS
The extraction of lipids and preparation of phospholipids were as described previously 4. The major part of the lipids was extracted at room temperature by stirring with methanol-chloroform (1 : 2, v/v), and the remaining few per cent by 3 h Soxhlet extraction with the same solvent in the presence of an antioxidant (hydroquinone). After purification from non-lipid material on a column of powdered cellulose, the lipids other than phospholipids were separated by dialysis in petroleum ether (b.p. 40-60~ through a rubber membrane 10. In this non-polar solvent the phospholipids form micelles (consisting of about seventy molecules) which are too large to pass through the membrane. The concentration of the mixture in the dialysis bag must not exceed 50 mg/ml, otherwise cholesterol associates with the micelles and is not completely dialysed. J. Atherosder. Res., 1 (1961) 3 6 - 4 6
38
c.j.F.
BOTTCHER, C. M. VAN GENT
General precautions in the handling of the lipid factions to avoid oxidation and other degradation of unsaturated fratty acids were as described in a previous publication 11. Phospholipids from individual aorta preparations were subjected to chromatography on silica-impregnated paper; for column chromatography aorta preparations from each of the four stages were pooled to yield sufficient quantities of lipid material.
Chromatography of the phospholipids on silica-impregnated paper. This method for the quantitative estimation of phospholipid fractions was introduced by LEA, RHODES AND STOLL12 and developed by MARINETTI et al. 6. The impregnation of the paper (Schleicher and Schfill, 2043b Mgl) and elution of the spots with di-isobutylketoneacetic acid-water (8 : S : 1, v/v) followed the instructions of the latter authors. To each of six spots 5 cm from the lower edge of an impregnated sheet 15 cm wide by 25 cm high were applied Sttl of a 2% solution of the phospholipid mixture in chloroform. After 3 to 4 h of ascending development at room temperature the solvent front had reached a height of 15 cm; the chromatogram was removed from the chamber and dried in the air. Longer development is unnecessary since the separation is not improved and one increases the risk of hydrolysing the plasmalogens. The completed chromatograms were immersed for 16 h in a solution of 0.01 N HC1 containing 0.005% Ponceau Red (Supra Ponceau RS, Edicol) and 0.2% uranyl nitrate. This staining procedure 7 is simpler than that employing rhodamine 6G and subsequent determination of phosphorus. Red spots are obtained only for compounds having amphoteric character, so that any lipid contaminants other than phospholipids do not stain. The four spots (corresponding to cephalins, lecithins, sphingomyelins and lysolecithins) were outlined in pencil, the paper was dried in the air and the spots were accurately cut out and eluted with 3 ml of 0.6 N HC1/tert-butyl alcohol (prepared by mixing equal volumes of t-butanol and 1.2 N aqueous HC1). After 3 h extraction the extinctions at S10 m# were measured against the same solvent and expressed as percentages of the total of the extinctions for the four spots. Satisfactory independent standards are not available. Background colour, which is very slight, does not have to be determined because the presence of lipid on a spot completely prevents the weak reaction of the staining reagent with the fibres. Silicic-acid column chromatography. Mallinckrodt silicic acid, 100 mesh (13g) was made up as a slurry in methanol-chloroform (1 : 1, v/v) into a short, wide column (3 cm in diameter and 3.5 cm high). A ring of filter paper and 0.5 g of glass helices on top of the column prevented disturbance of the surface during additions. The adsorbent was activated by passing 200 ml of dry methanol-chloroform (1 : 1, v/v) through it, followed by 200 ml chloroform to remove the methanol. All solvents mentioned were subjected to a preliminary redistillation from Drierite. The phospholipid mixture (200-250 mg) was applied in a small volume of chloroform and subjected to linear gradient elution with increasing proportions of methanol in chloroform (0 to 80%). Fractions of 15 ml, 120 in number, were collected and evaporated to dryness in vacuo in weighed vessels, the weights determined and the J. Atheroscler. Res., 1 (1961) 36-46
PHOSPHOLIPID FATTY ACIDS IN HUMAN AORTIC WALL
39
fractions re-dissolved in chloroform for storage at low temperature under nitrogen. Fractions were combined according to their composition as judged by qualitative "thin-layer" chromatography on silica-coated glass plates (using the same developing solvent as for the paper chromatography described above). The fractions were as follows: (a) First "cephalin" fraction:contained at least 4 components, including most of the ethanolamine plasmalogens. (b) Second "cephalin" fraction: contained at least 3 components, with RE values different from those in the first cephalin fraction and including cerebrosides. (c) Lecithins plus most of the sphingomyelins, small amounts of inositides, possibly lyso-cephalins, and choline plasmalogens. (d) Lyso-lecithins plus a small percentage of the sphingomyelins, but no lecithins. Better separation of the lecithins and sphingomyelins can be achieved on longer columns, but at the expense of much longer running times. We considered it more satisfactory to obtain the fatty acids of the different types of phospholipids by subjecting the above-mentioned fractions to differential hydrolysis as described below. By this means the fatty acids of the plasmalogens, which are eluted together with the corresponding cephalins and lecithins in all known systems of column chromatography, are also separately obtained. The different sensitivities to saponification of glycerophosphatides (hydrolysed by mild alkali at 37~ plasmalogens (aldehydes split off by aqueous acid at room temperature) and sphingomyelins (fatty acids removed only by methanolysis) were discovered by SCHMIDT, BENOTTI, HERSHMANN AND THANNHAUSER 13, but methods for the quantitative recovery of the various products, especially water-insoluble products, have been newly developed for this investigation.
Differential hydrolysis. For fractions containing alkali-labile phospholipids, plasmalogens and alkali-stable phospholipids (i.e. sphingomyelins), the procedure is as follows: (1) Mild alkaline hydrolysis (N KOH at 37~ for 16 h) to saponify the alkalilabile compounds. (2) In the solution so obtained, 1 h acid saponification at room temperature to split the aldehydes from the plasmalogens, leaving the corresponding lyso-phospholipids. (3) Addition of water to this solution to retain the glycerophosphoric acid and other water-soluble fragments, and extraction of the rest (free fatty acids, aldehydes, lyso-compounds, and sphingomyelins) with chloroform. (4) Chromatography of the chloroform-extractable compounds over silicic acid to yield aldehydes plus fatty acids ("first fraction"), and lyso-compounds plus sphingomyelins ("second fraction"). (5) Extraction of fatty acids from the "first fraction" with aqueous alkali. (6) Mild alkaline hydrolysis of the "second fraction", acidification, chloroform extraction and separation of the fatty acids originating from plasmalogens from the sphingomyelins by silicic-acid chromatography. (7) Methanolysis of the sphingomyelins. In the two "cephalin" fractions (a and b), where no sphingomyelins occur, and in the lyso-lecithin fraction, where plasmalogens are virtually absent, appropriate steps J. Atheroscler. Res., 1 (1961) 36-46
40
C. J. F. BOTTCHER, C, M, VAN GENT
can be omitted. Plasmalogen f a t t y acids from fractions a and b were combined; sphingomyelins derived from fractions c and d were also combined. Since lysorcephalins are known to be eluted with the lecithins, i.e. in fraction c, it is impossible by this procedure to avoid contamination of lecithin fatty acids with lyso-cephalin acids, but this contamination is unlikely to be serious. The details concerning the various steps in the differential hydrolysis are: (1) "fhe phospholipid mixture, 5 to 100 mg, was warmed in a centrifuge tube of 50 to 100 ml capacity with 5 ml N methanolic K O H at 35 40~ for 16 h. (2) To the cooled solution were added 6 ml N aqueous HC1 and the mixture was kept at room temperature for 1 h. The addition of mercury salts, recommended by SCHMIDTet a l ) 4 for the complete removal of aldehydes from plasmalogens, has been avoided because of their possible oxidative effect on highly unsaturated acids. (3) Water (15 ml) and chIoroform (10 ml) were added and the contents of the tube vigorously shaken and centrifuged (3,0003,500 • g for 10 min). The chloroform layer was removed with a pipette into a second centrifuge tube. The saponification mixture was extracted twice more in the same manner, using chloroform containing 10% methanol. The combined chloroform extracts were washed free of acid by three extractions, carried out similarly, each with 10 ml of 20% methanol in water. The chloroform solution was dried over sodium sulphate and evaporated to small volume. (4) Silicie acid (13 g) was brought into a 3-cm diameter column as a slurry in 20% methanol in chloroform and activated with a further 100 ml of the same solvent. The chloroform solution was applied and the i a t t y acids and aldehydes were eluted with 20% methanol in chloroform (50 ml). Lyso-compounds (from plasmalogens) and sphingomyelins were eluted ("second fraction") with 150 ml methanol-chloroform-water (80 : 18 : 2, v/v). (5) To the 20% methanol-chloroform solution were added 30 ml 0.05 N aqueous KOH, the whole was shaken and centrifuged, and the f a t t y acids were recovered from the (upper) aqueous layer b y acidification and extraction with petroleum ether in the usual way. Aldehydes in the chloroform layer were stored for later examination. (6) The "second fraction" was evaporated to dryness and steps (1) (followed by acidification and immediate extraction), (3) and (4) were carried out. The first eluate from the silicicacid column, which this time contained only fatty acids (from plasmalogens), was inethylated. The second eluate contained only sphingomyelins. (7) Sphingomyelin mixtures obtained in this way from fraction c and fraction d of the original silicic-acid chromatography (see above) were combined before being subjected to methanolysis as described below. Methylation and methanolysis. Fatty-acid mixtures were methylated by refluxing them for 30 minutes with 0.05 N methanolic HC1. F a t t y acids of the sphingomyelins were obtained as their methyl esters by refluxing the sphingomyelin fraction for 3 h with 3 N methanolic HC1. Hydroquinone (l rag) was added to all refluxing solutions. The risk of oxidation can be still further guarded against by replacing the air condenser with a ground-glass stopper and gently heating the tubes for the specified times in a thermostaticaIIy controlled water-bath at 65-70~ To the cooled methanolic soluJ, Atheroscler. Res., 1 (1961) 36-46
PHOSPHOLIPID FATTY ACIDS IN HUMANAORTICWALL
41
tions 60% of distilled water was added and the methyl esters were extracted with petroleum ether. Gas chromatography. Although satisfactory analyses of many fatty-acid methyl ester mixtures can be made using solely a polar stationary phase, this is impossible for the fatty acids derived from phospholipids, since the acids above C20 present a complex picture which is hard to interpret without comparison with the pattern given by the same mixture when a non-polar phase is used. This results partly from the fact that a single peak on either phase may contain more than one ester. The combinations of such esters differ, however, depending on the phase employed. The comparison also enables one to decide whether late peaks eluted from the polar phase are long-chain esters or hydroxy-esters of shorter chain-length. We have some slender evidence that hydroxy-acids do occur, but to the extent of not more than 2 30/0. Apiezon L (10% on Celite 545, 50-80 mesh) was used as non-polar stationary phase at 203~ We have continued to find the most satisfactory polar stationary phase to be the co-polyester MAE (20% on Chromosorb, 80-100 mesh) previously described 4, 11; chromatography on this phase was carried out at 193~ The ester mixture was introduced in the liquid state (100-150#g) onto columns 5 mm by 120 cm, and eluted wifh argon at an inlet over-pressure of 60 cm of mercury (exit pressure atmospheric). Flow rates were 62 and 33 ml/min for Apiezon and MAE columns respectively. Under these conditions the retention time of lignoceric acid (C24) was 200 rain on Apiezon and 120 rain on the MAE colunm. Detection was by means of a fl-ray ionization detector. RESULTS
In Table I are shown the percentage compositions of the phospholipids at different stages of atherosclerosis obtained by chromatography on silica-impregnated paper. TABLE
I
PERCENTAGES OF MAJOR FRACTIONS IN TIlE PHOSPI-IOLIPIDS OF AORTA AT DIFFERENT STAGES OF ATHEROSCLEROSIS (Results o b t a i n e d b y c h r o m a t o g r a p h y on s i l i c a - i m p r e g n a t e d paper. F o r l i m i t a t i o n s of t h e m e t h o d see t e x t )
Number Stage
Average age
of Samples
Percentage Composition Cephalins
Lecithins
Sphingomyelins
Lysolecithins
0
7 (3-12)
5
17.9 (15.6-22.8)
42.7 (39.5-46.8)
34.8 (29.4-38.5)
4.6 (1.7- 7.7)
I
29 (14-55)
6
7.4 (3.5-13.4)
32.4 (27.2-37.9)
53.3 (47.6-56.5)
6.9 (4.1-10.0)
II
51 (25-82)
6
9.5 (6.7-12.8)
26.7 (13.4-36.3)
59.4 (47.2-75.2)
4.4 (2.3- 7.8)
III
61 (49-75)
5
9.2 (6.8 13.9)
23.4 (16.1-31.0)
62.5 (54.3-69.2)
4.9 (3.8- 6.1)
j . Atheroscler. Res., 1 (1961) 36-46
42
c.j.F.
BOTTCHER, C. M. VAN GENT
I t must be pointed out t h a t the m e t h o d of estimation described does not detect cerebrosides, inositides or phosphatidyl serines. Further investigations into these constituents are in progress, but they are known to be present in relatively small amounts. Ethanolamine plasmalogens and choline plasmalogens are included in the cephalin and lecithin percentages respectively. Quantitative data on the variation of plasmalogen content with degree of atherosclerosis will be published shortly. The main feature discernible in Table I is the increase in sphingomyelin percentage with degree of atherosclerosis, with corresponding decreases in lecithin and (to a lesser extent) in cephalin contents. The distinction between apparently undiseased tissue (stage 0) and stage I is particularly sharp. I t must be stressed t h a t decreases in percentage, where t h e y occur, are relative. As can be seen from our previous results (Table I of ref. 4), the average phospholipid content of the dry tissue increases b y a factor of 2.5 from stage 0 to I I I , so that the tissue percentages of lecithins and cephalins actually increase in the course of the disease. For reasons of space limitation it is impossible to show the detailed fatty-acid compositions of all seven of the fractions obtained at all four stages of atherosclerosis. I n a n y case the details would not be of great significance, as the results relate to single examples of each stage; the object of this investigation was to examine major TABLE II LECITHINS AND SPHINGOMYELINS OF AORTA AT DIFFERENT STAGES ATHEROSCLEROSIS. PERCENTAGE FATTY-ACID COMPOSITIONS*
Lecithins Acids +
Total phospholipids
Sphingomyelins
Stage II
III
o
I
II
III
Stage III
28 20 18 5
26 17 28 9
29 21 24 8
19 12 8 2
22 11 7 2
28 14 7 2
32 8 2 0
31 14 9 3
23 0
14 0
5 0
5 0
7 10
3 9
1 7
0 10
4 6
0 0
0 0
0 0
0 0
14 12
17 14
1I 11
13 19
7 11
37 19 44
46 22 32
47 36 17
54 29 17
60 24 16
65 25 lO
69 25 6
71 25 4
65 23
o
I
16 : 0 18 : 0 18:1 18:2
21 14 18 7
20:4 22:0 24 : 0 24 : 1
Saturated Monounsaturated Polyunsaturated
OF
Stage
12
* Percentages, determined to one decimal place, have been rounded off to the nearest whole number for simplicity of presentation. + The figure before the colon denotes chain-length; that after it, the number of double bonds. trends. The lecithin and sphingomyelin fractions, which together make up about 80% of the phospholipids at all stages of the disease, have, however, been selected to illustrate the m a r k e d difference between their fatty-acid characteristics. The percentages of the quantitatively most i m p o r t a n t individual acids are shown in Table II. J. Atherosder. Res., 1 (1961) 3 ~ 6
PHOSPHOLIPID
FATTY
ACIDS IN HUMAN
AORTIC
WALL
43
The high degree of s a t u r a t i o n a n d t h e high c o n t e n t of C22 a n d Ce4 acids in sphingomyelins is evident. T h e f a t t y - a c i d composition of the u n f r a c t i o n a t e d stage I I I phospholipids is shown in t h e last column. F o r c o m p a r i s o n of the differences between stages of atherosclerosis for t h e seven p h o s p h o l i p i d fractions, t h e f a t t y - a c i d p e r c e n t a g e s h a v e been s u m m e d i n t o t h r e e groups; s a t u r a t e d , m o n o u n s a t u r a t e d a n d p o l y u n s a t u r a t e d , a n d are shown in T a b l e I I I . W h i l e t h e changes w i t h increasing stage of atherosclerosis are n o t regular, certain TABLE I I I S A T U R A T E D , M O N O U N S A T U R A T E D AND P O L Y U N S A T U R A T E D ACIDS AS P E R C E N T A G E S OP T H E P A T T Y ACIDS OP P H O S P H O L I P I D S OP AORTA AT D I P P E R E N T STAGES OF A T H E R O S C L E R O S I S
Saturated
Phospholipid fraction Cephalins 1 Cephalins 2 Lecithins Sphingomyelins Lysolecithins Plasmalogens in the cephalin fractions Plasmalogens in the lecithin fraction
Monounsaturated
Stage II I I I
0
I
42 25 37 60 25
42 31 46 65 42
-52 47 69 43
44
69
Polyunsaturated
Stage II
III
0
I
18 15 22 25 22
-30 36 25 34
26 21 29 25 26
39 52 44 16 55
40 54 32 10 36
-18 17 6 23
30 37 17 4 14
20
33
36
25
11
22
8
30
36
36
27
0
I
44 42 54 71 60
19 23 19 24 20
45
56
31
34
37
Stage II I I I
t r e n d s are e v i d e n t w h i c h are c o m m o n to all fractious, n a m e l y increasing c o n t e n t s of s a t u r a t e d a n d decreasing a m o u n t s of p o l y u n s a t u r a t e d acids in the f a t t y - a c i d m i x t u r e . T h e decrease in p o l y u n s a t u r a t e d a c i d c o n t e n t in lecithins a n d s p h i n g o m y e l i n s is acc o u n t e d for largely b y t h e decrease in the p e r c e n t a g e of arachidonic acid (Table II), t h e differences in linoleic a c i d c o n t r i b u t i n g little if a n y t h i n g to t h e changes. T h e lysolecithin f a t t y acids closely resemble those from the lecithins (Table uI). This w o u l d n o t be t h e case if these c o m p o u n d s were p r e d o m i n a n t l y a- or p r e d o m i n a n t l y fl-lysolecithins, since it is k n o w n from studies of p h o s p h o l i p i d s from o t h e r sources is t h a t t h e former contain r e l a t i v e l y s a t u r a t e d , the l a t t e r r e l a t i v e l y u n s a t u r a t ed f a t t y acids. I n s e r u m phospholipids, for example, t h e lysolecithin f a t t y acids are m u c h m o r e s a t u r a t e d t h a n those from t h e lecithins is. Aortic lysolecithins are therefore p r o b a b l y a r a n d o m m i x t u r e of a - a n d fl-forms. T h e plasmalogens also show no over-riding preference for s a t u r a t e d or u n s a t u r a t e d acids. T h e results o b t a i n e d confirm t h e h y p o t h e s i s t h a t t h e o b s e r v e d changes in f a t t y - a c i d composition of t h e t o t a l p h o s p h o l i p i d s from i n t i m a - p l u s - m e d i a p r e p a r a t i o n s of the aortic wall are l a r g e l y d u e to a n increase in s p h i n g o m y e l i n content. I t is also r e v e a l e d t h a t t h e f a t t y - a c i d p a t t e r n of aortic sphingomyelins is similar to t h a t found in sphingomvelins from o t h e r sources, so far as these h a v e been r e p o r t e d , including those f o u n d
J. Atherosder. Res., 1 (1961) 36-46
44
C.J.F.
BOTTCHER, C. M. VAN GENT
in blood plasma. Furthermore, the results show that the compositions of the fatty acids from the phospholipid fractions isolated change with the degree of atherosclerosis. For the most part these changes take place in the same direction in the various fractions, namely towards a lower content of polyunsaturated and a higher content of saturated acids in the more diseased aortic wall. These observations are in accordance with our previous findings 4 on the fatty-acid composition of the unfractionated phospholipids, although the differences in the present examples are more pronounced. DISCUSSION
Certain criticisms of the chemical methods used in this first attempt at a detailed analysis of aorta phospholipids must be mentioned. No proof has been presented of the identity and purity of the fractions from which fatty acids have been isolated other than their behaviour on silicie acid columns and on differential hydrolysis. Control experiments with the purest available preparations of individual phospholipids (cephalins and lecithins) supported these identifications. Detailed analytical data will be published at a later date. It is clear already from our elution curves, the silicaimpregnated paper chromatography patterns and other evidence that the "cephalin" fractions are complex mixtures which include cerebrosides and possibly as yet undescribed phospholipids. On the other hand the combination of the procedures of chromatography and differential hydrolysis ensures that the fatty acids from lyso-lecithins and plasmalogens and from the two major fractions, lecithins and sphingomyelins, have been obtained reasonably free from one another. Evidence for the effective separation of lecithins and sphingomyelins is provided by a comparison of the fatty-acid compositions found for these phospholipids with those reported for the same phospholipids from other sources. The C22 and C24 acids, long known 17 to be a typical feature of the sphingomyelin (and cerebroside) acids, are completely absent from the lecithin analyses. Further, NELSON AND FREEMAN18 found for phospholipid fractions from serum lipids that "pahnitic is the most abundant fatty acid of lecithin and sphingomyelin, whereas the primary fatty acid of the phosphatidyl ethanolamine-serine (i.e. our "cephalin" fractions) appears to be stearic. The principal unsaturated fatty acids are palmitoleic, oleic, linoleic and arachidonic, with linolenic acid almost completely absent". All these statements are true of the corresponding aortic phospholipid fractions, which is remarkable in view of the probable fact that these phospholipids are independently synthesized. Within this framework of characteristic fatty-acid patterns for each phospholipid class there can occur, however, rather marked differences in the compositions according to the degree of atherosclerosis (Table III). The trends are the same in almost all the different fractions, and show decreasing percentages of polyunsaturated acids, especially arachidonic acid, with increasing degree of atherosclerosis. This is balanced by increasing percentages of saturated acids, especially palmitic acid, while the monounsaturated acid contents show no regular tendency. The largest quantitative change is the decrease of arachidonic acid percentage in J. Atheroscler. Res., 1 (1961) 3 6 - 4 6
PHOSPHOLIPID FATTY ACIDS IN HUMAN AORTIC WALL
45
the lecithins. We have the impression that in bIood phospholipids this percentage also shows a decrease with atherosclerosis. The results obtained to date can be fitted in to any of the existing theories concerning the source of the lipids in atherosclerotic vessels. If one accepts the theory that the lipids enter the wall by infiltration into the intima, the observations require that preferential deposition of relatively saturated phospholipids from the lumen takes place, or that there is indiscriminate deposition followed by preferential removal of relatively unsaturated phospholipids. Neither of these alternatives is intrinsically impossible, but the infiltration theory is not supported by the results of ZILVERSMIT AND MCCANDLESS 19, who demonstrated that virtually all the phospholipids in rabbit aorta are synthesized in situ. Similar experiments in man point in the same direction. Another hypothesis, based on the view held by many authors that essential fatty acid (EFA) deficiency of the whole organism leads to the onset of atherosclerosis, would be that such a shortage would lead to a decreased percentage of EFA in the phospholipids which have been synthesized - whether in the arterial wall or elsewhere during the development of the disease. Some light can be thrown on these questions by examining the changes of phospholipid and phospholipid fatty acid composition in the early stages of atherosclerosis in the intima and media separately, and investigations along these lines are proceeding. -
ACKNOWLEDGEMENTS
The expert assistance in some of the experimental work of Mrs. H. A. M. ROTHUISTER HAAR is gratefally acknowledged. Our thanks are due to Dr. C. Ch. TER HAAR ROMENY-WACHTER and Mr. L. BARNARD for the preparation of the aorta material and to the Laboratory of Pathological Anatomy, University Hospital, Leiden, for granting facilities for this work. The research was supported by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and the Netherlands Organization for Health Research (T.N.O.).
SUMMARY
The increasing percentage of sphingomyelins in the phospholipids of human aorta (intima plus media) with increasing degree of atherosclerosis has been confirmed. A method employing silicic-acid chromatography, differential hydrolysis, and gas-liquid chromatography is described which enables the fatty-acid composition of seven phospholipid fractions (two "cephalin" fractions, ethanolamine plasmalogens, lecithins, choline plasmalogens, sphingomyelins and lyso-lecithins) to be determined. It has been established by this means that the different fractions have characteristic fatty-acid compositions and that these compositions change lin the direction of increasing saturation with increasing degree of atherosclerosis. This is to be attributed mainly to a decline in the percentages of polyunsaturated acids, especially arachidonic acid. Possible reasons for these changes are discussed. J. Atheroscler. Res., 1 (1961) 36-46
46
c . J . F . BOTTCHER, C. M. VAN GENT RI~SUM]~
Les a u t e u r s o n t confirm6 que le p o u r c e n t a g e de sphingomy61ines d a n s les p h o s p h o lipides de l ' a o r t e h u m a i n e (intima plus media) a u g m e n t e lorsque le degr6 d ' a t h 6 r o s cl6rose augmente. Ils d6crivent une m 6 t h o d e c o m b i n a n t la c h r o m a t o g r a p h i e sur acide silicique et l ' h y d r o l y s e diff6rentielle qui p e r m e t de d 6 t e r m i n e r la composition en acides gras de sept fractions phospholipidiques (deux fractions de "c6phalines", 6thanolamineplasmalog&nes, 16cithines, choline-plasmalog~nes, sphingomy61ines et lysol6cithines). I1 a 6t6 6tabli p a r cette voie que les diff6rentes fractions ont des compositions caract6ristiques en acides gras et que ces compositions c h a n g e n t dans le sens d ' u n e s a t u r a t i o n a u g m e n t a n t e lorsque le degr6 d'athdroscl6rose augmente. I1 faut a t t f i b u e r ceci s u r t o u t ~ une d i m i n u t i o n des p o u r c e n t a g e s d'acides poly-nonsatur6s, p a r t i c u l i ~ r e m e n t de l ' a c i d e arachidonique. Les a u t e u r s d i s c u t e n t des raisons possibles de ces changements. ZUSAMMENFASSUNG Es w u r d e bestiitigt, dass bei z u n e h m e n d e r Atherosklerose der P r o z e n t s a t z der Sphing o m y e l i n e in den P h o s p h o l i p o i d e n der menschlichen A o r t a (intima plus media) z u n i m m t . L i n e aus S i l i c i u m o x y d c h r o m a t o g r a p h i e u n d Differentialhydrolyse bestehende M e t h o d e wurde beschrieben die es erm6glicht, die Fetts~iurenzusammensetzung von sieben P h o s p h o l i p o i d f r a k t i o n e n (zwei " C e p h a l i n " - F r a k t i o n e n , A t h a n o l a m i n p l a s malogene, Lezithine, Cholinplasmalogene, S p h i n g o m y e l i n e u n d Lysolezithine) zu b e s t i m m e n . So wurde festgestellt, dass die verschiedenen F r a k t i o n e n c h a r a k t e r i s t i s c h e F e t t s ~ u r e n z u s a m m e n s e t z u n g e n h a b e n u n d dass sich diese Z u s a m m e n s e t z u n g e n bei z u n e h m e n d e r Atherosklerose n a c h z u n e h m e n d e m S ~ t t i g u n g s g r a d hin ~ndern. Dies ist haupts/ichlich a u f eine A b n a h m e der m e h r f a c h unges~ittigten S~uren insbesondere der A r a c h i d o n s ~ u r e zuriickzufiihren. M6gliche Grfinde ftir diese A n d e r u n g e n werden er6rtert. REFERENCES 1 S. V~?EINHOUSEAND E. F. HIRSCH, A.M.A. Arch. Pathol., 29 (1940) 31. 2 j. M. STEELE AND H. J. KAYDEN, Trans. Assoc. Am. Phys., 68 (1955) 249. 3 E. B. SMITH, Lancet, i (1960) 799. 4 C. J. V. B6TTCHER, F. P. WOODFORD, C. CH. TER HAAR ROMENY-WACHTER, E. BOELSMA-VAN
HOUTE AND C. M. VAN GENT, Lancet, i (1960) 1378. 5 H. E. CARTER, in I. H. PAGE (ed.) Chemistry of Lipides as related to Atherosclerosis, Charles C. Thomas, Springfield, Ill., 1958, p. 85. 6 G. V. MARINETTI, J. ERBLAND AND J. NOCHEN, Fed. Proc., 16 (1957) 837. 7 G. HOOGHWINKELAND H. VAN NIEKERK, Proc. k. ned. Akad. Wetenschap., 63 B (1960) 469. 8 R. C. BUCK AND R. J. ROSSITER, A.M.A. Arch. Pathol., 51 (1951) 224. 9 World Health Organisation, Tech. Repts. Ser. No. 143 (1958) 4. 10 G. J. VANBEERS, H. DE IONGHAND J. BOLDINGH,in H. M. SINCLAIR(ed.), Essential Fatty Acids, Butterworths Sci. Publ., London, 1958, p. 43. 11 C. J . F . BOTTCHER, F. P. WOODFORD, E. BOELSMA-VAN HOUTE AND C. M. VAN GENT, Rec. trav. chim., 78 (1959) 794. 12 C. H. LEA, D. N. RHODES AND R. D. STOLL, Biochem. J., 60 (1955) 353. 13 G. SCHMIDT, J. BENOTTI, B. HERSHMANN AND S. THANNHAUSER, J. Biol. Chem., 166 (1946) 505. 14 G. SCHMIDT, B. OTTENSTEIN, W. A. SPENCER, K. NECK, R. BLIETZ, J. PAPAS, D. PORTER, M. L.
LEVIN AND S. J. THANNHAUSER, Amer. J. Dis. Child., 97 (1959) 691. 15 D. J. HANAHAN, H. BROCKERHOFF AND E. J. BARRON, J. Biol. Chem., 235 (1960) 1917. 16 E. GJONE, J. F. BERRY AND D. A. TURNER, Biochim. Biophys. Acta, 34 (1959) 288.
17 p. _A..LEVENE, J. Biol. Chem., 24 (1916) 69; E. KLENK, Z. physiol. Chem., 221 (1933) 259. 18 G. J. NELSON AND N. K. FREEMAN, J. Biol. Chem., 235 (1960) 578. 19 D. B. ZILVERSMIT AND E. L. MCCANDLESS, J. Lipid Res., 1 (1959) 118. J. Atheroscler. Res., 1 (1961) 36-46