The effect of the infusion of synthetic dihexanoyl phosphatidyl ethanolamine on experimental atherosclerosis in the rabbit

Journal of Atherosclerosis Research

Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

T H E E F F E C T OF T H E I N F U S I O N OF SYNTHETIC D I H E X A N O Y L P H O S P H A T I D Y L E T H A N O L A M I N E ON E X P E R I M E N T A L A T H E R O SCLEROSIS IN T H E R A B B I T

J. MAURUKAS, J. B. PILLEMER AND C. V. HOLLAND Research Department, Elyria Memorial Hospital, Elyria, Ohio (U.S.A.)

(Revised, received October 21, 1965)

INTRODUCTION FRIEDMAN et al. 1 demonstrated marked resolution of atheromatous plaques as a result of infusions of animal phospholipids in previously cholesterol-fed rabbits. In this laboratory, it has been shown 2 that the infusion of synthetic DE-a-(dimyristoyl)phosphatidyl choline reduced cholesterol-induced atheromatous plaques. SACHS and co-workers 3 injected alcohol-insoluble soybean phosphatides in rabbits after the cessation of a high cholesterol diet and were unable to detect resolution of atheromatous plaques. VAN HANDEL4 found no effect on the production or resolution of atherosclerotic plaques when either alcohol-insoluble (primarily phosphoinositides) or alcohol-soluble (primarily phosphatidyl choline) fractions of soybean were fed to rabbits. Since available evidence seemed to indicate that different phospholipids might have different biological effects, it appeared to be of interest to investigate the effect of additional phospholipid classes on atherosclerosis. Accordingly, the work described here using synthetic dihexanoyl phosphatidyl ethanolamine was undertaken. Dihexanoyl phosphatidyl ethanolamine was chosen for this work because it had been shown to be relatively more soluble in aqueous media than long-chain fatty acid phosphatidyl ethanolamines which had been synthesized in this laboratory 5. MATERIALAND METHODS Animals

Twenty male New Zealand white rabbits, 5-6 weeks old, weighing 3.5-4.5 pounds, were used.

This work was supported by grant No. HE-06408-02 of the United States Public Health Service and by the United Foundation of greater Elyria. j . Atheroscler. Res., 6 (1966) 386-391

D I H E X A N O Y L P H O S P H A T I D Y L E T H A N O L A M I N E AND E X P T L . A T H E R O S C L E R O S I S

387

Diet The regular diet consisted of regular Purina Rabbit Chow Checkers. Purina Rabbit Chow containing 1.5 % cholesterol and 2 % cottonseed oil was used to develop atherosclerosis 3.

Analytic methods Plasma cholesterol was determined by tile method of PEARSON et al. 6, plasma lipid-P b y the method of ZILVERSMITAND DAVIS7. Tissue extracts were prepared and analyzed as described b y MAURUKAS AND THOMAS2. An estimate of the percent of surface area of the aorta covered by plaques was made independently b y two individuals by examination of color photographs taken at the time of autopsy. There were only two cases in which these individuals were not in agreement in their estimates. Transverse sections of the aortic arch, stained with hematoxylin and eosin, were examined microscopically using a 10 • eyepiece fitted with a Bausch and L o m b micrometer eyepiece graduated to 0.05 mm. Random measurements of width of plaques were made so as to include areas of differing thickness. Measurements made b y two individuals on each section were averaged. Infusion Two-hundred and eighty mg of synthetic DL-a-(dihexanoyl)-phosphatidyl ethanolamine synthesized in this laboratory 5 were dissolved with shaking in 100 ml sterile, normal saline. The rabbits were placed in a bleeding box, 0.5 g morphine was injected intravenously, and infusion of the phosphatidyl ethanolamine solution was carried out via the marginal ear vein using a 25 gauge hypodermic needle attached by tubing to a 50 ml syringe held in a kymograph. The infusion was carried out over a period of 1-2 hours. Control animals received iniections of 0.5 g morphine only. Statistical analysis of data The data obtained were tested for significance b y analysis of variance. PROCEDURE

Table I shows the time schedule which was followed in administering diet and phosphatidyl ethanolamine (PE) injections. Group N (normal, 10 rabbits) received the normal diet for 12 weeks; Group A (atherosclerostic, 10 rabbits) received the high-cholesterol diet for 12 weeks. Group A was returned to the normal diet at this time and b o t h groups continued to receive the normal diet for one month. (From this time on, all animals received normal diet only.) After both groups had received the normal diet for one m o n t h the animals in each group were subdivided into groups of 5 animals each: N1 and N2; A1 and A2. Groups N1 and A1 constituted normal and atherosclerotic controls and received no infusions; each rabbit of Groups N2 and A2 received a weekly infusion of 100 ml of a solution containing 280 mg of dihexanoyl phosphatidyl ethanolamine in normal saline for a period of 7 weeks. By the end of the

j. Atheroscler. Res., 6 (1966) 386-391

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J. MAURUKAS,J. B. PILLEMER, C. V. HOLLAND

TABLE I TIME SCHEDULE FOLLOWED IN ADMINISTERING DIET AND PHOSPHATIDYL ETHANOLAMINE (P]~) INJECTIONS TO RABBITS

Period of time (weeks)

Treatment Group N (normal) (I0)*

Group A (atherosclerotic) (10)*

12 4

Normal diet Normal diet

High cholesterol diet Normal diet

N1 (5)

N2 (5)

A1 (5)

A2 (5)

7

Normal diet

Normal diet

2

Normal diet

Normal diet + PE injections Normal diet

Normal diet + PE injections Normal diet

Normal diet

* Numbers in parentheses indicate the number of animals per group. infusion period, each of the animals in Groups N2 and A2 had received a total of 2 g of dihexanoyl phosphatidyl ethanolamine. Two weeks following the last infusion, the animals were bled by cardiac puncture and sacrificed by intracardiac injection of air. Aorta and liver were removed immediately and color photographs were taken of the aorta. A 5-cm section of aorta was frozen for subsequent chemical analysis. A portion of the arch was placed in formaldehyde for the preparation of microsections. Slices of liver were washed with cold normal saline and frozen for chemical analysis. Liver and aorta were analyzed for cholesterol, lipid-P and total lipid. Blood samples were taken at 4, 8 and 12 weeks; before phosphatidyl ethanolamine infusions were begun; and at sacrifice. Blood samples were analyzed for total cholesterol and lipid-P. RESULTS Table I I presents a comparison of mean values of body weight, plasma cholesterol and plasma lipid-P in the four groups of rabbits after the first two months of the experiment, before the start of infusions of phosphatidyl ethanolamine in groups N2 and A2, and at sacrifice. At the end of the first two months of the experiment, the body weight of the animals in all groups was comparable. Both plasma cholesterol and lipid-P were elevated in both of the atherosclerotic groups. After the high cholesterol diet had been discontinued for one month and before infusions of phosphatidyl ethanolamine were begun, the body weights of the atherosclerotic groups were lower than those of the normals. At this period, the blood cholesterol and lipid-P of groups A1 and A2 were declining; however, neither value had reached a normal level. At the conclusion of the experiment, after the phosphatidyl ethanolamine infusions had been given, body weight of both normal groups had continued to increase, whereas that of both atherosclerotic groups remained stationary. At this time, plasma lipid-P had returned to normal, but the plasma cholesterol of both atherosclerotic groups was elevated as compared to normal animals (groups N1 and N2). J. Atkeroscler. Res., 6 (1966) 386-391

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The a p p a r e n t difference in mean plasma cholesterol between A1 and A2 was not significant when analyzed statistically. Table I I I shows values of total lipid, cholesterol, and lipid-P for aorta and liver. Total lipid and cholesterol were increased above normal in the aortas of groups A1 and A2, b u t these two groups did not differ significantly from one another in these measures. Essentially no difference in content of lipid-P could be detected among the aortas of the 4 groups. TABLE II[ M E A N V A L U E S OF T O T A L L I P I D , C H O L E S T E R O L , A N D L I P I D - P

Group

Aorta

OF A O R T A A N D L I V E R

Liver

total lipid

cholesterol

lipid-P

total lipid

cholesterol

lipid-P

N1 chow diet N2 chow diet + PE* infusions

28 (20-41) 24 (10-39)

0.26 (0.21-0.30) 0.26 (0.17-0.33)

0.06 (0.053-0.063) 0.06 (0.033-0.079)

15 (12 19) 17 (13-20)

0.67 (0.58-1.01) 0.79 (0.56-1.08)

0.33 (0.2l-0.42) 0.39 (0.24-0.46)

A1 cholesterol diet A2 cholesterol diet + PE infusions

68 (54-85) 58 (33-87)

6.4 (1.2-11.8) 6.0 (3.9-10.8)

0.11 (0.065-0.21) 0.10 (0.052-0.179)

16 (12.1-21.2) 12 (8-17)

1.78 (1.09-2.87) 0.97 (0.067-1.41)

0.40 (0.30-0.56) 0.29 (0.21-0.41)

* PE: phosphatidyl ethar~olamine. ** All values are expressed as mg/100 mg dry weight. The range of values is given in parenthese.~ The lower liver cholesterol and lower liver total lipids in group A2 are significant at a level of P < 0.05 but there is no significant difference in liver lipid-P between groups A1 and A2. Average width of a t h e r o m a t o u s plaques in group A1 was 322 # ; in group A2, 338 #. E s t i m a t e d surface area covered b y plaques in b o t h groups A1 and A2: 75-100% arch; 50-75 ~o thoracic portion. No plaques were observed in the aortas of normal animals (N 1 and N2) either grossly or microscopically. DISCUSSION J u d g i n g from available evidence, dimyristoyl phosphatidyl choline is capable of reducing the severity and extent of pre-established a t h e r o m a t o u s plaques in experimental atherosclerosis in rabbits 2 and dihexanoyl phosphatidyl ethanolamine is not. PILGERAM AND GREENBERG8 have suggested t h a t phosphatidyl choline m a y be necessary for cholesterol metabolism and t h a t species differences in susceptibility to cholesteroMnduced atherosclerosis m a y be due to ability or inability to form phosphatidyl choline. R a t and pigeon liver have been shown to be capable of conj . Atheroscler. Res., 6 (1966) 386-391

DIHEXANOYL PHOSPHATIDYL ETHANOLAMINE AND EXPTL. ATHEROSCLEROSIS

391

v e r t i n g p h o s p h a t i d y l e t h a n o l a m i n e to p h o s p h a t i d y l choline 9. I n tile e x p e r i m e n t r e p o r t e d here, it would appear t h a t this conversion did n o t occur to a n appreciable e x t e n t , since p h o s p h a t i d y l e t h a n o l a m i n e was n o t as effective in the t r e a t m e n t of e x p e r i m e n t a l atherosclerosis as p h o s p h a t i d y l choline. BYERS et al. lo observed no difference in the cholesterol c o n t e n t of the liver of n o r m a l rats a n d of normal, phospholipid-infused rats, i n spite of the hypercholesteremia which developed in the latter. T h e y are of the opinion t h a t cholesterol is mobilized from the liver a n d other tissues or t h a t the r e t u r n of cholesterol from the blood to the tissues is p r e v e n t e d following phospholipid infusion. The r e s u l t a n t tissue deficiency of cholesterol is t h e n c o m p e n s a t e d for b y increased cholesterol synthesis. TOMKINS et al. 11 have shown t h a t the a b i l i t y of the liver of cholesterol-fed r a b b i t s to synthesize cholesterol is depressed. Thus, in our a n i m a l s which h a d previously received a high cholesterol diet a n d in which liver synthesis of cholesterol was pres u m a b l y depressed, m o b i l i z a t i o n of tissue cholesterol m i g h t n o t have been compensated for b y increased synthesis. SUMMARY

I n f u s i o n s of s y n t h e t i c DL-a-(dihexanoyl) p h o s p h a t i d y l e t h a n o l a m i n e in r a b b i t s in which atherosclerosis h a d previously been produced b y diet, had no effect on the e x t e n t or composition of a t h e r o m a t o u s plaques or u p o n plasma cholesterol or lipid phosphorus. The infusions did, however, result in significantly lower liver cholesterol a n d t o t a l lipid in atherosclerotic a n i m a l s as compared to non-infused controls.

REFERENCES 1 M. FRIEDMAN, S. O. BYERS AND R. H. ROSENMAN,Proc. Soc. Exptl. Biol. Med., 95 (1957) 586. 2 j . MAURUKASAND R. G. THOMAS,J. Lab. Clin. Med., 56 (1960) 30. 3 B. A. SACHS,E. DANIELSONAND L. LEITER, J. Appl. Physiol., 15 (1960) 983. 4 E. VAN HANDEL, Nutrition, 73 (1961) 259. 5 j. MAURUKAS,S. KAIRYSAND C. V. HOLLAND, Biochemistry, 2 (1963) 397. 6 S. PEARSON, S. STERNAND T. H. MCGAVACK,Anal. Chem., 25 (1953) 813. 7 D. lB. ZILVERSMITAND A. 1s DAVIS, J. Lab. Clin. Med., 35 (1950) 155. 8 L. O. PILGERAMAND D. M. GREENBERG, Circulation Res., 3 (1955) 47. 9 C. ARTOMAND H. B. LOFLAND, Biochim. Bwphys. Res. Commun., 3 (1960) 244. lO S. O. BYERS, ~V[.FRIEDMANAND T. SUGIYAMA,J. Biol. Chem., 237 (1962) 3375. 11 G. M. TOMKINS, H. SHEPPARDAND I. L. CHAIKOFF, J. Biol. Chem., 201 (1953) 137. j . Atheroscler. Res., 6 (1966) 386-391