Atherosclerosis, 31 (1978) 205-215 @ Elsevier/North-Holland Scientific

PYRIDINOLCARBAMATE Correlation

EN.

The George

W.N. KASSIRA of Anatomy

Washington

and Biochemistry,

ATHEROSCLEROSIS

and Antiatherogenic

*, R. MUESING

University,

(Received 5 April, 1978) (Revised, received 15 June, (Accepted 21 June, 1978)

Ltd.

AND EXPERIMENTAL

of Hypocholesterolemic

ALBERT,

Departments

Publishers,

** and G.V. VAHOUNY

School

Washington,

Effects

of Medicine

and Health

Sciences,

DC (U.S.A).

1978)

Summary Rabbits were maintained for 12 weeks on either a control or hypercholesteremit dietary regime, or on comparable diets supplemented with pyridinolcarbamate (PDC) at a level of 30 mg/kg body weight/day. Blood was obtained from all rabbits prior to study and at two-week intervals for analysis of serum cholesterol, phospholipid phosphorus and triglycerides. Animals from each group were sacrificed at 4-week intervals for quantitative assessment of the degree of atherosclerotic involvement of the aorta. All animals in the four groups consumed their entire daily allowance (100 g) of their respective diets, and weight gains throughout the feeding period were comparable in the 4 groups. PDC given with the control chow diet had no effect on serum cholesterol levels but did result in persistent decrease in serum triglycerides and a variable decrease in serum phospholipids during the 12-week feeding period. None of the rabbits on the chow diet, with or without PDC, had any evidence of aortic lesions during the experimental period. Rabbits fed 1% cholesterol administered with chow exhibited markedly elevated levels of serum cholesterol and phospholipids, while serum triglycerides were not significantly different than in the control group. In these animals there was a rapid and progressive increase in aortic atherosclerosis throughout the study, and at 12 weeks plaque involvement was 74 f 8% of the aortic surface. Addition of PDC to the 1% cholesterol--chow diet resulted in significantly This study was supported in part by grants from the U.S.P.H.S. HL 12557 (E.N.A.) and HL 02033 (G.V.V.). * Present address: Department of Anatomy, Bagdad Medical School, Bagdad, Iraq. * * Director. Lipid Research Clinic Laboratory, Department of Medicine. Address for correspondence: Dr. G.V. Vabouny, Department of Biochemistry, The George Washington Universitv. 2300 Eye Street, N.W.. Washington. DC 20037, U.S.A.

206

lowered levels of serum cholesterol and phospholipids, but these remained elevated compared to the control levels. There was also a dramatic reduction in the rate and extent of aortic plaque formation. Thus, after 12 weeks on diet, only 27 * 6% of the aortic surface showed evidence of atheroma. The data suggest that PDC significantly decreases the hypercholesteremia resulting from feeding 1% cholesterol to rabbits, and that this may be largely responsible for the antiatherogenic effect of this drug. Keywords:

Athcroma

~~ Lipoproteins

-- Pyridinolcarbamate

~ Serum

lipids

Introduction Pyridinolcarbamate (PDC) [2,6_bis(hydroxymethylpyridine) di-N-methylcarbamate] was first shown by Shimamoto [ 11 to inhibit acute vascular injury of the arterial wall induced by a chemical stress, such as that observed by cholesterol feeding in rabbits. In 1966, these workers also reported the inhibitory effect of PDC on the progression of experimental atherosclerosis in rabbits [ 21. There have since been numerous studies on the vascular and metabolic effects of this compound [3-13,25-271. However, reports on the effects of PDC on the development of experimental atherosclerosis in a variety of species have been contradictory. Shimamoto and co-workers have shown repeatedly [2--41 that levels of 10 mg/kg/day of PDC has no effect on blood lipid levels in rabbits but effectively prevents against atherosclerosis induced by feeding 1.5% cholesterol in the diet. Comparable results have been reported by others in rabbit and chickens [5,6], although in these later studies, higher levels of PDC were employed. However, others using PDC levels as high as 100 mg/kg [7] have reported no effects of this compound in the rabbit [8--101 or monkey [ 111, not only with respect to blood and liver lipid levels [8], but also to the severity of aortic atherosclerosis [ 8,9] or to regression rates of the disease [9]. This inconsistency of positive and negative effects of PDC has also been reported from the same laboratory [5,13]. It has been suggested [8] that differences in the results obtained with PDC may be related to species, to method of drug administration, or to the presence and type of fat in the diet. We have re-investigated the effect of PDC on experimental atherosclerosis in the rabbit using a dietary sequence which has been employed in our laboratories in previous studies [14,15]. It was considered that an effective protection against cholesterol-induced atherosclerosis might allow a more critical biochemical and morphological evaluation of the mechanisms of induction and progression of this disease. In the studies reported here, we have obtained data on the effect of dietary PDC (30 mg/kg/day) on the blood lipid levels of control rabbits and rabbits fed 1% cholesterol, and on the progression and severity of aortic atherosclerosis. Morphological correlates, obtained by scanning electron microscopy, will be reported elsewhere *. * Pyridinolcarbamate and Experimental Atherosclerosis: Transmission and Scanning copy of Aorta, E.N. Albert, W.L. Kassira and G.V. Vahouny, In preparation.

Electron

Micros-

207

Materials and Methods Animals and diets All diets consisted of commercial rabbit pellets (Gleco Co., Charlottesville, VA) and were prepared in the same manner. The control diet (Group 1) was prepared by slowly adding 180 ml of anhydrous ethyl ether : ethanol (2 : 1, v/v) over the pellets (1 kg) with continuous stirring in a commercial diet mixer. The solvents were evaporated at room temperature in a fume hood. Experimental diets were prepared in the same manner except that crystalline cholesterol (10 g) was dissolved in the ethyl ether, and/or PDC * (equivalent to 30 mg/kg rabbit/day) was dissolved in the ethanol prior to addition of the mixture of solvents to the pellets. Diets were prepared twice weekly and stored at 5°C. Young male albino rabbits (1.5-2.0 kg) were divided into 4 groups and allowed 100 g of their respective diets each per day. All animals consumed their entire allowance. Group 1 received only the treated pellets with no further supplementation; Group 2 received pellets, plus 30 mg/kg/day PDC; Group 3 received pellets, plus 1 g/day cholesterol; and Group 4 received pellets containing both PDC and cholesterol. Animals were weighed at weekly intervals throughout the study, and the dietary intake of PDC was adjusted at the expense of pellet weight. Blood samples were collected from the ear vein of all animals prioi, to experiment, and from the animals remaining in each group at the intervals indicated throughout the 12-week study. Serum was prepared immediately after collection of blood samples. Serum cholesterol and triglycerides were analyzed simultaneously on isopropanol extracts using an Auto-Analyzer TM II (AA II) ** and the protocol developed for the Lipid Research Clinic Program [16]. The procedure involved extraction of 0.5 ml serum with 9.5 ml isopropanol, removal of phospholipids by treatment of the extract with a Zeolite mixture [17], and hydrolysis of esterified lipids with alcoholic KOH. Glyceride glycerol was analyzed by periodate oxidation and reaction with acetyl-acetone to yield a fluorescent product, 3,5-diacetyl-4-dihydrolutidine. Cholesterol was analyzed with Lieberman-Burchard reagent (sulfuric acid-acetic anhydride). Lipid phosphorus was determined on chloroform-methanol [18] extracts of serum by the method of Bartlett [19]; phospholipid was calculated as concentration of lipid phosphorus X 25. For the determination of the extent and progression of aortic atheroma, four rabbits were sacrificed prior to feeding, and thereafter, three rabbits on each of the dietary regimes were sacrificed at 4, 8, and 12 weeks of feeding. Prior to sacrifice, the animals were anesthetized and perfused for 30 min with Karnovsky’s fixative in cacodylate buffer [20], and the fixative was allowed to remain in situ for two hours. The entire thoracic and abdominal aorta was removed, split longitudinally, rinsed with saline, and stained with eosin for three seconds. Photographs were made of each aorta, and the intima was graded for the extent of atheromatous plaque involvement using a grid stencil method [ 14,211. As shown in Fig. 1, this involved tracing the plaque * A gift of Banyu Pharmaceutical Company, * * Technicon Instruments Corp., Tarrytown,

Ltd., Tokyo, NY.

Japan

pattern from each aorta onto a grid stencil, prepared on graph paper. The grids were then scored as percentage involvement of the aorta surface. All values represent the means + standard error. Apparent differences between means of all groups were analyzed for significance by the Student t-test [ 221. Results The dose of PDC (30 mg/kg body weight/day) employed in the present study has been used previously by others [9] and was chosen to optimize the effect of the drug in retarding aortic atherosclerosis in the rabbit. This dose appears to be well tolerated since all animals ate their entire daily dietary allowance and showed weight gains over the 1.2-week experimental period comparable to those observed in the groups not receiving the drug (Table 1). As shown in Table 1, the mean initial weight of the rabbits used in the present study was 1.88 + 0.04 kg. The average weight gains in the four groups ranged from 1.55 to 1.80 kg for the 12-week period and were comparable between groups during the entire feeding period. The serum cholesterol levels in animals fed the control diet (Group 1) remained constant throughout the feeding period (Fig. 2). The addition of PDC to the cholesterol-free chow (Group 2) did not significantly affect these levels, even after 12 weeks on diet. When animals were fed the diets containing 1% cholesterol (Groups 3 and 4), serum cholesterol levels increased sharply during the first four weeks and thereafter remained relatively constant. While animals fed cholesterol without the drug (Group 3) attained levels of 2,000-2,500 mg/ dl, the serum cholesterol in animals on the cholesterol--PDC diet reached maximum levels of 1,250-1,700 mg/dl. This difference was statistically significant

TABLE.

I

WEIGHT GAINS, AORTIC ATHEROSCLEROSIS AND EXPERIMENTAL RABBITS

.9ND FINAL

SERUM

LIPID

LEVELS

IN CONTROL

_.__~_~_ Addition chow

to control

Weight gain a

Serum lipids (mg/dl)

(kg)

Cholesterol __-____-

Triglvcerides ~~_ ~_ _

Phospholipids

Initial

Final

initial

Final

Initial

Final

83 t7

74 t11 44 215 2192 b,c i 111 1676 b3c.d *140

137 *I0

131 + .56 67 b -6 149 c ?32 152 C +6

279 + 16

193 f45 150 _tlO 740 b.c 2100 468 b.c.d

0

t79

+6

1. None

1.55

2. PDC. 30 mg/kg/day

1.80

3. Cholesterol,

1.75

1 g/clay

4. PDC + cholesterol

1.72

‘0 0 t0 74

b.c

+8 27 b.c.d

a Initial weight = 1.88 ?-0.04 kg (44 animals): all figures represent mean values + SEM. Initial values are from 44 animals.Values at 12 weeks arc for 4 animals on tiroups 1 and 2, and for 6 animals on Groups 3 and 4. b P < 0.05 compared to Group 1. c I’ < 0.05 compared to Group 2. d P < 0.05 compared to Group 3. e Percentage of total aortic surface containing plaques. Values represent means t SEM for 3 animals in each group

-__

________

2800 2600

SERUM

CHOLESTEROL

f

T

.

CONTROL

+ PDC

.ci CHOLESTEROL

0

2

4

6 WEEKS

8 ON

10

12

DIET

Fig. 2. Effect of pyridinol carbamate (30 mg/kg/day) on serum cholesterol levels in rabbits fed chow alone or chow containing 1% cholesterol (1 g/day). Each point represents means ? SEM. The numbers of animals represented by each point are: 34 at 0 weeks: B-11 at 2 and at 4 weeks: 6-9 at 8 weeks; 4--6 at 10 and 12 weeks.

SERUM

PHOSPHOLIPIOS

?? CONTROL+POC i ‘.

L

I

0

2

CHOLESTEROL

+ POC

~~~~~

1

,-

4

~

_ _

I

/

1

6

8

10

WEEKS

ON

T--

i

12

DIET

Fig. 3. Effect of pyridinol carbamate on serum phospholipids cholesterol. See Fig. 2 and text for details.

in rabbits

fed chow or chow containing

1%

from the 4th week to the end of the study (Table 1) (P < 0.01 at weeks 4 and 8;P < 0.05 at weeks 10 and 12). Changes in serum phospholipids during the 12-week feeding regime are shown in Fig. 3. These levels remained constant in animals fed chow alone (Group 1). Addition of PDC to the chow diet (Group 2) resulted in a lowering of serum phospholipids which remained significantly lower than the serum phospholipids in the chow-fed group at the 8th and 10th weeks of the feeding period (P < 0.01 at 8 weeks; P < 0.05 at 10 weeks). In Group 3 fed 1% cholesterol, the increase in serum phospholipids paralleled the rapid increase in serum cholesterol and attained a plateau after 4 weeks on diet. In contrast, serum phospholipids in the group fed cholesterol plus PDC were not dramatically elevated and remained statistically lower than those not given the drug from the 4th week on. These levels were, however, higher than those in the control animals fed drug alone (Group 2). Changes in serum triglycerides are summarized in Fig. 4. Serum triglycerides were not altered significantly in the control chow-fed group over the 12-week experimental period. However, the overall effect of PDC feeding was a lower serum triglyceride level which persisted throughout the study (upper panel, Fig. 4). Serum triglycerides were not significantly elevated in animals fed 1% cholesterol (lower panel, Fig. 4), although these levels fluctuated more than in the control group. Addition of PDC to the 1% cholesterol diet generally appeared to result in lower and more consistent serum triglycerides, but these (except at 8 weeks of feeding) were not statistically different from either cholesterol-fed animals (Group 3) or controls (Group 1).

SERUM

TRIGLYCERIDES

ZOO CONTROLS

1

i P

CHOLESTEROL

FED 7

r

/

] A CHOLESTEROL+PDC

oi--7-

I 6

1

2

1

4 WEEKS

I

8

I 10

I

12

ON DIET

Fig. 4. Effect of pwidinol carbamate on serum triglyceride 1% cholesterol. Details are given in the text and Fig. 2.

levels in rabbits fed chow alone or containing

Throughout the 12-week feeding period, none of the animals fed the chow diet with or without PDC had any grossly visible aortic lesions. On the other hand, both of the cholesterol-fed groups showed progressive atherosclerotic involvement. However, the extent of involvement was significantly lower in the group given PDC in addition to cholesterol. Figure 5 shows the comparison of the extent of aortic atherosclerosis in the two cholesterol-fed groups. By 4 weeks on diet, during which serum cholesterol levels were maximally elevated, rabbits on 1% cholesterol alone (Group 3) had aortic lesions ranging from O-6% (mean 3%). In contrast, animals fed cholesterol plus PDC were totally free of aortic atherosclerosis after 4 weeks on diet. After 8 weeks on diet, all of the animals receiving cholesterol alone showed extensive aortic plaque involvement ranging from 34~56% (mean 42 f 7). After 12 weeks on diet, the extent of aortic plaque involvement had rapidly increased to 74 + 8%. In contrast to this rapid increase in aortic atherosclerosis between 4-8 weeks of cholesterol feeding, the extent of aortic involvement in animals given the cholesterol plus drug slowly increased to 27 + 6% at 12 weeks. These levels were significantly less (P < 0.01) than in the animals fed cholesterol alone.

AORTIC

LESION

AREA

?? CHOLESTEROL ?? CHOLESTEROL

0

+PDC

4 WEEKS

8 ON

12

DIET

Fig. 5. Percentage of aortic atheromatous involvement in rabbits fed chow containing 1% cholesterol with and without 30 mg/kg/day of pyridinolcarbamate. The percentage of the aorta surface involved was quantitated using a grid stencil procedure [ 141 as shown in Fig. 1.

A composite of the data on weight gain, serum lipids and aortic plaque intensity from animals maintained on the experimental diets for 12 weeks is shown in Table 1. The addition of PDC to the diet did not influence weight gain in these rabbits and animals ate their entire daily allowance of chow. With animals fed cholesterol-free chow plus PDC, there was evidence of a hypolipemic effect of the drug (Figs. 2-4); however, by 12 weeks on diet, only triglyceride levels remained significantly lower than in the drug-free control group. Rabbits fed 1% cholesterol administered with chow showed markedly elevated levels of serum cholesterol and phospholipids, while serum triglycerides were not significantly elevated. The persistent elevation of serum cholesterol (and phospholipids) in this group resulted in extensive atherosclerotic involvement of the aortic intima (74 + 8%). Addition of PDC to the cholesterol-chow diet resulted in significantly lower levels of serum cholesterol and phospholipids, but these still remained elevated when compared to the control groups. This depression also was not associated with significant change in serum triglycerides. Under these conditions, there was also a dramatic reduction in aortic plaque formation with only 27% t 6% of the aortic surface showing evidence of atheromatous involvement after 12 weeks on the cholesterol-PDC supplemented diet. Discussion It is clear from the temporal studies reported of 30 mg/kg body weight/day, has a significant

here that PDC, given at a level antiatherogenic effect on cho-

213

lesterol-induced aortic lesions in rabbits. These data are, in general, comparable to those reported earlier [3,4,23] using PDC levels of 10 or 20 mg/kg body weight. However, in these earlier studies, there were not significant reductions in cholesterol-induced elevations in blood cholesterol levels, resulting in a search for other explanations for the antiatherogenic property of PDC. It was subsequently reported that PDC is an antagonist of bradykinin [24,25], decreases transendothelial transport of cholesterol [26], and inhibits ADPinduced platelet aggregation * [ 271. Although any or all of these effects may be involved in reducing the severity of aortic lesions, PDC feeding in the present study also clearly resulted in a reduction in the hypercholesterolemia resulting from cholesterol feeding. This effect was evident during the final eight weeks of study, and must be considered as directly correlated to the reduced progression of cholesterol-induced aortic atherosclerosis observed in drug-fed rabbits. Although the hypocholesterolemic effect of PDC observed in the present study has not previously been a uniform finding, this effect was not completely unexpected. Kritchevsky et al. [8] reported that PDC administration to rats resulted in decreased absorption of Lholesterol and a reduced level of hepatic cholesterol genesis. These effects were not reflected in marked changes in plasma cholesterol levels due to the efficient homeostatic mechanisms regulating blood cholesterol in this species. However, the rabbit is more responsive to dietary cholesterol with respect to elevations in plasma cholesterol, and similar effects of PDC on sterol absorption or synthesis would likely be directly reflected in changes in circulating cholesterol levels. As mentioned earlier, variable effects of PDC on plasma lipid levels in earlier studies might be due to the level or type of dietary fat included in the hypercholesteremic diet. The degree of hypercholesteremia in cholesterol-fed rabbits decreases with increasing amounts of triglyceride in the diet [ 281. Under these conditions the triglyceride : phospholipid ratio and the size of rabbit lymph chylomicrons is significantly increased [28]. Approximately 60-80s of the lymph cholesterol is transported in the form of chylomicrons and very low density lipoproteins (VLDL) while the remainder is associated with lipoproteins of d > 1.019 g/ml [29]. The proportion of cholesterol transported among these lipoproteins in the rabbit also varies with the quantity of dietary fat. Chylomicron formation occurs in response to the amount of triglyceride absorption, while VLDL formation tends to be more constant [ 301. It had been reported [29] that the proportion of cholesterol transported in rabbit chylomicrons is increased by polyunsaturated fat, and that in VLDL is increased by saturated fat. Similar findings have been reported in the rat [31]. Redgrave and Dunne [32] have recently reported on the comparative effects of dietary oleic acid, coconut oil or peanut oil on the proportion of cholesterol transported in the various lipoproteins of rabbit lymph. When the triglyceride output in lymph was 1 mg/ml, cholesterol was transported largely in the form of VLDL; at triglyceride levels of 5 mg/ml, most of the lymph cholesterol was transported in chylomicrons [ 321. These differences in the level and lymphatic transport forms of cholesterol in diets containing or lacking dietary fat may be,

* E.N.

Albert,

J.M.

Bailey,

A.

Makheja.

Unpublished

data.

214

in part, responsible for the variable effects of PDC on plasma lipid levels in studies with rabbits. There are no direct data in the present study on the mechanism of the hypocholesterolemic effect of PDC. In the control animals fed chow and PDC, there are indications of reduced levels of serum phospholipids and triglycerides. In the cholesterol-fed rabbit. given PDC, the hypolipidemic effect of the drug is even more dramatic. Since the rise in serum cholesterol and phospholipid is a direct response to the addition of 1% cholesterol to the diet, it seems possible that one major effect of PDC is on absorption (and/or transport) of dietary cholesterol, as has been shown in the rat [S]. Furthermore, the data in Figs. 2 and 3 indicate that cholesterol-fed animals given PDC had a lower level of both serum cholesterol and phospholipids after 4 weeks on diet, than was observed in animals fed cholesterol in the absence of PDC. This can be accounted for by either an effect of the drug on cholesterol absorption or to other effects on lipoprotein metabolism (i.e. decreased synthesis, increased catabolism or both). The mechanism(s) of this hypocholesterolemic effect of PDC is (are) currently under study. References 1 Shimamoto, T.. The relationship of edematous arterial reaction in arteries to atherosclerosis, Atheroscler. Res.. 3 (1963) 87. 2 Shimamoto, T., Numano, F. and Fujita. T., Atherosclerosis-inhibiting effect of an anti-bradykinin agent. pyridinol carbamate, Amer. Heart J., 71 (1966) 216. of experimental and 3 Shimamoto, T.. Numano, F., Fujita, T., Ishioka, T. and Atsumi, T., Treatment human atherosclerosis with pyridinol carbamate, Asian Med. J., 8 (1965) 12. 4 Shimamoto, T., Atsumi, T., Numano, F. and Fujita. T., Treatment of atherosclerosis with pyridinol carbamate, Progr. Biochem. Pharmacol.. 4 (1968) 597. 5 Pick, R., The effect of pyridinol carbamate on the induction and regression of aortic and coronary atherosclerosis in cholesterol fed cockerels. In: T. Shimamoto and F. Numano (Eds.), Atherogenesis. Vol. 1, Excerpta Medica. Amsterdam, 1969, p. 57. 6 Martens de Oliveira, J., The effects of pyridinol carbamate on experimental atherosclerosis. In: T. Shimamoto and F. Numano (Eds.), Atherogenesis, Vol. 1. Excerpta Medica. Amsterdam, 1969, P. 53. 7 MBttGnen. M., Pantio, M. and Nieminen. L., Enzyme histochemical observations on the effects of pyridinol carbamate on cholesterol-induced atherosclerosis, Atherosclerosis, 15 (1972) 77. 8 Kritchevsky, D., Koligmaga, J.T., Kim, H.K. and Tepper. S.A., Influence of pyridinol carbamate on cholesterol metabolism in rats and rabbits. In: T. Shimamoto, F. Numano and G.M. Addison (Eds.), Atherogenesis, Vol. 2. Excerpta Medica, Amsterdam, 1972, p. 129. 9 Parevaresch, M.R. The anti-atherogenic effect of pyridinol carbamate. In: T. Shimamoto, F. Numano and G.M. Addison (Ed%). Atherogenesis. Vol. 2, Excerpta Medica, Amsterdam, 1972, P. 86. 10 Kipshidze. N.N., Tkeskelashvili, L.K., Vadachkoria, G.A., Chkhatarashveli, D. and Javakhishvcli. N.. The role of pyridinol carbamate in the prevention and reversal of the development of experimental cholesterol atherosclerosis. In: T. Shimamoto, F. Numano and G.M. Addison (Eds.). Atherogenesis, Vol. 2. Excerpta Medica. Amsterdam, 1972, p. 76. 11 Malinow. M.R., Perley, A. and McLaughlin, P., The effect of pyridinol carbamate on induced atherosclerosis in the monkey, J. Atheroscler. Res.. 8 (1968) 55. 12 Malinow, M.R., McLaughlin, P. and Perley, A., The effect of pyridinol carbamate on induced atherosclerosis in cynomolgus monkeys, Atherosclerosis, 15 (1972) 13. 13 Pick, R., Further studies on the effect of pyridinol carbamate on cholesterol-induced atherosclerosis in cockcrels. In: T. Shimamoto, F. Numano and G.M. Addison (Eds.j, Atherogenesis. Vol. 2, Excerpta Medica, Amsterdam, 1972, p. 92. 14 Chung, T.H., Vahouny, G.V. and Treadwell, C.R.. Dietary inhibition of experimental atherosclerosis in rabbits by a-ethyl-n-caproic acid, J. Atheroscler. Res., 10 (1969) 217. 15 Bailey, J.M. and Butler. J.. Anti-inflammatory drugs in experimental atherosclerosis, Part 1 (Relative potencies for inhibiting plaque formation), Atherosclerosis, 17 (1973) 515. 16 Lipid Research Clinics Manual of Laboratory Operations. Lipid and lipoprotein analysis. HEW No. NIH 75-628, U.S. Government Printing Office, Washington. DC. Vol. 1. 1974.

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Van Handel,

E. and Zilversmit,

D.. Micromethod

J. Lab. Clin. Med., 50 (1957) 152. 18 Folch. J., Lees, M. and Sloane-Stanley, 19 20 21

22 23 24 25

26

27

28 29 30

31 32

G.H.,

for the direct

A simple method

determination

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for the isolation

triglyceride.

and purification

of

total lipides from animal tissues. J. Biol Chem., 226 (1975) 497. J. Biol. Chem., 234 (1959) 466. Bartlett, G.R.. Phosphorus assay in column chromatography, Karnovsky, J.A., A formaldehyde--glutaraldehyde fixative of high osmolality for use in electron microscopy, J. Cell. Biol., 27 (1965) 137a. Bailey. J.M. and Butler, J., Synthetic cholesterol ester antigens in experimental atherosclerosis. In. N.K. Diluaio and R. Paoletti (Eds.), The Reticuloendothelial System and Atherosclerosis, Plenum Press. New York, NY, 1967, P. 433. Fisher, R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh, 1938. P. 120. Wu, C., Huang. T. and Hsu, C. Prevention of experimental atherosclerosis with pyridinol carbamatr, Amer. Heart J., 77 (1969) 657. Shimamoto, T., Maezawa, H., Yamazaki, H.. Atsumi. T., Fujila, T., Ishioka. T. and Sunaga. T.. Pvridino1 carbamate, ;I bradykinin antagonist in veins, Amer. Heart J., 71 (1966) 297. Willoughby. D.A., Lykke, A.W. and Ryan, G.B.. A study of the anti-inflammatory action of pyridinol carbamate (anginin). In: T. Shimamoto and F. Numano (Eds.), Atherogenesis. Vol. 1, Excerpta Medica. Amsterdam, 1969, p. 133. Shimamoto, T. and Sunaga, T., The contraction and blebbing of endothelial cells accompanied by acute infiltration of plasma substances into the vessel wall and their prevention, In: T. Shimamoto, F. Numano and G.M. Addison (Eds.), Atherogenesis, Vol. 2. Excerpta Medica. Amsterdam, 1972, p. 3. Yamazaki, H., Sane, T.. Kobayashi, I., Takahashi, T. and Shimamoto. T., Enhancement of ADPinduced platelet aggregation by adrenaline and cholesterol in viva and its prevention. In: T. Shimamote, F. Numano and G.M. Addison (Eds.), Atherogenesis, Vol. 2. Excerpta Medica, Amsterdam, 1972, p. 177. Fraser. R.. Cliff, W.J. and Courtice, F.C., The effect of dietary fat load on the size and composition of rhylomicrons in thoracic duct lymph, Quart. J. Exp. Physiol., 53 (1968) 390. Zilversmit, D.B.. Courtice, F.C. and Fraser, R., Cholesterol transport in thoracic duct lymph of the rabbit, J. Atheroscler. Res., 7 (1967) 319. Rudel. L.L., Morris, M.D. and Felts, J.M., The transport of exogenous cholesterol in the rabbit, Part 1 (Role of cholesterol ester of lymph chylomicra and lymph very low density lipoproteins in absorption). J. Clin. Invest., 51 (1972) 2686. Ockner, R.K., Hughes. F.B. and Isselbacher. Very low density lipoproteins in intestinal lymph - Origin, composition and role in lipid transport in the fasting state. J. Clin. Invest., 48 (1969) 2079. Redgrave. T.G. and Dunne, K.B., Chylomicron formation and composition in unanaesthetized rabbits, Athuosclerosis, 22 (1975) 389.