Studies on new nicotinic acid ester derivatives

Journal of Atheroscierosis Research

391

Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

STUDIES ON NEW NICOTINIC ACID E S T E R DERIVATIVES

P A R T 6. S E R U M C H O L E S T E R O L L E V E L L O W E R I N G E F F E C T OF 2,2,6,6-TETRAKIS(NICOTINOYLO XYMETHYL) CYCLOHEXANOL (K-31) Y. ASO, Y. ABE, K. HIGO, T. N A R U K E AND T. I R I K U R A

Department of Biochemistry, Chiba University School of Medicine, Chiba, and Department of Biochemistr% Kyorin Chemical Laboratory, Tokyo (Japan) (Received July 7th, 1969)

SUMMARY

The effects of 2 , 2 , 6 , 6 - t e t r a k i s ( n i c o t i n o y l o x y m e t h y l ) cyclohexanol (K-31) on cholesterol-fed rabbits were compared with those of meso-inositol hexanicotinate and nicotinic acid. In the regressive studies using oral K-31 after inducing experimental atherosclerosis, the drug suppressed the elevation of serum total cholesterol, phospholipid and triglyceride levels, and also decreased the deposition of total cholesterol and phospholipids in the liver. In the progressive studies, oral K-31 exhibited a marked serum cholesterol lowering effect. The effect of K-31 on intestinal absorption of cholesterol was studied in thoracicduct fistula rats administered labeled cholesterol. K-31 significantly depressed the absorption of [4-14CJcholesterol into lymph, and caused more absorbed [4-14C]cholesterol to appear as ester. The hypoeholesterolemic action of K-31 is thought to be due to inhibition of exogenous sterol absorption.

Key words: E x p e r i m e n t a l atherosclerosis in rabbits - Hypocholesterolemic

drug -

K - 3 1 - N i c o t i n i c acid ester derivative - Thoracic-duct fistula rat

INTRODUCTION

In studying the effects of nicotinic acid ester derivatives, we found that 2 , 2 , 6 , 6 - t e t r a k i s ( n i c o t i n o y l o x y m e t h y l ) cyclohexanol (K-31) (see Fig. 1) substantially lowers serum cholesterol without causing toxicity 1. This paper describes the effect of K-31 on experimental atherosclerosis in j. Atheroscler. Res., 1969, 10:391-401

392

Y. ASO, Y. ABE, K. HIGO, T. NARUKE, T. I R I K U R A

9

9 C34H3209 N4

mo[. wt. 640.63

Fig. 1. C h e m i c a l s t r u c t u r e of K-31.

cholesterol-fed rabbits, and the drug's action on cholesterol absorption in thoracicduct fistula rats. This investigation was undertaken to elucidate the hypocholesterolemic action of K-31. MATERIALS AND METHODS

Experimental atheroselerosis in rabbits Experimental atherosclerosis in rabbits was induced by feeding a cholesterol diet prepared by adding cholesterol to the basal diet for rabbits; the tested compounds were added to the cholesterol diet.

Studies of regressive experiments Male rabbits, weighing 2.0-2.3 kg, were divided into 5 groups of 6 each. The normal control group received the basal diet for 100 days. The basal diet was a commercial rabbit chow (CR-1; Nippon Haigo Shiryo Co., Ltd., Tokyo*). The remaining 4 groups were fed for 50 days with a cholesterol-enriched diet containing 10 % oleic acid, 1 % cholesterol and 0 . 1 % cholic acid added to CR-1. At the 50th day of feeding, rabbits whose total serum cholesterol level was more than 650 rag/100 ml were selected. One of 4 groups containing selected animals was then continuously fed with the cholesterol diet as a placebo control for a further 50 days. The remaining 3 groups were given a cholesterol-enriched diet containing 2 % of K-31 hydrochloride, nicotinic acid (hereafter abbreviated to NA) or meso-inositol hexanicotinate (hereafter abbreviated to IHN) for a further 50 days to examine the therapeutic effect of these agents.

* R a b b i t pellet chow (CR-I) a n d r a t pellet c h o w (CE-2) are c o n s t i t u t e d as follows:

GR- 1

CE-2

20.5 51.0 3.0 11,5 8.0 6.0

24.0 56.0 3.5 4.5 6.0 6.0

(%)

Protein Carbohydrate Lipid Fibre Ash Water

dr. Atheroscler. Res., 1969, 1 0 : 3 9 1 - 4 0 1

(%)

NICOTINIC ACID ESTER DERIVATIVE

393

Studies of progressive experiments Thirty-five male rabbits weighing 2.5 kg were divided into 4 groups. Group I was fed CR-1 for 120 days as a normal control group. Group II was fed with the cholesterol diet containing 1 % cholesterol added to CR-1 as a placebo control group. Group III was given the cholesterol diet containing 2 % K-31 for 120 days to study the prophylactic effect of K-31. Group IV was fed with the cholesterol diet for 62 days and then given the cholesterol diet containing 2 % K-31 for the next 58 days in order to reconfirm the therapeutic effects of K-31. In both regressive and progressive experiments, the food intake in all groups of animals was 150 g daily. During the period of these experiments, body weight, serum total cholesterol, esterified cholesterol, phospholipid and triglyceride levels were determined at stated intervals. At the end of the experiment, hepatic lipids were also determined. Serum was extracted with ethanol-ether (3 : 1, v/v) by BLOOR'S technique 2, and liver homogenates were extracted with 95 ~o ethanol. Total cholesterol and cholesterol ester were determined by the method of ZAK AND DICKENMANa and digitonin precipitation, phospholipids by ALLEN'S method 4 and triglycerides by VAN HANDEL'S method 5. Thoracic-duct fistula rats Male Wistar-strain rats weighing 250-310 g were fasted for 12-16 h with free access to water before operation. Under ether anesthesia, by a modification of the procedure of BOLLMANN et al. n, the beveled tip of a U-shaped polyethylene tube (1.33 mm diam.) was cannulated into the thoracic lymph-duct. After the operation, rats were kept in restraining cages 6, and were fed dried milk and allowed free access to 0.9 ~o saline. At the 24th h after operation rats with a free flow of lymph were selected and examined. Group I was fed water, dried milk and [4-14C]cholesterol and formed the control group. Group II received a commerical rat chow (CE-2: Nippon Haigo Shiryo Co., Ltd.) containing 0.5 ~o K-31 for 1 week up to the time of the operation and thereafter were given oral K-31 (300 mg/kg) suspended in water, dried milk and [4-14C]cholesterol. L4-14C]Cholesterol was prepared by dissolving 15 mg of carrier cholesterol and 2 ~C of [4-14C]cholesterol (obtained from the Radiochemical Centre, Amersham, Bucks., Great Britain) in 1 ml of olive oil. Thin-layer chromatography showed that the labeled cholesterol was 97 % radiochemically pure. Lymph was collected for 24 h, and blood was collected by cardiac puncture. The lipids in blood and lymph were extracted with chloroform-methanol (2 : 1, v/v) and washed with water by the method of FOLCHet al.7. An aliquot was evaporated and its residue was disolved in n-hexane. For separating esterified cholesterol and free cholesterol, an aliquot of n-hexane was applied to a thin-layer plate of silicagel (Merck A); the plate was then developed in the solvent mixture (n-hexane-ethylether-acetic acid; 165 : 15 : 1, v/v/v) s. Labeled cholesterol in the developed plates J. Atheroscler. Res., 1969, 10:391-401

394

Y. ASO, Y. ABE, K. HIGO, T. NARUKE, T. IRIKURA

2000

4, /\ F,'~ ~.o

L

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Plecebo c o n t r o l IHN 2% A NA 2 %

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.

.

.

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Time (days)

16o

Fig. 2. Effect of K-31, NA and IHN on the serum total cholesterol level in cholesterol-fed rabbits (mean values of 5-6 rabbits).

was detected with a thin-layer chromatogram scanner (Aloka TRM-1 type), thereafter the pertinent areas were removed from the plate by scraping and transferred to scintillation vials. The total recovery rate of [4-14C~cholesterol was determined with a liquid scintillation counter (Beckman Model LS-100) by adding 10 ml of toluene scintillator to 1 ml of n-hexane solution. The compo3ition of the scintillator fluid was: toluene 1000 ml, 4 g of PPO (2,5-diphenyloxazole) and 0.4 g of dimethyl-POPOP (1,4-bis-2-I4-methyl-5-phenyloxazole l-benzene). Counting-efficiency for each sample was determined by the external standard quench-correction method. Statistical comparisons were carried out with Student's t test. RESULTS

Therapeutic effect (regressive experiments) Fig. 2 shows changes in the mean serum total cholesterol level in the groups of rabbits in the therapeutic study. The mean serum total cholesterol level of the placebo control group reached 1720 rag/100 ml serum at 100 days after beginning cholesterol feeding, while the mean of these values in the NA-treated group, IHNtreated group and the K-31-treated group were 1200 rag/100 ml, 1632 mg/100 ml and 698 rag/100 ml, respectively. Thus, K-31 markedly suppressed elevation of the serum cholesterol level. NA temporarily suppressed the elevation of the serum total cholesterol, but its effect was not durable. I H N did not suppress it.

J. Atheroscler. Res., 1969, 10:391-401

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Concerning the effects of these drugs on serum lipids, Table 1 shows serum cholesterol levels, Table 2 shows phospholipid and triglyceride levels. Free cholesterol was 36 % of the serum total cholesterol level in the normal group, 32 % in the placebo control group and 32-40 % in the drug-treated groups. The mean phospholipid levels for the placebo control group, NA-treated group and IHN-treated group were 805, 568 and 666 rag/100 ml, respectively, whereas that for the K-31-treated group was only 375 rag/100 ml at the 100th day. The serum triglyceride level remaind unchanged in normal control rabbits. The mean serum triglyceride levels for placebo control, NA- and IHN-treated groups were 263, 319 and 213 rag/100 ml, respectively, while that for K-31-treated group was 145 rag/100 ml at the 100th day. Table 3 shows the effect on the liver lipids. The hepatic mean total cholesterol level was 25 mg/g in the placebo control; K-31 reduced hepatic cholesterol to half of this level (13.9 rag/g), but NA and IHN had no such action. Liver phospholipid levels did not differ among the experimental groups.

Prophylactic effect (progressive experiments) Table 4 shows serum lipid levels and Table 5 shows liver lipid levels in the prophylactic study on K-31. When K-31 was fed at the 2 % level with the cholesterol diet throughout the experimental period, the serum total cholesterol level decreased to 55.8 % of the placebo control level at the 63rd day and to 25.8 % at the 120th day. The serum triglyceride and phospholipid levels fell markedly in K-31-treated animals at the 63rd day. The hepatic cholesterol ester level in the K-31-treated group was lower than the placebo control level, but liver phospholipid levels did not differ among these two groups. As shown in the therapeutic study, K-31 markedly suppressed the elevation of serum and liver lipid levels.

TABLE 3 EFFECTS OF K-31, N A AND I H N ON THE LIVER LIPIDS IN CHOLESTEROL-FED RABBITS AT THE END OF THE REGRESSIVE EXPERIMENTS N u m b e r s in p a r e n t h e s e s indicate n u m b e r of r a b b i t s used. Values r e p r e s e n t m e a n 4- s t a n d a r d deviation.

No treatment

Placebo control K-31-HCI

NA

IHN

(s)

(6)

(s)

(s)

T o t a l cholesterol (mg/g) 4.1 4- 0.5 Free cholesterol (rag/g) 2.8 4- 0.2 Phospholipids (mg/g) 41.6 4- 4.6 Liver weight ( g / 1 0 0 g b . w . ) 2.79 4- 0.35 Body weight (kg) --

(s)

25.9

4- 2.9

13.9

4- 2.9 a

23.9

4- 1.9

23.5

4- 4.1

12.7

4- 3.6

5.5

4- 0.5 a

7.8

4- 1.3

8.9

4- 2.8

43.0

4- 4.6

43.5

4- 4.1

42.5

4- 8.6

47.0

4- 4.0

4.58 4- 0.96

4.14 4- 0.56

4.89 4- 0.94

4.39 4- 0.54

3.45 4- 0.21

3.38 4- 0.56

3.26 4- 0.63

3.49 4- 0.51

a Significantly different f r o m placebo c o n t r o l ( P < 0.01).

J. Atheroscler. Res., 1969, 1 0 : 3 9 1 - 4 0 1

398

Y. ASO, Y. ABE, K. HIGO, T. NARUKE, T. IRIKURA

TABLE 4 EFFECTS

OF

K-31 ON S E R U M

LIPIDS

IN CHOLESTEROL-FED

RABBITS

Numbers in parentheses indicate number of rabbits used. Values represent mean 4- standard deviation.

Days

Total cholesterol (mg/100ml) Esterifiedcholesterol (mg[100 ml) Triglycerides (mg/100 ml) 1Jhospholipids (mg/100 ml)

No treatment (5)

0 63 120 0 63 120 0 63 120 0 63 120

59 39 38 35 26 24 98 88 77 I09 62 55

• ~ • • • • • • • i • :~

11 4 11 7 4 11 28 19 15 29 19 9

Placebo control K-31 (4) prophylactic

58• 18 1544 i 305 1274 & 248 37 ~ 12 1 0 1 3 i 160 8 2 2 • 195 82• 41 1 2 3 ~ 54 1 8 6 • 76 87• 37 622 • 137 534 t 101

therapeutic

(5)

(5)

48~ 8 684 4- 326 a 945 • 176 32 4- 7 4 1 9 i 69 b 5 8 9 ~ 98 80• 29 9 1 ~ 41 2 2 0 • 89 76• 18 314 • 102 b 463 4- 86

1691 ~ 716 • 36 41101 • 451 • 72i 126• 136• 96i 658 • 370 •

56•

20 318 254 c 15 148 156 29 43 42 26 198 101 a

a Signifcantly different from placebo control (P < 0.05). b Significantly different from placebo control (P < 0.01). c Significantly different from placebo control (P < 0.025).

TABLE 5 EFFECTS

OF

K-31 ON L I V E R

LIPIDS

IN CHOLESTEROL-FED

RABBITS

AT T H E E N D

OF THE EXPERIMENT

Values represent mean ~z standard deviation

No treatment

Total cholesterol (rag/g) Esterified chomsterol (mg/g) Phospholipids (mg/g) Body weight (kg)

Placebo control

K-31 prophylactic

therapeutic

6.28 • 1.58

24.61 4- 3.61

23.19 5= 2.87

17.59 • 4.02~

3.45 • 0.45

10.09 • 2.25

7.76 ~z 1.39

6.66 • 0.91 b

42.10 ~z 4.91

39.50 • 8.61

37.70 ~z 9.56

36.80 ~ 4.46

3.86 • 0.55

3.44 • 0.40

3.05 -4- 0.36

3.29 ~ 0.59

Significantly different from placebo control (P < 0.05). b Significantly different from placebo control (P < 0.025).

Table 3 shows that body weight gain did not differ among the groups in the regressive experiments: at autopsy less fat was deposited in animals given K-31. As shown in Table 5, the average body weight and growth rate were essentially the same among the treated and control groups. J. Atheroscler. Res., 1969, 10:391-401

399

NICOTINIC ACID ESTER DERIVATIVE

TABLE 6 T H E E F F E C T OF K - 3 1

ON INTESTINAL

CHOLESTEROL ABSORPTION

IN THORACIC-DUCT

FISTULA RATS

Rats were given an oral dose of E4-14C]cholesterol(1S mg 2/~C per rat) dissolved ilx 1 ml of olive oil. K-31 was suspended in water. 14C-activityof 24 h lymph lipids was analyzed. Values are mean + standard error. Numbers in parentheses indicate number of rats used. K-31 dose (mg/kg)

0 300

(5) (4)

[ 4-14C]cholesterol recovered (%)

Cholesterol ester (%)

43.9 4- 4.2 29.2 • 2.4a

64.8 ~= 2.5 72.3 ~ 1Ab

~,b Significantly different from untreated group (P < 0.05).

Thoracic-duct l y m p h fistula rats

15 mg of ~4-14CJcholesterol (2 ~C/rat) were given orally to thoracic-duct fistula rats. No serum [4-14C]cholesterol was recovered from the rats in both control and K-31-treated groups at 24 h after administration. As shown in Table 6, the [4-14C]cholesterol recoveries in the 24 h lymph samples was 29.2 -4- 2.4 % for the K-31treated group and 43.9 ~: 4.2 ~o for the control group. Administration of K-31 significantly reduced [4-14C]cholesterol absorption from the intestine (P < 0.05). The recovery of E4-14C]cholesterol from the lymph in the group administered K-31 decreased to about 66.5 % of the control. From thin layer chromatographic studies on lymph [4-14C]cholesterol, it was found that the percentage of absorbed [4-14C]cholesterol present as ester in the K-31-treated groups was higher than that in the controls. Administration of K-31 did not cause severe diarrhea, and the survival rate of K-31-treated animals was the same as the controls. DISCUSSION IRIKURA et aL 1 previously showed that 2,2,6,6-tetrakis(nicotinoyloxymethyl) eyclohexanol (K-31) significantly decreased serum total cholesterol in experimentally hyperlipemic mice. In the present study, the therapeutic and prophylactic effects of K-31 were investigated in cholesterol-fed rabbits--ANITSCHKOW AND CHALATOW'S model 9 for atheroma. K-31 was compared with nicotinic acid (NA) and nleso-inositol hexanicotinate (IHN) in this study. NA is well known for its antilipemic lo and hypocholesterolemic n effect in man; FONTENOT et al. 12 reported a similar action for NA in rabbits. As shown in our therapeutic (regressive) studies, K-31 markedly suppressed the elevation of serum total cholesterol, triglyceride and phospholipid levels, and also substantially lowered the hepatic total cholesterol. On the other hand, I H N did not suppress the elevation of serum cholesterol, and NA suppressed it only temporarily. The liver total cholesterol and phospholipid levels among the placebo control group J. Atheroscler. Res., 1969, 10:391-401

400

Y. ASO, Y. ABE, K. HIGO, T. NARUKE, T. IRIKURA

and the IHN- and NA-treated groups did not differ. These results suggest that the effects of K-31 on experimental atherosclerosis in rabbits differ from those of NA and IHN. The progressive experiments show that K-31 has a prophylactic action on experimentally-induced hypercholesterolemia in rabbits. In both the therapeutic (regressive) and prophylactic (progressive) studies, aortic plaques were less severe in the K-31-treated animals than in the placebo controls; this might be related to the hypocholesterolemic action of K-31. Hypocholesterolemic agents may act b y one or more of the following mechanisms: inhibition of intestinal absorption or reabsorption of cholesterol, depression of cholesterol biosynthesis, increased cholesterol degradation and excretion, ~nd redistribution of cholesterol from blood to tissues. It has been firmly establishedl3,14 that cholesterol is mainly absorbed in the free form and is esterified during its passage through the intestinal epithelium. Thence, it is carried into the systemic circulation via the thoracic lymph duct and is then distributed into blood or other tissues after being attached to a lipoprotein vehicle by the hepatic parenchyma. Some hypocholesterolemic agents, such as fi-sitosteroP 5 or cholestyramine 16 are reported to inhibit cholesterol absorption. In our experiments, K-31 inhibited ~4-14C]cholesterol absorption from the intestine. However, the volume of lymph tended to fall in the K-31-treated group. In our previous pharmacological evaluation of K-3117, the drug was found to exert a choleretic action in laboratory animals: we now think that this choleretic action may play some role in inhibiting cholesterol absorption. But the percentage of esterified E4-14C~cholesterol recovered from the lymph of K-31-treated animals was higher than that in controls. Most sterols, including cholesterol, are esterified in the intestinal wall and then enter the lymph, but such esterification is not considered to be an essential condition for absorption. HERNANDEZ et al. Is and SWELL et al. 19 reported that epicholesterol was slowly absorbed, but was recovered from the lymph in its free form. DASKALAKIS AND CHAIKOEF 20 reported in his experiment on thoracic-duct lymph fistula rats that ergosterol inhibited cholesterol absorption, but the ester-ratio of lymph cholesterol remained the same as in controls. HYUN et al. 16 investigated cholestyramine and pectin which are thought to inhibit absorption of exogenous cholesterol from the digestive tracts; they concluded that esterification of cholesterol during absorption does not defend on the amount of cholesterol absorbed. GALLO-TORRESet al. 21 recently suggested that esterification may not be obligatory for cholesterol absorption. The increased ~4-14C]cholesterol ester content of lymph could be due to an effect of K-31 on intestinal mucosal cells. Thus, the hypocholesterolemic action of K-31 would depend on the inhibition of intestinal cholesterol absorption. ACKNOWLEDGEMENTS We wish to thank Dr. S. Ogiwara, President of Kyorin Pharmaceutical Co., ]. Atheroscler. Res., 1969, 10:-391-401

NICOTINIC ACID ESTER DERIVATIVE

401

Ltd., for his kind support, and Mr. S. Sato and Mr. K. Kasuga, Department of Organic Chemistry, Kyorin Chemical Laboratory, for the supply of K-31. We are grateful to Mr. T. Umezawa for his expert technical assistance.

REFERENCES 1 IRIKURA, T., Y. ABE AND K. HIGO, Studies on new nicotinic acid ester derivatives, P a r t 1 (Effect of 2,2,6,6-tetrakis(nicotinoyloxymethyl)cyclohexanol on t h e lipid metabolism and serum factors in blood), Pharmacometrics (Japan), 1968, 2: 237. 2 BLOOR, W. R., The oxidative determination of phospholipid (lecithin and cephalin) in blood and tissues, J. biol. Chem., 1929, 82: 273. 3 ZAK, B. AND R. DICKENMAN, R a p i d estimation of free and total cholesterol, Amer. J. clin. Path., 1954, 24: 1307. 4 ALLEN, R. T. L., The estimation of phosphorus, Biochem. J., 1940, 34: 858. 5 HANDEL, E. VAN, Suggested modification of t h e micro d e t e r m i n a t i o n of triglyceride, Clin. Chem., 1961,7: 249. 6 BOLLMAN, J. L., J. C. CAIN AND J. H. GRINDLAY, Technique for t h e collection of l y m p h from t h e liver, small intestine, or thoracic duct of the rat, J. Lab. clin. Med., 1948, 33: 1349. 7 FOLCH, J., M. LEES AND G. H. SLOANE-STANLEY, A simple m e t h o d for t h e isolation a n d purification of total lipids from animal tissues, J. biol. Chem., 1957, 226: 497. 8 ARAKI, E., Thin-layer c h r o m a t o g r a p h y of serum tipids, Jap. J. reed. Progr., 1963, 50: 85. 9 ANITSCKOW, N. AND S. CHALATOW, E x p e r i m e n t a l cholesterolsteatosis, and its significance for t h e etiology of certain pathological processes, Zbl. allg. Path. path. Anat., 1913, 24: 1. 10 CARLSON, L. A. AND P. R. BALLY, Inhibition of lipid mobilization. In: A. E. RENOLD AND G. F. CAHILL (Eds.), Handbook of Physiology, Vol. 5, American Physiological Society, Washington, D.C., 1965, p. 557. 11 ALTSCHUL, R., A. HOFFER AND J. D. STEHEN, Influence of nicotinic acid on serum cholesterol in man, Arch. Biochem. Biophys., 1955, 54: 558. 12 FORTENOT, R., H. REDETZKI AND R. DEUPREE, Effects of nicotinic acid and nicotinamide on serum cholesterol and e r y t h r o c y t e nicotinamide adenine dinucleotide levels of rabbits, Proc. Soc. ezp. Biol. ( N . Y . ) , 1965, 119: 1053. 1~ BIGGS, M. W., M. FRIEDMAN AND S. O. BYERS, Intestinal l y m p h a t i c t r a n s p o r t of absorbed cholesterol, Proc. Soc. exp. Biol. ( N . Y . ) , 1951, 78: 641. 14 CHAIKOFF, I. L., B. BLOOM, M. D. SIPERSTEIN, J. K. I~IYASU, W. O. REINHARDT, W. G. DAUBEN AND J. F. EASTHAM, C14-Cholesterol, P a r t 1 ( L y m p h a t i c t r a n s p o r t of absorbed cholesterol4-C14), J. biol. Chem., 1952, 194: 407. 15 HERNANDEZ, H. H. AND I. L. CHAIKOFF, DO soy sterols interfere with absorption of cholesterol? Proc. Soc. exp. Biol. ( N . Y . ) , 1954, 87: 541. 16 HYUN, S. A., G. V. VAHOUNY AND C, R. TREADWELL, Effect of hypocholesterolemic agents on intestinal cholesterol absorption, Proc. Soc. exp. Biol. (N. Y.), 1963, 112: 496. 17 IRIKURA, T., Y. KUDO, T. TAMADAAND R. IWAKI, Studies on new nicotinic acid ester derivatives, P a r t 4 (General pharmacological action of 2,2,6,6-tetrakis(nicotinoyloxymethyl)cyclohexanot (K-31)), Pharmacometrics (Japan), 1968, 2: 259. 18 HERNANDEZ, H. H., I. L. CHAIKOFF, W. G. DAUBEN AND S. ABRAHAM, The absorption of C T M labeled epicholesterol in t h e rat, J. biol. Chem., 1954, 206: 757. 19 SWELL, L., E. STUTZMAN, M. D. LAW AND C. R. TREADWELL, Intestinal metabolism of epicholesterol-4-C TM, Arch. Biochem., 1962, 97:411. 20 DASKALAKIS, E. G. AND 1. L. CHAIKOFF, The significance of esterification in t h e absorption of cholesterol from t h e intestine, Arch. Biochem., 1955, 58: 373. 21 GALLO-TORRES, H. E., O. N. MILLER AND J. G. HAMILTON, A comparison of t h e effects of bile salts on t h e absorption of cholesterol from t h e intestine of the rat, Biochim. biophys. A cta (Amst.), 1969, 176: 605.

J. Atheroscler. Res., 1969, 10:391-401