Effects of Sitosterol on the CholesterolConcentration in Serum andLiver in Hypothyroidism

By MAURICE MI. BEST, M.D., AND CHARLES H. DUNCAN, M.D.

With the Technical Assistance of Joan D. Wathen, B.S. and Ursula Jegher

The effects of the administration of the plant sterol, f3-sitosterol, on the elevated serum lipids ofhypothyroidism and on the cholesterol content of the liver have been investigated. In patientswith hypothyroidism sitosterol administration, without dietary restriction, resulted in a reductionof serum cholesterol and other lipids. In the hypothyroid rat the addition of sitosterol to thediet prevented the increase in serum lipids and in the cholesterol content of the liver which other-wise occurs on a high-cholesterol diet.

T HE plant sterol, j3-sitosterol, has beenreported to exert a hypocholesterolemiceffect in the cholesterol-fed chick,1 and

rabbit,2 and in man on a free diet.3 It presum-ably acts by interfering with the intestinalabsorption of cholesterol.4

In hypothyroidism, numerous observers havedemonstrated that the plasma cholesterol andother plasma lipids tend to be elevated.5 Inthe rat fed thiouracil, Rosenman, Byers, andFriedman6 observed a much reduced rate ofturnover of plasma cholesterol, both excretionand hepatic synthesis being reduced. Thesealterations in the handling of cholesterol inhypothyroidism might be expected to modifythe effects on plasma lipids of sitosterol ad-ministration.The present study compares the effects on

serum and liver lipids of sitosterol administra-tion to normal and to hypothyroid rats. Theeffect on serum lipids of sitosterol administra-tion to myxedematous human subjects has alsobeen studied.

From the Department of Medicine, Universityof Louisville School of Medicine, Louisville, Ky.

This investigation was supported by a grant fromthe American Heart Association. The 0-sitosterolwas generously supplied by Robert E. Shipley, M.D.,Eli Lilly & Company, Indianapolis, Ind.

Presented at the meeting of the American Societyfor the Study of Arteriosclerosis, Nov. 6, 1955, Chi-cago, Ill.

METHODSRat Studies. Mlale white rats (Holtzman) of ap-

proximately 210 Gm. were maintained for 30 dayson a commercial feed containing no added iodine(Pillsbury's mix-grain pellets), 1 per cent sodiumchloride solution, and tap water, provided ad libitum.The animals were then divided into 6 groups of 4 to7 each, the groups having similar mean weights.They were housed in mesh-bottomed cages andmaintained in an air-conditioned animal room atapproximately 75 F. for the duration of the experi-ment. At the end of this 30-day period of low-iodineintake, the 17 rats in 3 of the groups were subjectedto radiation destruction of the thyroid by radio-iodine. Each of the animals received 875 ,ue. ofcarrier-free J131 intraperitoneally. The 14 rats in theremaining 3 groups received no J131 and were con-sidered to be euthyroid.

Three experimental diets were prepared as fol-lows: low-cholesterol, Purina rabbit pellets plus 5 percent cottonseed oil; high-cholesterol, Purina rabbitpellets plus 5 per cent cottonseed oil plus 1 per centcholesterol-the cholesterol was dissolved in thewarmed cottonseed oil prior to its addition to therabbit pellets; cholesterol-sitosterol, Purina rabbit pel-lets plus 5 per cent cottonseed oil plus 1 per centcholesterol plus 5 per cent f-sitosterol the rabbitpellets were impregnated with the fl-sitosterol dis-solved in chloroform, the chloroform was removedby evaporation prior to the addition of the (otton-seed oil and cholesterol.

Each of the 3 diets was fed to 1 group of normaland to 1 group of 1131 treated rats. The special dietswere started 2 weeks after the administration of theradioiodine, and offered ad libitum for a period of 13days. Tap water was provided at all times. The dietswere well tolerated, none of the animals developeddiarrhea, and all appeared healthy throughout the

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BEST AND DUNCAN

TABLE 1.-Effects on Normal and Hypothyroid Rats of the Addition to the Diet of Cholesterol and ofCholesterol Plus Sitosterol*

Diet

Low cholesterol

1% Cholesterol

1% Cholesterol5% SitosterolLow cholesterol

1% Cholesterol

1% Cholesterol5% Sitosterol

* Values given are the mean, standard deviation (estimated using n - 1) and, in parentheses, the range.

period of study. On the thirteenth day of the specialdiets, the animals were anesthetized with intraperi-toneal amobarbital sodium, blood was obtained bycardiotomy, and the liver and thyroid were removed.The blood was permitted to clot, serum was sepa-

rated by centrifugation, and total cholesterol, totallipid, and lipid phosphorus were determined within24 hours. Cholesterol was determined by the methodof Abell and associates,7 lipid phosphorus by thatof Zilversmit and Davis,8 and total lipid by theturbidometric method of de la Huerga and co-

workers.9 The livers were blotted dry, weighed, anddigested over night in 20 per cent alcoholic potassiumhydroxide. Total cholesterol was then determined bythe method of Abell, and expressed as mg./100 Gm.of liver, wet weight.The destruction of the thyroid glands was con-

firmed by microscopic examination. Portions ofrepresentative livers were fixed in formalin, em-

bedded in gelatin, and stained for lipid with oil red 0.

Human Studies. Six patients with myxedema were

selected for study. Included were 2 patients withspontaneous myxedema, 2 with post-thyroidectomymyxedema, and 2 with radioiodine-induced myx-

edema. Beta sitosterol was administered before eat-ing in a total daily dosage of 20 to 25 Gm.; therewas no restriction as to the type or amount of food.A placebo preparation was administered during a

portion but not all of the control periods. Fastingserum lipids were determined by the previouslydescribed methods at intervals of 1 to 3 weeks dur-ing control and sitosterol periods.

RESULTS

The results of the animal experiments are

summarized in table 1. The addition of 1 per

cent cholesterol to the diet resulted in a signifi-

cant* increase in liver cholesterol in both nor-mal and hypothyroid animals. Some increase inserum total cholesterol and in total lipid wasalso observed with the 1 per cent cholesteroldiet. The increases were greater in the hypo-thyroid than in the normal rats, but in neithergroup were they statistically significant.The further addition of 5 per cent f3-sitosterol

to the 1 per cent cholesterol diet significantlyreduced the liver cholesterol in both normaland hypothyroid rats. In addition to the reduc-tion in liver cholesterol, the hypothyroid ratsalso displayed a significant lowering of serumcholesterol and serum total lipid as a result ofthe added sitosterol.Comparison of the cholesterol-sitosterol fed

groups with those on the low cholesterol dietreveals no significant differences in liver choles-terol or in serum lipids. This is true of bothnormal and hypothyroid groups.

Microscopic examination of the livers showeda pronounced increase in stainable lipid in theliver cells at the periphery of the lobules ofthe cholesterol-fed rats, both normal and hypo-thyroid. The further addition of sitosterol pre-vented this increase in stainable lipid, the sec-tions being indistinguishable from those ofanimals on the low cholesterol diet (fig. 2).For the final 13-day period during which the

* Difference of the means considered significantwhen p < 2.5 per cent as estimated by the t test.

Thyroidstate

Normal

Normal

Normal

Hypo

Hypo

Hypo

Rats

4

5

5

4

7

6

Weight change

Gin.

+33 4± 8.7(+22-+43)+35 i 5.0(+30-+42)+29 4t 10.2(+17-+43)-21 i1 2.7(-18-23)-24 i 9.3(-8-35)

-27 i 6.5(-18-40)

Serum totalcholesterol

mg.1100 inl.

72 i 5(64-76)

75 9(59-81)

68 i 6(59-74)

88 16(74-111)

119 i 25(81-151)

80 4: 13(66-95)

Serum lipidphosphorus

mg.1100 mld.

7.6 i .7(6.8-8.5)

6.6 i 1.5(5.3-7.9)

7.2 + .9(5.8-8.0)

7.6 i .6(7.1-8.4)

7.0 i 1.1(6.0-8.8)

7.2 i .9(5.9-8.3)

Serum totallipid

nig.1100 nil.

217 i 27.3(189-250)

254 i 42.0(192-310)

220 i 18.5(208-253)

246 i 63.5(192-336)

312 i 53.8(250-397)

233 26.6(189-266)

Liver totalcholesterol

mg./100 Gmn.

303 i 6(297-311)

1109 ± 285(872-1558)

271 i 39(237-333)

270 i 28(230-293)

1625 i 296(1044-1914)281 i 28(260-321)

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346 SITOSTEROL AND CHOLESTEROL CONCENTRATION IN HYPOTHYROIDISM

TABiE 2.-Effects on Serumn Lipids of the Administration of Stiosterol to Six Hypothyroid Patients*

Subject

S.M., JIll Induced

V.S., Surgical

G.K., Spontaneous

CM.XI., J131 Induced

L.H., Surgical

J.W., Spontaneous

Observation Total cholesterol L~ipid phosphorus Total Lipid(weeks) (mg.1100 ml.) (mg. '100 ml.) (mg. 100 ml.)

Control Sitosterol Control Sitosterol Control Sitosterol Control Sitosterol

24 42 363(+-41.5)

14 24 315(+-14.3)

8 24 563(+95.4)

17 16 246(+26.8)

54 4 268(i32.3)

3 3 257(i10.5)

305(+33.2)

266(i13.1)

415(+117)

216(+23.6)

216(+14.9)

175(+5.0)

16.8(+0.7)13.5

(+1.1)22.7

(+4.0)11.6

(+1.2)12.6

(i1 .8)12.4

(+0.7)

14.1(+0.7)12.0

(+4.4)18.6

(+4.9)10.9

(+1.2)10.6

(+0.6)8.9

(+0.5)

1039(+-99.6)879

(+68.5)1922

(+394)768

(+77.8)812

(+53.5)778

(+50.2)

869(+t84.4)767

(+59.9)1583

(+471)682

(+72.6)662

(+54.9)539

(+16.5)* Values given are the mean and, in parentheses, the standard deviation (estimated using n - 1).

M9Q /IOORI300,l

250

200-

150

30 mc.

4

209% 57%08% 26% 1131 UPTAKE

2- SITOSTEROL, ,2 II 14 16 19

O 16 7 8 9 16 11 1'2 1'3 14 15 16 19MONTHS

FIG. 1. Changes in the serum total cholesterolin C. MI. Following the administration of radioactiveiodine the patient developed clinical manifestationsof myxedema and elevation of serum cholesterol.Upon 3 separate occasions the administration ofsitosterol was accompanied by a reduction in serum

cholesterol (cross-hatched line).

special diets were fed, all euthyroid animalsgained weight (mean 2.47 Gm./day) while allthe J131 treated rats lost weight (mean 1.88Gm./day). These weight changes were notsignificantly influenced by the addition ofcholesterol or sitosterol to the diet.The patient observations are summarized in

table 2. The administration of sitosterol was

accompanied by a fall in the mean values ofserum total cholesterol, lipid phosphorus, andtotal lipid. The etiology of the hypothyroidismhad no apparent effect on the response tositosterol. Figure 1 depicts the effects on serum

cholesterol of 3 separate periods of sitosteroladministration to patient C.M., in whom hypo-

thyroidism was induced by J1lt in an attemptto alleviate intractable congestive heart failuredue to rheumatic heart disease with mitralinsufficiency.

DISCUSSIONThe administration of 3-sitosterol to 6 hypo-

thyroid patients was accompanied by a con-sistent reduction in serum cholesterol. Themean fall of 20.1 per cent of control level com-pares with the fall of 15.6 per cent observed in10 euthyroid hypercholesterolemic patients.10Human studies thus indicate that the hyper-cholesterolemia associated with hypothyroidismis reduced by the administration of sitosterolwithout dietary modification.That the greater fall in serum cholesterol in

the hypothyroid patients was not due purelyto chance is suggested by the results of the ratstudies. Under the experimental conditionsemployed, the addition of 1 per cent cholesterolto the diet of the rats resulted in a 3- to 6-foldincrease in the cholesterol level of the liver,the value being significantly higher in thehypothyroid than in the normal animal. Ifliver cholesterol is referred to total body weightinstead of to liver weight, the relative valuesfor the various groups are not appreciablychanged.Although in this small series the increase in

serum cholesterol occurring when hypothyroidrats were fed a high cholesterol diet was notstatistically significant, the report of Page and

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BEST AND DUNCAN

Brown11 suggests that it would be with a largerseries or more prolonged period of feeding. Theeuthyroid rat fed cholesterol demonstratedessentially no effect on serum cholesterol level.The greater accumulation of cholesterol inserum and liver occurring in the hypothyroidas compared to the euthyroid rat when fed ahigh cholesterol diet is presumed to be due tothe decreased rate of cholesterol excretion ordestruction. 6

FIG. 2. Stainable lipid in the liver of the hypo-thyroid rat: (top) 1 per cent cholesterol diet, (center)low cholesterol diet, (bottom) 1 per cent cholesteroldiet plus 5 per cent sitosterol.

The further addition of 5 per cent j-sitosterolto the diet prevented the increases in liver andserum cholesterol levels, in both normal andhypothyroid rats on a high cholesterol diet.This effect was more pronounced in the hypo-thyroid rat in which cholesterol feeding pro-duces greater increases. The effect was alsomuch more pronounced in the liver than in theserum.

In the hypothyroid rat, not only cholesterolexcretion, but also hepatic synthesis of choles-terol is greatly reduced.6 Under these conditionsof decreased rate of turnover of cholesterol, onewould expect changes in the amount of choles-terol absorbed from the intestine to have anaugmented effect on serum and liver levels.Since sitosterol interferes with the absorptionof cholesterol from the digestive tract,4 its in-creased hypocholesterolemic effect in hypo-thyroidism is understandable.

SUMMARYIn 6 patients with hypothyroidism the pre-

prandial administration of f-sitosterol in thetotal daily dosage of 20 to 25 Gm. withoutdietary restriction, resulted in a mean redue-tion of serum total cholesterol of 20.1 per centof the control level.

In both the normal and the hypothyroid rat,the addition of 5 per cent 3-sitosterol to thediet prevented the increase in liver cholesteroland stainable lipid which otherwise occurred ona 1 per cent cholesterol diet. Sitosterol alsoprevented the rise in serum total cholesteroland total lipid which occurs in the hypothyroidrat fed a 1 per cent cholesterol diet.The reduction in serunm and liver cholesterol

levels resulting from the interference by sitos-terol with the intestinal absorption of choles-terol is augmented by the decreased rate ofturnover of cholesterol in the hypothyroid state.

SUMMARIO IN INTERLINGUA

In sex patientes con hypothyroidismo leadministration preprandial de beta-sitosterol intotal dosages diurne de 20 a 25 g, sin restrictiondietari, resultava in un reduction median deltotal cholesterol seral de 20,1 pro cento delnivello de controlo.

In rattos normal e in rattos hypothyroide, le

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348 SITOSTEROL AND CHOLESTEROL CONCENTRATION IN HYPOTHYROIDISM

addition al dieta de 5 pro cento de beta-sitos-terol preveniva le augmento de hepatic choles-terol e lipido colorabile que occurreva altere-mente con un dieta a 1 pro cento de cholesterol.Sitosterol etiam preveniva le augmento deltotal cholesterol seral e del total lipido seralque occurre in rattos hypothyroide recipienteun dieta a 1 pro cento de cholesterol.Le reduction del nivellos seral e hepatic de

cholesterol, resultante ab le obstruction persitosterol in le absorption intestinal de choles-terol es augmentate per le reducite passage decholesterol que es characteristic del statohypothyroide.

REFERENCES1 PETERSON, D. W.: Effects of soybean sterols in

the diet on plasma and liver cholesterol inchicks. Proc. Soc. Exper. Biol. & Med. 78: 143,1951.

2POLLACK, 0. J.: Successful prevention of experi-mental hypereholesterolemia and cholesterolatherosclerosis in the rabbit. Circulation 7: 696,1953.

3BEST, M. M., DUNCAN, C. H., VAN LOON, IE. J.,AND WATHEN, RJ. D.: Effects of sitosterol onserum lipids. Am. J. Med. 19: 61, 1955.

4HERNANDEZ, H. H., PETERSON, D. W,. CHAIKOFF,I. L., AND DAUBEN, W. G.: Absorption of cho-

lesterol-4-C'4 in rats fed mixed soybean sterolsand beta-sitosterol. Proe. Soc. Exper. Biol. &Med. 83: 498, 1953.

DEUEL, H. J., JR.: The Lipids: Their Chemistryand Biochemistry. Vol. II. New York, Inter-science Publishers, Inc., 1955.

6 ROSENMAN, R. H., BYERS, S. 0., AND FRIEDMAN,MI.: Mechanism responsible for the alteredblood cholesterol content in deranged thyroidstates. J. Clin. Endocrin. & Metab. 12: 1287,1952.

7ABELL, L. L., LEVY, B. B., BRODIE, B. B., ANDKENDALL, F. E.: A simplified method for theestimation of total cholesterol in serum anddemonstration of its specificity. J. Biol. Chem.195: 357, 1952.

8 ZILVERSMIT, D. B., AND DAVIS, A. K.: Microdeter-mination of plasma phospholipids by trichloro-acetic acid precipitation. J. Lab. & Clin. Me(l.35: 155, 1950.

9DE LA HUERGA, J., YESINICK, C., AND POPPER, H.:Estimation of total serum lipids by a turbido-metric method. Bull. Reg. M. Tech. 23: 231,1953.

10 DUNCAN, C. H., AND BEST, M. M.: Effects ofsitosterol on serum lipids of hypercholester-olemic subjects. Abstracted, J. Clin. Invest.34: 930, 1955.

11 PAGE, I. H., AND BROWN, H. B.: Induced hyper-cholesterolemia and atherogenesis. Circulation6: 681, 1952.

Joyce, J. C.: Dextrocardia, Situs Inversus, and Twinning. Brit. M. J. 2: 950 (Oct. 15), 1955.Four instances of situs inversus are described in which, although none was a twin, a strikingly

high incidence of twinning occurred in the family: (1) both parents twins; (2) grandfather andgrandmother twins; a sister and uncle begat 1 and 3 sets of twins; (3) father a twin, paternal grand-mother had set of twins by each of 2 husbands; (4) mother twin, a first cousin begat 2 sets oftwins.

Reference is made to the work of Ruud and Spemann (1922) who produced situs inversusthrough division of amphibian eggs. The twin derived from the right side showed a special tendencyto develop this abnormality.

MIcKuslcK

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JegherMAURICE M. BEST, CHARLES H. DUNCAN, Joan D. Wathen and Ursula

HypothyroidismEffects of Sitosterol on the Cholesterol Concentration in Serum and Liver in

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1956 American Heart Association, Inc. All rights reserved.

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