Life Sciences Vol. 3, pp. 857-888, 1984. Pergamon Press, Inc. Printed in theUnited States .

DF~03ITION OF CALCIUM IN AORTA OF COCI~ItII.S

TREATm WITH TRIPARANOL *

Harry Y. C. Wong and Frank B. Johnsonl

Department of Physiology, Howard University College of Medicine,

and The Armed Forces Institute of Pathology snd the

Veterans Administration Central Laboratory for

Anatomic Patholo® and Research, Armed Forces Institute of Pathologyl ,

waahington, D. C.

(Received 22 June 1984)

3tudiee by Blohm et al . (1~ 2) have reported that administration of

(1-[p-(~iethylaminoethozy)~pheayl ]-1-(-(p-tolyl)-2-(p-chlorophenyl) ethanol

(triparsnol) can lower cholesterol levels in both serum and tiasne in rata and

monkeys . Other investigators (3-b) have reported that thin drug lowers blood

cholesterol in mn. Avigsn and associates (4) have de®onstrated that the

observed effect was caused by a block in a late stage of cholesterol synthesiy

namely, in reduction of deemosterol to cholesterol . Data from our laboratory

(8) showed that administration of tripsrsnol for 10 weeks to 10 week old

cockerels fed plain mash did not result in any significant ahasges in the

levels of cholesterol or phospholipid in the plasma. Unezpectedly, we ob-

nerved an increased incidence of microscopic aortic atherosclerosis in birds

treated with triparsnol in doses of 12.b mg or 25 ~kg of body weight tri-

parasol for 10 weeks on plain mash as compared to no lesions is the controls

on plain mash only.

Therefore, an eztended study for 20 weeks wsa later re-

ported from our laboratory (?) showing that the administration of triparsnol

resulted in an increase in plasma total sterols and desatosterol in plasma of

cockerels. Upon analysis of the sortaa~ we observed mrked deposits that

seemed to be cholesterol in the twits of the aortas of the tripsranol-treated

cockerels.

* Preliminary data were reported at the Federation of Americas Societies for~perisestal Biology held in Chicago, Illinois, April 12 - 17, 1984.

857

858

DEPOSITION OF CALCIUM IN AORTA

Vol. 3, No. 8

The present study ie of particular interest since it indicates that

accumulation of deamosterol is as atherogenic as cholesterol and, in addition,

causes deposition of calcium in the aorta of cockerels treated with triparanol

as compared to no lesions in the coc%erel on plain mash alone.

Methods

Day old Hy-Line cockerels were obtained from Heatwole Hatcheries, Madison,

Virginia, sad fed starter-grower mash.

1Khen they were 10 weeks old they were

divided into 3 groups of 8-11 birds to a group. Triparanol dissolved in olive

oil was injected subcutaaeously five times weekly at doges of 12 .0 and 2b.0

mg~kg body weight in each of two groups for 20 consecutive weeks.

The controls

were injected with 1 ml of olive oil .

The body weights and plasma samples

were taken at the beginning of the ezperiment and every 2 weeks thereafter .

was determined by the method of Sperry and Webb (8),

Fiske and Subbarow (9), and total lipid determination

The analysis of deeawsterol in the plasma and in

method of Avigan and associates (4) . After sacri-

aortas were opened, slit longitudinally, and ezamined

for the degree of severity of atheroscleroais according to the method of I(atz

snd Stamler (11), based on zero to four . Frozen histologic sections were made

of the thoracic aorta and stained for calcium deposit by the Alizarin Red S

method (12) .

Results

Figure 1 depicts the ehangea in body weight of triparanol-treated cock-

erels as compared to the control on plain mash only. There were no aigaifi-

cant differences in the body weight of the different groups, whether treated

with triparanol or not, as compared to the controls on plain

the course of the ezperiment.

Figure 2 summarizes the influence of trfparanol on the

in the plasma of the various groups of birds . There was a decrease in plasma

cholesterol of the cockerels treated with triparsnol at the onset of the ez-

Total plasma cholesterol

phoepholipids by that of

by that of Hragdon (10) .

aortic tissue was by the

ficing the cockerels the

mash alone during

cholesterol levels

Vol. 3, No . 8

DEPOSITION OF CALCIUM IN AORTA

859

periment ae compared to the control8 on plain mash alone . During the reme.in-

ing experimental period, however, the plasma cholesterol of the various groups

remained quite similar with the exception that the cockerels treated with 12 .5

mg of triparanol showed a decrease between the 14th and the 1Gth week and au

increase at the 18th week of treatment (28th week of age) .

axc~z

xc~w3

r

Ôm

250-

200

150-i~ 100-

50~

D

FIG. 1

PLAIN MASH CONTROLS

-- PLAIN MASH+12.5 TRIPARANOL

+- PLAIN MASH+25.OTRIPARANOL

FIG. 2

Changes in body weight of control cockerels or birds treated with triparanolfor 20 weeks.

- PLAIN MASH CONTROLS-- PLAIN MASH+12.5mq TRIPARANOL

- PLAIN MASH+25.Omq TRIPARANOL

AGE IN WEEKS

0 i

i

T

r

r

i-~

~

i--~--r10 12 14 16 18 20 22 24 26 28 30

Comparison of cholesterol in plasma of control cockerels with that in birdstreated with triparanol for 20 weeks.

860

DEPOSITION OF CALCIUM IN AORTA

Vol. 3, No. 8

Figure 3 ehawa that the phospholipid level of triparanol-treated cock-

erels was significantly elevated xhen compared to the controls . Also after

20 weeks of treatment (at 30 weeks of age) there was an increase of total

(p greater than 0 .01) sterol as well as a eigaificant increase in the amount

of 24 dehydrocholeeterol (desmosterol) in the plasma and sterols in the aorta

of the triparanol-treated cockerels ae compared to the controls on plain mash

(Table 1) .

300

250

200 "

100

30

r

PLAIN MASH CONTROLS-- PLAIN MASH+12.5 mq TRIPARANOL-.- PLAIN MASH+25.0 mq TRIPARANOL

12 14 16 18 20 22 24 26 28 30

AGE IN WEEKS

FIG. 3

Level of phoapholipids in control cockerels and in those treated with tri-paranol for 20 xeek$.

Table 2 summarizes the effect of administration of triparsnol on the

gross and microscopic grading of the aortas and coronary arteries. No gross

lesions were observed in the control group . In the group of 8 birds injected

with 12 .5 mg of triparanol~ however 3 had aortic atheroaclerosis, and in the

group of nine receiving 25 mg 3 had lesions . No lesions xere found micro-

scopically in the control cockerels, while those treated with 12 .5 or 25 mg

of Triparsnol showed a significant increase in aortic lesions. Birds receiv-

ing 12 .5 mg of the drug showed three out of eight had aortic lesions, while

in the group on 25 mg five ont of nine cockerels had lesions. Ae the dosage

Vol. 3, No. 8

DEPOSTTION OF CALCIUM IN AORTA

861

Comparison of Total Sterols and Desmosterol in Plasmaand Aorta of Control Cockerels and Birds Having 20 Weeks

of Treatatent With Triparanol

* Standard error of mean+ Not detectable

Only two aortas were analyzed.

TABLE 1

TABLE 2

Gross and Microscopic Grading of Aorticand Coronary Atheroaclerosis

* Based on zero to four.t Red Rose Starter Grower Mash John W, Eshelman & Sons, Sanford~ N, C, Analy~

sis is as follows: Min, crude fat, 4 .0~ mazimum crude fiber, 8,~ ; calciumcarbonate, 1~; defluorinated phosphate, l~; salty ~ of 1~.

Ingredients:

soybean meal ; wheat middlinge ; dehydrated and bone meal ; con-densed fish solubles; fish meal ; dried whey solnblea ; riboflavin ; calciumpantothenate; niacin; vitamin B12 supplement; procaine penicillin; cholinechloride ; HHT (butylated hydrozy toluene) ; a preservative ; vitamin A palmitate;D-activated animal sterol ; animal fat preserved with BHA (butylated hydrozyaui-sole) ; trace amounts of manganese ozide, iron carbonate s copper hydrozide~cobalt carbonate, potassium iodide, and zinc ozide .

Plasma Aorta

~Regirt~en ~ of ,~ ofNo, of Total Desmosterol Total DesmoaterolCockerels Sterol in Sterols Sterol in Sterols

mg~100m1 mg,~gControls onplain mash 11 80 t 13.0* N,D.+ 1 .1 t 0.1 N .D.+

12 .5 mg of tri-paranol~kg 8 120 t 8.7 BO t 1 .0 1 .8 f 0. 37.9 t 2 .0

25 mg of tri-paranol~kg 9 108 t 5.0 76 t 4.0 1 .1 # 67.5

Gross Grading* Microscopic Grading

Aortic Aortic Coronary arteryRegimen

Na.of No, with ~ No, with ~ No, withCockerels Lesions Lesions Lesions

Controls t 11 0 0 0 0 0 012,5 mg of tri-paranol~kg 8 3 37.5 3 37 .5 0 0

25 ag of tri-paranol~kg 9 3 33 .3 5 5b.b 0 0

862

DEPOSITION OF CALCIUM IN AORTA

Vol. 3, No. 8

was increased, there was an increased number of cockerels having calcium de-

posit in the aorta (Table 3) . Control birds had no deposits, while two out

of eight of those treated with 12 .5 mg of triparanol had calcium deposits and

four out of nine of those given the dose at 25.0 mg had medial calcification

of the aorta.

TABLE 3

Deposition of Calcium in Aorta

* Stained by Alizarin Red Method

Histologic sections of the control cockerels revealed no calcium

dsposita in the aorta, even then the tissue was stained ~ Alizarin Red

method (Fig . 4) .

The cockerels treated with 12 .b mg of triparanol, however,

had some calcium deposits in the media of the aortas (Fig . 5) . Birds receiv-

ing 25 mB of the drug showed cosiplete medial calcification of the sorts

(Fig. 8) . No coronary atheroscleroais wan observed in any of the cockerels,

whether or not treated with triparanol .

No. of Calcium Deposits*Regimen

CockerelsNo.

Controls 11 0 0

12.5 mB tri-paranol~kg 8 2 25

25 mg tri-psranol~kg 9 4 44 .4

Vol. 3, No. 8

DEPOSITION OF CALCIUM IN AORTA

863

FIG. 4

Hietologic section of aorta of control cockerel after 20 weeks on the ez-periment~ showing no calcium deposit. Aliaarin Red method. Z 11 .

"orts of cockerel treated with 12 .5

3ectfoa of sorts of cockerel treatedmB of tripsranol for 20 weeks, show-

with 2b AB of trfparsnol for 20ing calcim deposit in media of vessel .

weeks showing an increased calciumAlissria Red method. Z 11,

deposit in tLe sedia. Alfssria Redmethod. Z 11 .

Discussion

From onr atndies, it is evident that triparsnol-treated cockerels had

sn increase in the percentage of plssaa desoosterol, since there was cone

in the controls bnt 80~ sad T8% is birds injected with 12 .b or Eb og of the

884

DEPOSITION OF CALCIUM IN AORTA

Vol . 3, No. 8

drug, respectively. Alao~ there was a corresponding increase in the amount

of deamoaterol in the aortic tissue as the dosage of the drag was increased

from 12 .5 to 25 mg . Data from our studies show that an increase of deamoa-

terol by triparanol sdministration~ which we have noticed previously (B) also

may cause calcium deposits in the aorta of these birda~ as no lesions were

observed is the control on plain mash only.

In additions this drug may cause

other metabolic disturbances mediated through the endocrine system which

caused calcium deposition. Further studies ere in progress to determine the

effects of triparanol administration on calcium metabolism. The group of

cockerels treated with 25 mg of triparsnol had arteriosclerosis resembling

Mönckeberg'a type in that there is calcium deposition in the media of the

aorta (13) .

It has been observed by Avigan and Steinberg (14) that there is

a close similarity between cholesterol and deamoaterol in its chemical, bio-

logical~ and physical properties. These investigators have fed a rabbit as

atherogenic diet for 112 days consisting of 0 .056 de®oaterol~ 0.05 cho-

leaterol and 0 .16 triparanol which caused gross atheroaclerotic lesions in

the aorta. When the grossly normal areas of the aorta ae well as those arena

having lesions were snalyzed~ however the ratio between deamoaterol and cho

lesterol was nearly the same ae in the serum.

This is similar to what we

found in our studies reported previously (7) .

It has also been reported by

Harndon and Siperateia (15) that deamoaterol deposits as easily as does cho

lesterol .

The identification of deamoaterol in serum and tissues of animale

and man treated with triparanol was first reported b9 Avigan et al . (4) .

They reported that the administration of triparanol inhibited the conversion

of deamoaterol to cholesterol . We have no ezplanation at present for the

increase of phospholipids of the triparanol-treated cockerels as compared to

the controls oa plain sash.

Whitby et sl . (5), have recently pnbliahed a sethod for estimating

plasma cholesterol and deamoaterol . Their method for sterol deteraination

was similar to Heany'a procedure (lß) for plasma cholesterol which is a

Vol. 3, No. 8

DEPOSITION OF CALCIUM IN AORTA

865

modification of Bloor'a method (17) . The proposed method for deamosterol was

similar in principle to the procedure of Avigan et al . 1Ye have obtained

similar results on several specimens analysed at random using gas chromatog-

raphy with a Barber-Colman S.E 30 column .

Summe,rv

Subcutsaeous injections of 12.5 or 25.0 mg of tri~ranol~kg body weight

of lOrweek-old cockerels fed plain mash for 20 weeks resulted is the deposition

of calciva in the media of the aortas of some birds, while no deposition was

observed in the controls.

There were no significant changes in the body weight

or level of cholesterol in plasma. The triparaaol-treated cockerels had an

elevation of phoapholipids, however, as compared to the controls on plain mash

only. In birds treated for 20 weeks with 2b.0 mg of triparanol, the total

sterol level in plasma was 106100 ml as compared to 80 mg~100 ml for the

controls.

In the cockerels treated with triparanol, the level of desmoaterol

was significantly elevated.

Triparanol was generously provided by the William S . Aierrell Compaq,

Cincinnati, Ohio. We wish to acknowledge the technical assistance of Sara

Geer and Frances Fsrrow.

Supported by grants HE-02420-07 and HE-08649-01, Public Health Service;

American Heart Association ; and Hunt Foods and Industries Incorporated .

References

1 .

T. R. HLOH6f, T. KARIYA~ and 1f . W . LAUGHL]:N, Arch . Hiochea., 8b, 250 (19b9) .

2 . T. R. HLO®f, and R. D . 1fACJ~TZIE, Arch. Biochem., 85, 24b (19b9) .

3 .

W. HOd.LANDER, and A. CHOBANIAN, B.bi.Q . (Boston üed.Quarterly), 10, 37

(1959) .

4. J . AVIGAN, D. STEIhTHEIfG, H. E . VROIfAN, Lf . J . THO1IpSON, and E. lAOSSETTIG,

J . Hiol . Chea., 235, 3123 (1960) .

b . L . G . WHITHY, R. G. GOLD, E. G . KITCHIiN, and H. A. FLElSIIdG, Hrit. Heart

8.

J., 26, 274 (1984) .

Acknowledgement

H. Y . C . Wong, and F. B. JOHNSON, Circ . Rea .~ >~ 843 (1982) .

866

DEPOSITION OF CALCIUM IN AORTA

Vol. 3, No. 8

7 . H. Y. C. WONG, J . AYIGAN, R . L. RAIFORD~ A. HUTLER, AA~ H. E. VßONeW,

J. Lia Res.,4~ 47? (1983) .

8. W. Iü . SPERRY, and lf . WEBH~ J. Hiol . Chem.~ 187~ 97 (19b0) .

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C. H . FISKE, end Y. 5ITHBAROW~ J. Hiol . Chem., 88, 375 (182b) .

10. J . H . BItA(iDON~ J . Hiol . Chem., 190~ 513 (1961) .

11 . L. N . KATZ~ and J . STALILER~ Fsperimentel Atheroacleroaia p . 138 . Charlea

C. Thom~s, Springfield~ Illinoia (1953) .

12 . 3 . Id . IdCGEE-RiTSSELL~ J. Hiatochem, and C~tochem.~ 8, 22 (1958) .

13. W. A. D. ANDERSON, Patholo~ p. 544 . C . V. hosby~ St . Louis~ bfissouri

(1981) .

14 . J . AVIGAN~ aad D . STEINHERG, Lsncet.~ 1, b72 (1982) .

lb . J . H . HFZiNDON, and 1f . D . SIPPIiSTEIN, Circ. Res ., 12 . 228 (1983) .

18 . A . P . KIIJNY~ Biochem. J., ~ 811 (1952) .

17 . W . R . HLOOR~ 1 Biol . Chem., 29~ 437 (1917) .