Atherosclerosis,
22 (1975) 299-3 11
299
(0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECTS OF PERIMENTAL
DEXTROTHYROXINE ATHEROSCLEROSIS
ON HYPERLIPIDEMIA IN BEAGLE DOGS
AND
EX-
RAJIVA NANDAN, JERRY D. FISHER, EARL P. TOWERY, DAVID R. BROWN, CHARLEXS E. GANOTE AND ROBERT B. JENNINGS Baxter Laboratories, Inc., Morton Grove, IN. 60053, and Department Uaiversity Medical School, Chicago, III. 60611 (U.S.A.)
of Pathology,
Northwestern
(Received October 4th, 1974) (Accepted February 2nd, 1975)
SUMMARY
Beagle dogs, 24 3 6 months old, fed a thiouracil-free semi-synthetic diet containing hydrogenated coconut oil and cholesterol (SS diet) for 12 months, developed marked hyperlipidemia and severe atherosclerosis. SS diet produced a marked elevation of serum cholesterol, triglyceride, phospholipid, and /?-lipoprotein and severe atherosclerosis in large and small arteries. Intimal fatty lesions were always present in the abdominal aorta and many of its branches. Large and small coronary arteries showed similar lesions. The degree of atherosclerosis was directly related to circulating lipid levels. Dextrothyroxine, at dose levels of 0.1 (equivalent to normal human dose) and 0.5 mg/kg
body weight,
produced
a significant
dose related
lowering
of serum
lipids and was associated with a markedly decreased severity of aortic and coronary artery lesions. Untreated control dogs that were maintained on Purina dog meal developed neither hyperlipidemia nor atherosclerosis.
lesion - Cholesterol (total and ester) -Key words : Atherosclerosis - Atherosclerotic Dextrothyroxine (Choloxin) - Free fatty acid - Hematocrit - Hemoglobin - Hypocholesterolemia - Lipid - /3-Lipoprotein - Phospholipid - Planimetq - Triglyceride
INTRODUCTION
The purpose of this study was to test the effects of Dextrothyroxine (D-T4) on hyperlipidemia and experimental atherosclerosis in Beagle dogs. A canine model for experimental atherosclerosis, with reproducible, significant localized lesions was
R. NANDAN
300 developed taining
by feeding
Beagle dogs Malmros’t
thiouracil-free
semisynthetic
hydrogenated coconut oil and cholesterol (SS diet). In 1968, Malmros and Sternbyt reported that hypercholesterolemia
sclerotic
lesions could be produced
within
Steiner
diet conand athero-
a year in dogs fed the SS diet. It was not
necessary to suppress thyroid function with thiouracil if the diet contained and saturated fat in the form of hydrogenated coconut oil. Earlier,
et d.
et al.213demonstrated
the production
cholesterol
of atherosclerotic
lesions
in dogs fed thiouracil and cholesterol, a combination reported to be effective by Bevans et a1.4. The production of experimental canine atherosclerosis has been also reported by Crisp, Gonzalez, Sabiston, DiLuzio, Stephenson, Suzuki, Geer, Schenk et al.5-13. Therapeutic measures to reduce plasma cholesterol concentrations are frequently undertaken to retard the development of atherosclerosis and possibly to cause a mobilization of cholesterol from existing lesions into the plasma. Sodhi et al.14
and Rawitscher
studied
the effect of hypocholesterolemic
agents in man. They reported
duction in plasma cholesterol concentration cholesterol from tissues into plasma. Plasma the bile. MATERIALS
AND
that the re-
was associated with mobilization cholesterol was ultimately excreted
of in
METHODS
Twenty-six beagle dogs, 24 f 6 months old, were obtained from the same source and divided into four groups. Group I consisted of 7 dogs (4 male, 3 female) maintained on a thiouracil-free semisynthetic diet containing hydrogenated coconut oil and cholesterol (SS diet). Group II consisted of 9 dogs (4 male, 5 female) maintained on SS diet plus single daily dose of Dextrothyroxine (D-T& 0.1 mg/kg body weight in tablet form. This dose was equivalent to the quantity usually administered to humans for treatment of hypercholesterolemia. Seven dogs (3 male, 4 female) in group III were given SS diet plus a single daily dose of D-T4, 0.5 mg/kg body weight in tablet form. Three untreated control dogs (1 male, 2 female) in Group IV were fed Purina dog meal. Dogs were housed in individual cages and received diet and water ad libitum. They were weighed monthly. The dogs were maintained for 2 months for pretreatment conditioning. Pretreatment samples of blood were taken from each dog for hematocrit and hemoglobin determinations. Total and ester cholestero115J6, triglycerideIT, phospholipidls, /3-lipoprotein19320 and free fatty acidzl were also measured on pretreatment fasting serum samples. The dogs in each group were then fed and maintained on their respective diets for twelve months. Hematology and serum lipids were determined once every month on blood drawn from each dog after overnight fasting. At the end of 12 months all dogs were anesthetized with sodium pentobarbital and euthanatized by exsanguination. The cranial, thoracic, and abdominal organs were examined grossly, and the major organs were weighed. The heart, aorta, and major arterial branches were removed. The aorta was opened longitudinally and fixed in 10%
& S.E.
& S.E.
31 32 33 57 34 35 36 58 59 Mean
37 38 56 40 41 60 61 Mean
45 47 48
~.~S.E.
rllcl?7beu
M F F
M M M F F F F
M M M M F F F F F
M M M F F F M
Se.u
diet diet diet diet diet diet diet diet
diet diet diet diet diet diet diet diet diet diet
diet diet diet diet diet diet diet diet
untreated untreated untreated
SS SS SS SS SS SS SS SS
SS SS SS SS SS SS SS SS SS SS
SS SS SS SS SS SS SS SS
!
I + -1 + + + +
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
control control control
i + + + $ -I+ f T -:
AND EXPERIMENTAL
1.0 2.0 0.5 0.2 0.0 0.5 0.5 0.0 0.0 0.5 t 0.0 0.0 0.0 0.0 I.0 0.0 0.3 0.2 & 0.1 0.0 0.0 0.0
17.5 28.1 1.6 2.4 0.3 1.4 22.6 0.7 0.4 8.3 -’ 3.7 0.0 0.0 0.0 0.0 13.0 0.0 8.6 3.1 + 5.4 0.0 0.0 0.0
0.2
3.5 3.0 2.0 2.5 2.5 2.3 2 0.3
1.o
1.5
21.7 12.2 71.0 76.3 59.8 69.6 75.6 55.2 zk 10.1
.scale (O-5)
IN BEAGLE
DOGS
0.0 0.0 0.0
0.0 0.0 0.0 17.9 6.5 0.0 3.3 4.0 & 2.5
0.0 2.7 7.9 0.0 16.2 6.7 0.5 0.0 0.0 3.8 + 1.9
49.8 12.8 74.8 54.2 34.0 71.3 44.6 48.8 $ 8.1
involven7rr2t iktima ( “h)
0.0 0.0 0.0
0.0 0.0 0.0 9.1 2.8 0.0 0.7 1.8 :- 1.3
0.0 0.0 0.0 0.0 12.1 0.0 0.0 0.0 0.0 1.3 -:- 1.3
42.6 1.3 84.9 32.7 43.9 68.2 3.5 39.6 :’ 11.7 0.4
0.0 0.0 0.0
0.0 0.0 0.0 1.1 0.0 0.0 0.4 0.2 ‘1 0.2
0.0 0.3 0.4 0.0 0.9 0.1 0.1 0.0 0.0 0.2 1: 0.1
2.7 0.5 4.3 2.2 2.1 3.1 2.5 2.5
i/?volvemr/7t visuul grade media ( O,,) scale (O-5)
Coronary atherosclerosis
ATHEROSCLEROSIS
visual grude
Aortic atherosclerosis
ON HYPERLTPIDEMIA
Treatment D-T4 (tt1glkg)
OF DEXTROTHYROXlNE
1
9 I1 14 21 23 30 54 Mean
Dog
EFFECTS
TABLE
120 212 131
226 198 216 516 1404 176 593
I266 772 392 228 1068 654 1130 313 103
1244 3062 2116
1744 1016 2554 2232
Serum cholesterol i 12 months) (mg/dl)
0
;i g Fz 2 2!
r:
R.
302 neutral
buffered formalin.
The aorta The distribution following (a)
The coronary
et d.
with buffered formalin.
was stained with Sudan IV in acetone and 70 % alcohol (1 :l, v/v). and severity of gross atherosclerotic lesions was estimated by the
two techniques.
The observer
Visual estimation
of the proportions
arteries were perfused
NANDAN
of involvement:
of aortic intimal
in Table 1. (b) Planimetric
had no knowledge
determinations:
Gross
lesions
surface involvement The involved
of the source of the aorta. were graded
on the basis
on a scale of 0 to 5 as shown
surface
of aorta
was drawn
on
transparent paper and the area of involvement was determined by planimetry. Cross sections of thoracic and abdominal aorta were taken at sites of branching as well as in areas showing gross intimal changes, when present, for light and phase contrast microscopy. Aorta specimens for phase microscopy were dissected and fixed in 1 ‘A phosphate buffered glutaraldehyde solution at 4°C for 30 min, rinsed three times in phosphate buffer and postfixed in 1% phosphate buffered osmium tetroxide at 4°C for one hr. Following
dehydration
epoxy resin embedding was carried and stained with toluidine blue and examined
in ascending
concentrations
of ethanol,
l-pm thick sections of the specimen by phase contrast microscopy.
were
Samples were also taken for light and phase contrast microscopy from the right and left coronary arteries of each dog. Six segments of coronary arteries were selected (Fig. 1). These were (1) left common coronary ,(2) proximal circumflex branch (under left atria1 appendage),
(3) proximal
(one mm from origin), (5) ventral the branch) and (6) right coronary
circumflex
obtuse,
(4) ventral
interventricular
interventricular at apical branch (one mm from artery (one mm from origin). Sections for light
Fig. 1. A diagram of the main coronary arteries in beagle dogs (sternocostal aspect). Segments obtained for histological examination are shown as shaded areas: 1 = left common coronary, 2 = circumflex branch, 3 = circumflex obtuse (not visible), 4 = ventral interventricular (one mm from origin), 5 = ventral interventricular at apical branch (one mm from the branch), 6 = right coronary artery (one mm from origin).
OF DOGS FOR THE
0.5 O.b-2.0 9
1.6 0.3-28.1 9
0.0 0.0-l 3.0 7
0.0 0.0 3
SS diet + 0.1 D-T4
SS diet + 0.5 D-T4
Untreated control
* b c d
7
2.5
0.0 0.0 3
0.0 0.0-17.9 7
0.5 0.0-16.2 9
49.8 12.8-74.8 7 0.1 0.0-0.9 9 0.0 0.0-1.1 7 0.0 0.0 3
0.0 O&9.1 7 0.0 0.0 3
2.5 0.554.3 7
0.0 0.0-12.1 9
7
1.3-68.2
42.6
involvement involvement intima” ( O,,) mediaa ( Oh)
0.0 0.0 3
0.0 0.0-29.2 6
0.0 0.0-42.2 9
37.2 5.1-47.5 4
involvement intimaa ( “I)
0.0 0.0 3
0.0 0.0-l 5.2 6
0.0 0.0-53.1 9
27.8 0.0-66.7 4
involvement mediaa ( “$)
0.0 0.0 3
0.0 0.61.3 6
0.0 0.c2.0 9
1.7 0.3-3.0 4
visaalgrade scale O-5”
Athero.&rosi.c in common carotid, popliteal femoral junction, radial-ulnar junction
GROUP
visualgrade scale 0-5a
BY TREATMENT
154 120-212 3
226 176-1404 7
392 103-1266 9
2116 1016-3062 7
Serum cholesterol (12 months)=.
22 (1975) 299-3 11
299
(0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECTS OF PERIMENTAL
DEXTROTHYROXINE ATHEROSCLEROSIS
ON HYPERLIPIDEMIA IN BEAGLE DOGS
AND
EX-
RAJIVA NANDAN, JERRY D. FISHER, EARL P. TOWERY, DAVID R. BROWN, CHARLEXS E. GANOTE AND ROBERT B. JENNINGS Baxter Laboratories, Inc., Morton Grove, IN. 60053, and Department Uaiversity Medical School, Chicago, III. 60611 (U.S.A.)
of Pathology,
Northwestern
(Received October 4th, 1974) (Accepted February 2nd, 1975)
SUMMARY
Beagle dogs, 24 3 6 months old, fed a thiouracil-free semi-synthetic diet containing hydrogenated coconut oil and cholesterol (SS diet) for 12 months, developed marked hyperlipidemia and severe atherosclerosis. SS diet produced a marked elevation of serum cholesterol, triglyceride, phospholipid, and /?-lipoprotein and severe atherosclerosis in large and small arteries. Intimal fatty lesions were always present in the abdominal aorta and many of its branches. Large and small coronary arteries showed similar lesions. The degree of atherosclerosis was directly related to circulating lipid levels. Dextrothyroxine, at dose levels of 0.1 (equivalent to normal human dose) and 0.5 mg/kg
body weight,
produced
a significant
dose related
lowering
of serum
lipids and was associated with a markedly decreased severity of aortic and coronary artery lesions. Untreated control dogs that were maintained on Purina dog meal developed neither hyperlipidemia nor atherosclerosis.
lesion - Cholesterol (total and ester) -Key words : Atherosclerosis - Atherosclerotic Dextrothyroxine (Choloxin) - Free fatty acid - Hematocrit - Hemoglobin - Hypocholesterolemia - Lipid - /3-Lipoprotein - Phospholipid - Planimetq - Triglyceride
INTRODUCTION
The purpose of this study was to test the effects of Dextrothyroxine (D-T4) on hyperlipidemia and experimental atherosclerosis in Beagle dogs. A canine model for experimental atherosclerosis, with reproducible, significant localized lesions was
R. NANDAN
300 developed taining
by feeding
Beagle dogs Malmros’t
thiouracil-free
semisynthetic
hydrogenated coconut oil and cholesterol (SS diet). In 1968, Malmros and Sternbyt reported that hypercholesterolemia
sclerotic
lesions could be produced
within
Steiner
diet conand athero-
a year in dogs fed the SS diet. It was not
necessary to suppress thyroid function with thiouracil if the diet contained and saturated fat in the form of hydrogenated coconut oil. Earlier,
et d.
et al.213demonstrated
the production
cholesterol
of atherosclerotic
lesions
in dogs fed thiouracil and cholesterol, a combination reported to be effective by Bevans et a1.4. The production of experimental canine atherosclerosis has been also reported by Crisp, Gonzalez, Sabiston, DiLuzio, Stephenson, Suzuki, Geer, Schenk et al.5-13. Therapeutic measures to reduce plasma cholesterol concentrations are frequently undertaken to retard the development of atherosclerosis and possibly to cause a mobilization of cholesterol from existing lesions into the plasma. Sodhi et al.14
and Rawitscher
studied
the effect of hypocholesterolemic
agents in man. They reported
duction in plasma cholesterol concentration cholesterol from tissues into plasma. Plasma the bile. MATERIALS
AND
that the re-
was associated with mobilization cholesterol was ultimately excreted
of in
METHODS
Twenty-six beagle dogs, 24 f 6 months old, were obtained from the same source and divided into four groups. Group I consisted of 7 dogs (4 male, 3 female) maintained on a thiouracil-free semisynthetic diet containing hydrogenated coconut oil and cholesterol (SS diet). Group II consisted of 9 dogs (4 male, 5 female) maintained on SS diet plus single daily dose of Dextrothyroxine (D-T& 0.1 mg/kg body weight in tablet form. This dose was equivalent to the quantity usually administered to humans for treatment of hypercholesterolemia. Seven dogs (3 male, 4 female) in group III were given SS diet plus a single daily dose of D-T4, 0.5 mg/kg body weight in tablet form. Three untreated control dogs (1 male, 2 female) in Group IV were fed Purina dog meal. Dogs were housed in individual cages and received diet and water ad libitum. They were weighed monthly. The dogs were maintained for 2 months for pretreatment conditioning. Pretreatment samples of blood were taken from each dog for hematocrit and hemoglobin determinations. Total and ester cholestero115J6, triglycerideIT, phospholipidls, /3-lipoprotein19320 and free fatty acidzl were also measured on pretreatment fasting serum samples. The dogs in each group were then fed and maintained on their respective diets for twelve months. Hematology and serum lipids were determined once every month on blood drawn from each dog after overnight fasting. At the end of 12 months all dogs were anesthetized with sodium pentobarbital and euthanatized by exsanguination. The cranial, thoracic, and abdominal organs were examined grossly, and the major organs were weighed. The heart, aorta, and major arterial branches were removed. The aorta was opened longitudinally and fixed in 10%
& S.E.
& S.E.
31 32 33 57 34 35 36 58 59 Mean
37 38 56 40 41 60 61 Mean
45 47 48
~.~S.E.
rllcl?7beu
M F F
M M M F F F F
M M M M F F F F F
M M M F F F M
Se.u
diet diet diet diet diet diet diet diet
diet diet diet diet diet diet diet diet diet diet
diet diet diet diet diet diet diet diet
untreated untreated untreated
SS SS SS SS SS SS SS SS
SS SS SS SS SS SS SS SS SS SS
SS SS SS SS SS SS SS SS
!
I + -1 + + + +
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
control control control
i + + + $ -I+ f T -:
AND EXPERIMENTAL
1.0 2.0 0.5 0.2 0.0 0.5 0.5 0.0 0.0 0.5 t 0.0 0.0 0.0 0.0 I.0 0.0 0.3 0.2 & 0.1 0.0 0.0 0.0
17.5 28.1 1.6 2.4 0.3 1.4 22.6 0.7 0.4 8.3 -’ 3.7 0.0 0.0 0.0 0.0 13.0 0.0 8.6 3.1 + 5.4 0.0 0.0 0.0
0.2
3.5 3.0 2.0 2.5 2.5 2.3 2 0.3
1.o
1.5
21.7 12.2 71.0 76.3 59.8 69.6 75.6 55.2 zk 10.1
.scale (O-5)
IN BEAGLE
DOGS
0.0 0.0 0.0
0.0 0.0 0.0 17.9 6.5 0.0 3.3 4.0 & 2.5
0.0 2.7 7.9 0.0 16.2 6.7 0.5 0.0 0.0 3.8 + 1.9
49.8 12.8 74.8 54.2 34.0 71.3 44.6 48.8 $ 8.1
involven7rr2t iktima ( “h)
0.0 0.0 0.0
0.0 0.0 0.0 9.1 2.8 0.0 0.7 1.8 :- 1.3
0.0 0.0 0.0 0.0 12.1 0.0 0.0 0.0 0.0 1.3 -:- 1.3
42.6 1.3 84.9 32.7 43.9 68.2 3.5 39.6 :’ 11.7 0.4
0.0 0.0 0.0
0.0 0.0 0.0 1.1 0.0 0.0 0.4 0.2 ‘1 0.2
0.0 0.3 0.4 0.0 0.9 0.1 0.1 0.0 0.0 0.2 1: 0.1
2.7 0.5 4.3 2.2 2.1 3.1 2.5 2.5
i/?volvemr/7t visuul grade media ( O,,) scale (O-5)
Coronary atherosclerosis
ATHEROSCLEROSIS
visual grude
Aortic atherosclerosis
ON HYPERLTPIDEMIA
Treatment D-T4 (tt1glkg)
OF DEXTROTHYROXlNE
1
9 I1 14 21 23 30 54 Mean
Dog
EFFECTS
TABLE
120 212 131
226 198 216 516 1404 176 593
I266 772 392 228 1068 654 1130 313 103
1244 3062 2116
1744 1016 2554 2232
Serum cholesterol i 12 months) (mg/dl)
0
;i g Fz 2 2!
r:
R.
302 neutral
buffered formalin.
The aorta The distribution following (a)
The coronary
et d.
with buffered formalin.
was stained with Sudan IV in acetone and 70 % alcohol (1 :l, v/v). and severity of gross atherosclerotic lesions was estimated by the
two techniques.
The observer
Visual estimation
of the proportions
arteries were perfused
NANDAN
of involvement:
of aortic intimal
in Table 1. (b) Planimetric
had no knowledge
determinations:
Gross
lesions
surface involvement The involved
of the source of the aorta. were graded
on the basis
on a scale of 0 to 5 as shown
surface
of aorta
was drawn
on
transparent paper and the area of involvement was determined by planimetry. Cross sections of thoracic and abdominal aorta were taken at sites of branching as well as in areas showing gross intimal changes, when present, for light and phase contrast microscopy. Aorta specimens for phase microscopy were dissected and fixed in 1 ‘A phosphate buffered glutaraldehyde solution at 4°C for 30 min, rinsed three times in phosphate buffer and postfixed in 1% phosphate buffered osmium tetroxide at 4°C for one hr. Following
dehydration
epoxy resin embedding was carried and stained with toluidine blue and examined
in ascending
concentrations
of ethanol,
l-pm thick sections of the specimen by phase contrast microscopy.
were
Samples were also taken for light and phase contrast microscopy from the right and left coronary arteries of each dog. Six segments of coronary arteries were selected (Fig. 1). These were (1) left common coronary ,(2) proximal circumflex branch (under left atria1 appendage),
(3) proximal
(one mm from origin), (5) ventral the branch) and (6) right coronary
circumflex
obtuse,
(4) ventral
interventricular
interventricular at apical branch (one mm from artery (one mm from origin). Sections for light
Fig. 1. A diagram of the main coronary arteries in beagle dogs (sternocostal aspect). Segments obtained for histological examination are shown as shaded areas: 1 = left common coronary, 2 = circumflex branch, 3 = circumflex obtuse (not visible), 4 = ventral interventricular (one mm from origin), 5 = ventral interventricular at apical branch (one mm from the branch), 6 = right coronary artery (one mm from origin).
OF DOGS FOR THE
0.5 O.b-2.0 9
1.6 0.3-28.1 9
0.0 0.0-l 3.0 7
0.0 0.0 3
SS diet + 0.1 D-T4
SS diet + 0.5 D-T4
Untreated control
* b c d
7
2.5
0.0 0.0 3
0.0 0.0-17.9 7
0.5 0.0-16.2 9
49.8 12.8-74.8 7 0.1 0.0-0.9 9 0.0 0.0-1.1 7 0.0 0.0 3
0.0 O&9.1 7 0.0 0.0 3
2.5 0.554.3 7
0.0 0.0-12.1 9
7
1.3-68.2
42.6
involvement involvement intima” ( O,,) mediaa ( Oh)
0.0 0.0 3
0.0 0.0-29.2 6
0.0 0.0-42.2 9
37.2 5.1-47.5 4
involvement intimaa ( “I)
0.0 0.0 3
0.0 0.0-l 5.2 6
0.0 0.0-53.1 9
27.8 0.0-66.7 4
involvement mediaa ( “$)
0.0 0.0 3
0.0 0.61.3 6
0.0 0.c2.0 9
1.7 0.3-3.0 4
visaalgrade scale O-5”
Athero.&rosi.c in common carotid, popliteal femoral junction, radial-ulnar junction
GROUP
visualgrade scale 0-5a
BY TREATMENT
154 120-212 3
226 176-1404 7
392 103-1266 9
2116 1016-3062 7
Serum cholesterol (12 months)=.
