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Clot-strength and clot-lysis in rats fed thrombogenic diets
EXPERIMENTAL
AND
MOLECULAR
Clot-strength KYU
TAIK
and LEE,
Department
R.
PATHOLOGY
Clot-lysis
FOSTER
(1962)
in Rats
SCOTT,
of Pathology,
151-161
1,
DONC
Albany
Received
Fed
NACK Medical
KIM, College,
January 20,
Thrombogenic AND Albany,
WILBUR New
Diets’ A.
THOMAS
York
1962
INTRODUCTION In previous experiments with rats it has been demonstrated that intra-arterial thrombi and myocardial and renal infarcts can be produced by diets containing among other ingredients butter, cholesterol, bile salts, and propylthiouracil (Thomas and Hartroft, 1959; Thomas et al., 1960). Thrombosis has usually occurred in the absence of significant arteriosclerosis suggesting to us that it results from alterations in clotting (or clot-lysis) factors in the blood. Substitution of corn oil for butter in these diets has resulted in loss of the thrombogenic properties of the diet. Using in vitro studies involving a complex lytic system in a thrombelastograph we have demonstrated that clots formed from plasma of rats fed the above butter-containing thrombogenic diet have a prolonged “lysis time” as compared with clots formed from plasma of rats fed either the corresponding corn oil diet or an ordinary stock diet (Scott et al., 1961; Tillman et al., 1960). Coagulation studies have shown the stypven, one stage prothrombin and two stage serum and plasma prothrombin times to be decreased in rats receiving similar butter-containing thrombogenic diets (Davidson et al., 1961). The pertinence of these experiments for man is not known. However, it seems at least possible that the elucidation of the mechanism of dietary-induced clotting alterations and thrombosis in experimental animals might provide some information that will be useful in the understanding of arterial thrombosis and infarction in man. The prolongation of the clot-lysis times of rats receiving a thrombogenic diet demonstrated in previous experiments could have been the result of: (1) formation of a “stronger” clot, more resistant to lysis, due to physical or chemical alterations in the structure of the clots; or (2) inhibition or other direct changes in the lytic system that is responsible for dissolution of the clot. In the current experiment our purpose has been to obtain some information on the strength of clots formed from plasma of rats on thrombogenic and nonthrombogenie diets. We have used as an index of one aspect of strength, the “binding power” of a clot in a thrombelastograph. In order to place the information on binding power in appropriate perspective we have also carried out studies of clot lysis on the same samples of plasma using techniques somewhat modified from those of previous experiments. 1 This study was supported the National Heart Institute,
by Public Health Bethesda, Maryland.
Service
151
Grants
H-4727,
H-5189
and H-4791
from
152
K. T. LEE,
R. F. SCOTT,
MATERIALS
D. N. KIM,
AND
AND W. A. THOMAS
METHODS
Ninety male Wistar rats were divided into three groups of thirty each, all in the same general weight range. The rats were housed in individual wire-bottomed cages, offered water and the diets listed in Table I ald Zibitum. Individual dietary intake was measuredby the weigh-back method, and the rats were weighed weekly. The “stock” diet in column 1 of Table I contains what we consider to be the normal required TABLE COMPOSITION
Ingredient
BY PER
CENT
WEIGHT
BUTTER,
AND
CORN
OIL
Stock diet
Butter diet
Corn oil diet
2
40
40 -
20
20
6
6
20 6
65.8 2
19.7 2
19.7 2
Corn oil Butter Casein Alphacel Sucrose Vitamin rnix(l Choline chloride Salt mix6 Cholesterol Propylthiouracil Sodium cholate
I
OF STOCK,
-
0.2
1
1
4
4
4
5
5
0.3
0.3
2
2
DIETS
a The vitamin mixture used was choline chloride free Vitamin Diet Fortification Mixture, Nutritional Biochemical Corporation, Cleveland, Ohio. b Wesson modification of Osborne and Wendel Salt Mixture, Nutritional Biochemical Corporation, Cleveland, Ohio.
nourishment for adult rats. The “butter” diet listed in column 2 of Table I contains 40% butter by weight, and when fed to rats in previous experiments has resulted in an inhibition of clot-lysis within less than 30 days, and in a substantial number of renal or myocardial infarcts in 4 or 5 months. The “corn oil” diet listed in column 3 is identical to the butter diet except that 40% corn oil is substituted for 40% butter; this diet has not produced infarcts in rats receiving it for 4 to 5 months. The diets listed in Table I were prepared by mixing them thoroughly in a Hobart food blender, after which individual portions were weighed out in plastic cups and kept in a refrigerator until used. The animals in each of the three groups (stock, butter, corn oil) were numbered consecutively and each rat in each group was paired with a rat in another group. Thus there were thirty sets of rats, each set containing one rat receiving each of the 3 is identical to the butter diet except that 4070 corn oil is substituted for 40% in the set were discarded. Because of anesthetic deaths, three sets of animals were lost to the experiment. After the animals had been on the 3 diets for 30 days they were killed and bled. In preparation for bleeding, each animal was anesthetized with 5 mg of pentobarbital sodium (Abbott) per 100 gm body weight. The peritoneal cavities were opened and blood obtained by aortic puncture using a 21-gauge needle attached to plastic tubing. The blood flowed through the tube in a 15 ml polyethylene test tube containing 0.1 ml of 3.87* sodium citrate solution per 1 ml of blood collected. Immediately after drawing, the blood was centrifuged at 2000 r.p.m. for 10 minutes at 4“ C. The plasma was
CLOT-STRENGTH
AND CLOT-LYSIS IN RATS
153
then drawn off, divided into three portions, placed in siliconed glass tubes and stored in liquid nitrogen at -195.8” C until strength of clot, clot-lysis, and recalcification time measurementswere carried out. If plasma samplesare stored in liquid nitrogen, the lytic enzyme system appears to remain stable (Scott et al., 1961). The animals were killed in sets; i.e., one control, one butter, one corn oil animal were killed together rather than killing all of one dietary group in sequence. The killing of the entire ninety rat experimental group took a period of 2 days. STRENGTH
OF CLOT STUDIES
All studies of clot-strength were carried out with the thrombelastograph which has been described in detail in previous reports (Scott et al.? 1961; Tillman et al., 1960). In brief, plasma mixed with suitable ingredients, which will be described below, is placed in a cup which rotates back and forth constantly through an arc of 4O and 45 seconds.Three samplescan be tested simultaneously in one machine and in this experiment plasma from a control, butter, and corn oil rat were tested at one time; that is, each sample from a set was put in one of the three cups of the thrombelastograph and subjected to the sameprocedures.A pin suspendedby a torsion wire is immersedin the plasma in each cup, and the surface of the plasma is covered with a layer of paraffin oil to prevent drying. As long as the contents are fluid the pin does not move with rotation of the cup. When the plasma filling the space between the pin and cup clots, it clings to the torsion pin and walls of the cuvette! binding them together, and the pin (with torsion wire) begins to rotate with the cup. The degree of rotation of the pin depends upon physical characteristics of the clot such as adhesivenessand viscosity, all of which can be grouped together under the term “binding power.” The greater the binding power of the clot, the greater will be the amount of rotation of the torsion pin suspendedwithin the cuvette. The degree of rotation is reflected by measurement of maximum amplitude on the photographic paper. With lysis of the clot, the pin and torsion wire gradually decrease in degree of rotation, as the viscosity and adhesivenessof the clot decreasesand finally motion stops. A mirror mounted on the torsion wire transmits, by meansof a beam of light, any motion of the wire to a slowly moving roll of photographic paper. The paper moves at a constant rate so that time can be measuredas distance. Only the two extremes of rotation are recorded; all three samplesare recorded on a single roll of paper simultaneously. Overlapping of the records on the paper is easily separated by the eye since each has a different center point. MEASUREMENT
OF BINDING
POWER
Preparation of Ingredients to be Mixed with Plasma Plasma. One set of plasma (stock, butter, corn oil) was taken out of liquid nitrogen and thawed by placing the glasstest tubes in water at 37” until the plasma became liquid. Imid&zzole Bugler. This was prepared at pH 7.2 dissolving 17.2 gm of imidiazole in 8.0 ml of concentrated hydrochloric acid and adding distilled water to make a total of 1OOOml.
154
K. T. LEE,
R. F. SCOTT,
D. N. KIM,
AND
W.
A. THOMAS
Calcium Chloride. Anhydrous calcium chloride was added to 1000 ml of distilled water until a specific gravity of 1.0835 was obtained at 20” C indicating a 10% solution. This 10% solution was then diluted to 1.29% and used. Mixing
of Various Ingredients 0.1 ml plasma (stock, butter or corn oil) 0.2 ml imidiazole buffer
This mixture was placed in the cuvette of the thrombelastograph. After 5 minutes when the mixture had reached 37’, the rotation mechanism of the thrombelastograph was started and 0.06 ml of calcium chloride added. The pin was then immediately lowered into the cuvette and paraffin oil layered over the top of the plasma. Without the addition of agents which promote clot-lysis, rat plasma clots formed in this fashion never lyse completely. We measured the binding power of the clot as reflected by maximum amplitude at 45 minutes. A control, butter and corn oil plasma were run together so that direct comparisons of effect of the three diets on lot strength could be made. CLOT-Lvsrs STUDIES All studies of clot lysis were also carried out with the thrombelastograph. The difference between the clot-lysis studies and the strength-of-clot studies was that in the former an active lytic system was developed in the cuvette together with the plasma and calcium chloride. Preparations of Ingredients to be Mixed with Plasma Streptohinase.” A 0.5 ml vial containing 100,000 units of a purified streptokinase preparation was diluted with 10 ml of imidiazole buffer so that 1 ml of solution contained 10,000 units of streptokinase. Diagnostic Plasma.3 A 0.5 ml vial of lyophilized whole human plasma was diluted with 0.6 ml of distilled water. The plasma, imidiazole buffer, and calcium chloride used in the clot-lysis studies were prepared in an identical manner to the same materials used for the binding power studies. Mixing of Various Ingredients The various ingredients were mixed in a different manner than in previous periments in order to gain better standardization of clot-lysis times. Mix A.
0.5 ml (5000 units) of streptokinase 0.5 ml of diagnostic plasma
This mixture was incubated in a glass test tube for 1 hour at 37’ Mix B.
0.5 ml of Mix A 1.5 ml imidiazole buffer
Mix C.
0.5 ml of rat plasma 0.08 ml of imidiazole buffer 0.08 ml of Mix B
2 The streptokinase 3 Sold by Warner
was made available Chilcott Laboratories,
through Morris
the courtesy Plains, New
of Lederle Jersey.
Laboratories.
ex-
CLOT-STRENGTH
AND
CLOT-LYSIS
IN
155
RATS
As a last step, 0.3 ml of Mix C was placed in the cuvette of the thrombelastograph, and after being allowed to reach 37” C through letting it remain in the cuvette for 5 minutes, the rotating mechanism of the machine was started, including the turning of the paper, and 0.06 ml of calcium chloride solution (1.29%) was added to each cup. Immediately afterward, the pin attached to the torsion wire was lowered into the cup and recording started. Paraffin oil was quickly layered over the top of the plasma to prevent drying. Measurements made on the photographic paper were maximum amplitude and lysis time, the latter defined herein as the time from the first movement of light until the end of movement of the light. This measurement gives an objective recording of physical state of the plasma mixture from fluid to clot to fluid again. Plasma cholesterol was measured on plasma in all surviving rats by the method of Zak (Zak, 1957); each determination was done in duplicate to within 5% accuracy. RESULTS The results are shown in Table II through IV. The animals receiving butter and corn oil diets ate less and lost weight compared with rats receiving the stock diet (Table II). As expected from previous experiments, even in one month the rats fed MEAN
INITIAL
TABLE II AND FINAL WEIGHTS, DAILY INTAKE AND PLASMA CHOLESTEROL OF STOCK, BUTTER, AND CORN OIL GROUPS
Stock Initial Final Daily Daily Plasma
weight (gm) weight (gm) intake (pm) intake (calories) cholesterol (mg%)
LEVELS
diet
Butter diet
Corn oil diet
242 313 21.7 78.3 161
241 214 8.5 44.1 1248
294 266 7.3 37.9 4S6
the butter diet became very lipemic, with a mean cholesterol value of 1248 mg% while the cholesterol values for the corn oil and stock-fed animals were 486 rng?) and 161 rngs respectively (Table II). The differences between cholesterol values of animals fed stock vs butter diet, stock vs corn oil diet, and butter vs corn oil diet were all significant at the 0.001 level using Student’s t test. BINDING
POWER
STUDIES
Using no streptokinase or lyophilized human plasma to promote clot lysis, the binding power (maximum amplitude) measurements and recalcification times of plasma from each animal in the experiment are shown in Table III, and illustrated in Fig. 1. The mean maximum amplitude times for the stock, corn oil, and butter-fed groups were 8.9, 23.2, and 24.0 mm respectively. The differences between the stock and butter group, and between the stock and corn oil group are significant at the 0.001 level; no significant difference in maximum amplitude was found between the butter and corn oil groups. When preincubated streptokinase and lyophilized human plasma were added to the system, thus inducing lysis in the clot, the mean maximum amplitude for the
156
RECALCIFICATION
K. T. LEE, R. F. SCOTT,
D. N. KIM,
W. A. THOMAS
TABLE III TI~VIE AND MAXIMUM AMPLITUDE OF STOCK-, CORN OIL-, WITHOUT ADDITION OF ACTIVE LYTIC SYSTEM Stock
Mean
AND
AND BUTTER-FED
Corn oil
RATS
Butter
Recal. timea (min.)
Max. amp1.b (mm)
Recal. time (min.)
Max. ampl. (mm)
Recal. time (min.)
Max. ampl. (mm)
5 1.0 2.0 3.0 2.0 4.5 2.5 2.5 1.5 2.0 2.5 2.0 2.S 2.5 1.5 4.0 3.0 1.5
20 26 6.5 8.5 10 8 6 6 7 8 4.5 9 6 10 9.5 5 6 5
5 1.5 2.0 3.5 3.0 I.5 2.0 2.5 1.5 3.0 1.0 1 .o 1.5 2.0 1.5 2.5 3.5 1 .o
SO.5 41 21 10 22 24 18 10 14 19 33.5 24 27 22 20.5 17 24 20.5
.S 1 .o 2.5 2.5 4.0 1.5 2.0 1.5 3.0 3.0 1.5 2.0 2.5 2.0 2.5 4.0 3.0 2.5
51.5 41 14 23 23 24 2s 20 19.5 20 21.5 16 19 27 17 26 14 30
2.3
8.9
1.9
23.2
2.3
24.0
Q Recal. time = recalcification time. b Max. ampl. = maximum amplitude.
stock, corn oil, and butter-fed rats were 17.4, 43.3, and 43.6 mm respectively (Table IV and Fig. 2). The differences between the stock and the corn oil and the stock and the butter-fed rats were significant at the 0.001 level; there was no significant difference in the maximum amplitude times of the corn oil and butter-fed rats.
FIG. I. Thrombelastogram without addition of active lytic system. The thrombelastograph stopped at 45 minutes after initiation of recording and maximum amplitude was measured at point. The upper, middle, and lower tracings are from corn oil-, butter-, and stock-fed respectively. The binding power of the clot from the corn oil- and butter-fed rats are similar, both are greater than the binding power of the clot from the stock-fed rat,
was this rats and
TIME
1.0
0.9
.5
1.0
.5 .5 1.5
1.5
1.0
.5 1.0 .5
.5
1.0
21.5
28 25
20.5 23
18.5 25.5 22.5
13 15
20.5 21.5
21.5 18.5 12.5
23.5 20
.5
.5 .5 .5
18
1.5 1 .o
22
and
17.4
10.5
16 13.5
15.5 17
16 9.5
23 11 11.5
19
10
18 27
21
12.5 20
23 13 16
18
30.5 19.5 25
2.0
1.0 .5
1.0
1.0
24 21
23 24
22.5 26
30 26.5
10.5 21
1.0 .5
14 21
Max. ampl.” (mm)
MAXIMUM
1.5
AND
Lysis time (min.)
Stock
LYSIS
Recal. time” (min.)
TOME,
(1 Preincubated lyophilized human plasma b Recal. time = recalcification time. (’ Max. ampl. = maximum amplitude.
Mean
REC.~LCIFICATION
streptokinase
AMPLITUDE
TABLE
IV
See text
36 36
.5 .5 .5
added.
0.6
.5
.5 1 .o 1.0
for
32.1
36.5
36 21 48
41 27
24 37.5 22
.5 .5 .s
26.5 65
.5 .5
details.
43.3
57 47
50.5 28.5
43
51
32
44 45.5 32
59 30
41.5 40
50
38
38 29
41.5 47
29 50.5
51.5
40.5
40
Max. ampl. (mm)
BUTTER-FED
34 24.5 28 23.5 28
.5
AND
42 51
oil
OIL-,
30.5 31
47.5 21
19.5 48
23 27.5
23.5
Lysis time (min.)
Corn
CORN
.5 .5
1.5 1.0
.5 .5 .5
1.0 .5
1.0 1.0
1.0 .5
.5
Recal. time (min.)
OF STOCK-,
RATS
85 40.8
1.0
54 30.5
0.7
.5 1.5 1.0
40 40
30 42 .5
32.5
50
36.5
46.5
40 27.5 39.5
26 3.5
50.5 36
56 51.5
33.5 39.5 32
50.0
25.5 39.5
51.5
Lysis time (min.)
Butter
Ly~1c
.5 .5
ACT~TE
.5
.5 .5
.5
1.0
.5
.5 .5
2.0
.5 .5 .5
.5
1.0 .5
1.0 1.0
.5
1.0
.5
Recal. time (min.)
WITH
S~STEMU
43.6
63.5
42.5 49 30
56.5 40
42 38
35 51
46.5 50
38 31
44 38 43
51.5
45 42.5
38 47 46
50
46
44
28
Max. ampl. (mm)
PRESENT
158
K. T. LEE,
R. F. SCOTT,
D. N. KIM,
CLOT-I.YSIS
AND W. A. THOMAS
STUDIES
The lysis times for each stock, butter and corn oil diet animal are shown in Table IV; the mean lysis times of the stock, butter, and corn oil diet groups were 21.5, 40.8, and 31.2 minutes respectively. The difference in lysis times between the stock and butter group was significant at the 0.001 level; the difference between the butter and corn oil group was significant at the 0.01 level.
Fro. 2. Thrombelastogram showing the simultaneous recording, starting with the solid black line at the left. The distance from the start to the point where the solid black lines separate represents the recalcification time. Maximum amplitude is measured at the point of widest separation of the lines. Lysis time is measured as the distance from the initial separation of the lines to the point at which they rejoin as a single line. The upper, middle, and lower tracings are from butter-, corn oil-, and stock-fed rats respectively. The maximum amplitude (binding power) of clots from the butterand corn oil-fed rats is similar, but the clot&is times are different. RECALCIFICATION
TIMES
In the absence of an active lytic system, mean recalcification times for the stock, corn oil, and butter-fed groups were 2.3, 1.9, and 2.3 minutes respectively (Table III and Fig. 1) . When an active lytic system was present, the mean recalcification times for the stock, corn oil, and butter-fed groups were 0.9, 0.6, and 0.7 minutes respectively. No significant differences in recalcification times in the three groups of animals with or without the presence of an active lytic system, could be demonstrated. DISCUSSION Rats receiving a diet containing, among other important ingredients, 40% butter will develop intra-arterial thrombi and myocardial and renal infarcts in 4 or .5 months. This same diet will result in a prolonged clot-lysis time in rats within 30 days. If corn oil is substituted for butter in these diets with other ingredients such as cholesterol and propylthiouracil unchanged, no infarcts are produced, and the clot-lysis time is significantly shorter than in the butter-fed rats. The prolonged clotlysis time in the butter-fed rats could have been produced by the formation of a stronger clot, by alterations of the Iytic system, or by a combination of both factors.
CLOT-STRENGTH
AND
CLOT-LYSIS
IN
RATS
159
In an attempt to elucidate the mechanism responsible for the prolonged clot-lysis times in butter-fed rats, in this experiment we have compared one aspect of clotstrength (binding power in a thrombelastograph) and clot-lysis times in groups of animals receiving a 40% butter, a 40% corn oil (each also containing cholesterol and other ingredients), and a control or stock diet. The 40% butter and 405% corn oil diets after 30 days produced clots of equal strength, both of which had much greater binding power than the clots produced by the stock diet. The results were similar, whether an active lytic system was present or not when the binding power measurements were made. Binding power most probably reflects either the viscosity* or the adhesiveness (or both) of a clot. The fact that a clot has greater binding power than another suggests that it is stronger and hence might be more resistant to lysis by plasmin. While it appears that the 40% butter and 40% corn oil diets both greatly increase the binding power of a clot, compared with a low fat stock diet, the mechanism of action of the butter and corn oil diets leading to increased clot-strength is a subject which requires further investigation. One possibility is that the polymer structure of the fibrin clot is changed by some constituents of these diets and the different molecular structure of fibrin accounts for its different viscosity or adhesiveness. Another possibility is that the molecular structure of the fibrin is unaffected, but that some component of the plasma associated with the clot produces an adhesive film which binds the clot to surrounding structures. The fact that the greater binding power of clots from butter and corn oil-fed rats compared to clots of stock-fed rats was demonstrable in the presence or absence of a lytic system suggests that this index of clot-strength is at least partially independent of clot-lysis. The length of time a clot will resist lysis depends on the nature of the clot itself (how resistant it is to lytic agents) and the effectiveness of the lytic system acting on the fibrin of the clot. Clots formed from the plasma of corn oil- and butter-fed rats appear to have equal binding power and possibly equal resistance to plasmin lysis, but the clots of butter-fed rats have a significantly longer clot-lysis time than do those of corn oil-fed rats. This suggests as one possibility that the lytic systems in corn oil- and butter-fed rats are different from each other and are presumably influenced by the diets fed to the two groups of rats. The nature of the alteration in the lytic system of corn oil- and butter-fed rats is not apparent from this experiment, but is at present being investigated using a casein-lysis method. Also the binding power of a clot is only one measurement of clot-strength. In order to fully evaluate the possible importance of this factor, other methods of measuring the strength of clots in rats fed butter, corn oil, and stock diets should be investigated. It would be of interest to know if the urea solubility and tensile strength of the different types of clot varied. If these further measurements of clot strength show that there is no difference in the strength of clots formed from the plasma of butter- and corn oil-fed rats, it would suggest that the differing thrombogenic properties of butter and corn oil diets in rats is due to alterations of the lytic system and not simply to changes in clot structure produced by these two fats. 4 Viscosity of a substance has been defined as the tangential force per unit horizontal planes at unit distance apart, one of which is fixed while the other velocity, the space being filled with the substance (Hodgman, 1958).
of either of two moves with unit
160
K. T. LEE, R. F. SCOTT,
D. N. KIM,
AND W. A. THOMAS
In addition to the fact that the clots of corn oil- and butter-fed rats may be more resistant to clot-lysis, other characteristics of these clots suggested by their increased binding power may be of importance. If clots formed in viva in corn oil- and butterfed rats are more viscous than those formed from stock animals, they may more effectively resist breakup by the physical action of the flow of blood within the coronary and renal arteries of the rat. It is also possible that increased adhesiveness may enable them to cling to vessel walls more tenaciously, thus preventing them from being swept along and broken up as readily as the thrombi in stock-fed rats. Each of these properties if present would seem to enhance the development of myocardial and renal infarcts. Whether or not these findings of the similar effect of butter and corn oil on clotstrength in the rat are applicable to man is not known. We believe that investigation of clot-strength in humans (in addition to measurement of clot-lysis time) following ingestion of various fatty meals might be of great value in elucidating the possible relationship of dietary fats and arterial thrombosis. Although reports are conflicting (Hougie and Ayers, 1960; Bronte-Stewart, 1961), some studies have suggested that dietary fats in humans will alter the clotting mechanism (Buckell and Elliott, 1959; Greig, 1956). It would be of interest to know that if such alterations do occur they are accompanied by physico-chemical changes resulting in increased clot-strength in humans. SUMMARY Butter appears to have a different biologic effect than corn oil regarding the development of intra-arterial thrombi and infarcts in rats. Rats receiving a diet containing 40% butter (plus cholesterol, propylthiouracil, and other ingredients) develop thrombi with myocardial and renal infarcts, together with an increased clot-lysis time. If corn oil is substituted for butter no thrombi or infarcts have been found and the clot-lysis times are shorter than that of the butter-fed animals. In order to elucidate the mechanism of the thrombogenic action of butter when fed to rats, the comparative strengths of clots formed from the plasma of rats fed butter or corn oil (plus other ingredients) or a stock diet, were measured using binding power in a thrombelastograph as one index of clot-strength. In addition, clot-lysis and plasma cholesterol determinations were done on all groups. The binding powers of the clots formed from the plasma of butterand corn oil-fed rats were not significantly different, but both fats produced clots of far greater binding power than did the low fat, stock diet. The clot-lysis times of the same butter-fed group of rats was significantly greater than that of the corn oil group. Both fat-fed groups had a longer clot-lysis time than did the stock diet group of rats. The finding of equal binding powers but different clot-lysis times in corn oil- and butter-fed rats suggests that the thrombogenic property of the 40’3% butter diet is due either to alterations in the lytic system secondary to the butter diet, or to aspects of clotstrength not reflected by binding power. REFERENCES B. (1961). Lipids and atherosclerosis. Federation Proc. 20, 127. M., and ELLIOTT, F. A. (1959). Effect of butter lipemia on the rate of clot lysis in normal males. Lancet i, 662. DAVIDSON, E., HOWARD, A. N., and GRESHAM, G. A. (1961). Blood coagulation studies in rats given diets which produce thrombosis or atherosclerosis. Brit. J. Exptl. Pathol. 42, 195. GREW, H. B. W. (1956). Inhibition of fibrinolysis by alimentary lipemia. Lancet ii, 16. HODGMAN, C. D. (1958). “Handbook of Chemistry and Physics.” 2154 p. Chemical Rubber Publishing Co., Cleveland, Ohio. HOUGIE, E., and AYERS, F. (1960). Lipemia and fibrinolytic potentiality. Lancet ii, 186. BRONTE~TEWART, BUCKELL,
CLOT-STRENGTH
AND
CLOT-LYSIS
IN
RATS
161
R. F., ALOUSI, K., and THOMAS, W. A. (1961). Chemico-pathologic studies of dietary lipids, thrombosis, and clot-lysis I: In vitro methods, utilizing liquid nitrogen refrigeration and the thrombelastograph for demonstrating with rats the antilytic effect of butter-containing atherothrombogenic diets. Arch. Pathol. 71, 594. THOMAS, W. A., and HARTROFT, W. S. (1959). Myocardial infarcts in rats fed diets containing high fat, cholesterol, thiouracil and sodium cholate. Circulation 19, 65. THOMAS, W. A., O’NEAL, R. M., and HARTROFT, W. S. (1960). Modification of diets responsible for induction of coronary thrombosis and myocardial infarction in rats. J. Nlctrition 69, 325. TILLMAN, R. L., O’NEAL, R. M., THOMAS, W. A., and HIXON, B. B. (1960). Butter, corn oil and fibrinolysis in rats. Circ&!ion Research 8, 423. ZAK, B. (1957). A simple rapid technique for serum total cholesterol, Anz. J. Clin. Pathol. 27, 583. SCOTT,
AND
MOLECULAR
Clot-strength KYU
TAIK
and LEE,
Department
R.
PATHOLOGY
Clot-lysis
FOSTER
(1962)
in Rats
SCOTT,
of Pathology,
151-161
1,
DONC
Albany
Received
Fed
NACK Medical
KIM, College,
January 20,
Thrombogenic AND Albany,
WILBUR New
Diets’ A.
THOMAS
York
1962
INTRODUCTION In previous experiments with rats it has been demonstrated that intra-arterial thrombi and myocardial and renal infarcts can be produced by diets containing among other ingredients butter, cholesterol, bile salts, and propylthiouracil (Thomas and Hartroft, 1959; Thomas et al., 1960). Thrombosis has usually occurred in the absence of significant arteriosclerosis suggesting to us that it results from alterations in clotting (or clot-lysis) factors in the blood. Substitution of corn oil for butter in these diets has resulted in loss of the thrombogenic properties of the diet. Using in vitro studies involving a complex lytic system in a thrombelastograph we have demonstrated that clots formed from plasma of rats fed the above butter-containing thrombogenic diet have a prolonged “lysis time” as compared with clots formed from plasma of rats fed either the corresponding corn oil diet or an ordinary stock diet (Scott et al., 1961; Tillman et al., 1960). Coagulation studies have shown the stypven, one stage prothrombin and two stage serum and plasma prothrombin times to be decreased in rats receiving similar butter-containing thrombogenic diets (Davidson et al., 1961). The pertinence of these experiments for man is not known. However, it seems at least possible that the elucidation of the mechanism of dietary-induced clotting alterations and thrombosis in experimental animals might provide some information that will be useful in the understanding of arterial thrombosis and infarction in man. The prolongation of the clot-lysis times of rats receiving a thrombogenic diet demonstrated in previous experiments could have been the result of: (1) formation of a “stronger” clot, more resistant to lysis, due to physical or chemical alterations in the structure of the clots; or (2) inhibition or other direct changes in the lytic system that is responsible for dissolution of the clot. In the current experiment our purpose has been to obtain some information on the strength of clots formed from plasma of rats on thrombogenic and nonthrombogenie diets. We have used as an index of one aspect of strength, the “binding power” of a clot in a thrombelastograph. In order to place the information on binding power in appropriate perspective we have also carried out studies of clot lysis on the same samples of plasma using techniques somewhat modified from those of previous experiments. 1 This study was supported the National Heart Institute,
by Public Health Bethesda, Maryland.
Service
151
Grants
H-4727,
H-5189
and H-4791
from
152
K. T. LEE,
R. F. SCOTT,
MATERIALS
D. N. KIM,
AND
AND W. A. THOMAS
METHODS
Ninety male Wistar rats were divided into three groups of thirty each, all in the same general weight range. The rats were housed in individual wire-bottomed cages, offered water and the diets listed in Table I ald Zibitum. Individual dietary intake was measuredby the weigh-back method, and the rats were weighed weekly. The “stock” diet in column 1 of Table I contains what we consider to be the normal required TABLE COMPOSITION
Ingredient
BY PER
CENT
WEIGHT
BUTTER,
AND
CORN
OIL
Stock diet
Butter diet
Corn oil diet
2
40
40 -
20
20
6
6
20 6
65.8 2
19.7 2
19.7 2
Corn oil Butter Casein Alphacel Sucrose Vitamin rnix(l Choline chloride Salt mix6 Cholesterol Propylthiouracil Sodium cholate
I
OF STOCK,
-
0.2
1
1
4
4
4
5
5
0.3
0.3
2
2
DIETS
a The vitamin mixture used was choline chloride free Vitamin Diet Fortification Mixture, Nutritional Biochemical Corporation, Cleveland, Ohio. b Wesson modification of Osborne and Wendel Salt Mixture, Nutritional Biochemical Corporation, Cleveland, Ohio.
nourishment for adult rats. The “butter” diet listed in column 2 of Table I contains 40% butter by weight, and when fed to rats in previous experiments has resulted in an inhibition of clot-lysis within less than 30 days, and in a substantial number of renal or myocardial infarcts in 4 or 5 months. The “corn oil” diet listed in column 3 is identical to the butter diet except that 40% corn oil is substituted for 40% butter; this diet has not produced infarcts in rats receiving it for 4 to 5 months. The diets listed in Table I were prepared by mixing them thoroughly in a Hobart food blender, after which individual portions were weighed out in plastic cups and kept in a refrigerator until used. The animals in each of the three groups (stock, butter, corn oil) were numbered consecutively and each rat in each group was paired with a rat in another group. Thus there were thirty sets of rats, each set containing one rat receiving each of the 3 is identical to the butter diet except that 4070 corn oil is substituted for 40% in the set were discarded. Because of anesthetic deaths, three sets of animals were lost to the experiment. After the animals had been on the 3 diets for 30 days they were killed and bled. In preparation for bleeding, each animal was anesthetized with 5 mg of pentobarbital sodium (Abbott) per 100 gm body weight. The peritoneal cavities were opened and blood obtained by aortic puncture using a 21-gauge needle attached to plastic tubing. The blood flowed through the tube in a 15 ml polyethylene test tube containing 0.1 ml of 3.87* sodium citrate solution per 1 ml of blood collected. Immediately after drawing, the blood was centrifuged at 2000 r.p.m. for 10 minutes at 4“ C. The plasma was
CLOT-STRENGTH
AND CLOT-LYSIS IN RATS
153
then drawn off, divided into three portions, placed in siliconed glass tubes and stored in liquid nitrogen at -195.8” C until strength of clot, clot-lysis, and recalcification time measurementswere carried out. If plasma samplesare stored in liquid nitrogen, the lytic enzyme system appears to remain stable (Scott et al., 1961). The animals were killed in sets; i.e., one control, one butter, one corn oil animal were killed together rather than killing all of one dietary group in sequence. The killing of the entire ninety rat experimental group took a period of 2 days. STRENGTH
OF CLOT STUDIES
All studies of clot-strength were carried out with the thrombelastograph which has been described in detail in previous reports (Scott et al.? 1961; Tillman et al., 1960). In brief, plasma mixed with suitable ingredients, which will be described below, is placed in a cup which rotates back and forth constantly through an arc of 4O and 45 seconds.Three samplescan be tested simultaneously in one machine and in this experiment plasma from a control, butter, and corn oil rat were tested at one time; that is, each sample from a set was put in one of the three cups of the thrombelastograph and subjected to the sameprocedures.A pin suspendedby a torsion wire is immersedin the plasma in each cup, and the surface of the plasma is covered with a layer of paraffin oil to prevent drying. As long as the contents are fluid the pin does not move with rotation of the cup. When the plasma filling the space between the pin and cup clots, it clings to the torsion pin and walls of the cuvette! binding them together, and the pin (with torsion wire) begins to rotate with the cup. The degree of rotation of the pin depends upon physical characteristics of the clot such as adhesivenessand viscosity, all of which can be grouped together under the term “binding power.” The greater the binding power of the clot, the greater will be the amount of rotation of the torsion pin suspendedwithin the cuvette. The degree of rotation is reflected by measurement of maximum amplitude on the photographic paper. With lysis of the clot, the pin and torsion wire gradually decrease in degree of rotation, as the viscosity and adhesivenessof the clot decreasesand finally motion stops. A mirror mounted on the torsion wire transmits, by meansof a beam of light, any motion of the wire to a slowly moving roll of photographic paper. The paper moves at a constant rate so that time can be measuredas distance. Only the two extremes of rotation are recorded; all three samplesare recorded on a single roll of paper simultaneously. Overlapping of the records on the paper is easily separated by the eye since each has a different center point. MEASUREMENT
OF BINDING
POWER
Preparation of Ingredients to be Mixed with Plasma Plasma. One set of plasma (stock, butter, corn oil) was taken out of liquid nitrogen and thawed by placing the glasstest tubes in water at 37” until the plasma became liquid. Imid&zzole Bugler. This was prepared at pH 7.2 dissolving 17.2 gm of imidiazole in 8.0 ml of concentrated hydrochloric acid and adding distilled water to make a total of 1OOOml.
154
K. T. LEE,
R. F. SCOTT,
D. N. KIM,
AND
W.
A. THOMAS
Calcium Chloride. Anhydrous calcium chloride was added to 1000 ml of distilled water until a specific gravity of 1.0835 was obtained at 20” C indicating a 10% solution. This 10% solution was then diluted to 1.29% and used. Mixing
of Various Ingredients 0.1 ml plasma (stock, butter or corn oil) 0.2 ml imidiazole buffer
This mixture was placed in the cuvette of the thrombelastograph. After 5 minutes when the mixture had reached 37’, the rotation mechanism of the thrombelastograph was started and 0.06 ml of calcium chloride added. The pin was then immediately lowered into the cuvette and paraffin oil layered over the top of the plasma. Without the addition of agents which promote clot-lysis, rat plasma clots formed in this fashion never lyse completely. We measured the binding power of the clot as reflected by maximum amplitude at 45 minutes. A control, butter and corn oil plasma were run together so that direct comparisons of effect of the three diets on lot strength could be made. CLOT-Lvsrs STUDIES All studies of clot lysis were also carried out with the thrombelastograph. The difference between the clot-lysis studies and the strength-of-clot studies was that in the former an active lytic system was developed in the cuvette together with the plasma and calcium chloride. Preparations of Ingredients to be Mixed with Plasma Streptohinase.” A 0.5 ml vial containing 100,000 units of a purified streptokinase preparation was diluted with 10 ml of imidiazole buffer so that 1 ml of solution contained 10,000 units of streptokinase. Diagnostic Plasma.3 A 0.5 ml vial of lyophilized whole human plasma was diluted with 0.6 ml of distilled water. The plasma, imidiazole buffer, and calcium chloride used in the clot-lysis studies were prepared in an identical manner to the same materials used for the binding power studies. Mixing of Various Ingredients The various ingredients were mixed in a different manner than in previous periments in order to gain better standardization of clot-lysis times. Mix A.
0.5 ml (5000 units) of streptokinase 0.5 ml of diagnostic plasma
This mixture was incubated in a glass test tube for 1 hour at 37’ Mix B.
0.5 ml of Mix A 1.5 ml imidiazole buffer
Mix C.
0.5 ml of rat plasma 0.08 ml of imidiazole buffer 0.08 ml of Mix B
2 The streptokinase 3 Sold by Warner
was made available Chilcott Laboratories,
through Morris
the courtesy Plains, New
of Lederle Jersey.
Laboratories.
ex-
CLOT-STRENGTH
AND
CLOT-LYSIS
IN
155
RATS
As a last step, 0.3 ml of Mix C was placed in the cuvette of the thrombelastograph, and after being allowed to reach 37” C through letting it remain in the cuvette for 5 minutes, the rotating mechanism of the machine was started, including the turning of the paper, and 0.06 ml of calcium chloride solution (1.29%) was added to each cup. Immediately afterward, the pin attached to the torsion wire was lowered into the cup and recording started. Paraffin oil was quickly layered over the top of the plasma to prevent drying. Measurements made on the photographic paper were maximum amplitude and lysis time, the latter defined herein as the time from the first movement of light until the end of movement of the light. This measurement gives an objective recording of physical state of the plasma mixture from fluid to clot to fluid again. Plasma cholesterol was measured on plasma in all surviving rats by the method of Zak (Zak, 1957); each determination was done in duplicate to within 5% accuracy. RESULTS The results are shown in Table II through IV. The animals receiving butter and corn oil diets ate less and lost weight compared with rats receiving the stock diet (Table II). As expected from previous experiments, even in one month the rats fed MEAN
INITIAL
TABLE II AND FINAL WEIGHTS, DAILY INTAKE AND PLASMA CHOLESTEROL OF STOCK, BUTTER, AND CORN OIL GROUPS
Stock Initial Final Daily Daily Plasma
weight (gm) weight (gm) intake (pm) intake (calories) cholesterol (mg%)
LEVELS
diet
Butter diet
Corn oil diet
242 313 21.7 78.3 161
241 214 8.5 44.1 1248
294 266 7.3 37.9 4S6
the butter diet became very lipemic, with a mean cholesterol value of 1248 mg% while the cholesterol values for the corn oil and stock-fed animals were 486 rng?) and 161 rngs respectively (Table II). The differences between cholesterol values of animals fed stock vs butter diet, stock vs corn oil diet, and butter vs corn oil diet were all significant at the 0.001 level using Student’s t test. BINDING
POWER
STUDIES
Using no streptokinase or lyophilized human plasma to promote clot lysis, the binding power (maximum amplitude) measurements and recalcification times of plasma from each animal in the experiment are shown in Table III, and illustrated in Fig. 1. The mean maximum amplitude times for the stock, corn oil, and butter-fed groups were 8.9, 23.2, and 24.0 mm respectively. The differences between the stock and butter group, and between the stock and corn oil group are significant at the 0.001 level; no significant difference in maximum amplitude was found between the butter and corn oil groups. When preincubated streptokinase and lyophilized human plasma were added to the system, thus inducing lysis in the clot, the mean maximum amplitude for the
156
RECALCIFICATION
K. T. LEE, R. F. SCOTT,
D. N. KIM,
W. A. THOMAS
TABLE III TI~VIE AND MAXIMUM AMPLITUDE OF STOCK-, CORN OIL-, WITHOUT ADDITION OF ACTIVE LYTIC SYSTEM Stock
Mean
AND
AND BUTTER-FED
Corn oil
RATS
Butter
Recal. timea (min.)
Max. amp1.b (mm)
Recal. time (min.)
Max. ampl. (mm)
Recal. time (min.)
Max. ampl. (mm)
5 1.0 2.0 3.0 2.0 4.5 2.5 2.5 1.5 2.0 2.5 2.0 2.S 2.5 1.5 4.0 3.0 1.5
20 26 6.5 8.5 10 8 6 6 7 8 4.5 9 6 10 9.5 5 6 5
5 1.5 2.0 3.5 3.0 I.5 2.0 2.5 1.5 3.0 1.0 1 .o 1.5 2.0 1.5 2.5 3.5 1 .o
SO.5 41 21 10 22 24 18 10 14 19 33.5 24 27 22 20.5 17 24 20.5
.S 1 .o 2.5 2.5 4.0 1.5 2.0 1.5 3.0 3.0 1.5 2.0 2.5 2.0 2.5 4.0 3.0 2.5
51.5 41 14 23 23 24 2s 20 19.5 20 21.5 16 19 27 17 26 14 30
2.3
8.9
1.9
23.2
2.3
24.0
Q Recal. time = recalcification time. b Max. ampl. = maximum amplitude.
stock, corn oil, and butter-fed rats were 17.4, 43.3, and 43.6 mm respectively (Table IV and Fig. 2). The differences between the stock and the corn oil and the stock and the butter-fed rats were significant at the 0.001 level; there was no significant difference in the maximum amplitude times of the corn oil and butter-fed rats.
FIG. I. Thrombelastogram without addition of active lytic system. The thrombelastograph stopped at 45 minutes after initiation of recording and maximum amplitude was measured at point. The upper, middle, and lower tracings are from corn oil-, butter-, and stock-fed respectively. The binding power of the clot from the corn oil- and butter-fed rats are similar, both are greater than the binding power of the clot from the stock-fed rat,
was this rats and
TIME
1.0
0.9
.5
1.0
.5 .5 1.5
1.5
1.0
.5 1.0 .5
.5
1.0
21.5
28 25
20.5 23
18.5 25.5 22.5
13 15
20.5 21.5
21.5 18.5 12.5
23.5 20
.5
.5 .5 .5
18
1.5 1 .o
22
and
17.4
10.5
16 13.5
15.5 17
16 9.5
23 11 11.5
19
10
18 27
21
12.5 20
23 13 16
18
30.5 19.5 25
2.0
1.0 .5
1.0
1.0
24 21
23 24
22.5 26
30 26.5
10.5 21
1.0 .5
14 21
Max. ampl.” (mm)
MAXIMUM
1.5
AND
Lysis time (min.)
Stock
LYSIS
Recal. time” (min.)
TOME,
(1 Preincubated lyophilized human plasma b Recal. time = recalcification time. (’ Max. ampl. = maximum amplitude.
Mean
REC.~LCIFICATION
streptokinase
AMPLITUDE
TABLE
IV
See text
36 36
.5 .5 .5
added.
0.6
.5
.5 1 .o 1.0
for
32.1
36.5
36 21 48
41 27
24 37.5 22
.5 .5 .s
26.5 65
.5 .5
details.
43.3
57 47
50.5 28.5
43
51
32
44 45.5 32
59 30
41.5 40
50
38
38 29
41.5 47
29 50.5
51.5
40.5
40
Max. ampl. (mm)
BUTTER-FED
34 24.5 28 23.5 28
.5
AND
42 51
oil
OIL-,
30.5 31
47.5 21
19.5 48
23 27.5
23.5
Lysis time (min.)
Corn
CORN
.5 .5
1.5 1.0
.5 .5 .5
1.0 .5
1.0 1.0
1.0 .5
.5
Recal. time (min.)
OF STOCK-,
RATS
85 40.8
1.0
54 30.5
0.7
.5 1.5 1.0
40 40
30 42 .5
32.5
50
36.5
46.5
40 27.5 39.5
26 3.5
50.5 36
56 51.5
33.5 39.5 32
50.0
25.5 39.5
51.5
Lysis time (min.)
Butter
Ly~1c
.5 .5
ACT~TE
.5
.5 .5
.5
1.0
.5
.5 .5
2.0
.5 .5 .5
.5
1.0 .5
1.0 1.0
.5
1.0
.5
Recal. time (min.)
WITH
S~STEMU
43.6
63.5
42.5 49 30
56.5 40
42 38
35 51
46.5 50
38 31
44 38 43
51.5
45 42.5
38 47 46
50
46
44
28
Max. ampl. (mm)
PRESENT
158
K. T. LEE,
R. F. SCOTT,
D. N. KIM,
CLOT-I.YSIS
AND W. A. THOMAS
STUDIES
The lysis times for each stock, butter and corn oil diet animal are shown in Table IV; the mean lysis times of the stock, butter, and corn oil diet groups were 21.5, 40.8, and 31.2 minutes respectively. The difference in lysis times between the stock and butter group was significant at the 0.001 level; the difference between the butter and corn oil group was significant at the 0.01 level.
Fro. 2. Thrombelastogram showing the simultaneous recording, starting with the solid black line at the left. The distance from the start to the point where the solid black lines separate represents the recalcification time. Maximum amplitude is measured at the point of widest separation of the lines. Lysis time is measured as the distance from the initial separation of the lines to the point at which they rejoin as a single line. The upper, middle, and lower tracings are from butter-, corn oil-, and stock-fed rats respectively. The maximum amplitude (binding power) of clots from the butterand corn oil-fed rats is similar, but the clot&is times are different. RECALCIFICATION
TIMES
In the absence of an active lytic system, mean recalcification times for the stock, corn oil, and butter-fed groups were 2.3, 1.9, and 2.3 minutes respectively (Table III and Fig. 1) . When an active lytic system was present, the mean recalcification times for the stock, corn oil, and butter-fed groups were 0.9, 0.6, and 0.7 minutes respectively. No significant differences in recalcification times in the three groups of animals with or without the presence of an active lytic system, could be demonstrated. DISCUSSION Rats receiving a diet containing, among other important ingredients, 40% butter will develop intra-arterial thrombi and myocardial and renal infarcts in 4 or .5 months. This same diet will result in a prolonged clot-lysis time in rats within 30 days. If corn oil is substituted for butter in these diets with other ingredients such as cholesterol and propylthiouracil unchanged, no infarcts are produced, and the clot-lysis time is significantly shorter than in the butter-fed rats. The prolonged clotlysis time in the butter-fed rats could have been produced by the formation of a stronger clot, by alterations of the Iytic system, or by a combination of both factors.
CLOT-STRENGTH
AND
CLOT-LYSIS
IN
RATS
159
In an attempt to elucidate the mechanism responsible for the prolonged clot-lysis times in butter-fed rats, in this experiment we have compared one aspect of clotstrength (binding power in a thrombelastograph) and clot-lysis times in groups of animals receiving a 40% butter, a 40% corn oil (each also containing cholesterol and other ingredients), and a control or stock diet. The 40% butter and 405% corn oil diets after 30 days produced clots of equal strength, both of which had much greater binding power than the clots produced by the stock diet. The results were similar, whether an active lytic system was present or not when the binding power measurements were made. Binding power most probably reflects either the viscosity* or the adhesiveness (or both) of a clot. The fact that a clot has greater binding power than another suggests that it is stronger and hence might be more resistant to lysis by plasmin. While it appears that the 40% butter and 40% corn oil diets both greatly increase the binding power of a clot, compared with a low fat stock diet, the mechanism of action of the butter and corn oil diets leading to increased clot-strength is a subject which requires further investigation. One possibility is that the polymer structure of the fibrin clot is changed by some constituents of these diets and the different molecular structure of fibrin accounts for its different viscosity or adhesiveness. Another possibility is that the molecular structure of the fibrin is unaffected, but that some component of the plasma associated with the clot produces an adhesive film which binds the clot to surrounding structures. The fact that the greater binding power of clots from butter and corn oil-fed rats compared to clots of stock-fed rats was demonstrable in the presence or absence of a lytic system suggests that this index of clot-strength is at least partially independent of clot-lysis. The length of time a clot will resist lysis depends on the nature of the clot itself (how resistant it is to lytic agents) and the effectiveness of the lytic system acting on the fibrin of the clot. Clots formed from the plasma of corn oil- and butter-fed rats appear to have equal binding power and possibly equal resistance to plasmin lysis, but the clots of butter-fed rats have a significantly longer clot-lysis time than do those of corn oil-fed rats. This suggests as one possibility that the lytic systems in corn oil- and butter-fed rats are different from each other and are presumably influenced by the diets fed to the two groups of rats. The nature of the alteration in the lytic system of corn oil- and butter-fed rats is not apparent from this experiment, but is at present being investigated using a casein-lysis method. Also the binding power of a clot is only one measurement of clot-strength. In order to fully evaluate the possible importance of this factor, other methods of measuring the strength of clots in rats fed butter, corn oil, and stock diets should be investigated. It would be of interest to know if the urea solubility and tensile strength of the different types of clot varied. If these further measurements of clot strength show that there is no difference in the strength of clots formed from the plasma of butter- and corn oil-fed rats, it would suggest that the differing thrombogenic properties of butter and corn oil diets in rats is due to alterations of the lytic system and not simply to changes in clot structure produced by these two fats. 4 Viscosity of a substance has been defined as the tangential force per unit horizontal planes at unit distance apart, one of which is fixed while the other velocity, the space being filled with the substance (Hodgman, 1958).
of either of two moves with unit
160
K. T. LEE, R. F. SCOTT,
D. N. KIM,
AND W. A. THOMAS
In addition to the fact that the clots of corn oil- and butter-fed rats may be more resistant to clot-lysis, other characteristics of these clots suggested by their increased binding power may be of importance. If clots formed in viva in corn oil- and butterfed rats are more viscous than those formed from stock animals, they may more effectively resist breakup by the physical action of the flow of blood within the coronary and renal arteries of the rat. It is also possible that increased adhesiveness may enable them to cling to vessel walls more tenaciously, thus preventing them from being swept along and broken up as readily as the thrombi in stock-fed rats. Each of these properties if present would seem to enhance the development of myocardial and renal infarcts. Whether or not these findings of the similar effect of butter and corn oil on clotstrength in the rat are applicable to man is not known. We believe that investigation of clot-strength in humans (in addition to measurement of clot-lysis time) following ingestion of various fatty meals might be of great value in elucidating the possible relationship of dietary fats and arterial thrombosis. Although reports are conflicting (Hougie and Ayers, 1960; Bronte-Stewart, 1961), some studies have suggested that dietary fats in humans will alter the clotting mechanism (Buckell and Elliott, 1959; Greig, 1956). It would be of interest to know that if such alterations do occur they are accompanied by physico-chemical changes resulting in increased clot-strength in humans. SUMMARY Butter appears to have a different biologic effect than corn oil regarding the development of intra-arterial thrombi and infarcts in rats. Rats receiving a diet containing 40% butter (plus cholesterol, propylthiouracil, and other ingredients) develop thrombi with myocardial and renal infarcts, together with an increased clot-lysis time. If corn oil is substituted for butter no thrombi or infarcts have been found and the clot-lysis times are shorter than that of the butter-fed animals. In order to elucidate the mechanism of the thrombogenic action of butter when fed to rats, the comparative strengths of clots formed from the plasma of rats fed butter or corn oil (plus other ingredients) or a stock diet, were measured using binding power in a thrombelastograph as one index of clot-strength. In addition, clot-lysis and plasma cholesterol determinations were done on all groups. The binding powers of the clots formed from the plasma of butterand corn oil-fed rats were not significantly different, but both fats produced clots of far greater binding power than did the low fat, stock diet. The clot-lysis times of the same butter-fed group of rats was significantly greater than that of the corn oil group. Both fat-fed groups had a longer clot-lysis time than did the stock diet group of rats. The finding of equal binding powers but different clot-lysis times in corn oil- and butter-fed rats suggests that the thrombogenic property of the 40’3% butter diet is due either to alterations in the lytic system secondary to the butter diet, or to aspects of clotstrength not reflected by binding power. REFERENCES B. (1961). Lipids and atherosclerosis. Federation Proc. 20, 127. M., and ELLIOTT, F. A. (1959). Effect of butter lipemia on the rate of clot lysis in normal males. Lancet i, 662. DAVIDSON, E., HOWARD, A. N., and GRESHAM, G. A. (1961). Blood coagulation studies in rats given diets which produce thrombosis or atherosclerosis. Brit. J. Exptl. Pathol. 42, 195. GREW, H. B. W. (1956). Inhibition of fibrinolysis by alimentary lipemia. Lancet ii, 16. HODGMAN, C. D. (1958). “Handbook of Chemistry and Physics.” 2154 p. Chemical Rubber Publishing Co., Cleveland, Ohio. HOUGIE, E., and AYERS, F. (1960). Lipemia and fibrinolytic potentiality. Lancet ii, 186. BRONTE~TEWART, BUCKELL,
CLOT-STRENGTH
AND
CLOT-LYSIS
IN
RATS
161
R. F., ALOUSI, K., and THOMAS, W. A. (1961). Chemico-pathologic studies of dietary lipids, thrombosis, and clot-lysis I: In vitro methods, utilizing liquid nitrogen refrigeration and the thrombelastograph for demonstrating with rats the antilytic effect of butter-containing atherothrombogenic diets. Arch. Pathol. 71, 594. THOMAS, W. A., and HARTROFT, W. S. (1959). Myocardial infarcts in rats fed diets containing high fat, cholesterol, thiouracil and sodium cholate. Circulation 19, 65. THOMAS, W. A., O’NEAL, R. M., and HARTROFT, W. S. (1960). Modification of diets responsible for induction of coronary thrombosis and myocardial infarction in rats. J. Nlctrition 69, 325. TILLMAN, R. L., O’NEAL, R. M., THOMAS, W. A., and HIXON, B. B. (1960). Butter, corn oil and fibrinolysis in rats. Circ&!ion Research 8, 423. ZAK, B. (1957). A simple rapid technique for serum total cholesterol, Anz. J. Clin. Pathol. 27, 583. SCOTT,