Dietary lipids and thrombosis

EXPERIMENTAL

AND

MOLECULAR

Dietary Study

4, 581-596

PATHOLOGY

Lipids

of Factors

and

Thrombosis

in Thrombogenic

Accounting

for

Increases

Cholesterol KYU TAIK

DONG NACK KIM, Department

(1965)

of Pathology,

Albany

Received

Diets

Fed to Rats

in Fibrinogen

and

Levels’ LEE, AND WILBUR A. THOMAS Illedical College,

September

Albany,

Xew

York

29, 1964

Myocardial and renal infarcts associated with arterial thrombi can be produced in rats fed a complex diet containing, among other ingredients, butter, cholesterol, propylthiouracil, sodium cholate and choline chloride (Thomas and Hartroft, 1959). When corn oil is substituted for butter, the thrombogenic and infarct-producing properties of this diet are virtually abolished (Thomas and Hartroft, 1959; O’Neal et al., 1959). In both butter-cholesterol and corn oil-cholesterol-fed rats serum lipids and various coagulation factors, including plasma fibrinogen and prothrombin, factors V, VIII, and fibrin stabilizing factor, are significantly elevated as compared to stockfed rats (Kim et al., 1963). Serum cholesterol, plasma fibrinogen, and prothrombin levels began to rise as early as within 1 week on high-fat diets (Kim et al., 1964). The exact mechanismof increased production of these factors is not clear. Extensive fatty change in the liver, which is a common feature in rats fed these high-fat diets, may cause basic changes in protein and carbohydrate metabolism and may be related to the increased production or decreaseddegradation of many factors. The infarct-producing diet used in our experiments is a complex mixture of several chemical components added to basic ingredients of a stock diet (Table I). Which specific chemical constituent (or combination of constituents) is responsible for the significant elevation of serum lipids and various coagulation factors observed is not known. In the current experiment an attempt has been made to investigate the effect of four individual chemical components in the infarct-producing diets, as well as combinations of these components, on plasma fibrinogen and serum cholesterol levels. Sixteen groups of 10 rats each were fed various combinations of the major chemical ingredients of the infarct-producing diet. Plasma fibrinogen was chosen as reflecting one aspect of protein metabolism, serum cholesterol as reflecting lipid metabolism. In addition, serum triglyceride and lipid phosphorus determinations, and fatty acid analyses of various serum lipid fractions were carried out on pooled samples. The experiment was designed to be completed at the end of 4 weeks because from previous experience with this diet, fibrinogen and serum lipid levels are markedly 1 This

work

was

supported

by

USPH

grants

HE-07155 581

and

Tl-GM

477.

582

D. N.

KIM,

K.

T. LEE,

AND

Vv’. A. THOMAS

elevated by this time. However, because of the short duration no infarcts were expected or found. MATERIALS Initially, were divided group), and binations of

AND

of the experiment,

METHODS

170 male Wistar albino rats in the same weight range ( 120 gm * ) into 17 groups of 10 rats each (16 experimental groups and one stock were housed individually in wire-bottomed cages and fed various comdiets shown in Fig. 1 and Table I. The amount of diet offered to each

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50

t kfi

25

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0

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&q

100

88 .

50 0

FOOD lnnnl

CONSUMED lnnnrlnnnnn

I -11

FIG. 1. Amounts of food consumed and changes in body weight in various dietary groups. The top figure shows the changes in body weights. The shaded bars represent the weight changes at the end of 2 weeks and the whole bars represent the weight; changes at the end of 4 weeks. The bottom figure represents selected chemical ingredients included in various groups.

rat in all groups were restricted to 10 gm per day. In our previous experiments when similar types of diets were offered ad Zibitum, the amount of diet consumed by rats varied widely depending upon the ingredients of the diet; the usual amount of the infarct-producing diet consumed was 4 to 8 gm per day and 20 to 30 gm per day of the stock diet. Although the infarct-producing diet contains a large quantity of butter and the caloric value per gram of the diet is far greater than that of the stock diet, the total caloric intake of rats on the infarct-producing diet is much less than that of rats on the stock diet. Restricting the diet of every rat to 10 gm per day in this experiment does not make the caloric intake of every rat equal but it at least maintains the caloric intake of all groups closer to that of the group on infarct-producing diets than would be the case otherwise. Animals were weighed weekly and at the termination of the experiment, and the amount of food consumed by each rat was measured by the weigh-back method. Since in the original experiment cholesterol and fibrinogen levels in BB group (Table I) were not consistent with other results suggesting possible experimental error, the experiment with this group was repeated twice. As a further safeguard, the experiment with groups BG, BI, BJ, BL, and BG that are indicated by asterisks in Fig. 2 were also repeated at the same time. Results of the two repeated experiments with BB group were similar to each other but unlike the result in the original experiment. Thus with BB group the results from the original experiment were dis-

40.0 40.0 40.0 40.0 40.0

40.0 40.0 40.0 40.0 40.0

40.0

40.0 40.0 40.0 40.0

-

BB BC BD BE BE

BG BH BIa BJ BK

BL

BM BN BO BP

CC”

a Infarct-producing b Stock diet.

40.0

BA

Butter

diet.

2 .o

-

-

-

-

-

-

Corn oil

-

0.3 0.3 0.3 0.3

0.3

0.3 0.3 0.3

-

-

-

-

Propylthiouracil

-

2.0 2.0 2.0 -

-

-

2.0 2.0

2.0 2.0 2.0

-

-

Sodium cholate

COMPOSITION

0.2

1.0 1 .o

1 .o

1 .o 1.0 1.0 -

1 .o 1.0 -

-

Choline chIoride

-

5.0 5.0

-

5.0 5.0 5.0 5.0

5.0 5.0

-

Cholesterol

TABLE I OF 16 EXPERIMENTAL DIETS

20.0

20.0 20.0 20.0 20.0

20.0

20.0 20.0 20.0 20.0 20.0

20.0 20.0 20.0 20.0 20.0

20.0

Casein

AND STOCK DIET

2.0

2.0 2 .o 2.0 2.0

2.0

2 .o 2.0 2 .o 2.0 2 .o

2.0 2 .o 2.0 2.0 2.0

2.0

Vitamin mixture

4.0

4.0 4.0 4.0 4.0

4.0

4.0 4.0 4.0 4.0 4.0

4.0 4.0 4.0 4.0 4.0

4.0

Salt mixture

-.

6.0

6.0 6.0 6.0 6.0

6.0

6.0 6.0 6.0 6.0 6.0

6.0 6.0 6.0 6.0 6.0

6.0

.4lphacel

65.8

25.7 24.7 20.7 21.7

26.7

22.0 20.0 19.7 27.7 22.7

23.0 27.0 26.0 25.0 21.0

28.0

Sucrose

-.

$ 2

2

$

i P E z

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r

584

D. N.

KIM,

K.

T. LEE,

AND

W.

A.

THOMAS

carded and the combined results from the two repeated experimems were used instead. Results of repeated experiments with five other groups were similar to the original values and combined with those of the original experiments. Before being bled the animals were fasted for 16 hours. Rats were anesthetized by intraperitoneal injection of sodium barbital, 5 mg per 100 gm of bodyweight. The abdomen was opened and blood was obtained by aortic puncture with siliconized 18-gage disposable needle attached to plastic tubing. Blood was oxalated (1 volume of 3.8% sodium oxalate per 9 volumes of blood) and centrifuged at 1600 X g for 10 minutes at 1°C to obtain plasma.

Fl8RlNOGEN

No. OF RiiTS

FIG. 2. Plasma fibrinogen and serum cholesterol levels and degree of fatty changes in the liver. In 6 groups that are indicated by asterisks at the bottom of the upper figure, the experiment has been repeated and the data from those groups were combined with the data from the original 6 groups as explained in text. The middle figure shows the average fatty changes in the liver. Fatty changes are classified into 4 categories: Normal (0), slight (+), moderate (++), and severe (+++). Fairly consistent correlation was observed between the degrees of fatty changes in the liver and levels of cholesterol and fibrinogen.

Plasma fibrinogen levels were determined by the method of Ware et al. (1947) and serum cholesterol levels by the method of Zak et al. (1954) in individual rats. In addition, cholesterol, triglyceride, and phospholipid levels were determined on pooled serum in each group by the method of Loeffler (1959), Van Handel and Zilversmit (1957)) and Bartlett (1959)) respectively. Fatty acid patterns of each lipid fraction were examined by gas phase chromatography (Scott et aE., 1964). The composition of the infarct-producing and stock diets is shown in Table I. The various combinations of four of the main ingredients of the infarct-producing diet (propylthiouracil, sodium cholate, choline chloride and cholesterol) were added to the basic 40% butter diet (BA) and 16 such combinations were obtained (Table I). When any of the ingredients was added, an equal amount of sugar was removed from the diet to maintain 100% weight. Figures shown are in per cent. The caloric values of 40% butter (BI) and stock diets were 5.8 and 3.8 calories per gram, respectively. Statistical analyses were made using Student’s “t” test.

FIBRINOGEN

AND

CHOLESTEROL

IN

RATS

585

RESULTS Food intake The amount of food consumed and changes in the body weight at the end of 4 weeks for each group are shown in Fig. 1. The average food intake ranged from 4.7 gm per day to 9.2 gm per day. The fat and protein content of each diet (except for stock diet) is identical, and hence, food intake reflects differences in the total amount of fat and protein consumed and the actual amount of various other chemical ingredients consumed. The body weight in various groups has a general relationship to the amount of food consumed. In general, rats that ate the most had the greatest increase in body weight. However, even among the rats that consumed all the food offered, there were considerable variations in body weight. The BA group whose diet contained 40% butter but no toxic chemical ingredients (such as propylthiouracil or sodium cholate) gained the most. When any of the four selected chemical ingredients under study was added to the diet (even excessive choline chloride alone), the weight gain was much less than that of the BA group. The stock group gained very little but calories of this group were restricted to 38 daily as compared with 58 in the BA group. There was decreased food intake and weight loss when both sodium cholate and propylthiouracil were present in the diets but neither one alone resulted in weight loss. The most dramatic changes were observed in the group which had all four major ingredients (BI). In this group the dietary intake was less than 50% of food offered (4.7 gm daily) and the average body weight was 70%’ (84 gm) of original body weight (120 gm) at the end of 4 weeks. Thus not only protein and fat intakes were lowest but also actual amounts of all individual major chemicals were lowest in this group. In all groups except for BN more than 70yr of rats survived at the end of 4 weeks. Chemistry

values

The serum cholesterol and plasma fibrinogen levels and the degree of gross fatty change in the liver in various groups are shown in Fig. 2. The fibrinogen and cholesterol levels of the stock group were 189 and 100 mg per cent, respectively and those of the infarct-producing diet group (Bl) were 509 and 1860 mg per cent, respectively. Values for the rest of the groups ranged between these two extremes. There were no changes in the fibrinogen and cholesterol levels in the group fed the basic 40% butter diet (BA) without major chemical ingredients under investigation when compared with the stock diet group (CC). The effects of the addition to the basic 40% butter diet of individual major ingredients singly or in various combinations on serum cholesterol are shown in Figs. 3-6 and on plasma fibrinogen levels in Figs. 7-10. When average values of the plasma fibrinogen and serum cholesterol of each group were plotted on the log-log scale, there was a linear relationship between those two values (Fig. 11). The addition of propylthiouracil to the diet, either singly or in combination with other major chemical ingredients, resulted in significant elevation of the serum cholesterol level in most instances (p between 0.001 and 0.025), except for one instance (between BE and BN groups) (Fig. 3). The effect of the addition of sodium cholate (Fig. 9) was similar to that of propyl-

586

r~. N. KIbf,

FIG. 3.

FIG. 4.

FIG.

5.

The

effect

The

The

effect

effect

K.T.

LEE,

of propylthiouracil

of sodium

of choline

cholate

chloride

AND

w.

on the

A. THOMAS

serum

on the serum

on the

serum

cholesterol

level

cholesterol

eve1 .

cholesterol

level.

FIBRINOGEN

FIG.

6.

The

effect

I

FIG.

The

8.

The

IN

on the serum

587

RATS

cholesterol

level.

WITH

p 600

7.

CHOLESTEROL

of cholesterol

8cil

FIG.

AND

effect

effect

-

of propylthiouracil

of sodium

cholate

on the

plasma

on the plasma

fibrinogen

tibrinogen

level.

level.

588

D. N.

KIM,

K.

8 9.

The

effect

of choline

A. THOMAS

chloride

on the plasma

fibrinogen

level.

WITH

6 600

10.

W.

WITHOUT

0

8

FIG.

AND

WITH

d, 600 F

FIG.

T. LEE,

The

effect

of cholesterol

on the plasma

fibrinogen

level.

8 p/J::i..;-; l -40% o--STOCK 100 50

I 100 TOTAL

FIG. 11. The relationship between perimental diet groups and stock diet scale. A linear relationship is seen.

BUTTER DIET

I I I( 200 300 400 500 CHOLESTEROL

serum group.

I 1000 mg. %

DIETS r 2000

cholesterol and plasma fibrinogen levels in 16 exThe average for each group is plotted on a log-log

FIBRINOGEN

AND

CHOLESTEROL

IN

RATS

589

thiouracil and the magnitude of increase was far greater than any other major ingredients under investigation (0.001 < p < 0.005). When choline chloride was added singly or in various combinations with the three other ingredients, the effect was different between different dietary groups (Fig. 5). Adding choline chloride alone to the basic 40% butter diet (BA 78 mg per cent) did not alter the cholesterol level (BC 75 mg per cent). No significant changes were observed when choline chloride was added to the diet containing sodium cholate (BD 150 to BE 169 mg per cent) or added to the diet containing propylthiouracil (B J 126 to BL 151 mg per cent). When choline chloride was added to the diet containing propylthiouracil and cholesterol there seemed to be a tendency toward a decrease in cholesterol level (BK 247 to BP 205 mg per cent). A slight to marked increase in cholesterol values was observed when choline chloride was added to the diet containing the following ingredients: cholesterol (BB 83 to BG 113 mg per cent, p < O.OS), sodium cholate and cholesterol (BF 340 to BH 476 mg per cent (0.10 > p > 0.05) propylthiouracil, sodium cholate, and cholesterol (BO 541 to BI 1860 mg per cent, p < O.OOl), or propylthiouracil and sodium cholate (BM 275 to BN 425 mg per cent, 0.1 > p > 0.05). The effect of cholesterol (Fig. 6) when added to the basic 40% butter diet was negligible (BA 78 to BB 83 mg per cent). However, when cholesterol was added in the presence of other chemical ingredients, there was significant increase in the cholesterol level in all instances (0.025 > p > 0.001). The effects of various ingredients on plasma fibrinogen levels were similarly analyzed and are presented in Figs. 7-10. The range of values for fibrinogen levels in various groups was from 194 to 508 mg per cent and this was much narrower than the range of cholesterol values which was from 75 to 1860 mg per cent. The effect of propylthiouracil (Fig. 7) onthe fibrinogen level was slightly different from that on the cholesterol level. When propylthiouracil was added to the diet containing cholesterol (BC 231 mg per cent), or sodium cholate (BD 264 mg per cent), or sodium cholate and cholesterol (BF 319 mg percent) there were no significant increases in fibrinogen levels (BL 234 mg per cent, BM 267 mg per cent, and BO 265 mg per cent, respectively). But propylthiouracil singly or in combination with other ingredients exclusive of above combinations resulted in significant increase in fibrinogen levels (0.01 > p > 0.001). The effect of propylthiouracil (Fig. 7) on the fibrinogen level was slightly different from that on the cholesterol level. There was no significant difference in the fibrinogen levels when sodium cholate was added to the diet containing propylthiouracil (BJ 225 to BM 267 mg per cent) and to the diet containing propylthiouracil and cholesterol (BK 264 to BO 265 mg per cent), but in all other combinations there was significant increase in the fibrinogen levels (p < 0.001) . The effect of choline chloride (Fig. 9) on the fibrinogen level was least striking as compared to other chemical ingredients. Significant changes were observed when it was added alone to the basic 405% butter diet (BA 194 to BC 231 mg percent, p < O.Ol), to that containing sodium cholate (BD 264 to BE 298 mg per cent, p > 0.05) and to that containing propylthiouracil, sodium cholate and cholesterol (BO 265 to BI 434 mg per cent, p < 0.005). Similar differences were observed when it was added to the diet containing propylthiouracil and sodium cholate (BM 267 to BN

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FIBRINOGEN

AND

CHOLESTEROL

IN

RATS

591

334 mg per cent) and to the diet containing sodium cholate and cholesterol (BF 3 19 to BH 369 mg per cent). Significant increases in the fibrinogen levels were noted when cholesterol (Fig. 10) was added to the diets containing sodium cholate (BD 264 to BF 319 mg per cent, p < O.Ol), or propylthiouracil and choline chloride (BE 234 to BP 290 mg per cent, p < O.OOl), or sodium cholate and choline chloride (BE 298 to BH 369 mg per cent, p < 0.001) or propylthiuoracil, sodium cholate and choline chloride (BN 334 to BI 505 mg per cent, p < 0.001). However, the addition of cholesterol did not result in SERUM

CHOLESTEROL

CHOLESTEROL

TOTAL

ARACHIDONIC

ARACHIDONATE

ACID

IN CHOLESTEROL

ESTER

FIG. 12. Serum choksterol and per cent and absolute amount of cholesterol arachidonate of 16 experimental diet groups. Sixteen groups were arranged in the order of their serum cholesterol levels and the percentage and total amount of cholesterol arachidonate was plotted. The percentage of the cholesterol arachidonate appears to be inversely proportional to the serum cholesterol level for most groups.

an increasein fibrinogen level when it was added alone to the basic 40% butter diet (BA 194 to BB 200 mg per cent) or to the diets containing choline chloride (BC 231 to BG 224 mg per cent), or propylthiouracil and sodium cholate (BM 267 to BO 265 mg per cent), or propylthiouracil only (B J 255 to BK 274 mg per cent). The degree of fatty change of the liver of each rat was evaluated by both weight and grossappearance and graded as normal (0) , slight ( + ), moderate (+ +) , and severe (+++). The average grade of each group is shown in Fig. 2. There was fairly consistent correlation between the degreesof fatty change in the liver and levels of serum cholesterol and plasma fibrinogen in each group. The triglyceride, phospholipid, and cholesterol values and esterified fatty acid patterns of pooled serum of each group is shown in Table II. When sixteen groups were arranged in the order of their cholesterol levels (Fig. 12) and the percentages of cholesterolarachidonate were plotted, the percentages of the cholesterol-arachidonate were inversely proportional to the serum cholesterol levels in most groups.

592

D. N.

KIM,

K.

T. LEE,

AND

W.

A. THOMAS

DISCUSSION In experimental nutritional studies there are many methods of feeding and every method provides certain information which other methods do not. In the current experiment the amount of food offered to each rat was restricted to 10 gm per day. We have chosen this method of feeding to restrict the intake of the rats on less toxic diets (without thiouracil or sodium cholate) and yet to offer all they would eat to rats on the most toxic diets. In general, the average intake was over 85% of the amount of food offered in every group except for groups whose diets contained both sodium cholate and propylthiouracil. Since the per cent by weight of fat and protein and that of various individual chemical ingredients when they are included in the diets are identical in all diets under investigation (except stock), the amounts of these ingredients consumed are directly proportional to the amount of diets consumed. A greater amount of food provides more calories from protein and fat but also contains a larger amount of individual chemicals under investigation. These factors should be considered in evaluating the data obtained in this experiment. The group which had the lowest food intake (BI) also had the lowest body weight, and yet the levels of serum cholesterol and plasma fibrinogen were highest in this group. Retardation of growth or loss of weight may well be related to the relative protein deficiency due to decreased food intake. However, to what extent this protein deficiency is responsible for the significant increase in fibrinogen and cholesterol levels is difficult to assess with available data. This relationship has been suggested by several investigators (Renaud and Allard, 1964) including ourselves, and experiments exclusively designed to investigate this relationship are being conducted in our laboratory. We noted that in most instances when serum cholesterol levels were increased, plasma fibrinogen levels were also increased. This suggests that mechanisms involved in increasing serum cholesterol may be related to those affecting plasma fibrinogen levels and reminds us of the well known fact that lipid and protein metabolism are interrelated. The chemical ingredients investigated are known to be closely related to lipid metabolism but not much information is available regarding their relationship to protein metabolism. Since Anitschkow (1913) produced foam cell lesions in the aorta of rabbits by feeding cholesterol, numerous investigators used cholesterol in dietary experiments, and extensive data have been accumulated. Cholesterol and its esters appear to be essential constituents of living mammalian cells and there are constant synthesis and degradation of cholesterol proceeding at closely related rates in the living organism. Rate of degradation of cholesterol in certain disease conditions, such as hypothyroidism, is an important factor in the resultant hypercholesterolemia. In the current experiment, addition of cholesterol alone even in the presence of 40% butter by weight in the diet did not raise either cholesterol or fibrinogen levels. Only when cholesterol was given in combination with other chemical ingredients was there increase in cholesterol and fibrinogen levels. Mechanisms for such increase are not clear but they may be related to the rate of absorption, synthesis, or degradation. Sodium cholate was the most potent hypercholesterolemic agent in our experiment when it was added to the diet singly or in combination with other chemical ingredients. Bile acids are potent surface active agents and known to have a striking

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effect on the absorption of both dietary and entero-hepatically circulating cholesterol and other lipids (Block et aZ., 1943; Zabin and Barker, 1953). Feeding of bile acids to animals, which overloads the entero-hepatic cycle of bile acids and represses cholesterol oxidation in the liver, causes an increase in blood cholesterol levels (Brown and Page, 1952). The effect of sodium cholate was greatly enhanced when given in combination with other chemical ingredients, particularly with propylthiouracil or cholesterol. On the other hand, loss of bile acids by draining from the intestine by re-implantation of the bile duct (Beyers and Friedman, 1958) or by feeding any agent sequestersbile acids in the intestinal lumen increasesthe rate of cholesterol oxidation and lowers the blood cholesterol (Tennent et al., 1962). The effect of the addition of propylthiouracil to the diet was similar to that of sodium cholate. Rats are sensitive to propylthiouracil and the amounts added to the diets in the current experiment are considered to be sufficient to produce hypothyroidism in rats. In man serum cholesterol levels tend to be high in hypothyroidism and low in hyperthyroidism (Mason et al., 1930; Hurxthal, 1932; Peters and Man, 1943). Thyroidectomy has been shown to raise serum cholesterol levels in rabbits (Turner et al., 1938), rats (Handler, 1948), dogs (Parhon and DCrkvici, 1926)) and in drakes (Benoit and Bogdanovitch, 1937) and the same effect has been achieved by thiouracil in chickens (Fleischmann and Fried, 1945), dogs (Parodi and DeGregori, 1948), and rats (Rosenmanet al., 1952). Administration of thyroid preparations lowered serum cholesterol levels in these animals (Benoit and Bogdanovitch, 1937; Friedland, 1933; Parhon and DCrCvici, 1931). The cholesterol changes in hyper- and hypothyroid states do not appear to be accounted for by the basal metabolic rate alone (Cutting et al., 1934). The rate of cholesterol synthesis, studied with deuterium (Marx, 1953) or tritium (Byers et al., 1953)) is above normal in the hyperthyroid rat and below normal in the hypothyroid animal. However, hyperthyroidism is associatedwith increased destruction and increased intestinal excretion of cholesterol (Rosenman, 1952) without affecting intestinal absorption (Rosenman and Friedman, 1956). Conversely there is a marked decreasein catabolism and excretion of cholesterol in hypothyroidism, but here again intestinal absorption is unaffected, In the current experiment propylthiouracil exerted a significant effect on raising serum cholesterol and plasma fibrinogen in most instances and when it was combined with sodium cholate its effect was markedly augmented. In the current experiment choline chloride exerted different effects in different combinations with other chemical ingredients for unknown reasons.Choline has been reported to have no effect on cholesterol absorption (Rice et al., 1956), but when it was added in excessiveamounts to diets of rats fed cholesterol in certain experiments it raised serum cholesterol (Wilgram et al., 1957) but reduced liver cholesterol levels (Moyer et al., 1956). Artom (1960) suggestedthat the lipotropic action of choline is due in part to its effect on enhancing the rate of fatty acid oxidation in the liver. However, its action can not be explained solely as a result of enhanced oxidation. Another suggestionis that choline may promote the formation of some lecithin-containing lipoproteins in the liver which may be involved in transport of fatty acids from the liver to the depots. The diet group in the current study in which all four of the selected chemical ingredients were added (BI) had the lowest food intake but this group had the highest

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serum cholesterol and the lowest relative percentage of cholesterol ester arachidonate, and this latter fell in the lower range of that of human. There was no consistent change in the absolute amount of arachidonic acid. Previously we have reported that the infarct-producing diets produced severe hyperlipemia and significant change in the pattern of esterified fatty acids in rats (Scott et al., 1964). The most striking change was the decrease in the relative percentage of cholesterol arachidonate without appreciable change in its absolute amount. The absolute amount of linoleic acid, the precursor of arachidonic acid however was increased considerably. The suggestion has been made that those rats fed the infarct-producing diet may have limited ability to convert linoleic to arachidonic acid. SUMMARY The feeding of a complex infarct-producing serum lipids and many coagulation factors arterial thrombosis within 3 to 6 months.

diet to rats in the past has resulted in elevation of within 4 weeks, and vascular changes in the aorta, and

In the current experiment an attempt has been made to investigate with this infarct-producing diet the effect of four individual chemical components as well as combinations of these components on plasma fibrinogen and serum cholesterol levels after 4 weeks on the diets. In most instances when the serum cholesterol level was increased, the plasma fibrinogen level was also increased suggesting that mechanisms involved in increasing serum cholesterol are related to those affecting plasma fibrinogen levels. The highest values were obtained in rats fed all toxic ingredients, although the food intake (and hence doses of individual items) was lowest in these rats. Propylthiouracil and sodium cholate exerted a significant effect on raising serum cholesterol and plasma fibrinogen levels and in most instances when they were combined, their effects were markedly augmented. The addition of cholesterol alone, even in the presence of 40% butter in the diet, did not affect either cholesterol or fibrinogen levels. Only when cholesterol was given in combination with other chemical ingredients was there a significant increase. Choline chloride exerted different effects on different combinations on cholesterol and fibrinogen levels. Rats fed all four toxic ingredients showed a significant decrease in the relative percentage of serum cholesterol arachidonate without appreciable change in its absolute amount, while the absolute amount of cholesterol linoleate, the precursor of arachidonic acid, increased considerably. The fatty acid pattern of rats receiving the infarct-producing diet is somewhat similar to that of humans and such changes in fatty acid patterns may be important in regard to the development of atherosclerotic lesions in these rats. REFERENCES N., and CHALATOW, S. (1913). Uber experimentelle cholesterinsteatose und ihre bedeutung fur die entstehung einiger pathologischer prozesse. Zen&a. A&em. Pathol. Pathol. Anal., 24, I-9. ARTOM, C. (1960). Mechanism of action of choline. Am. J. CZin. Nut?. 8, 303-305. BENOIT, J., and B~CDANOVITCH, S. B. (1937). Sur la teneur du sang en acides gras, phosphore lipidique et cholesterol chez le canard domestique, apres injection d’extraits prehypophysaires et apres thyroidectomie. Corn@. Rend. Sot. BioZ. 125, 891-894. BLOCH, K., BERG, B. N., and RITTENBERG, D. (1943). Biological conversion of cholesterol to cholic acid. J. BioZ. Chem. 149, 511-517. BYERS, S. O., ROSENMAN, R. H., FRIEDMAN, M., and Bmos, M. W. (1952). Rate of cholesterol synthesis in hypo- and hyperthyroid rats. J. Exptl. Med. 96, 513-516. BYERS, S. O., and FRIEDMAN, M. (1958). Changes in intestinal absorption and plasma concentration of various lipids after ileal reimplantation of bile duct. Am. J. PhysioZ. 192, 427-431. CUTTING, W. C., RYTAND, D. A., and TAINTER, M. L. (1934). Relationship between blood cholesterol and increased metabolism from dinitrophenol and thyroid. J. CEn. Invest. 13, S47552. ANITSCHKOW,

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PETERS, J. P., and MAN, E. B. (1943). Interrelations of serum lipids in patients with thyroid disease. J. Clin. Invest. 22, 715.720. RENAUD, S., and ALLARD, C. (1964). Effect of dietary protein level on cholesterolemia, thrombosis, atherosclerosis and hypertension in the rat, J. ,Vutr. 33, 149.157. RICE, E. H., HUNGERFORD, G. F., and MARX, W. (1956). Lack of effect of dietary choline on cholesterol absorption in rat. Proc. Sot. Exptl. Biol. Med. 92, 754-756. ROSENMAN, R. H., BYERS, S. O., and FRIED&IAN, M. (1952). The mechanism responsible for the altered blood cholesterol content in deranged thyroid states. J. Clin. Endocrinol. Metab. 12, 12871299. ROSENMAN, R. H., FRIEDAWN, M., and BYERS, S. 0. (1952). Observations concerning the metabolism of cholesterol in the hypo- and hyperthyroid rat. Circulation 5, 589-593. ROSENMAN, R. H., and FRIEDMAN, M. (1956). Effect of hyperand hypothyroidism on intestinal absorption of cholesterol in rats. 187, 381-382. SCOTT, R. F., MORRISON, E. S., THOMAS, W. A., JONES, R., and NAM, S. C. (1964). Short-term feeding of unsaturated versus saturated fat in the production of atherosclerosis in the rat. &ptl. Mol. Pathol. 3, 421-443. TENNENT, D. M., HASHIM, S. -4., and VAN~TALLIE, T. B. (1962). Bile-acid sequestrants and lipid metabolism. Federation Pro<-. 21, No. 4, Suppl. 11, Pt. 2, 77-80.

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high fat,

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