LINOLEIG ACID COMPONENT IN THE HYPERLIPIDEMIA INDUCED BY TRITON***

LINOLEIC

ACID

COMPONENT INDUCED

YOSHIO

IMAI

IN BY

THE

HYPERLIPIDEMIA ***,

TRITON

AND YOSHITOMO

ARAMAKI

Research Laboratories, Takeda Chemical Industries, Ltd., Kgashiyodogawa-ku, Osaka Received for publication

November 6, 1963

Many reports have been published on the genesis of hyperlipidemia

induced by

parenterally administered Triton WR-1339 (Oxyethylated tertiary octylphenol formalde hyde polymer, Winthrop Laboratories, New York), one of the surface-active agents, but the interests were paid especially on hypercholesterolemia (1-10). Frantz et al. (2) and Hirsch et al. (3) described an enhanced rate of hepatic synthesis of cholesterol as the causal factor for the rise of plasma cholesterol level after Triton injection. As an evidence of enhanced cholesterogenesis, further, Hirsch et al. (4) recognized an increase of body total cholesterol could be induced by Triton in mice without any exogenous sources. Meanwhile, Friedman et al. (6) considered the mobilization of cholesterol from body pool was the primary response in this hypercholesterolemia. At present it is a problem to be solved to determine the origin of plasma excess triglyceride after Triton injection, since this change is greater than that of cholesterol itself and the elevation of plasma cholesterol induced by sustained hypertriglyceridemia has been shown by Friedman et al. (5, 6). The authors in this paper studied on the origin of this plasma excess triglyceride with the use of gaschromatographical analysis of fatty acid composition. METHODS The rats (Sprague-Dawley) or mice (deutsche-denken-Takeda) weighing 160-200 or 17-25 g, respectively, were maintained on a stock chow (Diet No. TH-1), but in some special cases the animals were fasted or given a cholesterol-free synthetic diet (Diet No. T-6) prior or after Triton injection to avoid the dietary influences. Diet No. T-6 was composed of sucrose 68%, cotton seed oil 8%, casein 18%, salt mixture 4%, cellulose powder 1.5% and complete vitamin mixture in glucose 0.5%. Triton was given intravenously as a saline solution in 12.5 or 20% in mice or rats, respectively. To control groups vehicle only was given. For the measurement of body total chole sterol of mice, the whole body was hydrolysed with five volumes of alcoholic potassium hydroxide (20% w/v KOH in 90% v/v EtOH) on a boiling water bath for two hours. Standing overnight at room temperature and adding water by 50%, cholesterol was 今 井 祥 雄 ・荒 蒔 義 知 * This paper constitutes Part X of series entitled ** This study was presented at the 22nd Kinki Japan,

Osaka

(Oct.

1962).

"Studies Regional

on lipid Meeting

metabolism" . of Pharmacological

Society

of

extracted by petroleum ether, purified through digitonide formation and determined colorimetrically by the Liebermann-Burchard reaction. In order to investigate the sequential changes of plasma or liver lipid levels, each group of five animals was bled by decapitation at various intervals. For the analysis of lipids, the methods of Sperry Webb (11), Abell et al. (12), King (13) and Carlson-Wadstrom (14) were employed for the determination of liver cholesterol, plasma cholesterol, lipid phosphorus and trigly ceride, respectively. The analyses of fatty acid pattern of triglyceride were performed on the pooled sample of five rats in each group, but 20 rats were used for the analysis of normal plasma. The plasma and tissue lipids were extracted three times with chloroform methanol mixture (2: 1 v/v), then the triglyceride fraction was separated from the crude lipid extract by Florisil column chromatography according to the method of Carroll (15). But in the case of adipose tissue, such as perirenal fat, this procedure was omitted because this tissue consisted almost of triglyceride (16). The triglyceride samples described above were hydrolysed with alcoholic potassium hydroxide (5% w/v KOH in 95% v/v EtOH) for one hour at 70°C on a water bath and added water by 50%. After removing the contaminants by repeated washing with petroleum ether, the water layer was sub jected to remove ethanol under nitrogen stream, diluted with water and acidified with concentrated sulfuric acid. Then, fatty acids were extracted five times with petroleum ether. The fatty acid treated with diazomethane were analysed by gaschromatography (Yanagimoto, GCG-2). For measuring the effect on the lipemia clearance in vivo, the rats pre-treated with Triton 5 minutes before were injected 1 ml of 30% coconut oil emulsion and the blood samples were collected from the tail vein at each time under ether anaesthesia. For in vitro experiment 3 ml of the incubation mixture, containing post-heparin rat plasma 0.5 ml (1 mg/kg i.v.; taken 10 minutes after injection), 0.1 M phosphate buffer solution 1 ml (pH 7.5), 0.101ococonut oil emulsion 1 ml and distilled water 0.5 ml, was kept in 37TC bath time. rat tilled

throughout

Triton

the

solution

plasma

was

water.

plasma sample of incubation

incubation

or post-Triton

substituted

The

optical

for

dis

density

of

diluted with saline mixture was read

or at

660 m,a. RESULTS

1) Sequential changes of total cholesterol in whole body, blood and liver after Triton injection in mice Forty-two seven groups

mice were divided into and

maintained

on a

FIG. 1. Effect of Triton'on body total cholesterol in mice (sequential change). Each point represents the mean (* *S.E.) of 6 male mice (body wt. 17-25 g). Triton : 1,250 mg/kg i.v.

T-6 diet for one week prior to the experiment.

Triton

intravenously

was given

in one dose level

of 1,250 mg/kg and the total body cholesterol

was determined

one

as shown

week

There tion, within FIG. 2. Effect of Triton on body total cholesterol in mice (dose-response). Each point represents the mean (I *S.E.) of 6 male mice (body wt. 17-25 g).

three

in Fig. 1.

was an apparent

body total cholesterol reaching

concentra

The response

at

dose level at this

time is illustrated cholesterol liver were

rise in

to its maximum

2 days. different

for

in Fig. 2. The

level in blood and shown in Fig. 3. A

time-lag of onset of rise in liver was observed.

2) Sequentialchangesof lipid com

FIG. 3. Sequential changes of blood and liver total cholesterol after Triton injection in mice. Each point represents the mean of 6 male mice (body wt. 17-25 g). Triton : 1,250 mg/kg i.v. BTC : blood total cholesterol, LTC : liver total cholesterol.

ponentsin plasma and liverafter Triton injectionin rats The rise of plasma lipids after an intravenous injection of 500 mg/kg of Triton in intact rats is illustrated in Fig. 4. The value represents the mean of five rats. The elevation of triglyce ride level was more obvious than the change of cholesterol. The increasing rate of plasma phos pholipid appeared to be earlier than that of cholesterol which showed the plateau between 24 and 48 hours. The dietary factor was tested in a further experi ment in which the rats were fasted after Triton injection or

FIG. 4. Sequential changes in plasma lipids after Triton injection in rats. Each point represents the mean of 5 rats (a , ; body wt. 160-200 g). Triton : 500 mg/kg i.v. TG : triglyceride, PL : phospholipid, TC : total cholesterol.

placed upon a T-6 diet for one week prior to injection. As shown in Figs. 5 and 6, the pre cedence of rise of triglyceride to cholesterol happened regard less whether the animal took a

food or not. On the other hand, the hepatic concentration of cholesterol in treated rats exhibited a decrease at 4 hours after Triton injection, while an increase at 24 hours as shown in Table 1. We reemphasize a difference of response in cholesterol and trigly ceride in Triton-hyperlipidemia. 3) Linoleicacid contentin triglycerideof post-Triton plasma or tissues in rats Table 2 illustrates the composi tion of higher fatty acids. It is evident that the triglyceride-fatty acids of

FIG. 5. Changes of plasma lipids after Triton injec tion in rats fed a cholesterol-free diet. Each point represents the mean of 5 male rats (body wt. 160-200 g). Triton : 500 mg/kgdi.v.

post-Triton plasma showed almost the same pattern to that of normal rat plasma. As discussed later, this result suggests the origin of plasma excess triglyceride largely depends upon the mobilization rather than its enhanced biosynthesis. 4)

Inhibitory effect of Triton on lipemia clearance in vivo and in vitro Though

rapid

the intact

disappearance

rats showed a from

blood

stream of emulsified coconut oil given intravenously, the

in Triton-treated

fat particles

even

in

the

diminished

presence

heparin

and

Typical

disappearance

the degree

of

slowly injected

of delay curves

FIG. 6. Changes of plasma lipids after Triton injec tion in fasted rats. Each point represents the mean of 5 female rats (body wt. 160-200 g). Triton : 500 mg/kg i.v.

rats

are

was shown shown

to be in proportion

in Fig. 7. Further,

to the

dose of Triton.

in in vitro experiment

TABLE 1. Changes of total cholesterol and triglyceride content in plasma and liver after Triton injection in rats.

* mean value±standard error, **0.001>P, 160-200 g), Triton : 500 mg/kg i.v. TC : total cholesterol, TG : triglyceride.

***0.02>P>0.01,

rats : SD a (body wt.

Triton

TABLE

2.

Fatty

acid

pattern

of

triglyceride

in

plasma

and

tissues

in

rats.

* fatty acid with a carbon number 14-16. ** fatty acid with a carbon number 16-18. *** fatty acid with a carbon number over 18. PF : perirenal depot fat.**** MF : mesentery depot fat.**** L : liver triglyceride.**** SW : triglyceride of small intestinal wall.**** NB : normal rat plasma triglyceride. 4B : plasma triglyceride 4 hours after Triton injection. 24B : plasma triglyceride 24 hours after Triton injection. rats : SD a (body wt. 160-200_g), Triton : 500 mg/kg i.v., **** of normal rats.

FIG. 7. Inhibitory effect of Triton on lipemia clea rance in rats in vivo. Each point represents the mean of 3 female rats (body wt. 160-200 g). Dilution of plasma : x 50. A : 30% coconut oil emulsion (C.O.E.) 1 ml/rat plus 4% Triton 0.3 ml/rat i.v. B : 30% C.O.E. plus 2% Triton 0.3 ml/rat i.v. C : 30% C.O.E. plus 1% Triton 0.3 ml/rat i.v. D : 30% C.O.E. plus 4% Triton 0.3 ml/rat and Heparin 1 mg/kg i.v. H : 30% C.O.E. plus Heparin 1 mg/kg i.v. N : 30% C.O.E. i.v.

FIG. 8.

Inhibitory

ing

activity

effect of

of

Triton

post-heparin

on

lipemia

plasma

exhibited an inhibition on the so-called "lipemia clearance" of incubation mix ture

not only made up of coconut oil

emulsion, but also of the lipemic plasma obtained

from rat fed oil (Fig. 8). DISCUSSION

The

genesis

of Triton-induced

clear

in vitro.

hy

percholesterolemia has been understood along the lines of biosynthesis and mobiliza tion. Frantz et al. (2) described the acceleration of cholesterol biosynthesis, especially

in liver, as the causal factor in this phenomenon, while Friedman et al. (6) considered the "mobilization" of cholesterol from tissues sufficiently enough to explain the origin of plasma excess cholesterol. The rise of hepatic cholesterogenesis was confirmed using 14C-acetate (2 , 3, 17, 18) and the decreased response of elevation of plasma cholesterol in partially hepatectomized animal (4) was observed. The increase of total body cholesterol of mice which were maintained on a chole sterol-free synthetic diet throughout the experimental period was reafirmed in this ex periment probably by the enhanced cholesterogenesis or by the retarded catabolism. In the results of Frantz et al. (2), Hirsch et al. (3), Garrattini et al. (18), and our experi ment (19) the cholesterogenesis of rat liver was enhanced significantly in a stage of hy percholesterolemia, in which the plasma cholesterol level still continued to elevate. As the hepatic concentration reaches to its maximum after the preceded high blood chole sterol level has begun to decline, the excess cholesterol mobilized in plasma would be taken up by liver in the later stage. However, we must consider this phenomenon in connection with the fact present ed by Friedman et al. (5, 6) that in intact or hepatectomized rats the elevation of plasma triglyceride or phospholipid was followed by a rise of cholesterol concentration. In our present result the rise of plasma triglyceride concentration preceded to that of chole sterol about four times in the ratio of increase. Linoleic acid contained in tissues is diet-dependent and can not be synthesized by the rat body (20). If the origin of excess triglyceride in post-Triton plasma consisted in the newly biosynthesized fatty acids, the linoleic acid content in triglyceride would be declined to the very low level, because the increase of plasma triglyceride reaches about forty times as large as normal level at twenty-four hours after Triton injection. Our result in this paper showed almost no difference in the proportion of linoleic acid between normal and Triton-treated groups. This suggests that the "mobilization" is a primary factor for the elevation of plasma triglyceride level. This supports strongly the data of isotopic experiment (19), in which the fatty acid synthesis of liver slice ex cised from Triton-treated rats was not accelerated compared to that of control animals . Though it does not preclude the possibility of fatty acid-transfer from phospholipid in tissues. The diminution of lipid clearance from the blood stream may be also a pos sible factor for Triton-hyperlipidemia, as already Schotz et al. (7) and Waddell et al. (21) demonstrated. Kellner et al. (22) described similar pattern of plasma lipid change in the experiment of rabbit given intravenously so-called "Lipid Mobilizing Factor" prepared from bovine pituitary, which showed to have an inhibitory activity on the lipoprotein lipase. This may be referred to Triton's action. Authors believe from these observations the "primary" factor for "Triton-hyper cholesterolemia" consists in the "mobilization" from tissues.

SUMMARY In the present study concerning with the genesis of Triton-induced hyperlipidemia in rats, we tried to study the origin of plasma excess triglyceride and measured the linoleic acid content of triglyceride in plasma and tissues. There was no significant difference in the linoleic acid content of plasma triglyceride before and after Triton injection, though the plasma triglyceride level rose significantly above the normal value. The result suggests that the rise of plasma triglyceride may be introduced by the mobilization of triglyceride from tissues, but not depended upon the stimulation of its biosynthesis, since linoleic acid is known to be not synthesized in the rat body. We also recognized that Triton injection increased the body total cholesterol content in mice and had an inhibitory activity on the lipemia clearance in vivo and in vitro. Acknowledgement : We are indebted to Dr. S. Tatsuoka (Directorof the laboratories)and Dr. K. Kanazawa(Vice-director)for their encouragementand to Dr. T. Kobayashifor his helpful advice. Thanks are also due to Mr. H. Matsumura for technical assistanceand to Mr. T. Shima for gas chromatographicalanalysis of fatty acid. REFERENCES 1) FRIEDMAN,M.: J. exp. Med. 97, 117 (1953) 2) FRANTZ, I.D. AND HINKELMAN,B.T. : Ibid. 101, 225 (1955) 3) HIRSCH, R.L. AND KELLNER,A.: Ibid. 104, 1 (1956) 4) HIRSCH, R.L. AND KELLNER,A.: Ibid. 104, 15 (1956) 5) FRIEDMAN,M. AND BYERS,S.O.: Amer. J. Physiol. 186, 13 (1956) 6) FRIEDMAN,M. AND BYERS,S.O.: Ibid. 190, 439 (1957) 7) SCHOTZ,M.C., ScANU, A. AND PAGE, I.H.:

Ibid. 188, 399 (1957)

8) BYERS,S.O., CADY, P. AND FRIEDMAN,M.: Ibid. 199, 833 (1960) 9) JANICKI, B.W. AND ARON, S.A. : Proc. Soc. exp. Biol., N.Y. 109, 507 (1962) 10) JANICKI,B.W., LEAHY,W.V.C., MCNICKLE,T.F. AND ARON, S.A. : Amer. J. Physiol. 202, 367 (1962) 11) SPERRY,W.M. AND WEBB, M. : J. biol. Chem. 187, 97 (1950) 12) ABELL,L.L., LEVY, B.B., BRODDIE,B.B. AND KENDALL,F.E.: Ibid. 195, 357 (1952) 13) KING, E.J. : Biochem.J. 26, 292 (1932) 14) CARLSON,L.A. AND WADSTROM,L.B. : Glin. chim. acta 4, 197 (1959) 15) CARROLL,L.A. : J. Lip. Res. 2, 135 (1961) 16) MILLER,J.P. AND COOPER, J.A.D. : Biochem. biophys.acta 27, 141 (1958) 17) GARATTINI,S., PAOLETTI,P. AND PAOLETTI,R. : Biochemistryof Lipids, edited by POPJAK, p. 184, Pergamon Press, Oxford (1960) 18) GARATTINI,S., PAOLETTI,R., BIZZI, L., GROSSI, E. AND VERTUA, R. : Drugs Affecting Lipid Meta bolism, edited by GARATTINI,S. AND PAOLETTI, R., p. 144, Elsevier Publishing Amsterdam (1961) 19) IMAI, Y. AND ARAMAKI,Y. : unpublished

Company,

observation

20) HORNING, M.G., WILLIAMS,E.A., MALING,H.M. AND BRODIE,B.B. : Biochem.Biophys. Research Com muns. 3, 635 (1960) 21) WADDELL,W.R., GEYER, R.P., SASLAW,I.M. AND STARE, F.J.: Amer. J. Physiol. 174, 39 (1953) 22) KELLNER, A., HIRSCH, R.L. AND FREEMAN,E.B. :J. exp. Med. 112, 1 (1960)