The Journal of Nutrition 1965 Volum 86 No.4

Cardiovascular Lesions, Blood Lipids, Coagulation and Fibrinolysis in Butter-induced Obesity in the R at* 1 SHAPUR NAIMI, GEORGE F. WILGRAM, MARTIN M. NOTHMAN AND SAMUEL PROGER Pratt Clinic-New England Center Hospital and the Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts ABSTRACT A study was made of obesity in the rat, induced by feeding a high butter fat diet over a long period and possible effects of obesity on the development of cardiovascular lesions. A group of 17 male W istar albino rats was fed a diet that contained 40% butter by weight (providing about 65% of the calories). To evaluate the independent contribution of obesity to the development of cardiovascular disease, repeated measurements were made of other parameters that might be affected by fats, namely, blood lipids, coagulation and fibrinolysis. The animals became grossly obese but did not develop any significant changes in blood lipids, coagulation and fibrinolysis throughout the period of the experiment (average of 428 days). Nor were there any significant cardiovascular lesions at the end of this period, beyond those normally observed in the aging rat. It was concluded that under the conditions of this experi ment where no significant changes occurred in blood lipids, coagulation and fibrino lysis, butter-induced obesity in the rat does not materially predispose to the develop m ent of cardiovascular lesions. Obesity has been implicated frequently tion and fibrinolysis over a period of several in the development of cardiovascular dis months, it was necessary to compare the ease (1, 2), and yet the clinical evidence measurements in the experimental group for this is by no means incontrovertible with that of a normal group to safeguard (3-6). Moreover, in view of the emphasis against any variation in the activity of placed on this aspect of obesity in man, reagents used at different periods of the long-term studies of the atherogenic effect experiment. This was particularly im of experimental obesity have not received portant for coagulation studies where dif the attention they might. It was there ferent batches of substrates and reagents fore considered worthwhile to study ex may show considerable variation in activ perimental obesity in the rat as induced ity. The normal group selected for this by a high fat diet; and in addition, to study purpose was a chow-fed group, since in the independent effect of obesity on the de our previous studies (7), we have had velopment of atherosclerosis by the meas extensive experience with the level of blood urement of some other parameters that lipids, coagulation, fibrinolysis and the might be affected by fats, such as blood normal variation thereof in this group, and lipids, coagulation and fibrinolysis. Also, since this is the accepted normal diet of a high butter fat diet was selected for the laboratory rats about which extensive data study because of the presumed atherogenic have accumulated over the years. effect of saturated fats. The statistical study both in the initial and the terminal phases of the experiment MATERIALS AND METHODS was carried out between the experimental Two groups of rats were used. Each group and the normal group. However, group consisted of 17 male Wistar albinos. this was used only as an indirect way to The experimental group received a diet detect the possible changes in the level of the composition of which appears in table blood lipids, coagulation and fibrinolysis in 1. The butter content of this diet was Received for publication March 8, 1965. 40% by weight which provided about 65% 1 Supported by Public Health Service Research of the calories. Since the studies involved Grants no. HE-03753 and GM-9726 from the National Institutes of Health, and by a Training Grant from the measurement of blood lipids, coagula the National Heart Institute (5 T1 HE 5391). J. N utrition, 86 : ’65 325 326 S. NAIMI, G. F. WILGRAM, M. M. NOTHMAN AND S. PROGER TABLE 1 used for 2 frozen sections. Thus, a mini Composition of the diet of the experimental group mum of 10 frozen sections, not more than 5 a thick, was prepared from each rat heart zvt % so that as many cardiac lesions as possi Powdered cellulose 1 6 .0 ble would be detected. The aortas were C a s e in 2 2 0 .0 Choline chloride 0 .2 first stained in the gross and examined for Salt m ixture 3 4 .0 lesions with a magnifying lens. They were S u c ro s e 2 7 .8 then cut longitudinally on the freezing mi Vitamin mixture 4 2 .0 crotome, mounted, and searched for le 4 0 .0 B u tte r sions under the microscope. The frozen 1 Alphacel, Nutritional Biochemicals Corporation, sections were stained with oil red O in C leveland. 2 Casein (purified), Nutritional Biochemicals Corpo triethylphosphate. Livers and kidneys were ra tio n . 3 The salt mixture (Nutritional Biochemicals Corpo embedded in paraffin and stained rou ration) was the Wesson modification of the Osborne tinely with hematoxylin and eosin. and Mendel salt mixture. The composition in per cent by weight is as follows: calcium carbonate, 21.000; Blood coagulation and lipid studies and copper sulfate ( 5 H 2O), 0.039; ferric phosphate, 1.470; manganous sulfate (anhyd.), 0.020; magnesium sul methods. The studies of blood lipids, fate (anhyd.), 9.000; potassium aluminum sulfate, coagulation, and fibrinolysis were carried 0.009; potassium chloride, 12.000; potassium dihydro gen phosphate, 31.000; potassium iodide, 0.005; sodium out in both groups simultaneously at chloride, 10.500; sodium fluoride, 0.057; tricalcium phosphate, 14.900. monthly intervals, in both the early phase 4 Each kilogram of the vitamin mixture contained the following triturated in dextrose: vitamin A con of the experiment, that is, the first 4 centrate (200,000 U /g), 4.5 g; vitamin D concentrate months, and the late phase, that is, the (400,000 U/g), 0.25 g; a-tocopherol, 5.0 g; ascorbic acid, 40.5 g; inositol, 5.0 g; niacin, 4.5 g; riboflavin, last 4 months of the experiment. Rats 1-0 g; pyridoxine-HCl, 1.0 g; thiamine-HCl, 1.0 g; Ca were fasted for 16 hours before blood was pantothenate, 3.0 g; biotin, 20 mg; folic acid, 90 mg; and vitamin B 12, 1.35 m g. collected from the femoral vein. Since a number of blood coagulation and lipid de the experimental group over the many terminations require relatively substantial months of the experiment. As such the volumes of blood, it was not feasible to normal group should not be considered as obtain enough blood from a single animal a control group. The studies on the ex (unless killed) for all the studies. How perimental group in the initial phase of ever, to minimize the possible errors from the experiment provide the true control for this source, the same number and type of the studies in this same group toward the blood coagulation and fibrinolysis tests end of the experiment. were made in both groups at each veni The average starting weight of the ani puncture. Each rat was anesthetized with mals in the normal group was 410 g and ether, and blood was drawn with a 21- in the experimental group 390 g (the gauge siliconized needle and syringe by a small differences were not statistically sig 2-syringe technique. The volume of blood nificant). All animals were housed in in drawn at each venipuncture varied from dividual cages and were fed ad libitum. 3 to 6 ml. Three per cent sodium citrate Pathological studies and methods. Six was used as the anticoagulant throughout, rats in the experimental group were killed and 9 volumes of blood were added to 1 for pathological studies after they had re volume of citrate. Blood was centrifuged ceived the diet for 6 months. The rest of at 2500 rev/min for 10 minutes. Blood the animals in both groups were main and plasma were kept in melting ice tained in the experiment for an average of throughout. Observations were made in 428 days (range 200 to 580 days). Complete duplicate. Since the trends were similar autopsies were done on all animals, but only both in the early and the late phase of the heart, aorta, liver and kidney were sub experiment, in each phase of the study re mitted to histological preparations. These sults were pooled for statistical purposes. organs were removed and were fixed in 10% calcium formalin after death or kill Coagulation methods ing of the animals. Hearts and aortas were Prothrombin time. The one-stage method then embedded in gelatin for the prepara of Quick (8) was used except that a saline tion of frozen sections. Each heart was cut extract of acetone-dried human brain was into at least 5 slices, each of which was used as a source of thromboplastin. STUDIES IN BUTTER-INDUCED OBESITY 327 Stypven time. Russell viper venom2 lary tube. The end point was taken at the was diluted 1:10,000 with distilled water, instant that the liquid level in the capil then diluted 1:100,000 with 0.9% saline. lary tube reached the upper surface of the Platelet-poor plasma, 0.1 ml, was incubated clot. with 0.1 ml of freshly prepared venom Platelet count. The method of Brecher for 30 seconds at 37°; then 0.1 ml of and Cronkite (15) was used. 0 . 0 2 m calcium chloride was added and the Plasma fibrinogen. The method of clotting time determined. Ratnoff and Menzie as modified by Holburn Prothrombin levels. Plasma prothrom (16) was used.

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