Elsevier Publishing Company, Amsterdam- Printed in The Netherlands EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS FED CHOLESTEROL-FREE DIETS

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1 Journal of A therosclerosis Research Elsevier Publishing Company, Amsterdam- Printed in The Netherlands EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS FED CHOLESTEROL-FREE DIETS PART 2. INFLUENCE OF VARIOUS CARBOHYDRATES D. KRITCHEVSKY*, P. SALLATA AND S. A. TEPPER Wistar Institute of Anatomy and Biology, Philadelphia, Pa. (U.S.A.) (Received 23 January, 1968) SUMMARY It has been shown that the addition of saturated fat to a semi-synthetic, cholesterol-free ration is atherogenic for rabbits. Experiments were carried out in which the carbohydrate component, which comprised 40 % of the diet, was either glucose, sucrose, starch or hydrolyzed starch. This diet was fed for 33 weeks. Starch was the most hypercholesteremic and atherogenic of the carbohydrates used and glucose was the least. Hydrolyzed starch resulted in higher serum cholesterol levels than did sucrose, but in slightly lower atheromata. Serum fl-lipoprotein cholesterol levels were elevated in all four dietary groups, least in the glucose-fed rabbits. The glucose group exhibited normal serum triglyceride and phospholipid levels. Serum phospholipid levels were only slightly elevated in the other dietary groups. Serum triglycerides were twice normal in the sucrose-fed rabbits and were elevated 3- and 5-fold in the starch and hydrolyzed starch groups, respectively. Liver cholesterol levels were elevated (4-6-fold) in all four carbohydrate-fed groups. Aortic cholesterol levels were elevated in all the experimental groups, and the aortic free/ester cholesterol ratio was much lower than that observed in the aortas of normal levels. The biliary bile acids were determined and showed deoxycholic acid to be the predominate component (over 90 %) in all groups. The data suggest that, in rabbits fed semi-synthetic diets, glucose is the least and starch the most atherogenic carbohydrate. In all rabbits fed the semi-synthetic diet, hypercholesteremia, elevations in serum fl-lipoprotein cholesterol and atheromata were observed. INTRODUCTION Our studies on the factors which cause a semi-synthetic, cholesterol-free diet to be atherogenic for rabbits, whereas a chow diet of similar protein-fat-carbohydrate * Wistar Professor of Biochemistry, Division of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (U.S.A.).

2 698 n. KRITCHEVSKY, P. SALLATA, S. A. TEPPER composition is notl, 2, suggest that the carbohydrate portion of the diet may be an important factor. Earlier studies on the semi-synthetic, atherogenic diet used glucose 3, sucrose 4 or glucose-starch~ as the carbohydrate source, but there has been no direct comparison of these different carbohydrates. Reports on the effects of different carbohydrates upon cholesteremia and atherosclerosis in rabbits6-s have been carried out, using cholesterol-containing diets. This report describes the effects of glucose, sucrose, starch and hydrolyzed starch upon the induction of atheroscierosis in rabbits fed a semi-synthetic, cholesterol-free diet. MATERIALS AND METHODS Male rabbits of the Dutch-belted strain (average starting weight 1740 g) were fed one of four experimental diets for a period of 33 weeks. The basic diet consisted of casein (25), carbohydrate (40), salt mix, USP XIV (5), cellulose (15), vitamin mix (1) and hydrogenated coconut oil (14). The rabbits were divided into four groups whose dietary carbohydrate component was either glucose (G), sucrose (S), starch (T) or a partially hydrolyzed starch (H). The latter was a commercial product (Amidex) prepared by acid hydrolysis of corn starch. A fifth group of rabbits (CO) was maintained on the common atherogenic regimen of rabbit chow, augmented with 2 % cholesterol and 6 % corn oil. The semi-synthetic diets were prepared to our specifications by General Biochemicals, Chagrin Falls, Ohio. At the end of the feeding period the rabbits were bled and killed and the livers and aortas removed. Aortic atheromata were graded visually on a 0-4 scale. The serum cholesterol was isolated as the digitonide using the SPERRY-WEBB 9 procedure, and color development was carried out, using the MANN 1~ reagent. Serum triglycerides 11 and phospholipids 12 were also determined. The serum a- and fl-lipoproteins were separated by dextran sulfate precipitation 1~ and analyzed for total cholesterol 9. Aliquots of the liver were saponified in 15 % Alc-KOH and the cholesterol was extracted and assayed colorimetricallyl0. The aortas of each group were pooled, extracted with chloroform-methanol (2:1, v/v) and the extract analyzed for free and total cholesterolg, 1~ The gall bladder bile of each group was pooled, deproteinized, and, after hydrolysis of the bile acid conjugated with NaOH under pressure, the bile acids were purified by the FeC13 method of DOUBILET 14. The bile acid content was assayed by gas-liquid chromatography using the triple component column described by OKISHIO AND NAIR 15. RESULTS AND DISCUSSION The autopsy results are presented in Table 1. We have included data obtained from 6 normal rabbits who were killed at the same time, but who had not been maintained in our colony for the duration of this experiment. The greatest mortality was observed in the cholesterol-fed group (CO), whereas the survival rate of all the carbohydrate-fed groups was similar. The rabbits in the four carbohydrate groups main- j. Atheroscler. Res., 1968, 8:

3 EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS FED CHOLESTEROL-FREE DIETS 699 TABLE1 AUTOPSY DATA ON RABBITS FED SPECIAL DIETS (33 WEEKS) Diet Survival Weight Liver Liver A theromata ratio change wt. (g) cholesterol arch thoracic (g) (g/100 g) Glucose (G) 7/ Sucrose (S) 6/ Starch (T) 6/ Hydr. starch (H) 8/ Cholesterol (CO) 3/ Normal (N) t tained their starting weight, within limits, but did not show the increases one might expect over an 8-month period. There is no simple explanation for the mortality rates observed in the course of this experiment. In an earlier experiment involving semi-synthetic diets 1 the mortality in most groups was much lower. The weight losses observed in the surviving animals are not large enough to suggest malnutrition and the greatest mortality was observed in the group (CO) whose survivors showed greatest weight gain. Average liver weights of the four carbohydrate-fed groups were slightly above those found in the normal rabbits. The average weight of the normal rabbits used was 2140 g and the liver weight represented 3.00 % of the body weight. The liver weights as percentage of body weight for the carbohydrate-fed groups were: G: 4.49 %; S: 4.15 %; T: 3.56 % and H: 4.32 %. The liver size of the cholesterol-fed group was much larger, % of body weight. The liver cholesterol levels of all the carbohydrate-fed groups were 4-6 times normal, with the lowest level observed in the sucrose-fed group. The liver cholesterol levels of group S (sucrose) were lower than those of groups G (glucose) and T (starch), below the 5 % level of probability (0.0s > p > 0.01). The atheromata were most severe in the group of rabbits fed cholesterol, as expected. Among the groups fed different carbohydrates, that fed starch exhibited the most severe atheromata, the glucose fed group exhibited the least. The arch atheromata in these groups were glucose, t- 0.25; and starch, 2.25 :k 0.31 (0.05 > P > 0.01). The atheromata observed in the groups fed sucrose and hydrolyzed starch were intermediate between those of the glucose- and starch-fed rabbits. The level of arch atheromata in the sucrose group (1.92 ~ 0.55) was quite high, but the difference between this group and the glucose group was not statistically significant. The level of arch atheromata in group H was 1.69 _t_ The finding that sucrose is more atherogenic than glucose is not surprising, since GRANT AND FAHRENBACH 6 had demonstrated a similar difference in atheromata in rabbits fed diets containing cholesterol and glucose or fructose. The even higher atherogenicity of starch has not been reported previously, but this material has been shown to be more hypercholesteremic than either glucose or sucrose in cholesterol- j. Atheroscler. Res., 1968, 8:

4 700 D. KRITCHEVSKY, P. SALLATA, S. A. TEPPER carbohydrate-fed rabbits 8, and we, too, found this diet to be the most cholesteremic. Whether this is generally true or whether the starch-induced hypercholesteremia is peculiar to the rabbit must await further investigation. PORTMAN et al. 16 found starch to be less cholesteremic than either glucose or sucrose in rats fed cholesterol and cholic acid. Similar results were obtained by STAUB AND THIESSEN 17, but they also found that potato starch was more cholesteremic than either wheat or corn starch. We observed is when rats were fed the identical diets as were the rabbits, the cholesterol levels of the starch, sucrose and glucose groups were not significantly different. STAUB AND THIESSEN 17 made a similar observation when these carbohydrates were fed to rats in cholesterol-free diets. The data reviewed in the foregoing paragraph emphasize how various animal species may react differently to similar dietary situations. The results which we and others have communicated cannot, at present, be directly related to human atherosclerosis. Continuing studies in various animal species and observation in man will show eventually where there are general similarities in response to high carbohydrate diets and where the various species diverge. TABLE 2 AORTIC LIPIDS OF RABBITS ON SPECIAL DIETS (33 WEEKS) Group No. Atheromata Cholesterol (mg] 100 g) arch thoracic total free FIE Triglyceride Phospholipid (rag/1 O0 g) (rag/1 O0 g) G I S T H CO N lost Table 2 summarizes the lipid spectrum of the pooled thoracic aortas of the various groups of rabbits. The free/ester cholesterol ratio is usually between 10 and 20 in normal rabbit aorta2,19, z~ whereas in cholesterol-fed rabbit aortas it is between 0.4 and 1.0. Similar differences have been observed between the aortas of young children and adults 21. Our present findings bear out the earlier reports. The total cholesterol levels of the thoracic aortas were roughly parallel to the visual grading. The aortic triglycerides were higher than normal in all of the groups, with only the starch group exhibiting a gross elevation. The aortig triglyceride content in rabbits is generally constant during the course of cholesterol feeding and does not exhibit the gross elevation observed in the aortic cholesterol levels 22. The aortic phospholipid was elevated only in the cholesterol- and glucose-fed groups. The rabbit aorta synthesizes phospholipid during the development of cholesterol-induced atherosclerosis23. The elevations in aortic phospholipid observed in the aortas of the carbohydrate-fed rabbits may also reflect a response to cholesterol deposition. The serum lipids of the rabbits used in this experiment are presented in Table 3. The serum cholesterol levels of the starch-fed group were significantly higher than those of the glucose (P < 0.001) or sucrose (0.05 > P :> 0.01) fed groups. The serum

5 EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS FED CHOLESTEROL-FREE DIETS 701 TABLE 3 SERUM LIPIDS OF RABBITS FED SPECIAL DIETS (33 WEEKS) Diet No. Cholesterol (mg/100 ml) total free ester alpa Triglyceride Phospholipid %a a/fl (mg/lo0 ml) (mg/looml) G ~ 31b S :[: T H CO 3 I N S 52+_ :I: _ 2 a alp ---- serum a-lipoprotein. b Standard error. cholesterol level of the group fed hydrolyzed starch was also significantly higher than that of the glucose group (0.05 :> P :> 0.01). The serum cholesterol levels of the carbohydrate fed groups are roughly in the order of the severity of their atheromata. All of these groups also showed elevations in serum fl-lipoprotein cholesterol levels and reductions in the ratio of serum a-/fl-lipoprotein cholesterol compared to the normal rabbits. The a-/fl-lipoprotein cholesterol ratios of groups G and N are identical with those observed in an earlier experiment 2. These results confirm the observation of many investigators that elevations of fl-lipoprotein cholesterol predispose to atherosclerosis. This is true even with the relatively modest cholesteremias found in this experiment. The serum triglycerides of the carbohydrate-fed rabbits were higher than normal levels, but with the exception of group H the elevations were 2-3 times normal, whereas cholesterol levels were 4-6-fold higher. The triglyceride level of group H was significantly higher than that of group G (P < 0.01). The serum phospholipid levels of group G were normal and those of the other groups were not significantly elevated. Since dietary carbohydrates have been shown to affect biliary cholic acid levels 24 and bile flow 25 in rats, we determined the bile acids present in the pooled gall bladder bile of the rabbits in the various dietary groups. Table 4 shows that, as PORTMAN et al. 24 found in the rat, glucose and sucrose did reduce the level of cholic TABLE 4 BILIARY BILE ACIDS (%) OF RABBITS FED SPECIAL DIETS (33 WEEKS) Diet Deoxycholic acid Cholic acid G 100 S 98 2 T 91 7 H 96 4 CO 93 7 N 97 3

6 702 D. KRITCHEVSKY, P. SALLATA, S. A. TEPPER acid when compared with starch. The level of cholic acid was highest in the two groups (T, CO) which had the most severe atheromata, but in view of the small amount of cholic acid found in all of the samples it is doubtful whether this plays a role in the development of atherosclerosis in the rabbit. These experiments indicate that the type of carbohydrate present in a semisynthetic diet which is atherogenic for rabbits, may significantly affect the cholesteremia and severity of atherosclerosis. These experiments are being extended to include other carbohydrates. The effect of fructose will be of special interest. ACKNOWLED GEM ENTS This work was supported by a Grant (No. 2-R01-HE ) and a Research Career Award (4-K06-HE ) from the National Heart Institute, U.S. Public Health Service and by a grant-in-aid from the National Dairy Council. We are grateful to Dr. P. P. Nair for assistance and advice in the experiments involving gas-liquid chromatography of the bile acids. We are indebted to Dr. D. Rathmann, Corn Products Co., for the generous gift of the hydrolyzed starch (Amidex) used in formulating the diets used in this experiment. REFERENCES 1 KRITCHEVSKY, D. AND S. A. Tt~PPER, Factors affecting atherosclerosis in rabbits fed cholesterolfree diets, Part 1 (Influence of chow components), J. Atheroscler. Res., 19(~8, 8: KRITCHEVSKY, D. AND S. A. TEPPER, Experimental atherosclerosis in rabbits fed cholesterolfree diets: Influence of chow components, J. Atheroscler. Res., 1968, 8: LAMBERT, G. F., J. P. MILLER, R. T. OLSEN AND D. V. FROST, Hypercholesterolemia and atherosclerosis induced ill rabbits by purified high fat rations devoid of cholesterol, Proc. Soc. exp. Biol. (N.Y.), 1958, 97: GRESHAM, G. A. AND A. N. HOWARD, Atherosclerosis produced by semisynthetic diet with no added cholesterol, Arch. Path., t962, 74: MALMROS, H. AND G. WIGAND, Atherosclerosis and deficiency of essential fatty acids, Lancet, 1959, ii: GRANT, W. C. AND M. J. FAHR~;NBACH, Effect of dietary sucrose and glucose on plasma cholesterol in chicks and rabbits, Proc. Soc. exp. Biol. (N. Y.), 1959, 100: WELLS, W. W. AND S. C. ANDERSON, The increased severity of atherosclerosis in rabbits on a lactose containing diet, J. Nutr., 1959, 68: POLLAK, O. J., Cholesteremia of rabbits fed carbohydrate diets, J. Amer. Geriat. Soc., I961, 9: SPERRY, W. M. AND M. WEBB, A revision of the Schoenheirner-Sperry method for cholesterol determination, J. biol. Chem., 1950, 187: MANN, G. V., A method for cholesterol in blood serum, Clin. Chem., 1961, 7: VAN HANDEL, E. AND D. B. ZILVERSMIT, Micromethod for the direct determination of serum triglycerides, J. Lab. clin. Mcd., 1957, 50: FISKE, C. H. AND Y. SUBBAROW, The colorimetric determination of phosphorus, J. biol. Chem., 1925, 66: CASTAIGNE, A. AND A. AMSIELEM, t~tude de Ia r6partition des deux fractions libre et esterifi~e du cholest6rol entre les a et /5 lipoprot6ines, Ann. Biol. clin., 1959, 17" DOUBILET, H., Differential quantitative analysis of bile acids in bile and in duodenal drainage, J. biol. Chem., 1936, 114: OKISHIO, T. AND P. P. NAIR, An improved column for gas-liquid chromatography of substituted cholanic acids, Anal. Biochem., 1966, 15:

7 EXPERIMENTAL ATHEROSCLEROSIS IN RABBITS FED CHOLESTEROL-FREE DIETS PORTMAN, O., E. Y. LAWRY AND D. BRUNO, Effect of dietary carbohydrate on experimentally induced hypercholesteremia and hyperbetalipoproteinemia in rats, Proc. Soc. exp. Biol. (N.Y.), 1965, 91: STAUB, H. W. AND R. THIESSEN, jr., Dietary carbohydrate and serum cholesterol in rats, Fed. Proc., 1967, 26:490 (abstract). 18 KRITCHEVSKY, D. AND S. A. TEPPER, Unpublished observation. 19 SWELL, L., M. D. LAW AND C. R. TREADWELL, Tissue cholesterol ester and triglyceride fatty acid composition of rabbits fed cholesterol diets high and low in linoleic acid, J. Nulr., 1962, 76: ~ PARKER, F. AND G. F. ODLAND, A correlative histochemicai, biochemical and electron microscopic study of experimental atherosclerosis in the rabbit aorta with special reference to the myo-intimal cell, Amer. J. Path., 1966, 48: SCOTT, R. F., R. A. FLORENTIN, A. S. DAOUD, E. S. MORRISON, R. M. JONES AND M. S. R. HUTT, Coronary arteries of children and young adults. A comparison of lipids and anatomic features in New Yorkers and East Africans, Exp. molec. Path., 1966, 5: NEWMAN, H. A. I. AND D. B. ZILVERSMIT, Accumulation of lipid and non-lipid constituents in rabbit atheroma, J. Atheroscler. Res., 1964, 4: SHORE, M. L., D. t~. ZILVERSMIT AND R. F. ACKERMAN, Plasma phospholipid deposition and aortic phospholipid synthesis in experimental atherosclerosis, Amer. J. Physiol., 1955, 181: PORTMAN, O. W., G. V. MANN AND A. P. WYsocKI, Bile acid excretion by the rat: nutritional effects, Arch. Biochem., 1955, 59: HEGGENESS, F. YV., Galactose and bile flow, Proc. Soc. exp. Biol. (N.Y.), 1959, 101: J. A theroscler. Res., 1968, 8: