Effects of Dietary Proteins, Methionine and Vitamins on Plasma Lipids and Atherogenesis in Cholesterol-Fed Cockerels

Transcription

1 Effects of Dietary Proteins, Methionine Vitamins on Plasma Lipids Atherogenesis in Cholesterol-Fed Cockerels By JEREMIAH STAMLER, M.D., RUTH PICK, M.D. AND LOUIS N. KATZ, M.D. With the assistance of Mrs. D. Century Mr. P. Johnson Downloaded from by on August 8, 8 by the total dietary pattern. An hypothesis was put forward, suggesting that dietary imbalance excess of some nutrients (e.g., total calories, total fats, saturated fats, refined carbohydrates) inadequacy (absolute or relative) of others (e.g., proteins, s, minerals, essential fatty acids) may be particularly pernicious in inducing hyperemia atherosclerosis. The present experiments were undertaken to test this hypothesis in chicks, particularly respect to inter-related effects of lipid,, amino acids, proteins s. METHODS Four series of experiments were accomplished, utilizing a total of 8 cockerels ( birds per ) (table ). The department's established technics for studies on experimental atherosclerosis From the Cardiovascular Department, Medical Beaearch Institute, Michael Reese Hospital, Chicago,. This department is supported by the Michael Reese Research Foundation. Supported by grants from the National Heart Institute, TJ. S. Public Health Service (H-76), from the Chicago Heart Association. Originally presented before the Federation of American Societies for Experimental Biology. Work done during Dr. Stamler's tenure of an Established Investigatorship of the American Heart Association. Received for publication February 6, 958. Methionine deficiency in the presence of a high-fat, high- diet aggravates atherogenesis in the cockerel. High-protein, high- supplementation suppresses the atherogenic effect of a -fat-containiiig diet. High-protein supplementation alone had a suppressive effect; high- supplementation had no effect. However, the combined protein supplement was the most effective. RECENT work in this department led were used throughout. One-day-old Hy-line to the tentative conclusion that patterns chicks were obtained from a commercial hatchery of emia atherogenesis in man reared in a battery brooder. The experi- experimental animals may be influenced mental periods were ages 6 to, 4 to, 8 to 5, 3 to 3 weeks in series 39, 4, 46, 47 respectively. In all series chicks were fed a diet supplemented 5 per cent cottonseed oil (,,, to per cent in series 39, 4, 46, 47 respectively). Over-all, two types of diets were utilized, one commercial chick starter mash as its base, the other purified ingredients (table ). In the latter, two types of purified basal proteins were used, casein-gelatin (series 39, 4, 46) or soy protein (series 46). In the control s on purified ration, the protein level was 35 per cent (s D, table ); in the high-protein s on purified ration, it was increased to 63 per cent (s 3 3D); in the reduced-protein, it was decreased to per cent ( 4, series 39). The sulfhydril amino acid d,l-methionine was incorporated in the purified rations of all s, except the low-methionine s (table ). In the control s, it was given at the level of.5 to.75 per cent of the ration; in the highprotein s,.5 to.5 per cent. Choline inositol (. to.3. to. per cent, respectively, for the control s on purified ration) were increased to. to.5 per cent.5 to. per cent, respectively, in the highprotein s. In the high- s, premises of multiple fat-soluble water-soluble s were incorporated in the ration at levels five times as great as for the control s on purified ration. Complete details concerning the salt mixtures utilized, as well as other aspects of the diets, are available from the authors upon request. In series 39, the effects of high -high 44 Circulation Research, Volume VI. July 9SS

2 DIETARY PROTEINS, METHIOXINE AND VITAMIXS 443 Downloaded from by on August 8, IC-O 4 -C-O 46 --C- 47f -.5 C-O TABLE. General Experiviental Design Group D 3D 3M 5D 3M 6 7 Over-all dietary characterization Mash Purified Purified Reduced protein Purified Low methionine Purified Mash Purified Purified Mash Purified Casein gelatin Puri fied Soy protein (D) Purified (D) Mash Borderline Low methionine Purified (D) t Mash Mash High protein Mash Mash *.5 C-O, C- C- are mash or purified feed supplemented.5, or per cent plus 5 per cent cottonseed oil. In series 46, mash birds ate C-O, purified diet chicks C-O. t In this series, each was made up of 7 or 8 birds at the start of the experimental feeding regimen. Two birds from each were sacrificed after 5 weeks. t The purified ration of this contained the usual soy protein (not ot-protein) at the 35 per cent level, but methionine supplementation was omitted. fat combined low protein, low methioninc or high protein-high diets were explored (table ). Low raethionine diet was accomplished by use of a-protein (5 per cent), a soy protein low in this ainino acid, plus omission of methionine in the ration (series 39, 5, table ). High protein-high experiments were repeated in series In series 46, high -high fat combined high protein-high was also studied, using commercial mash as the ration base. 47 repeated this design, extending it to include TABLE. Feed Intake, Body Weight Plasma Lipids in Cockerels on Purified Diets Serirt ^ D 46-3D 46-5D Type diet High protein Reduced protein Low methionine High protein Soy protein High protein (Soy) High Borderline methionine Feed intake (Gm./ chick/ day)* weight (Gm.) 9±75f ± ±43 465±68 7 ±8 355 ±4 Seriee oil supplement* total (mg. %) 5±8.9 96±68 36 ± ±65 8±. 557 ±77 63 ± ±38 3 ±.6 95 ±.8 46±5 C/P ratio 76±3 5 ± ±4. * Data collected on as a whole. t Phosphohpids were determined on pooled specimens of plasma. The C/P ratio is the ratio total phospholipids t Stard error of mean. analysis of the separate effects of high proteins high s individually (table ). In these experiments commercial mash, the protein supplement was casein, soy protein, defatted liver, defatted fish meal, Brewer's yeast, 7 per cent of each; the supplement was identical that used in the purified ration. RESULTS Effects of Reduced Protein or Low Methionine Content in a Purified Ration Containing Cholesterol Oil. These dietary combinations were studied in series 39 46, s 4, 5 5D (tables 3). The reduced protein diet (series 39, 4) was associated a lower feed intake weight gain. Plasma lipid levels were essentially similar in the reduced protein control s. A higher incidence of thoracic aorta was observed in the reduced protein

3 444 STAMLEE, PICK AND KATZ Downloaded from by on August 8, 8 TABLE 3. Atherogenesis in Cockerels on Purified Diets no D 46-3D 46-5D Per cont Grots thoracic aorta atherogenesifl grade or > 5. Mean grade hirds t.8±..3±..7±..3±.8± 5.3±.3±.3± Microscopic coronary atherogeneris of bird* of vessels (all birds) t.9±.9.±.3 9.±..±.9.±. 5.8±.7.8±. 4.7±.7 * Lesions in the thoracic aorta were graded on an arbitrary scale from -4. t This is the average of the grades for those birds ; chicks graded were excluded in calculating this mean. t Two stard Sudan-etained sections of the heart were examined microscopically, a count was made of all arterioles arteries visualized, the percentage of those exhibiting atherosclerotic plaques. This is the mean per cent for the, is an index of severity of atherosclerotic involvement. ; atherogenesis was otherwise essentially similar in the two s.* The low methionine exhibited a level of feed intake weight gain comparable to that of the control. Cholesterolemia, /phospholipid (C/P) ratio, incidence of thoracic coronary, severity of coronary were all increased in the low methionine. The borderlineadequate methioniue (series 46, 5D) was not significantly different from its matched control. Effects of High Protein-High Vitamin *It should be noted that the control on purified ration ingested protein at the 35 per cent level (5 per cent casein + per cent gelatin). The reduced protein consumed per cent protein ( per cent casein -f per cent gelatin). In later experiments, lowering of protein to to 5 per cent a frank low protein ration resulted in marked increase in liyperemia atherogenesis in cockerels on a high fat-high ration.* TABLE 4. Feed Intake, Body Weight Plasma Lipids in Cockerels on Mash Diet M M Type diet High protein High High protein High High protein Feed intake (Cm/ chick/ day) weight (Gm.) 4±7 49 ±3 86 ±87 total (nig. %) 447 ±66 8±9 5 ±3 978 ±88 55± 6 ±63 33 ±57 658± 74±34 C/P ratio Supplementation of a Purified Ration Containing Cholesterol Oil. This experiment was accomplished in series 39, 4, 46 (tables 3). Feed intake weight gain tended to be less in high protein-high s, compared controls. In of the 3 series of experiments, hyperemia was less marked in the high proteinhigh s. Except for one bird in series 46 a minimal lesion, thoracic aorta atherogenesis was absent in high protein-high chicks of all series. No coronary were noted in these birds in series 39 46, in contrast to the controls. Coronary atherogenesis was also less marked in the experimental s in series 4 (table 3). Effects of Soy Protein vs. Casein-Oelaiin in a Purified Ration Containing Cholesterol Oil. Peed intake weight gain were greater 35 per cent soy protein than the same level of casein-gelatin (series 46, s D). Hyperemia, aorta coronary atherogenesis tended to be less marked the soy than the caseingelatin protein. Effects of High Protein-High Vitamin Supplementation of Commercial Mash Containing Cholesterol Oil. Feed intake tended to be slightly less in the high protein-high s, as was to be expected in view of this ration's greater caloric content (table 4). Gain in weight was greater in

4 DIETAEY PROTEINS, METHIONINE AND VITAMINS 445 Downloaded from by on August 8, 8 the experimental s. Hypereholesterolemia, aorta coronary atherogenesis were less marked in the high protein-high supplemented. Effects of Separate Supplementation ivith High Protein or High Vitamin in Commercial Mash Containing Cholesterol Oil. This single supplementation resulted in lower feed intakes weight gains (series 47, s 6 7) (table 4). in contrast to the high protein-high (3M), both the high protein-alone the high -alone s had levels of hyperemia as high as or higher than the controls. Correspondingly, the high -alone had coronary atherogenesis as marked as the control (table 5). In the supplemented high protein-alone, incidence of coronary atherogenesis was markedly lower than in the controls, was similar to that of the high protein-high. Effects of Cholesterol OH Supplementation of Purified Ration vs. Commercial Chick Starter Mash. In all series, supplementation at the same (series 39 4) or twofold greater (series 46) levels in the purified, compared the commercial mash, yielded less marked hyperemia atherogenesis (, series 46, tables 4 5, series 46, tables 3*). DISCUSSION The findings of these experiments indicate that high protein-high supplementation suppressed hyperemia atherogenesis in -oil-fed cockerels. This was true both purified commercial mash diets. Vitamin supplementation alone had no such effect. Protein supplementation alone apparently suppressed coronary atherogenesis in a manner similar to high protein-high. However, protein alone apparently had less definitive clear-cut effects than the combination. It would seem, therefore, that the high protein diet is the decisive factor responsible for these results, but that high intake Data on the other s involved in this comparison are available from the authors upon request. TABLE 5. Atherogenesis in Cockerels on Mash Diet no M M Grc)S3 thoracic aorta atheroneneais 4 grade or> 6 Mean grade birds ± 4 ±. 4 ± 5 ± 3 ± Microscopic coronary atherogenesis of birds 9.9 SO.O of vessels lesionb (all birds).±4,3± 8 ±.8=fcl.9± 7.6* has an adjuvant, synergistic influence. These results are consistent the concurrent independent observations in chicks by other workers. 6 Antiatherogenic effects of protein have apparently not been hitherto reported. These experiments further demonstrate that inadequacy of methionine intake aggravated the hyperemic atherogenic effects of -oil ingestion. They also confirm in cockerels observations previously reported in monkeys rats. 8 ' The present results further indicate that addition of to the chick diet induced less hyperemia atherogenesis in a purified than in a regular mash ration. The mechanism of this effect is not apparent from the data of this experiment. It may be a resultant of the greater protein intake the purified ration (35 vs. approximately per cent). However, the results per cent protein in a purified ration (series 39, 4) do not support this suggestion, since enhancement of hyperemia was not observed. These data are difficult to interpret, however, in view of the differences in feed intake growth pattern. This problem was explored further. 5 Finally, the data of these experiments further suggest that different results may be obtained qualitatively different proteins in a -oil-supplemented purified ration. Thus, hyperemia atherogenesis were less marked soy protein than casein-gelatin (series 46, s D). Similar findings were obtained independently by other workers

5 446 STAMLER, PICK AND KATZ Downloaded from by on August 8, 8 The relevance (if any) of these findings for the problem of possible interrelationships among diet, emia atherogenesis in man is dealt elsewhere. 5 SUMMARY Inadequate methionine intake led to increased hyperemia, coronary aorta atherosclerosis in chicks on a oil-supplemented purified ration. High protein-high supplementation partially suppressed hyperemia atherogenesis in birds on oil rations, of both the purified commercial mash varieties. High protein supplementation alone tended to produce some of these effects, whereas high supplementation alone did not. Chicks fed a purified ration developed less marked hyperemia atherogenesis than birds on a commercial feed, even when the former ingested greater quantities of. In purified rations containing oil, emia atherogenesis tended to be less marked soy protein than casein-gelatin (both at the 35 per cent level). ACKNOWLEDGMENTS We are deeply indebted to Dr. H. M. Scott, University of Illinois, Urbana, Illinois for advice concerning the composition of the purified diets. It is a pleasure to express appreciation to the members of the experimental atherosclerosis technical team of the Cardiovascular Department, Miss Mildred Michael, Mrs. Charlene Thompson, Mrs. Eva Miller, Mrs. Montez Vankinscott (research technicinns, chemistry) Messrs. David Bryant Grady Crowley (laboratory assistants). SUMMABIO ix INTERLINGUA Inadequate nivellos dietari de methionina resultava in augmentos de hyperemia e de atherosclerosis coronari e aortic in gallettos recipiente un dieta purificate con supplementos de e oleo. Alte supplementos de proteina e de vitaniina resultava in un suppression partial del hyperemia e del atherogenese in aves recipiente dietas a e oleo, tanto del typo purificate como etiam de provenientia commercial. Alte supplementos de proteina sol (i.e. sin supplementation de as) tendeva a producer certes de iste effectos, durante que alte supplementos de a (sin supplementation proteinic) non monstrava un tal tendentia. Gallettos recipiente uu dieta purificate disveloppava minus marcate grados de hyperemia e de atherogenese que gallettos recipiente un dieta commercial, mesmo in casos in que le dieta purificate contineva plus alte quantitates de. In dietas purificate e supplementate per e oleo, le emia e le atherogenese tendeva a esser minus marcate con proteina de soja que con caseina e gelatina (in ambe casos a nivellos de 35 pro cento). REFERENCES. KATZ, L. N., STAMLER, J., AND PICK, R.: Nutrition atherosclerosis. Fed. Proc. 5: 885, STAMLER, J.: Basic research on atherosclerosis. J. Am. Pharni. Mfgs. A. 955, p : Current epideiniological, clinical, laboratory research findings on the etiology of atherosclerosis. Nebraska State M. J. 4: 75, KATZ, L. N., AND STAMLER, J.: Experimental Atherosclerosis. Springfield,., Charles C Thomas, STAMLER, J., PICK, R., AND KATZ, L. N.: Effects of dietary protein carbohydrate level on emia atherogenesis in cockerels on a high-fat, high- mash. Circulation Research 6: 447, KOKATAUR, M., RAND, N. T., KUMMEBOW, F. A., AND SCOTT, H. M.: Effect of dietary protein fat on changes of serum in mature roosters. Fed. Proc. 6: 389, GRANT, W. C, AND FAHRENBACH, M. J.: Influence of sucrose casein on plasma in animals on purified diets. Fed. Proc 6: 5, MANN, G. V., ANDRUS, S. B., MCXALLY, A., AND STARE, F. J.: Experimental atherosclerosis in Cebus monkeys. J. Exper. Med. 98: 95, FILLIOS, L. C, AND MANN, G. V.: Influence of sulfur amino acid deficiency on metabolism. Metabolism 3: 6, WlSSLER, R. "W., ElLERT, M. L., SCHROEDER, M. A., AND COHEN, L.: Production of lipomatous atheromatous arterial in the albino rat. Arch. Path. 57: 333, Li, T., AND FREEMAN, S.: Experimental lipemia hyperemia by protein depletion by feeding in dogs. Am. J. Physiol. 45: 66, 946.