Cystine Deficiency and Cholesterol Metabolism in Primates

Transcription

1 Cystine Deficiency and Cholesterol Metabolism in Primates By George V. Mann, Sc.D., M.D. A deficiency of sulfur amino acids impairs sterol metabolism in several species. 1 ' - The present work with primate species was directed at an explanation of this phenomenon. The role of dietary cholesterol in the regulation of blood cholesterol has been best documented for the rat* In that rodent, synthesis of cholesterol is reduced by the administration of dietary cholesterol. Gould et al. showed in the dog also that the presence of dietary cholesterol reduced the synthesis of cholesterol. 5 This effect of feeding cholesterol is more readily demonstrated in vitro with liver preparations than in the whole animal. The influence of exogenous cholesterol on sterol metabolism, known as the "feed back regulation" of cholesterol level, has been extrapolated freely to the human situation. Thus it has been argued that restriction of dietary cholesterol is of little practical importance in regulating the level of serum cholesterol in human beings because of the compensating effect of changing rate of synthesis. 0 Some important evidence from higher primates disagrees with this conclusion. Cox et al. showed that while dogs and Rhesus monkeys seemed to convert acetate-c' to cholesterol less well when hypercholesteremic on a high cholesterol diet, only one of seven human subjects showed suppression of this conversion when fed a diet rich in cholesterol." Taylor et al. have obtained supporting evidence in dogs and human beings 8 using the From the Division of Nutrition, Vanderbilt University School of Medicine, Nashville, Tennessee. Supported by research grants from the American Heart Association and Grant HE from the U. S. Public Health Service. This work was done while Dr. Mann was a Career Investigator of the National Heart Institute, (5K6-HE-888-0). Accepted for publication August 9, experimental design developed by Morris et al. 9 This procedure consists of feeding radiocholesterol at a constant level for several weeks and measuring the rise of specific activity of the serum cholesterol. If all the labeled cholesterol in the plasma were coming from the diet, the specific activity of cholesterol in plasma would come to approximate that of the diet. Thus the proportions of endogenous production and of accretion from diet are measured at equilibrium by the amount of labeled cholesterol in serum in relation to the activity of the cholesterol fed (fig-1). The interpretation of such experiments rests on several critical assumptions. The dietary radioactivity must be constant; a requirement not easily or even possibly met, because of the return of radioactive cholesterol to the intestinal contents via the bile. The biliary cholesterol increases in activity with cholesterol feeding and this material changes the specific activity of the intestinal cholesterol presented for absorption. This artefact would tend to produce an overestimate of endogenous accumulation, since the dilution of dietary Specific Activity as " m /mo n Serum Synthesis* S.A. as X of FIGURE 1 Graph to show theory of appraising source of cholesterol in serum by feeding labeled cholesterol until equilibrium is reached. 5

2 6 MANN cholesterol by biliary cholesterol of lesser specific activity would tend to reduce the actual specific activity of the cholesterol absorbed. The second assumption is that the low cholesterol diet contains only trace amounts of cholesterol of known specific activity. The biliary contribution is more critical for this assumption because its dilution effect is larger. Finally, it must be assumed that the clearance mechanisms for cholesterol are of similar nature in animals whether they are receiving a high or low level of cholesterol in the diet. Using this model Morris et al. 9 found in rats that the dietary cholesterol accounted for only 30% of plasma cholesterol on a diet with 0.05% cholesterol, but accounted for 85% of plasma cholesterol when the diet contained % cholesterol. The additional dietary cholesterol seemed to diminish the endogenous cholesterol by about 80%. Taylor et al. 8 and Cox et al. 7 fed human subjects 100 mg of cholesterol daily and found that unlabeled sources were accounting for 75% of the serum cholesterol. When seven subjects were fed TABLE 1 Description of Purified s for Monkeys * CO cioo SB100 Casein 1 CI assay protein- Sucrose 3 Lard-* Salt mix l\» Vit. mix" Cholesterol" *In grams of each. Commercial "purified" casein. -Archer Daniels Midland, 0.6% cystine, 1.0% methionine. s Commercial cane sucrose. Reliable Packing Company, pork fat. "'"Hegsted" salt mixture. fi A concentrate in dextrose added at a level of g/kilo of diet to supply/kg: thiamine HC1 mg, riboflavin mg, pyridoxine HC1 mg, niacin 100 mg, Ca pantothenate 30 mg, biotin 0. mg, folacin 5 mg, cobalamine.05 mg, inositol 0 mg, menadione 5 mg, vit. A concentrate,000 Int. units, vit. D concentrate 5,000 Int. units, ascorbic acid 500 mp, choline Cl,000 mg and o tocopheroi 00 mg. "Crystalline cholesterol purchased from Armour and Company. 00 mg of cholesterol daily, the endogenous sources still accounted for 70% of cholesterol in serum. Those workers showed no estimates of the net production of cholesterol in human beings when dietary cholesterol was very low but we assume this would be 90% or more of supply. Thus human beings seem to suppress the endogenous supply by about to 5% when fed cholesterol. The present data extend the work of Taylor et al. and Cox et al. 7-8 to primates of other species. The dynamics of cholesterol metabolism has been studied comparatively with the hypothesis that the metabolic lesion in hypercholesteremia is a result of an impairment of degradation of cholesterol, rather than an excessive synthesis. The production and the removal of cholesterol from serum have been examined in several subhuman primates in an effort to localize the biochemical lesion produced by limitation of the dietary sulfur amino acids. Methods The primates were fed the purified diets described in table 1. These diets were either complete when made with casein or, when made with a refined soy protein, moderately limited in cystine and methionine. Each type of diet was further varied to contain either 0.0% (CO and ) or 1.0% (C100 and SB100) cholesterol. The primates examined included Cebus, Rhesus and Lagothrix. The animals had been on the trial diets 6 to 36 months before the trace amounts of radioactive cholesterol were given so that an equilibrium state can be assumed. Nevertheless some unexplained variation of plasma level of cholesterol from week to week is usual. The radiocholesterol (G^-*: 13.7 mc/mmole) was dissolved in ethyl ether and added to a warm solution of the dietary fat. The diets containing added cholesterol were prepared by dissolving crystalline cholesterol with heat in the melted lard. The labeled diets were made to contain about 500 counts/min/mg of cholesterol, the activity being measured for each mix. The diets were kept refrigerated and prepared fortnightly. The equilibrium experiment was repeated on three occasions with a total of 18 monkeys. These trials were repeated on three monkeys, no. 1, 38, and 39, after a 1-month interval while continued on the same basal diet. A second series of experiments was done with Cebus monkeys equilibrated on different kinds of diet and given a single intravenous dose of cholesterol C H. This material (13.7 mc/mmole) was

3 AMINO ACIDS AND STEROLS IN PRIMATES 7 suspended in dilute ethanol with a small amount of Tween to give a faintly opalescent solution. Each animal was given 10 /xc of active cholesterol intravenously and blood samples were taken at intervals for seven weeks to follow clearance of radioactivity from the plasma cholesterol. The plasma total cholesterol was isolated and counted because in these experiments, lasting several weeks, we expected the specific activity of free and esterified cholesterol to be nearly equal, as Hellman and Rosenfeld found in human subjects. 10 This experiment with Cebus monkeys was also repeated on three occasions. The cholesterol in plasma and diet was isolated as the digitonide by the method of Kabara and McLaughlin. 11 The activity was measured by scintillation counting with quench correction. The levels of diet and plasma cholesterol were measured by the method of Mann. 1 It is convenient to express serum activity as per cent of diet activity although the activity of the several diets was essentially constant. Results In the first series of experiments the Cebus monkeys fed a constant amount of labeled cholesterol tended to reach an equilibrium of specific activity in two to four weeks (figs. and 3). The random variations of plasma cholesterol level could not be sensibly related to the specific activity. The equilibrium with cholesterol feeding (C100 and SB 100), which indicates about 60% of the plasma cholesterol was arising endogenously and 0% from diet, may have been reached a little earlier with the casein diet but this is uncertain. Animals fed the two kinds of diets (CO and ) with only minimal cholesterol (0.01%) equilibrated at lower specific activities indicating that about 90% of the plasma cholesterol was arising endogenously and 10% from the diet. The specific activity of the plasma cholesterol reached about the same proportion of diet specific activity in the Cebus fed 1% cholesterol, whether the protein was the sulfur deficient soybean protein or casein. The experiment was repeated with monkeys no. 1, 38, and 39 (fig. 3) after an interval of one year. The data suggest that the mechanism which leads to hypercholesteremia in the Cebus monkey when fed diets limited in the sulfur amino acids does not result from a disturbance of the feedback suppression of endogenous production of cholesterol. The data show also that while Cebus monkeys are able to suppress the endogenous supply of cholesterol when dietary cholesterol is furnished, the suppression is not complete but only a two-third reduction. This is in contrast to the findings in other primates and other species. CEBUS DIET CASEIN SOY PROTEIN I 80 8 WEEKS FIGURE Effects of diet protein and cholesterol upon the specific activity of plasma cholesterol with Cebus monkeys in equilibrium conditions. Circulation Research, Vol. Will, February 1966

4 8 MANN Rhesus monkeys fed cholesterol also suppressed their endogenous sources but only by about 30$ (fig. ). Lagothrix, the Brazilian "wooly" monkey, was found to have an 80: ratio of endogenous to diet in the source of plasma cholesterol and this was not influenced CEBUS DIET 100 CASEIN SOY PROTEIN 80 Specific Activity Percent of CIOO- SB I WEEKS FIGURE 3 Effects of diet protein and cholesterol upon the specific activity of plasma cholesterol with Cebus monkeys in equilibrium conditions. Specific Activity Percent of 0 T RHESUS T SOY PROTEIN SB 100 LAGOTHRIX W 31 W3 V SB WEEKS FIGURE Effects of feeding cholesterol on the suppression of "synthesis" of cholesterol in Rhesus and Lagothrix monkeys.

5 AMINO ACIDS AND STEROLS IN PRIMATES 9 by adding dietary cholesterol. Thus these animals did not suppress. While animal W3 was hypercholesteremic when this study was done, this finding was unusual because, animals of this species have generally been found resistant to the hypercholesteremic regimen in our hands. The summary of equilibrium experiments, shown in table, emphasizes the lack of effect of sulfur amino acid deficiency upon ability to suppress the endogenous supply of cholesterol. A second series of experiments was done to measure the effects of diet and hypercholesteremia upon the disposition of a single intravenous dose of radiocholesterol. This approach was intended to reveal an abnormality of the degradative and excretory mechnisms for sterols. Hellman et al. 10 have described similar experiments in human beings. While those authors thought the exponential decline too complicated to permit analyses, they found a biological half-life of to 5 days. They noted also the equivalence of serum cholesterol, whether derived from labeled acetate or fed as labeled cholesterol. The present experiments used a third alternative of injecting labeled cholesterol intravenously. The decline of specific activity in plasma cholesterol is described for several representative animals in figure 5. This decline shows at least two phases. The early phase, which is taken to represent mixing in at least two compartments, lasted about seven days and had a rapid rate of change. The second phase showed a half-life of 13 to 1 days (table 3). If the biological decay curves are Relation of the Specific Activity of Cholesterol in TABLE the to that of the Plasma at Equilibrium Genus Cebus Rhesus Lagothrix CO C100 SB 100 Animal 1a a 1b 0 38a 39a 38b 39b 0 1 Har SB100 5 W31 Concn mg% Plasma Cholesterol level Specific < activity Proportion Mean As per cent Mean "synthesis" ±SE of diet sp. act. ± SE diet mg % 11 % 190 ± ± ± 17 ± ± ± 6.6 J58_ 7. ± 0.7 _93_ ± Reduction "synthesis" SB100 W

6 10 MANN extrapolated in the second phase to zero time, one may calculate the theoretical pool size of cholesterol. The calculated pool size was found to be proportional to plasma cholesterol level (fig. 6), at least for the animals fed the complete diet. The values for the animals cluster around two lines, A and B in figure 6. This relationship seems to indicate that the pool size increases without a large increase of plasma level in animals receiving either the complete diet or the deficient diet containing added cystine. In the animals fed diets deficient in the sulfur amino acids, a pool size of about 8 g seemed to be a critical point because above that level there was an increase of plasma cholesterol level. One can suppose that at this pool size some compensating mechanism began to fail. CHOLESTEROL SPECIFIC, l»s8o5mg% 9*S8IOO-5lmg% " '" DAYS FIGURE 5 Disappearance of radiocholesterol from blood of Cebus monkeys maintained on several dietary regimens. s are described in table 1. and average level of cholesterol for each animal during the seven-week period are shown. TABLE 3 Sutmnary of Radiocholesterol Metabolism in Cebus CO C100 SB1OO Animal 1 H Weight g Average plasma level mg% Monkeys T y days Clearance Pool size mg %/day Amounts mg/day SB100 Cy* SB100 with 0.% Z-cystine added.

7 AMINO ACIDS AND STEROLS IN PRIMATES i PLASMA CHOLESTEROL 300 mg % POOL SIZE-g FIGURE Relation of the level of plasma cholesterol to the cholesterol "pool size" calculated from concentrations in the plasma after a single dose. Line A describes the relation for eight Cebus monkeys fed complete diets. Line B describes that for six Cebus monkeys fed a diet limited in sulfur amino acids. Animals 1, 39, and H, which were fed diets with a cystine supply, were able to handle a pool of over 1 g while still maintaining a low plasma level of cholesterol (table 3). Two critical parameters seem to be present, the pool size and the level of plasma cholesterol. Statistical analysis of group means for monkeys, arranged by diet, fail to show that the half-lives were different but the pool sizes were larger in the animals fed SB100 than were those fed diet (P < 0.001) as were the plasma levels of cholesterol. The differences of plasma level were not significant between the CO and the C100 groups, but the pool sizes were different. The clearance rate and the amount of cholesterol cleared from plasma per day were essentially the same in the hypercholestermic animals fed SB100 as they were in those animals that were fed complete diets C100 and SB100 with added cystine. Therefore the significant finding is that the deficient animals can accomplish the same degree of removal only by a disproportionate elevation of plasma cholesterol level in relation to pool size. Circulation Research, Vol. XVlll, February 1966 The pool size is linearly related to plasma level. Allowing for the slow equilibrium between free and esterified plasma cholsterol demonstrated by Hellman and Rosenfeld 10 and by Gould 1 the measurement of plasma cholesterol alone will, in well nourished animals, reflect pool size although in an insensitive manner because the plasma level is kept low over a wide range of pool sizes (fig. 6, line A). In an animal deficient in sulfur amino acids, the plasma level is allowed to rise when the pool size surpasses a critical level (line B). This seems to demonstrate the essential defect of a deficiency of sulfur amino acids in animals fed cholesterol. Discussion These experiments eliminate one possible explanation for the hypercholesteremia seen in primates fed diets limited in sulfur amino acid and containing cholesterol. While there are demonstrable interspecies differences in suppression of cholesterol accumulation produced by dietary cholesterol, a limitation of this suppression cannot explain the cholesteremia of the Cebus monkey deficient in

8 1 MANN sulfur amino acids. It is notable that suppression in primates is less complete than that of rats. The data from rats are not acceptable evidence that dietary cholesterol is inconsequential in primates. The present data indicate that a deficiency of sulfur amino acid impairs the efficiency of regulation of cholesterol in the blood. As the body pool of cholesterol is increased by dietary intake, well nourished animals increase their removal rate and maintain low plasma levels despite a large pool. Animals deficient in sulfur amino acids are unable to do this and as the pool increases above a critical level, the removal rate increases, but only with an associated high plasma level of cholesterol. This inefficiency of regulation of cholesterol seems to be the essential lesion of sterol metabolism produced by a deficiency of sulfur amino acids. Animals given cystine, or casein which is rich in methionine, were able to maintain their efficiency in the presence of dietary cholesterol. It is notable that there are at least two components of the hypercholesteremia produced by these means in monkeys. One is a small but measurable increase of plasma level with increase of pool size that occurs in normally nourished animals that are fed cholesterol. This is a kind of load effect. The other component, seen in animals restricted in sulfur amino acids, is more dramatic and might be considered a deficiency effect. In these animals normal cholesterolemia requires both the removal of dietary cholesterol and a supply of available cystine, a multifactorial treatment. It might be conjectured that there are other unidentified factors which also influence the pool size or the relation of plasma level to pool size. Summary Cebus and Rhesus monkeys suppress the in vivo production of cholesterol when fed cholesterol. This suppression is less than that observed in rats and it did not occur in Lagothrix monkeys. Cebus monkeys deficient in cystine lose their efficiency in removing cholesterol when the body pool is large. One result is an elevation of the plasma level of cholesterol. References 1. MANN, G. V., ANDBUS, S. B., MCNALLY, A., AND STARE, F. J.: Experimental atherosclerosis in Cebus monkeys. J. Exptl. Med. 98: 195, SEIDEL, J. C, NATH, N., AND HARPER, A. E.: and cholesteremia. V. Effects of sulfur containing amino acids and protein. J. Lipid Res. 1: 7, SIPERSTEIN, M. D., AND CUEST, M. J.: Studies on the site of the feedback control of cholesterol synthesis. J. Clin. Invest. 39: 6, TOMKINS, G. M., SHEPHARD, H., AND CHAIKOFF, I. L.: Cholesterol synthesis by liver. III. Its regulation by ingested cholesterol. J. Biol. Chem. 1: 137, GOULD, R. G., TAYLOR, C. B., HAGERMAN, L. S., WARNER, I., AND CAMPBELL, D. J.: Cholesterol metabolism. I. Effect of dietary cholesterol on the synthesis of cholesterol in dog tissue in vitro. J. Biol. Chem. 1: 519, KEYS, A., ANDERSON, J. T., MICKELSEN, O., ADELSON, S. F., AND FIDANZA, F.: and the serum cholesterol in man: Lack of effect of dietary cholesterol. J. Nutr. 59: 39, Cox, G. E., COUNTS, M., WOLSKI, J., ALVAREZ, J., AND TAYLOR, C. B.: The effects of dietary cholesterol upon the synthesis of plasma cholesterol in the human. Circulation 18: 93, TAYLOR, C. B., PATTON, D., Yocr, N., AND COX, G. E.: as a source of serum cholesterol in man. Proc. Soc. Exptl. Biol. Med. 103: 768, MORRIS, M. D., CHAIKOFF, I. L., FELTS, J. M., ABRAHAM, S., AND FAUSOH, N. O.: The origin of serum cholesterol in the rat: versus synthesis. J. Biol. Chem. : 1039, HELLMAN, L., AND ROSENFELD, R. S.: Isotopic studies of cholesterol metabolism in man. 7n Hormones and Atherosclerosis, ed. by G. Pincus. New York, Academic Press, 1959, pp KABARA, J. J., AND MCLAUGHLIN, J. T.: A microdibromide procedure for purifying radioactive cholesterol. J. Lipid Res. : 83, MANN, G. V.: A method for measurement of cholesterol in blood serum. Clin. Chem. 7: 75, BLOCH, K.: Biosynthesis of Cholesterol, chapt. 1, ed. by G. Pincus, New York, Academic Press, Goui-D, R. G.: The relationship between thyroid hormones and cholesterol biosyntheses and turnover. In Hormones and Atherosclerosis, chapt. 6, ed. by G. Pincus, New York, Academic Press, 1959.