ANEMIA CAUSED BY FEEDING CHOLESTEROL TO GUINEA PIGS

Transcription

1 ANEMIA CAUSED BY FEEDING CHOLESTEROL TO GUINEA PIGS BY RUTH OKEY AND VERA D. GREAVES (From the Division of Home Economics, College of Agriculture, University of California, Berkeley) (Received for publication, February 3, 1939) Much has become known during the past few years with regard to the function of compounds closely related in chemical structure to cholesterol; i.e., bile acids, sex hormones, and vitamin D. In contrast, very little is known of the metabolic activity of cholesterol itself. It would appear reasonable that a substance which is found in appreciable quantities in every animal tissue might have a specific function. This paper reports the production of a severe anemia with splenic enlargement in the guinea pig as a result of prolonged feeding of diets rich in cholesterol. This animal was used in the hope that the study of a species which we might expect to be especially sensitive to the effects of dietary cholesterol would give some evidence as to the mechanism of its functioning in the animal organism. Although guinea pigs are herbivorous animals and their usual diet does not contain cholesterol, there is evidence that cholesterol plays a part in their metabolic processes. Guinea pig tissues normally contain approximately the same concentration of cholesterol as those of rats. Moreover, unlike rats, but like human beings, these animals have gallbladders. Again, guinea pigs, also like human beings, require preformed vitamin C in the diet and this requirement has been reported to be associated with susceptibility to atherosclerosis (1). We make no assumption that the data here presented are directly applicable to interpretation of the human metabolic picture. The peculiar history of the cholesterol-fed animals offers at least a suggestion of the nature of the effect of variation in the cholesterol-lecithin content of blood and tissues on cell stability. 111

2 112 Anemia Due to Cholesterol EXPERIMENTAL Because the guinea pigs for this study became available in comparatively small numbers at any one time, the data represent a summation of findings from the study of a series of small groups of cholesterol-fed and control animals rather than the results of a study of a single control and a single experimental group. Series 1 and 2 were composed of guinea pigs purchased from a dealer. Subsequent series were from our own laboratory stock. Controls were litter mates where possible in the later series. Two additional series were not reported because of differences in age and size, rather than because of any essential difference in behavior. Diets-Comparatively little information was available on satisfactory synthetic diets for the guinea pig. For the purpose of the experiment it was desirable that the diet be adequate and capable of exact repetition rather than that each constituent be purified. Moreover, fat had to be fed in sufficient amount to facilitate cholesterol absorption. After a series of preliminary trials, it was found that with appropriate vitamin supplements the following was reasonably satisfactory for the guinea pigs which had attained a weight of approximately 300 gm., and quite satisfactory for guinea pigs weighing 350 to 400 gm.: raw casein 20 parts, fat (partially hydrogenated cottonseed oil, Primex) 15, corn-starch (raw) 46, Osborne-Mendel salts (2) 4, yeast (dry brewers ) 10, regenerated cellulose or alcohol-extracted bran 5. This was the control diet. When cholesterol was given, 1 gm. was dissolved in each 15 gm. of melted fat and thoroughly mixed into the diet, making a proportion of 1 gm. of cholesterol to 100 gm. of total food. Vitamin supplements were given by pipette. Carotene dissolved in coconut oil and mixed with a cottonseed oil solution of viosterol was used to supply a daily dose of approximately 75 I.U. of vitamin A and 12 I.U. of vitamin D in every case. 3 cc. of orange juice were given each animal daily. This was assayed by titration and enough additional ascorbic acid added to bring the total daily dose to 3 mg. except in the case of Series 2 in which the dose was adjusted to 30 mg. per day. Series 4 received 10 mg. of choline per day with the vitamin supplements. Growth and Behavior-Ordinarily the guinea pigs did not eat the experimental diet readily until after about a week. We found that they made better progress when a little whole wheat or wheat

3 R. Okey and V. D. Greaves germ was mixed with the new diet for the first few days. The time on the diet as given in Table I was computed from the day the synthetic diet was first offered. After 4 or 5 weeks on the cholesterol diet, the guinea pigs usually ceased t.o gain weight. Their muscles became noticeably flabby and there was lack of subcutaneous fat. Their abdomens became enlarged, and it was possible to note that the livers were also TABLE Weight and Blood Count Comparisons in Guinea Pigs on a Normal Diet (Controls) and Animals Made Anemic by Cholesterol Feeding I Series 1 Normal Anemic Series 2 Normal Anemic Series 3 Normal Anemic Series 4 Normal I Anemic I nitia wighi!3m Q ICY TIP minal wight gm I gm. Pm cent body weight gm i ' , 11.4! * Measurements made at the time the animals were killed. L - : houan& enlarged. The white guinea pigs from the cholesterol-fed group showed noticeably less pink color in their ears as the experiment progressed. Controls continued to grow and presented a normal healthy appearance throughout the time of study. Aut,opsy of the first series of animals showed that the cholesterolfed guinea pigs had fatty livers of approximately twice the weight of those of the controls and spleens approximately 10 times normal

4 TABLE II Lipid Content of Tissues of Control (Normal Diet) and Anemic (Cholesterol-Fed) Guinea Pigs TiSSUC? Livers No. of animals Moisture, To Total fatty acid 1 cholesterol Free Lecithin I Spleens No. of animals Moisture, jjo Total fatty acid TO moist weight cholesterol yo moist Free weight Lecithin Hearts No. of animals Moisture, % Total fatty acid cholesterol To moist Free weight Lecithin Series 1 - Anemic Series 2 Normal Anemic NOITWl Anemic NOrDIal Anemic Normal Anemic Series Series 4 - T Weighted average I 7

5 Lungs No. of animals Moisture, To Total fatty acid cholesterol % moist Free weight Lecithin Blood No. of animals Fatty acids 1 Totai cholesterol Free I w. % Lecithin I Adrenals No. of animals Fatty acids Total cholesterol % moist weight Free i Lecithin I F E b 7.1 * g I

6 116 Anemia Due to Cholesterol weight. The color of their blood was paler than that of the controls and its coagulation time seemed to be increased. Blood counts on later series of animals showed that at about the time the animals on the cholesterol diet began to lose weight there was a rapid decline in the number of their red blood cells. Methods The methods of investigation were the usual ones. Red and white cell counts were made with counting chambers calibrated by the United States Bureau of Standards. Slides prepared with Wright s stain served for microscopic examination and differential counts. Fragility tests were made by measuring the hemolysis in a series of concentrations of NaCl(3). Hemoglobin was estimated with a Bausch and Lomb calorimeter equipped with a Newcomer disk. At the end of the period of observation the animals were killed by decapitation. Whole organs were dissected out as quickly as possible and weighed. Samples were then taken for histological examination and others cut up finely with scissors and weighed for moisture determinations and lipid analyses respectively. When pooled samples were used, care was taken to secure thorough mixing of the samples before weighing. The methods used for lipid analyses have already been described (4, 5). Samples for histological study were placed in formalin solution. Later, both frozen and celloidin sections were made and studied. The data are summarized in Tables I and II. Fig. 1 shows the red cell count for the animals of Series 4. Microscopic Examination of Tissues We have included in this paper only the brief summary of the histological findings necessary to show the relationship which was observed between structural changes and changes in the lipid content of the tissues. Red cell counts were made at uniform and frequent intervals only for the later series of animals. Hence, it was impossible to correlate all our findings in one composite curve. The animals in Series 4 (Fig. 1) were a little older and heavier than those in the preceding groups. Onset of anemia in this group was consequently somewhat delayed. In every other respect, however, the picture presented by this series was like that of the preceding one.

7 I-t. Okey and V. D. Greaves 117 Blood-One very interesting characteristic of the anemia was the rapidity of onset. It was not unusual for the erythrocyte count of the cholesterol-fed animal to drop from 5.2 to 3.0 millions within 10 days. Nucleated red cells appeared at or just before the time of the rapid drop in total red cells. Leucocyte counts usually remained at or near the control level for at least 10 days following the drop in erythrocyte count. The guinea pigs often appeared too toxic to survive during this period, but the actual mortality was less than 10 per cent. About 10 days after the sharp fall in total red cell count most animals showed signs FIG. 1. Mean erythrocyte variation in guinea pigs (Series 4). Curve N represents the controls, fed a normal diet; Curve A, anemic animals, fed cholesterol. Like symbols represent counts for individual guinea pigs. of adjustment. The fur became smoother, the animals more alert. A considerable number of guinea pigs survived for from 2 to 3 weeks after the red cell count reached 3 millions. Erythrocyte counts remained relatively constant at this second stage, but there was a progressive increase in number of nucleated red cells. Stained slides showed a great diversity in the size of the red cells, some of which appeared to be undergoing decomposition. After a period of time which varied with the individual animal, however, the leucocyte count usually increased sharply. This came to be regarded as a danger signal, since several animals kept beyond this

8 118 Anemia Due to Cholesterol stage were found dead in their cages shortly after the high white cell count was observed. Autopsy showed the picture typical of the animals killed at this stage; i.e., enlarged livers and spleens but no evidence of bacterial infection. Data from animals found dead were not included in the tables because of the possibilities of tissue decomposition post mortem. In the very anemic animals the size of some of the red cells was larger than normal. This fact, as well as the presence of nucleated cells, suggested that the difficulty lay in the formation and destruction of red cells rather than of hemoglobin. In most of the cholesterol-fed animals hemolysis began in 0.56 to 0.50 per cent NaCl and was completed only in 0.32 to 0.30 per cent NaCI. For control animals the range was from 0.46 to 0.36 per cent NaCl. Some of the blood cells in the cholesterol-fed animals seemed to be, therefore, especially resistant to hemolysis in spite of the presence of others which were more fragile than normal; i.e., presumably necrotic. Spleen-The outstanding characteristic of the enlarged spleens seen in the cholesterol-fed animals was an engorgement with all types of blood cells; nucleated red cells in numbers which suggested that the spleen might be engaged in red cell formation, as well as masses of cells apparently in process of dissolution. Sections examined under a low power objective showed the extent of this infiltration clearly, in contrast with the sections from the spleens of the control animals. Frozen sections stained with Sudan III showed very little fat except when there was gross evidence of necrosis. Large necrotic areas, which were sometimes found after the white cell count had increased, seemed to have resulted immediately from thromboses. Liver-The livers of the cholesterol-fed animals were uniformly fatty and enlarged. They were whitish rather than brownish yellow in color. Gallbladders were occasionally full of slightly cloudy fluid but there were never concretions of any size nor was the bile deeper than normal in color. Sections of liver from animals fed cholesterol for 2 to 3 weeks showed deposits of quite large globules of material staining with Sudan III, but little apparent change in the liver cells themselves. This was much like the picture we had previously observed in the rat. Later on, however, there was unmistakable evidence of

9 R. Okey and V. D. Greaves 119 injury to the liver cells and sometimes grossly necrotic areas in the livers of the guinea pigs. The latter were never found until several weeks after severe anemia had been present. Bone Marrow-The bone marrow in the cholesterol-fed animals was hyperplastic. The celloidin sections showed few spaces for fat globules and a greater proportion of cells representing the early stages of erythrocyte formation than did the marrow from control animals. But the picture suggested rather the inadequate response of a primary anemia than the greater activity characteristic of an anemia caused by uncomplicated hemorrhage. The possibility that the fat-loaded liver cells may have failed to furnish some substance essential to the normal formation of erythrocytes cannot be ignored. This would; however, explain neither the rapid destruction of red cells nor the gross splenic enlargement in the cholesterol-fed animals. Chemical Analysis of Tissues Figures for lipid analyses are summarized in Table II. Data are reported only for animals which lived through the entire experimental period. Dietary supplements have already been reported. While Series 2 received 30 mg. of ascorbic acid daily instead of the 3 mg. supplied the others, and Series 4 had a 10 mg. daily dose of choline given in addition to the vitamin supplements, the effect of these dietary changes on the tissue composition of the cholesterol-fed animals was so nearly negligible that the figures are reported together. Control and experimental animals within individual series should, however, be used for comparisons. The findings were entirely consistent with those from two other series which received 3 mg. of ascorbic acid and no choline and which are not reported because of differences in the size and age of the animals. In order to save time, and in some cases to make tissue available for both chemical and histological study, pooled samples were used for analyses of part of the tissues. Hence statistical comparison of these data is not possible. Rlood Lipids-The animals were killed 14 to 18 hours after their last access to food. The digestive tracts were not entirely empty in all cases; hence some of the variations in the blood picture may be accounted for by differences in the state of absorption. However, all samples showed a consistently greater increase in

10 120 Anemia Due to Cholesterol free than in esterified cholesterol in the blood of the cholesterol-fed animals. Likewise while the percentage of blood lecithin was increased in the cholesterol-fed animals, this increase did not keep pace with that of free cholesterol. Hence, there was in the cholesterol-fed animals a decreased lecithin to cholesterol ratio as well as an increased destruction of the less resistant cellular elements in the blood; i.e., the erythrocytes. Spleen Lipids-In the spleens of the cholesterol-fed animals there was a slight decrease in percentage of lecithin coupled with a consistent decrease in the percentage of t.otal fatty acid. There was also an increase in percentage of free cholesterol which was greatest in the spleens which showed gross necrosis. The endresult was a decrease in the value of the average lecithin to cholesterol ratio from 4.6 to 3.1. This was accompanied by a considerable amount of cell degeneration within the tissue, in spite of the enlargement of the organ as a whole. Liver Lipids-The lipids of the guinea pig livers differed from those of the livers of cholesterol-fed rats in that the elevation of the total cholesterol was not quite so great as we might have expected from the content of total fatty acid. There was a consistent decrease in the liver lecithin of the guinea pigs as computed on the basis of per cent moist weight. This meant a distinct lowering of the value for the ratio, lecithin to free cholesterol. The mean for the controls was 10.8 and that of the cholesterol-fed animals 3.2. Computed on a neutral fat- and cholesterol ester-free basis, the difference in percentage of lecithin in the livers of the two groups was practically negligible but the difference in free cholesterol became exaggerated. When individual tissues were analyzed, there was a positive correlation between depression of Ohe lecithin to free cholesterol ratio and degree of liver injury. We have, therefore, in the guinea pig liver a tissue in which cellular destruction is definitely taking place, and in which it is possible to demonstrate a variation from the normal in both concentration of free cholesterol and ratio of lecithin to free cholesterol. In the liver of the cholesterol-fed rat, esterification of the cholesterol is more nearly complete, and there is little increase in percentage of free cholesterol or decrease in percentage of lecithin. Likewise, in the rat fed cholesterol there is little actual injury of liver cells and almost no demonstrable pathology resulting from

11 R. Okey and V. D. Greaves 121 changes in the lipids of tissues other than liver. This is probably to be taken as evidence that, in the liver at least, the ability of an animal to esterify and so hold inert large quantities of cholesterol is a defense mechanism. Heart, Muscle, Adrenals, Lung, and Kidneys-Heart tissue showed the same variation; i.e., a lecithin to free cholesterol ratio of approximately 10 for the control and 6 for the cholesterol-fed animals. The differences in lecithin values between the two groups were consistent but nevertheless probably within the limits of experimental error for the method. Here the histological picture was somewhat obscure. There was no clear evidence of cellular breakdown but rather a picture of poor nutrition. Perhaps this may be correlated with the fact that the actual concentrations of lipid in this tissue are so low as to produce a less marked effect in the short time of the duration of the experiment. Analyses of skeletal muscle showed an increase of 0.09 to 0.15 per cent in free cholesterol but practically no esterified cholesterol, even in the cholesterol-fed animals. The percentage of lecithin decreased very slightly in cholesterol-fed animals. We did not make a chemical study of the bone marrow, and our data for the lecithin content of lungs and adrenals are incomplete. The cholesterol content of adrenals was very nearly the same in the control and in the experimental animals. Fatty acid differences were probably to be accounted for by differences in the state of nutrition of the guinea pigs. Dissections were necessarily made roughly and quickly. While elimination of all adherent abdominal fat cannot be assumed, control studies have demonstrated that this fat is practically cholesterol- and lecithin-free and therefore negligible in computation of ratios between these substances. Lung tissue showed an increase in percentage of ester cholesterol which was proportionately greater than that in the blood of the cholesterol-fed animals. Free cholesterol was increased in about the same proportion as in liver tissue. It is difficult to judge whether this indicates functional activity in the esterification of cholesterol or mechanical accumulation in the capillaries. The kidneys, surprisingly enough, showed practically no differences in free or ester cholesterol or lecithin content between control and experimental animals.

12 122 Anemia Due to Cholesterol DISCUSSION These data indicate in the main that the chief changes in tissue produced by feeding cholesterol to guinea pigs are to be found in the organs which have to do most with production or destruction of red blood cells. The picture is one of liver enlargement, failure of the liver to esterify and hold back extra cholesterol. This free cholesterol presumably exerts a surface tension effect on the cell walls which is not balanced by increased lecithin concentration. Destruction of the non-nucleated and vulnerable red blood cells follows. Next there is hyperplasia of the bone marrow and extreme enlargement of an organ concerned in salvage of hemoglobin and probably, as an emergency measure, in formation of red cells; i.e., the spleen. Finally there is injury to the liver cells and necrosis of the spleen. Bloor et al. (6) have postulated that the phospholipid content of a tissue is a fun&ion of its physiological activity. They have suggested that the increased free cholesterol content of a growing tissue may likewise be an indication of increased activity. Yasuda (7) in Bloor s laboratory has found that in malignant tumors both lecithin and free cholesterol are present in higher concentration than in tumors of a benign type. We have here a story of forced increase in the cholesterol content of a series of tissues, unaccompanied by increased lecithin production, and there is every indication that cellular breakdown is a consequence. Further study will be required to decide whether the free cholesterol has a physicochemical effect on the cell walls which, when appropriately balanced by that of the lecithin, results in cell growth and otherwise produces cell destruct.ion. Certainly t,he data indicate that while esterified cholesterol is to be regarded as physiologically comparatively innocuous, cholesterol in a free state must have a very definite effect on cell structure and on the capacity of the cell to take UP nutrient substances from water solution. SUMMARY 1 per cent cholesterol fed to guinea pigs with a synthetic diet adequate for normal controls first produced enormous and fatty liiiers. After about 5 weeks of cholesterol feeding the animals became seriously anemic. Red cell counts sometimes fell 2 to 3 millions within 2 or 3 weeks. Splenic enlargement took place

13 R. Okey and V. D. G-reaves 123 rapidly, and within 7 to 9 weeks spleens were usually 10 times normal size. Nucleated red cells appeared at about the time the total red cell count dropped. Blood slides also showed cells in the process of breakdown. Analyses of tissues of guinea pigs killed after 7 to 9 weeks on the cholesterol diet showed an increased free cholesterol content of liver, spleen, heart, lungs, and blood. Ester cholesterol increases in liver were proportionately less and free cholesterol increases proportionately greater than in rats. There was also much more evidence of migration of cholesterol into tissues other than liver than in the rat. Lecithin values in the tissues when computed on the basis of moist weight were lower in cholesterol-fed than in control animals. Histological examination showed fatty infiltration and cellular destruction in the liver. There was engorgement with cellular debris and even gross necrosis in the spleen. The net effect of these changes was a decrease in the value of the lecithin to free cholesterol ratio in the blood and in the tissues in which cellular destruction has been demonstrated. The possible significance of this finding in connection with the functioning of cholesterol in metabolism is discussed. This study has been made possible by funds granted by the Research Board of the University of California. We are indebted to the Works Progress Administration for helpers both in the laboratory and in the care of the animals. We gratefully acknowledge the assistance in the chemical analyses given by Mrs. Jeannette Hobson Spencer, and the advice and help in the preparation and interpretation of the slides received from Dr. A. A. Koneff of the Department of Anatomy and Dr. C. L. Connor of the Department of Pathology. BIBLIOGRAPHY 1. Menten, M. L., and King, C. G., J. Nutrition, 10, 141 (1935). 2. Osborne, T. B., and Mendel, L. B., J. Biol. Chem., 37, 572 (1919). 3. Stitt, E. R., Clough, P. W., and Clough, M. C., Practical bacteriology, hematology and animal parasitology, Philadelphia, 9th edition (1938). 4. Okey, R., Gillum, H. L., and Yokela, E., J. Biol. Chem., 107,207 (1934). 5. Okey, R., Godfrey, L. S., and Gillum, F., J. Biol. Chem., 124,489 (1938). 6. Bloor, W. R., Okey, R., and Corner, G. W., J. Biol. Chem., 86, 291 (1930). 7. Yasuda, M., and Bloor, W. R., J. CZin. Inv., 11, 677 (1932).