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A comparison of the turnover of cholesterol in rabbits on a normal and high cholesterol diet

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A comparison of the turnover of cholesterol in rabbits on a normal and high cholesterol diet

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Content A COMPARISON OF THE TURNOVER OF CHOLESTEROL IN RABBITS j / : ON A NORMAL AND HIGH CHOLESTEROL DIET ' / • i A Thesis Presented to I the Faculty of the Department of Biochemistry i The University of Southern California i I j In Partial Fulfillment j of the Requirements for the Degree I : Master of Science I I Audrey Mildred Larack June 1951 UMI Number: EP41322 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI EP41322 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 t ■ * > ? $ i- p & fy tA J3.3 $ ( S f r o '5 i , ^ ^ 71 Au thesis, written by ...j^OC^u^L... under the guidance of hJU/Li.Facuity Committee, and approved by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill ment of the requirements for the degree of 1<U... , Faculty Committee ( y Chairman TABLE OP .CONTENTS PAGE INTRODUCTION..................... 1 HISTORICAL............... 4 EXPERIMENTAL PROCEDURE .................. 8 RESULTS............................. . 13 Background of Each Experimental Group ••••••••••••••• 13 Cholesterol Content of the Various Organs in the Normal Rahbit • • • • 13 Distribution of Cholesterol in the Various Organs in the Normal Rabbit • ••••••••••••« 15 Cholesterol Content of the Various Organs of Rabbits on a High Cholesterol Diet • •••••••• 15 Distribution of Cholesterol in the Organs of Rabbits on a High Cholesterol Diet • ••••.••• 17 The Deuterium Content of Cholesterol in the Various Organs of the Normal Rabbit............. 19 i i i PAGE The Relative Percent of Synthesis of Cholesterol in the Various Organs of the Normal Rabbit • •••••• 21 The Deuterium Content of Cholesterol in the Various Organs of Rabbits on a High Cholesterol Diet • • • • > • 21 : The Relative Percent of Synthesis of Cholesterol in the Various Organs of Rabbits on a High Cholesterol Diet ................ 22 DISCUSSION ••*•••#•••••••• 25 Effect of High Cholesterol Diet on the Content of Cholesterol in the Various Tissues of the Rabbit . • 25 The Effect of High Cholesterol Diet on the Metabolism of Cholesterol in the Rabbit .............. 30: SUMMARY AND CONCLUSIONS................ 42 BIBLIOGRAPHY......................... 45 LIST OP TABLES PAGE TABLE I* Background of Each Experimental Group • ••••••••••••« 14 II• Cholesterol Content of Normal Rabbit Organs • ••••••*•« 16 III. Cholesterol Content of Organs of Rabbits on a High Cholesterol Diet • • ........... 18 IV. Cholesterol Turnover in Normal Rabbits 20 V. Cholesterol Turnover in Organs of Rabbits on High Cholesterol Diet 24 VI# Comparison of Milligrams of Cholesterol Per Gram of Tissue in Rabbits Under Different Dietary Conditions ••••••• 29 VII. Comparison of Cholesterol Metabolism in Rabbits Under Different Dietary Conditions • • 34 LIST OP FIGURES PAGE FIGURE 1« Milligrams of Cholesterol Synthesized Per Gram of Tissue in the Various Organs • •. 35 2* Relative Percentages of* Cholesterol Synthesized in the Various Organs •«•••••• 39 INTRODUCTION Arteriosclerosis has been defined by MacCullum (1) as a disease of the artery causing loss of elasticity and changes in appearance and structure of the intima which result in dilation and deformity of the arterial wall* Marchand (2), in 1904, defined atherosclerosis as arter iosclerosis characterized by fatty lesions* The disease occurs in progressive stages of increasing localized yellow streaks of fatty material and wandering cells containing fat, followed by an overlaying of connective tissue which results in hard bluish-white plaques In the intima (1)• These lesions may ulcerate with subsequent calcification* Since atherosclerosis is an Important cause of death in man, there has been much research endeavoring to deter mine the etiology of the disorder* Recently, medical re search has rejected the idea that atherosclerosis is a disease of senility (4,5), and now considers it to be a metabolic disorder (4,6,7), involving either lipemia (8,9, 10), a lipoprotein complex as suggested by Gofman (11,12), a change in phospholipid-cholesterol ratio (13,14), a deficiency of lipotropic factors (15,16,17,18,19,20), or some combination of these with a localization of ather osclerotic lesions at pressure points* These arise from 2 the mechanical properties of the circulation such as zonal flow, continuous flow and pulsation intermissions of the arterial blood stream (21)* ; Analysis of the fatty plaques in 1907, revealed that they are composed largely of cholesterol (22), which suggested the idea that the etiology of atherosclerosis I might be primarily a disturbance in cholesterol metabolism* j This idea was supported by the fact that Anitschkow, six years later, was able to induce an experimental atheroscler osis in rabbits by feeding them cholesterol in oil (23)* His resuits have been substantiated by subsequent workers (7,24,25). The major Importance of Anitschkow’s experiment, how- ; ever, is the demonstration of an experimental animal in which the pathological changes under investigation can be induced, and the similarity between the experimental and the human disorders* That experimental atherosclerosis in rabbits is comparable with the human condition has been j demonstrated by several investigators (3,7,26,27)* Since Anitschkow’s work, attempts have been made to obtain atherosclerosis in animals other than the rabbit by choles terol feeding* These have met with varying degrees of ; success* 3 Altschul has produced similar lesions in guinea pigs by feeding egg yolk, rich in cholesterol (28)* Spontaneous and cholesterol-induced atheromas in chickens were demon strated by Dauber and his group (29,30,31)* and by other investigators (32,33). Steiner has produced experimental atherosclerosis in dogs fed cholesterol and thiouraeil (34) and Altschul and Goldman (35) still disagree as to the extent of atherosclerosis found in Golden Hampsters fed a diet rich in cholesterol* Attempts to obtain atheroscler osis in rats under various experimental conditions includ ing high cholesterol diet, however, have failed (36). The reasons for the varying degrees of resistance to dietary cholesterol can perhaps be determined by studying choles terol metabolism under different conditions. The purpose of the following experiments is the deter mination of the normal turnover of cholesterol in rabbits and a comparison of this with the turnover of cholesterol in rabbits on a high cholesterol diet. These data should indicate whether or not there is a disturbance of choles terol metabolism associated with the feeding of a diet high in cholesterol and the subsequent incidence of experi mental atherosclerosis in the rabbit. HISTORICAL Several investigators have studied the development of cholesterol atherosclerosis in rabbits in relation to his tological changes in the aorta and other organs at various periods from 30 to 380 days during the feeding of a high cholesterol diet (4,7,25,26,37)* Their results indicate that atheromas appear after a 30 day latent period in rabbits fed approximately one gram of cholesterol daily* The atheromas are associated with the intima only, and changes in the media are secondary* The degree of ather osclerosis is related to the period of feeding, and becomes progressively intensified from the 30 to 380 days studied* In later stages atheromas appear in the arteries of organs such as the kidney, liver, spleen adrenal, and the viscera* The lesions are initiated by the appearance of foam cells which are believed to arise locally in the intima or from the liver Kupffer cells* The latent period in the rabbit disorder is shorter than In the human disease where the later stages, there fore, may progress differently, although the early stages are comparable* The association of the extent of ather osclerosis with the duration of feeding was found to occur in chickens as well as In rabbits (38)• 5 Atherosclerosis in rabbits has also been studied in relation to the deposition of cholesterol in the various organs and changes in the cholesterol content of the blood (6,7,25,37)* The conclusions of these investigators are in agreement. The blood cholesterol level reaches a maximum at about three months of feeding a one percent cholesterol diet and thereafter declines* Dietary cholesterol is deposited principally in the aorta, liver and adrenals, and also in the lungs, kidneys and spleen* Although there is no direct correlation between the degree of hypercholester olemia and the degree of atherosclerosis, the physical state of the blood such as viscosity and hypercholester olemia are associated with the atherosclerosis found in rabbits* The experiments described in this paper were designed to ascertain more quantitative relationships between the normal content of cholesterol in the vari'ous tissues of the body, and the relative extent of deposition of exogenous cholesterol in these tissues in the animals fed a diet rich in that substance* This would indicate the relative im portance of the organs as they are involved in the storage and distribution of dietary cholesterol* The use of deuterium as a tracer in cholesterpl metab 6 olism was established by Rittenberg and Schoehheimer (39) who showed that the C-H linkages in cholesterol were stable to exchange with deuterium oxide even under drastic condi tions and, therefore, deuterium-labeled cholesterol isolat ed from an animal whose body fluids had been tagged with deuterium oxide is indicative of cholesterol synthesis* Subsequent experiments using deuterium as a tracer in the metabolism of cholesterol have demonstrated that biological turnover of cholesterol in animal organs takes place (39)5 that acetic acid is one of the precursors of cholesterol synthesis (40); that cholesterol is utilized in the biolog ical synthesis of cholic acid (41) and progesterone (42); and that synthesis of cholesterol takes place in surviving liver slices (43)* Popjak (44) has studied the synthesis ©f cholesterol in rabbit foetus* The use of other tracer elements has revealed extrahepatic synthesis of cholesterol (45)9 confirmed the work with deuteroacetate (46), and demonstrated the biological conversion of cholesterol to adrenal cortical hormones (47)* The experimental approach in tracer studies in this paper involves the labeling of the medium, which in this case is the body fluids, and the isolation of the substance under investigation at various periods of time to determine the incorporation of tracer into the substance by the organism* It was determined by Kamen (48) that application of the use of deuterium oxide is limited to an equilibrium value of not more than 2 to 5 percent in the intact animal* The maximum concentration in this study was 1.5 atoms per cent deuterium in the body fluids* This is below the toxic levels also reported by Barbour and Trace (49)* Previous studies concerning cholesterol turnover have been made by Bloch et al, in normal rat tissues in vivo (40), and by Waelsch et jal, in the same animal (50). The present studies with the rabbit were planned with a view towards determining the relative rapidity of cholesterol turnover in the various organs and whether any one specific organ is primarily concerned with synthesis or destruction of cholesterol; evaluation of the amount of deposition of excess exogenous cholesterol in the various organs and the effect of such deposition on the rate of replacement of cholesterol; and the determination of whether there is a stage in the cholesterol feeding at which a change in the endogenous metabolism of cholesterol can be noted. The organs studied included liver, lungs, brain, kidneys, adrenals, spleen, ovaries, aorta, and the blood plasma* EXPERIMENTAL PROCEDURE The first group of animals studied was made up of four control, normal, female albino rabbits, one month old on arrival. These were placed on stock diet of ground purina rabbit pellets containing 0.04 per cent cholesterol for one month. At the end of this time the animals were Injected intraperitoneally with a sufficient amount of 50 percent deuterium oxide to raise the body fluids to 1.5 percent deuterium oxide. The calculations were based upon a value for body fluids of 60 percent of body weight. This was the zero time of the experiment. Thereafter, for periods of 8 and 16 days, the animals were maintained on the stock diet plus drihking water containing 2.5 percent deuterium oxide, which, in a preliminary experiment with rats, had been determined to be sufficient to maintain the body water at a constant level (51) as was discussed by Bloch and Ritten- berg (39)• The second group of six female albino rabbits, one month old on arrival, was maintained on the stock diet for , one month. At that time their body fluids were labeled and maintained with deuterium oxide in the same manner as the controls. Thereafter, for periods of 8, 16 and 30 days, they were placed on a one percent cholesterol diet 9 prepared by mixing a water suspension of cholesterol, as described by Popjak (52), with the ground stock diet* Ike suspension was made by dissolving 10 grams of cholesterol in approximately 150 ml* of acetone and adding the solution to boiling water with vigorous stirring. After the acetone was evaporated off, the watery suspension was mixed with i the diet* The experimental dlet, therefore, was slightly moist when fed* It was stored in the cold room at 3°C* Only one or two kilograms of diet were mixed at any one time: to insure a fresh, unspoiled diet* At the end of each experimental period, two animals were sacrificed by exsanguination through heart puncture under nembutal anesthesia* The collected heparlnized blood, approximately 75 ml*, was Immediately processed for choles terol analysis by centrifugation to obtain plasma, followed by the addition of 14 ml. of a 1:1 mixture of cold alcohol- ; acetone per ml* of plasma to precipitate the proteins, which were then removed by centrifugation* The liver, lungs, brain, kidneys, adrenals, spleen, ovar ies, and aorta were removed as rapidly as possible and wei^i- i ed* After being ground by mortar and pestle, four aliquots of (each liver, two aliquots of each pair of kidneys and lungs, i and one aliquot of each brain end of each of the pooled spleens, ovaries, adrenals, and aortas were placed in tared i :Soxhlet thimbles and weighed# The samples were then ex tracted in Soxhlet extractors by a hot 3:2 mixture of aleohol-ether for eight hours, which had previously been shown to be sufficient for complete extraction of choles- ,terol# The extracts were placed in volumetric flasks and made up to volume at the time of cholesterol analysis# Choles- 'terol analyses were made on all the extracts, including that of the blood plasma, by the modification of the Schoen- heimer-Sperry method described by Nieft and Deuel (53). After cholesterol analysis, the tissue extracts were con- ,densed by distillation, and hydrolyzed with 33 percent KOH in a 1:1 aleohol-acetone mixture at 60°C for a period of one hour to free any esterified cholesterol# The amount of KOH was 0#2 ml. of 33 percent KOH per milligram of choles- !terol present# The hydrolyzed solution was neutralized to ■ the phenolphthalein end point with 15 percent acetic acid ;and 2 ml. of a 0*5 percent solution of digitonin in 50 per cent alcohol was added per milligram of cholesterol in order to precipitate the cholesterol as the digitonide# The precipitate was centrifuged, washed clean of supernatant by the alcohol-acetone solvent, and then washed 1 1 sufficiently with ether to remove any occluded fatty aeids. The digitonldes were dried in a vacuum desiccator and deuterium analyses were performed on all samples where possible, according to a method previously worked out in : this laboratory (54)* Deuterium values for cholesterol were calculated on the basis of three times the value : obtained for the digitonide since the hydrogen content of the digitonide is three times the hydrogen content of the cholesterol. Consequently, the deuterium concentration in : the digitonide is one third of that of the cholesterol I under analysis. In order to determine the exact deuterium content of ; the body fluids during the experimental period, pooled aliquots of the body carcasses were ground and lyophilized. ! The collected body water was distilled over alkaline per- i manganate, and the distillate collected from 99 to 102°C i j j was analyzed for deuterium. ' The animals were weighed at various intervals during j the pre-experimental period to ensure the use of healthy, growing animals. Weights were recorded at zero time and l ! at the termination of each experimental period since it was i recognized that there might be a relationship between ■ changes in the body weight and the incidence of ; atherosclerosis in rabbits* This has recently been discuss-! j ed by Firstbrook (55), and in addition, according to several other Investigators (56,57), there is a possibility of re- i t i sorption of the atherosclerotic plaques in malnutrition and 1 < wasting diseases* RESULTS Background of Each Experiment al Group Table I Illustrates the experimental details In the preparation of each experimental group* The amount of 50 percent deuterium oxide Injected to raise the body fluids to 1*5 percent and the amount of 2*5 percent deuterium oxide consumed by the animals in order to maintain that level are summarized* It can be seen that the animals on the cholesterol diet drank less water, probably due to the fact that the fresh cholesterol diet was slightly moist* This explains the deviation In the atoms percent deuterium in the body fluids of the animals on the high cholesterol diet from that of the control group* In all the groups the animals gained weight* Cholesterol Content of the Various Organs in the Normal Rabbit The Cholesterol contents and the weights of the tiss ues of the normal rabbit are shown in Table II* The weight of cholesterol per gram of tissue remains practically con stant over the experimental periods* The value Is highest in the adrenals, with the brain, lungs, ovaries, kidneys, spleen, liver, and aorta following in decreasing order* TABLE I BACKGROUND OF EACH EXPERIMENTAL GROUP Experi mental Animal Period days Wt. of Animal at Time of Injection gms • Ml. of 5056 D2° ' Injected Wt* of Animal at Sacrifice gms* Wt. Gain gms* Ml. of ! 2. 5* D20 ! Consumed ; Average/ 1 day/ I Animal j i Normal 8 2611* 1 * 8. 1 * 2790 176 1 i Diet 8 21*69 1*5.8 2657 188 2 1 * 6 i Normal 16 1952 36.2 2153 201 1 Diet 16 2128 39.5 2 1 * 6 8 31*0 268 I 1 5 6 Choles 8 1729 32.1 1883 151* | terol Diet 8 1 8 1 * 6 3U.3 1939 93 222 1% Choles 16 1581* 29.1* 2 1 1* 1 * 560 ; terol Diet 16 1619 30.0 2058 1*39 190 1% Choles 30 1661 30.9 2156 1*95 terol Diet 30 1670 31.0 2208 538 200 1 1 j * Calculated on basis of body weight x 60% s ml* body water ml* body water - x ml* * go x ml. “ T75 Where x Z ml. of 90% D2O injected to raise body fluids to 1.556 deuterium* 15 Distribution of Cholesterol In the Various Organs in the Normal Rabbit In Table II, the relative percents of the total choles terol per organ, based upon the total cholesterol content of the groups of organs studied, are listed. The percent ages for the liver, brain, lungs, adrenals, and kidneys are I based upon the total cholesterol found In those organs, ! whereas separate, additive totals were made for the spleen, ovaries, and aorta, respectively. This was done In order to facilitate comparisons with the turnover results, in which the first five organs constitute the main group studied. Comparison of the relative percent In the different organs clearly shows that the liver contains the bulk of the total cholesterol of the group, a value of about 50 percent. The brain, kidneys, lungs, spleen, ovaries, and aorta contain respectively less of the body cholesterol. The blood contains 0.62 to 0.64 milligrams of cholesterol per milliliter during both experimental periods. Cholesterol Content of the Various Organs of Rabbits on 1 £ High Cholesterol Diet Analogous data for the rabbits on a high cholesterol diet are shown in Table III, where it can be seen that the ' 16 TABLE I I CHOLESTEROL CONTEST OF NORMAL RABBIT ORGANS Organ Period Mg. Cholesterol Gm. Organ Wt. of Organ (gms.) Mg. Cholesterol Total Organ Relative Percents** of Total Cholesterol Per Organ Liver 8 2.05 109 223 49.6 ! 16 2.45 83 202 53.6 Lungs 8 4.36 9.73 42.41 9.4 16 4.38 7.89 34.55 9.2 Brain 8 15.8 6.96 110.0 24.4 16 13.8 5.51 74.99 20.0 Kidneys 8 3.53 17.05 60.19 13.4 ' 16 3.53 14.19 50.08 13.3 Adrenals 8 80.5 0.177 14.25 3.2 16 77.5 0.186 14.38 3.8 Spleen 8 2.58 0.728 1.88 0.4 16 2.78 0.494 1.37 0.4 Ovaries 8 4.26 0.235 1.00 0.2 16 4.15 0.160 0.665 0.2 Aorta 8 1.80 0.161 0.289 <0.1 16 - me m m Blood 8 0.62 “jj& l. 50 ml.* (31.90) (6.4) plasma 16 0.64 “9ml. 50 ml* (32.00) (6.8) Arbitrary to total value of plasma as described in text. Values in parentheses are calculated values, see*, ** Relative percent as defined in the text. 17 weight of cholesterol per gram of tissue is highest in the adrenals, with the brain, spleen, liver, lungs, kidneys, and aorta following in descreasing order. The cholesterol content per gram of tissue in the liver and lungs steadily increases during the experimental periods, whereas the adrenals appear to have reached a steady value (or a max imum peak) somewhere between 16 and 30 days. From the 1 values for total cholesterol content of the spleen, it appears that the cholesterol content of the spleen also does not continue to increase as the period on diet contin ues • The content per gram of tissue of the brain and aorta remains practically constant throughout the three periods. In the case of the ovaries, deposition of cholesterol occurs later in the feeding process. In the kidneys, there is a decrease during the 8 to 16 day period, with an in crease again by the end of 30 days. The cholesterol con- i tent of the plasma increases in value from 6.45 to 9.63 mg. per ml. during the 30 days• Distribution of Cholesterol in the Organs of Rabbits on a High Cholesterol Diet In Table III, the relative percents, calculated in the1 same manner as for the normal groups, show that the liver ■ 18 TABLE III CHOLESTEROL CONTENT OF ORGANS OF RABBITS ON A HIGH CHOLESTEROL DIET Organ Period Mg.Cholesterol Gm.Organ Wt. of Organ (s»») . . . Mg. Cholesterol Total Organ Relative Percents of Total Cholesterol > Per Organ ' Liver 8 8.96 74 663 75.1 16 10,8 93 1000 81.6 50 12.2 88 1074 82.0 Lung 3 8 5*60 6.45 36.11 4.1 16 6.68 7.58 50.63 4.1 30 8.09 7.91 63.97 4.9 Brain 8 14*0 7*52 106.3 11.9 16 15.7 6.35 99.74 8.2 30 13.3 5*91 78.56 6.0 Kidneys 8 5*50 12.54 68.96 7.8 16 4.47 11.97 53.50 4.4 30 5*90 11.86 69.95 5.3 Adrenals 8 94.0 0*106 9.983 1.1 16 146.3 0.148 21.62 1.8 30 145.4 0.178 25.81 2.0 Spleen 8 16.8 0.708 11.89 1.3 16 8.3 1.173 9.738 0.8 30 13.1 0.810 10.62 0.8 Ovaries 8 • • a » 16 3.24 0.107 0.345 C.l 30 8.74 0*092 0.800 <•1 Aorta 8 1.48 0.228 0.327 <•1 16 2,22 0.141 0.312 <•1 ! 30 1.47 0.164 0.241 Blood 8 6.45 “8ml* 50 ml. (322.5) (27)* plasma 16 9.48^1. 50 ml. (474.0) (28) 30 9*63 m8ml* 50 ml. (481.5) (28) Values in parentheses have been calculated on the basis of a total of 50 ml» of plasma per rabbit._______________ _ _________ 19 contains from 75 to 82 percent of the total cholesterol nnder conditions of high cholesterol diet. The brain, ■kidneys, lungs, adrenals, spleen, ovaries, ;and aorta 'follow in decreasing percentages, all of which are lower . than in the normal group# ! The Deuterium Content of Cholesterol in the Various Organs j of the Normal Rabbit The deuterium contents of cholesterol isolated fi»om the different tissues of the normal rabbit are shown in 1 Table IV# The actual values were calculated to 100 percent ; i deuterium in the body fluids. This value can be interpreted as an indication of the percent of cholesterol turned over : during those parts of the experimental periods in which the ;breakdown of labeled cholesterol can be considered neglig- i lible# This is probably true of all the organs during the zero to 8 day period. 1 It can be seen that the brain is turning over choles terol at the slowest rate, the liver at the highest rate, i !the lungs almost as rapidly as the liver, and the adrenals ! at an intermediate rate# The deuterium content of the J Iplasma cholesterol is comparable to that of the liver and J llungs. The kidneys appear to be turning over cholesterol [ I [ jat a greater rate during the 8 to 16 day period than during ' TABLE XV CHOLESTEROL TURNOVER IN NORMAL RABBITS Organ Period days Atoms Percent Deuterium in Body Fluids Cholesterol ■ Atoms % Excess D Actual Atoms % Excess D Corrected* Synthesis Gm. Organ Total Organ mg. mg. Relative % of Total Synthesis Liver 8 1.1*9 0.387 26.0 0.533 58.10 69.7 1 16 1.51 0.581* 38.7 0.91*0 78.05 66.0 Lungs 8 1.1*9 0.351 23.6 1,029 10.01 12.0 16 1.51 o.5i*o 35.7 1.561* 12.35 10.1* Brain 8 1.1*9 0.069 L.63 0.732 5.10 6.1 16 1.51 0.138 9.11* 1.21*3 6.85 5.8 Kidneys 8 1.1*9 0.201* 13.7 0.1*83 8.21* 9.9 i 16 1.51 0.525 3U.8 1.227 17.1*1 11*.7 Adrenals 8 1.1*9 0.201 13.5 10.87 1.92 2.3 I 16 1.51 0.381* 25.1* 19.69 3.65 3.1 Blood 8 1.1*9 0.333 22.3 0.138mg./ml. ( 6. 90)** (7.7) plasma 16 1.51 0.1*81 31.8 0.20i*mg./ml. (10.20) (8.2) * Corrected to 100 Atoms Percent Excess D in Body Fluids ]** Values in parentheses have been calculated for blood using $0 ml. plasma as total volume per rabbit. This was used to calculate relative percent in blood only. The other 5 organs are calculated in a separate group. g 21 the zero to 8 day period# The Relative Percent of Synthesis of Cholesterol in the Various Organs of the Normal Rabbit In Table XV can be found the relative percents of labeled cholesterol contained in the different organs# These are based upon the total amount of newly formed cholesterol found in all of the organs in the group# It can be seen that the liver normally synthesizes about 66 to 70 percent of all the cholesterol synthesized, with the lungs and kidneys, brain, and adrenals following in de creasing order# The relative percent formed in the kid neys during the 8 to 16 day period increases over that from the zero to 8 day period* The Deuterium Content of Cholesterol in the Various Organs of Rabbits on a High Cholesterol Diet The content of deuterium in the cholesterol of the various organs and the values calculated to 100 percent deuterium in the body fluids are shown in Table V# It can be observed that the percent of newly formed cholesterol in the different organs is decreased in comparison to the amount turned over in the normal organ except for the brain where it was shown that no cholesterol deposition 22 > took place* Table V also illustrates the amount of cholesterol synthesized per gram of organ. Here it can be seen that the actual synthesis of cholesterol per gram of liver is the same as under normal conditions* The uptake of deu- i terium into the cholesterol in the lungs, kidneys, and * adrenals on the per gram basis is depressed, however* There is an increase of labeled cholesterol per ml. of i plasma over the normal value* Synthesis in the brain is : the same as normal from zero to 8 days, with a slight increase during the 8 to 16 day period* The Relative Percent of Synthesis of Cholesterol in the Various Organs of Rabbits on a High Cholesterol Diet From Table V it can be seen that under conditions of I high cholesterol diet the liver is synthesizing from 79 to . 82 percent of the total newly formed cholesterol in the five organs, as compared with the 66 to 70 percent under 1 . i the normal conditions. The lungs contain about 3 percent, ! the brain 10 percent, and the adrenals approximately 1 1 percent. The kidneys during the zero to 8 day period con- ' ! tain 6*2 percent of the total but, during the 8 to 16 day i i period, only 3*8 percent. ' Similar comparison can be made for the blood if an Jarbitrary value of* 100 ml.of blood per rabbit, affording | 50 ml. of plasma, is taken as a basis for obtaining a total j with which to compare the relative percent of synthesized I cholesterol found in the plasma. It can be seen that upon Jthis basis, the relative percent is 13 for the zero to 8 : day period and 21 for the 8 to 16 day period* TABLE V CHOLESTEROL TURNOVER IN ORGANS OF RABBITS ON HIGH CHOLESTEROL DIET Cholesterol Organ Period days Atoms % D in Body Water Atoms % Excess D Actual Atoms % Excess Corrected Synthesis Gm. Organ Total Organ mg. mg. Relative % of Total Synthesis Liver 8 1 . 3 S > 0.088 6.33 0.568 U.97 79.0 16 1.00 0.088 8.80 0.916 87.98 82.2 30 1.36 0.126 9.26 1.128 99.26 81.1 Lungs 8 1.39 0.069 1.97 0.278 1.803 3.1 16 1.00 0.065 6.50 0.131 3.290 3.1 30 1.36 0.093 6,81 0.553 1.373 3.6 Brain 8 1.39 0.072 5.18 0.725 5.151 10.3 16 1.00 0.101 10.2 1.601 10.171 9.5 30 1.36 0.198 11.6 1.912 11.168 9.1 Kidneys 8 1.39 0.066 1.75 0.261 3.276 6.2 16 1.00 0.075 7.50 0.335 1.013 3.8 ■ ! 30 1.36 0U21 9.12 0.538 6.378 5.2 : Adrenals 8 1.39 0.078 5.62 5.283 0.561 1.1 f 16 1.00 0.071 7.10 10.387 1.535 1.1 i j 30 1.36 0.050 3.67 5.336 0.917 0.78 { ‘ Spleen 8 1.39 0.056 1.03 0.677 0.178 0.89 1 16 1.00 0.051 $.1 * 0 0.118 0.526 0.19 1 30 1.36 0.078 5.75 0.753- 0.611 0.50 Blood plasma 8 16 1.39 1.00 0.03U 0.055 m 0.157mgJnl. 0.521mg^ml. /(7.85) (26.05) S I P O tr * Corrected to 100 Atoms % D in body fluids. Discussion The results described above clearly Illustrate that there Is normally a turnover of cholesterol in all of the organs of the rabbit which were studied, and that dietary conditions of high cholesterol Intake have an effect on the cholesterol metabolism in that species. Effect of High Cholesterol Diet on the Content of Choles terol in the Various Tissues Of the Rabbit The effect of a high cholesterol diet on the content of cholesterol in the various organs is illustrated In Table VI. This contains a comparison of the weight of cholesterol per gram of tissue under the two dietary condi tions, and the percent of increase over the normal. There is a 300 to 400 percent increase per gram of tissue in the liver, which is a much greater percent In crease than for any other organ except the spleen. The comparable increase in the spleen is surprising. Schettler (58) related the storage of cholesterol In organs like the spleen to a fixation of cholesterol to protein and Its storage as a protoplasmic component. This storage would not necessarily involve histological changes according to Schettler, but It was observed in this experiment that histological changes can occur with storage since there was gross hyperplasia in the spleen of one of the experimental animals. This is in agreement with Altschul (59) who found that the histological effect of cholesterol feeding on the spleen is considerable, but lunruly*• Dietary cholesterol is deposited in all organs except the brain and aorta. It is interesting to note that the content of cholesterol per gram of tissue in the aorta does not increase during the period of feeding up to 30 days. This may indicate that deposition of cholesterol in the aorta does not occur primarily as a result of the presence of exogenous cholesterol in the body or the hypercholes terolemia thereby produced, but occurs instead as a change secondary to the effect of cholesterol in the other organs. This is consistent with the fact that there is a latent period in the development of arterial plaques upon feeding high cholesterol diets to rabbits. In recent discussions of experimental cholesterol atherosclerosis in rabbits by Weinhouse (24) and Kuntz (25), who conducted studies over extended periods of time, atheromas were found after ap proximately a thirty day latent period. Since in these experiments, there is no increase of cholesterol content in the aortas up to 30 days, it may be that once deposition 27 takes place it does so rapidly, or that the latent period varies somewhat under conditions in different laboratories* In relation to the deposition of cholesterol in the ovaries, it should be noted that ovarian developement takes place in animals of this age which might explain the delay in deposition to between the 16 and 30 day period* It can be observed in Table VI that the amount of cholesterol per gram of adrenal reaches an apparent limit since the values are the same for the 16 and 30 day periods. This could mean that a maximum point was reached between those periods or else that there is a limit which is maintained. The latter seems more probable since Altschul (60) reported that histologically there appears to be a limit to the amount of storage which takes place in the adrenal* In the ease of the kidney, there is a decline in the percent of cholesterol deposited from 56 percent during the zero to 8 day period to 27 percent during the 8 to 16 day period* At the 30 day point, the percent increase in kid ney cholesterol is less than that in all the other organs affected by the diet, although increased over the 16 day value* Perhaps some compensatory mechanism is operative up to a point and then less operative as the diet progresses* Altschul (61) has found histological abnormalities in the 28 renal duct epithelium very early in the cholesterol feed ing process. There were a great number of typical mitoses which were not present in sections from normal animals nor from animals which had been fed for long periods of time. It was also observed in this laboratory that atheromas occur in the renal arteries of animals on high cholesterol diet for a period of 4 to 5 months (62). By comparing the data in Tables II and III, it can be seen that the liver not only normally contains the largest relative percent, a value of 50 percent, but that this increases to 75 and 80 percent under conditions of excess exogenous cholesterol. All the other organs contribute to a much lower relative percent than normally (except for the spleen)• This, combined with the very high percent in crease of weight of cholesterol per gram of tissue, sug gests that the liver is the primary organ concerned with the storage of cholesterol from dietary sources. If the organs are responding to cholesterol diet by accelerating the destruction of cholesterol, this might mean that the liver is less able to do this than the other organs. This seems to be a less likely explanation in light of the histological results of Leary (6) who has observed that it is the liver cells (and also the cells in the adrenals) OF TABLE VI COMPARISON OF MILLIGRAMS OF CHOLESTEROL PER GRAM TISSUE IN RABBITS UNDER DIFFERENT DIETARY CONDITIONS 29 Organ Period days Mg* Cholesterol bin* Tissue Mg* Cholesterol Gm. tissue % Increase Mg. Cholesterol Gm. Tissue Normal High ** Based on average of normal values Liver 8 2*05 8.96 300 16 2 .1 *3 10.75 380 30 - 12.20 1 * 1 * 1 * Lungs 8 h.36 5.60 28 16 1*.38 6.68 53 • 30 - 8.09 85 Brain 8 15.8, , 1 1 * .0. , „ - 5 16 i5.7y ^ 7 30 m m 13.37 -10 Kidneys 8 3.53 5.50 56 ■ 16 3.53 l * . l * 7 27 30 - 5.90 6 1 * Adrenals 8 80.5 9U.0 19 16 77.5 11*6.3 85 30 - 1 1 * 5 . 1 * 85 Spleen 8 2.58 16.8 530 16 2.78 8.3* 210* 30 - 13.1 390 Ovaries 8 1*.26 16 i*.15 3.2 1 * - 2 1 * i 30 « ■ 8.71* 107 Aorta 8 1.80 1. 1 * 8. 16 - 2.22) 1.72 1 * 30 ** 1 .h r From weight of tissue and appearance, hyperplasia had occurred in the spleen during this period in this group* High cholesterol diet* 30 which esterify cholesterol and release the esters into the blood stream where they are transferred to other organs like the lungs* The globules of lipid containing choles terol, in animals on high cholesterol diet, remain in the cells until the cholesterol is esterified* In relation to the blood, the cholesterol content of plasma rises tenfold during the first 8 days and then in creases more slowly* It should be re-emphasized, however, that there is no direct correlation between blood choles terol levels and the degree of atherosclerosis in rabbits (3,63). The Effect of High Cholesterol Diet on the Metabolism of Cholesterol in the Rabbit The results of these experiments show that normally there are differences in the rate of turnover in the var ious organs studied* The liver has the most rapid turn over, and the brain a very much slower turnover which con firms the work of Waelsch and Sperry on non-saponifiable lipid in the brain (50)* The fact that the deuterium level in the blood plasma cholesterol is comparable with that of the liver and lungs in the normal rabbit indicates that normally most of the blood cholesterol is derived from one of these organs, 31 most likely the liver, unless there is turnover taking place independently in the blood* The idea that the liver is the primary source of blood cholesterol has been re ported very recently by Friedman (64,65) who demonstrated that the liver is the main organ concerned with restoring plasma cholesterol in rats which had been subjected to plasmaphoresis* This is consistent with these data and the results of Leary cited above* The apparent increase in the amount of synthesis taking place in the kidneys during the 8 to 16 day period in the normal rabbit may be explained in the same manner as for the blood, i*e., part of the labeled cholesterol found in the kidney is actually derived from some other source containing cholesterol which had been turned over at a rate greater than that actually occurring in the kidney* The level of cholesterol and newly synthesized choles terol in the blood is a measure of the absorption and mobilization of these substances as well as a measure of the balancing forces of destruction, excretion and de position. Thus, the constant content of cholesterol and the relatively high values of newly synthesized cholesterol in the blood of the normal rabbits during the experimental periods suggests that not only is there synthesis of 32 cholesterol in the "body, hut that there is also a constant movement of cholesterol to the blood from the organs con taining cholesterol which is relatively more hi^ily lab eled due to a more rapid turnover* In this respect, cholesterol synthesized in the liver might very well be going to the kidney via the blood, i since the kidney appeared to have a greater amount of 1 labeled cholesterol during the 8 to 16 day period than can be accounted for by the rate of synthesis during the zero to 8 day period, and since little cholesterol would be destroyed at the low level of cholesterol intake on the stock diet, as was pointed out in balance studies (66), At this point, a distinction between turnover and i • destruction should be made, in that turnover implies a constant amount of cholesterol in the tissues, which is ' constantly being synthesized and broken down in the con- i cept of dynamic equilibrium. Destruction, however, should Imply destruction of cholesterol without simultaneous re placement, i.e*, an absolute loss of cholesterol content. Under conditions of higjh cholesterol diet, the per- i cent of cholesterol turned over in the different organs studied is decreased with the exception of the brain. f t 1 This would be expected on the basis that deposition of | cholesterol is taking place, and in the ease of the brain, ; 33 no deposition took place. In order to compare the actual metabolism of choles terol under the different dietary conditions, therefore, it is necessary to compare the amount of cholesterol syn thesized per gram of tissue. This comparison is made in Table VII and Figure 1. Several facts stand out from this comparison. The amount of cholesterol synthesized per gram of liver is the same under the two dietary conditions. Thus, the liver Is turning over cholesterol at the same rate as normally despite the large amount of storage which is taking place at the same time. This places the role of < the liver as the primary organ of storage of cholesterol apart from its normal metabolism of that substance and supports the idea that storage of cholesterol is a normal function of that organ. Contrary to the condition in the liver, high eholes- ; terol diet causes depression of synthesis of cholesterol In all the other organs except the brain where no deposition takes place. These results may also be Interpreted in re- ■ lation to an increased rate of breakdown, or destruction of I V 1 V , cholesterol rather than a change in the rate of synthesis. * Deviations are found again in the metabolism of choles- 1 terol in the kidneys^ During the zero to 8 day period there I 31* ! ; i I i I TABLE VII ' . i COMPARISON OF CHOLESTEROL METABOLISM IN RABBITS j i UNDER DIFFERENT DIETARY CONDITIONS Organ Period days Cholesterol Metabolism Normal Diet Synthesis/gm. mg. High Cholesterol Diet Synthesis/gm. mg. Per cent Change from Normal/gm. Organ Liver 8 0.533 0.568 ♦ 6.6 16 0.9 1 * 0 0.9 1 * 6 ♦ 0.6 Longs 8 1.029 0.278 -73 16 1.561* 0 . 1 * 3 1 * -72 Brain 8 0.732 0.725 - 1 16 1.21*3 1.601 +29 Kidneys 8 0.1*83 0.261 -1 * 6 16 1.227 0.330 -73 Adrenals 8 10.868 5.283 -51 16 19.685 10.387 -1 * 2 Blood 8 0.138 mg/ml. 0.157 mg./ml. ♦13 plasma 16 0.20b mg/ml. 0.521 mg./ml. +155 i < v ; v Q O 3 / 7 SS' /l 30 M/\ftf£f 333 a jZ ’ / S J H J . M A g 7 0 3 . 7 / - S 3 7 0 //J 30 £/VVi/Ol7 7ff{/ 36 is a decrease of 46 percent in the amount of labeled choles terol per gram of tissue as compared with the normal, where as during the 8 to 16 day period a 76 percent decrease oc curs* The amount of labeled cholesterol in the blood plasma of animals on a high cholesterol diet is not only higher than normal, despite the excess of unlabeled cholesterol being ingested and hypercholesterolemia, but also does not increase constantly throughout the experimental periods* ihere is a 12 percent increase from zero to 8 days but a 155 percent increase from 8 to 16 days over the normal values* This is possibly explained if there is an in creased mobilization under conditions of high cholesterol diet and if it is the endogenous cholesterol, i.e., choles terol which has been turned over, which is mobilized to the blood. It was mentioned above that Leary has observed that dietary cholesterol undergoes a process in the liver which results in the formation of cholesterol esters which are then released into the blood stream. If the liver is mobilizing labeled cholesterol into t the blood stream, one would expect to find the same phen- ' I omenon occurring in the kidneys as under the normal condi tions, I.e., an apparent increase in the rate of synthesis . of labeled cholesterol. This would afford a depression 37 during the 8 to 16 day period of less than the 46 percent observed during the zero to 8 days. Since there is not only less depression, but actually a marked increase in depression of up to 76 percent, high cholesterol diet is . affecting the normal functioning of the kidneys. The possibility that the kidney is destroying the labeled cholesterol which is picked up from the blood is suggested, since it is known that destruction takes place to a large extent when there is a large excess of cholesterol in the ; animal (66). In these studies Schoenheimer and Breusch found that animals were able to destroy up to five times the body content of cholesterol in a month when large * amounts of cholesterol were fed. This and other balance studies (67), however, were unable to reveal where this destruction takes place or whether any particular organ is primarily concerned* The relationship of the kidney in rabbit cholesterol metabolism would be consistent with the findings that production of atherosclerosis In rabbits is accelerated when kidney damage was induced by pharmacological means , (68), if one of the causes of atherosclerosis in rabbits , is a loss of the kidney mechanism of destruction in later stages of the feeding process. (The low level of labeled cholesterol in the kidney could also be explained if the 38 kidney is not picking up the labeled cholesterol from the blood as It did in the normal animals*) It would be of value to determine the normal turnover of cholesterol in the kidney in the absence of the other organs, and whether perfusing a high concentration of cholesterol through the organ would result in destruction or a change in the turnover* This experiment is being planned in this laboratory at present* In addition, ex periments using animals which had been on a high choles terol diet for a period of four and a half months are being completed* Regardless of whether this explanation involving the kidney will prove valid or not,*the advisability of study ing the metabolism of cholesterol in the rabbit kidney more thoroughly is apparent from these results. In addi tion, the fact that mobilization complicates interpretation of data obtained from in vivo studies strongly suggests that studies designed to determine metabolism in any one organ should elimate the effects due to the presence of the other organs* From Figure 2, it can be seen that the relative per cents of synthesized cholesterol in the different organs under each dietary condition remain fairly constant throughout the periods studied except for the kidneys and 1 & O V ) * H j ° I * c > 4 J k t oo 90 80 70 60 ~ 4 O a : t u t o Q U J t y m On, Q So 3. . ‘ O'5" “ p ^i40 G S i < J « c U j 0. v > 30 20 )0 L IV E Ft H 16 H CHOLESTEROL DIET NO RM ft L D /E T BLOOD PLASMA LUNGS BRAIN R/DNEYS ADRENALS 16 & 16 8 /6 8 /6 PERIOD OF T IM E, DAYS ■ j-D— 8 !6 J _ 8 /& FIGURE 2. R E LA TIV E PERCENTAGES OF CHOLESTEROL S Y N T H E S IZ E D IN THE VARIOUS ORGANS 40 ' the blood plasma of the high cholesterol groups* A brief comparison of cholesterol turnover in the normal rabbit with that of the normal rat is of value since the rat is the species which is, insofar as deter mined, resistant to cholesterol induced atherosclerosis* The values for the rat are from unpublished data obtained ' in this laboratory (69)* Turnover of cholesterol in the rat liver and adrenals is markedly more rapid than in the corresponding organs of the rabbit* The turnover rates in the lungs and brain are only slightly faster in the rat* In the case of the kidneys, however, there is no apparent increase in the rate of uptake of deuterium in the rat during the 8 to 16 day period as was found in the rabbit* , The uptake in the kidneys during the zero to 8 day period is comparable In the two species, however* Comparing the relative percents of cholesterol and labeled cholesterol in the organs of the two species it can be seen that in the rabbit liver there is 50 percent of the total cholesterol and 68 percent of the total labeled cholesterol, whereas the corresponding values for the rat liver are 25 and 42 percent* The rabbit lungs contain 9 percent of the total cholesterol and 12 percent | of the total labeled cholesterol, while the rat lungs ' eontain 12 and 14 percent, correspondingly. The rabbit : 41 brain contains 22 percent of the total cholesterol and 6 percent of the total labeled cholesterol with corresponding values of 38 and 10 percent in the rat* The rat kidneys contain 13 percent of the total cholesterol and 10 percent of the total labeled cholesterol. The adrenals contain 4 percent of the total cholesterol in both species, and 3 and 5 percent of the total labeled cholesterol in the rabbit and rat respectively. SUMMARY A HD COHCLUSIOHS Cholesterol metabolism in normal rabbits and in rab bits placed on a one percent cholesterol diet was studied using deuterium as a tracer in the body fluids for periods of 8 and 16 days and 8, 16 and 50 days, respectively* Quantitative analyses for the cholesterol content of the liver, brain, lungs, spleen, adrenals, kidneys, ovaries, aorta, and the blood plasma were made. The isolated digitonides were analyzed for their deuterium content. The results can be summarized as follows: 1. The normal content of cholesterol per gram of tissue is highest in the adrenals, with the brain, lungs, ovaries kidneys, spleen, liver, and aorta following in decreasing order. The liver normally contains about 50 percent of the total cholesterol found in all the organs studied. 2. Cholesterol is deposited in all the organs except the brain and aorta during the periods studied on high choles terol diet. The liver contains about 75 to 80 percent of the total cholesterol tinder these conditions and consti tutes the main organ of storage of exogenous cholesterol. This storage appears to be a normal function of that organ. 3. Cholesterol turnover in the normal rabbit organs is 43 most rapid in the liver with the lungs, adrenals, and brain following in decreasing order. The kidneys appear to be turning over cholesterol at a faster rate during the second period than during the first period. 4. The amount of labeled cholesterol per gram of tissue decreased in all the organs in which cholesterol deposi tion took place except the liver where the amount of lab eled cholesterol per gram remained the same as normal. Either there is depressed synthesis or increased break down of cholesterol in the affected organs. 5. Hypercholesterolemia occurred in the animals on high cholesterol diet. Plasma cholesterol increased ten fold during the first period and then continued to increase more slowly. The amount of labeled cholesterol in the pla sma was not only greater in the animals on high cholesterol diet, but increased from 13 percent above normal during the zero to 8 day period to 155 percent above normal during the1 8 to 16 day period. ■ 6. The amount of labeled cholesterol per gram of kidney decreased to 54 percent of normal during the zero to 8 day period, but went down to 27 percent of normal during the f * second period. The results suggest the possibility that the kidney 44 might be destroying cholesterol which had been turned over in the liver and mobilized into the blood from Tafoich it is picked up by the kidney. The advisability of studying cholesterol metabolism in the kidney in vitro is discussed. BIBLIOGRAPHY BIBLIOGRAPHY 1. MacCallum, W. B., Textbook of Pathology, 7th edition, (Philadelphia: W. B* Saunders Co*, 1940) p* 324. 2* Marehand, F*, Verhandl, d, Kong, f, inn* Med*, 21, 23, (1904) in MacCallum, of*, 1, 3* Leary, T*, Arch, Path*, 21, 460 (1936), 4* Leary, T., Arch, Path,, 21, 419 (1936), 5, Morrison, L, M,, Ann. Western Med, Surgery, 4, 665 (1950), 6, Leary, T., Arch, Path., 32, 507 (1941), 7. Leary, T., Arch. Path., 47, 1 (1949), 8. Moreton, J, R., Science, 106, 190 (1947). 9# Becker, G. H., Meyer, J., and Nechelea, H,, Science, 110, 529 (1949). 10* Zinn, W* J,, and Griffith, G. C., Am* J* Med. Sci., 220. 597 (1950). 11. 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Creator Larack, Audrey Mildred (author)

Core Title A comparison of the turnover of cholesterol in rabbits on a normal and high cholesterol diet

Degree Master of Science

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), [illegible] (committee member), Alfin-Slater, R.B. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-777503

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