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The effect of incubation on the cholesterol content of rat liver homogenates

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The effect of incubation on the cholesterol content of rat liver homogenates

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Content THE EFFECT OF INCUBATION ON THE CHOLESTEROL CONTENT OF RAT LIVER HOMOGENATES A Thesis Presented to the Faculty of the Department of Biochemistry University of Southern California In Partial Fulfillment of the Requirements for the Degree Master of Science By Leslie Irene Rice August 1949 UMI Number: EP41309 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 EP41309 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 48106-1346 This thesis, written by .....Iiejali.ft..lJ!ene..JRi.©.e ....... under the guidance of hia r . . . Faculty Committee, and approved by a ll its members, has been presented to and accepted by the Council on G raduate Study and Research in p artial fu lfill ment of the requirements fo r the degree of M a ^ . t . e r . . . . . Q . f . . . . S . t t i e n . G . e ........... Emory S. Bogardus ....... Dean Date, August.li49r _____ Faculty Committee .. Chairman ...... TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION 1 II. HISTORICAL REVIEW 2 Synthesis of cholesterol 2 Precursors of cholesterol 6 Destruction of cholesterol 8 III. PLAN OF EXPERIMENT 16 IV. EXPERIMENTAL PROCEDURES 18 Treatment of animals 18 Preparation of liver homogenate 18 Incubation of homogenate 21 Extraction of cholesterol 22 Determination of cholesterol 36 V. RESULTS 38 Effect of pH 38 Effect of succinate 38 Effect of boiling the homogenate 40 VI. DISCUSSION 46 VII. SUMMARY 50 BIBLIOGRAPHY 52 LIST OP TABLES TABLE I. II. III. IY. V. VI. VII. VIII. IX. X. XI. XII. COMPOSITION OP DIET EXTRACTION OF CHOLESTEROL Effect of Temperature Effect of Volume of Solvent Method A Comparison of Methods A and B Method B Effect of Alkaline Digestion EFFECT OP pH ON THE CHANGE IN TOTAL CHOLES TEROL OF RAT LIVER HOMOGENATE DURING INCUBA TION COMPARISON OF THE EFFECTS' OF SUCCINATE BUFFER AND SALINE ON THE CHANGE IN TOTAL CHOLESTEROL OF RAT LIVER HOMOGENATE DURING INCUBATION EFFECT OF SUCCINATE ON THE CHANGE IN TOTAL CHOLESTEROL CONTENT OF RAT LIVER HOMOGENATES DURING INCUBATION EFFECT OF MALONATE ON THE CHANGE IN TOTAL CHOLESTEROL OF RAT LIVER HOMOGENATE DURING INCUBATION EFFECT OF BOILING AND INCUBATION ON THE CHANGE IN TOTAL CHOLESTEROL CONTENT OF RAT LIVER HOMO GENATES DURING INCUBATION CHAPTER X INTRODUCTION Balance experiments have shown that the animal organ ism Is capable of disposing of excess cholesterol. In view of the relation of cholesterol metabolism to atherosclerosis it was considered important to investigate the site and nature of the mechanism responsible for cholesterol destruc tion. Since a few reports in the literature indicate that the liver might possibly be involved in this process, It was decided to study the effects of incubation under various conditions on the cholesterol content of rat liver homogena- tes. CHAPTER II HISTORICAL REVIEW Synthesis of cholesterol* Cholesterol was first discovered by Poulletier around 1769 in gallstones* In the half-century which followed, its occurrence in higher ani mals and man was investigated extensively, and it was found to be present not only in gallstones but also in intestinal concretions, tumors, crude tubercle, and in the blood in various pathological conditions* These findings led the early Investigators to regard it as a product of patholo gical origin* This concept began to change with the dis covery by Couerbe in 1834 that cholesterol is a component of normal brain tissue and with its detection by other workers in normal blood* Numerous theories were advanced to explain the occur rence of cholesterol In animal tissues. Lehmann (1) regarded it as the decomposition product of some unknown precursor* Flint (2), in 1862, proposed that it was an excretion pro duct elaborated by the brain and nervous tissue, carried by the blood to the liver, excreted by the bile, and converted to coprosterol in the intestine* This theory was opposed by Beneke (3) who determined that cholesterol was present in the brain in larger quantities than would be predicted if it were a product of excretion* Since that time,the 5 origin and fate of cholesterol In the animal have heen the objects of numerous investigations. The complexity of the cholesterol molecule led at first to the assumption that the animal obtained this or a similar sterol from exogenous sources. This theory seemed logical at that time, since sterol syntheses had not been observed in animal tissues. Moreover, early experiments had indicated an inability of the animal to synthesize cho lesterol, and it had been shown (4) that dietary cholesterol is readily absorbed from the intestines. This concept was discarded when it was discovered that cholesterol is not an essential dietary component but can be synthesized by animals maintained on diets entirely free of cholesterol, as shown by relatively constant serum cholesterol levels and continued excretion of sterols in the feces. Some of the first experiments pertaining to the bio synthesis of cholesterol were performed on hen*s eggs early in this century. Mendel and Leavenworth (5), Ellis and Gardner (6), and Beumer (7) observed that the cholesterol content of newly-hatched chicks was not greater than that of the egg, thus indicating no synthesis. This finding has recently been substantiated by Rittenberg and Schoenheimer (8) with the aid of isotope techniques. Their failure to detect any deuterium in the cholesterol of chicks which had developed in eggs containing heavy water proved that cholesterol is not 4 synthesized during the development of hen’s eggs. Although the evidence against cholesterol formation in hen’s eggs was indisputable, balance studies (9) have shown the hen, itself, as well as many other species readily synthesize cholesterol. As early as 1914, Dezani (10) re ported that growing white mice fed on extracted diets over a period of several weeks did not lose cholesterol but actually increased their cholesterol content. Gamble and Blackfan (11) found a much larger excretion than intake of cholesterol in infants on a milk diet, while Gardner and Fox (12) observed a similar occurrence in adults on mixed diets. Beurner and Lehmann (13) showed that the cholesterol content of puppies increased twenty times during a four-week period on a choles terol-poor diet. Channon (14), in 1925, demonstrated that the quantity of cholesterol increased rapidly in young rats fed a diet free of cholesterol but complete in other nut rients. In the same year, Handles and Knudson (15) reported the results of an experiment in which female rats were placed on a cholesterol-free diet and allowed to reproduce. The offspring were raised on the same diet, and their bodies were found to contain an amount of cholesterol comparable to that of rats raised on normal diets. This work, and that of Channon proved beyond doubt that rats are able to synthesize their own cholesterol and can be maintained in good health on a diet free of cholesterol. Further proof that the animal body is able to syn thesize cholesterol was provided by the classical experiment of Schoenheimer and Breusch (16) in which mice fed on a diet of bread alone synthesized as much cholesterol in a month as they originally contained in their bodies. Since the first experimental indications of endogenous synthesis of cholesterol, the site and mechanism of this process have been repeatedly investigated. At present, the liver and adrenal cortex are the only organs in which this synthesis has been shown to occur. Various other organs have also been stuided. The discovery that cholesterol is a major component of brain tissue led Couerbe (17) in 1834 to suggest it was ela borated by the nervous system, a view which was later suppor ted by Flint (2) and has not yet been disproved. The adrenal gland is another organ rich in cholesterol, and many workers have considered it to play an important role in cholesterol metabolism. Grigaut (18), found in 1913 that unilateral adrenalectomy in the dog was followed by a short period during which the blood cholesterol remained constant. This latent period was succeeded by a hypercholesterolemia which later returned to normal. In a similar experiment, Baumann and Holly (19) detected no increase in blood choles terol levels of dogs after either unilateral- or bilateral- adrenalectomy, while Joelson and Shorr (20) found an increase in both situations* The latter workers concluded that this type of experiment provided insufficient evidence to draw any conclusions as to the role of the adrenals in cholesterol metabolism, although there was definite indication of a relationship between the two* The German School (20) regarded the adrenal as a storage depot for cholesterol, and numerous other theories have been advanced to explain its effects on blood choles terol. However, the only concrete evidence that the gland manufactures cholesterol is the recent report by Srere, Chaikoff and Dauben (21) that incubation of slices of beef adrenal cortex with C^-labeled acetate results in the in corporation of into the cholesterol molecule. Another organ which has been shown to synthesize cholesterol is the liver. In a series of reports, Artom (22, 23) claimed that there was an increase in cholesterol during either perfusion or autolyses of the livers from starved dogs. In 1946, Bloch, Borek, and Rittenberg (24) demonstrated that surviving rat liver slices synthesize cholesterol when incubated aerobically in the presence of deuterium-labeled.acetate. They also reported that no synthesis occurred in slices of kidney, spleen, testes, or G.I. tract. Precursors of cholesterol. In studies on the origin of cholesterol various substances have been considered as 7 possible precursors. Plant sterols were once thought to he changed to cholesterol in the body, but it has been proven by Schoenheimer (25) and others that they are not absorbed to any appreciable extent by animals. Another theory pos tulated the direct formation of cholesterol from fatty acids, but there is a considerable quantity of evidence against such a mechanism. In brief, little progress was made toward Identifying the substances from which cholesterol Is derived until the Introduction of isotdpic techniques. In 1937, Rittenberg and Schoenheimer (8) injected mice with enough DgO to raise the deuterium content of the body fluids to 15 atom per cent and maintained this concen tration for long periods by administering DgO in the drinking water. At intervals, groups of animals were killed and the deuterium content of the body cholesterol was measured. The proportion of deuterium found in the cholesterol indicated that half of the stable hydrogen atoms of the molecule were derived from those of the body fluids. This would not be possible if cholesterol were formed from any steroid in the food or by cyclization of a long-chain fatty acid; hence the authors concluded that in mammals it is prepared by the coupling of many small molecules. In further experiments, Bloch and Rittenberg (26) fed various deuterium-labeled substances to mice, in a search for precursors of cholesterol. They demonstrated that the 8 feeding of deufcerio-acetate led to the formation of choles terol containing deuterium in both the nucleus and side- chain. Propionic, butyric, succinic, pyruvic, and aceto- acetic acids were excluded as intermediates in the conver sion of acetate to sterol* Recently, these investigators (27) have obtained labeled cholesterol from animals.fed deuterio-acetone as well as from liver slices incubated with deuterio-acetone; they have suggested that acetone may be the source of the methyl groups of cholesterol. Since neither acetic acid nor acetone as such is an important dietary component, they must be derived from other substances before entering into the formation of choles terol. This infers that any material which forms acetone or acetic acid during its metabolism is a potential source of cholesterol. It is well known that both acetone and acetic acid are metabolic products of fats and ketogenic amino acids. Moreover, since carbohydrates are readily converted to fat in the body, any one of the three types of dietary constituents may act as a precursor of cholesterol. Thus, it would appear that attempts to control cholesterol formation in the body by limiting specific components of the diet would present great difficulties. Destruction of cholesterol. The information which has been accumulated with respect to the destruction of cholesterol in the body is even more limited than that concerned with its synthesis. It should he pointed out that the interpretation of the term "cholesterol destruction" depends upon the method used for determination of the sub stance. If, for instance, analysis is based on the formation of an insoluble complex with digitonin, any reaction which renders cholesterol unprecipitable by digitonin would be termed a destruction. (Conversely, any reaction by which a substance becomes precipitable by this reagent would be termed a synthesis.) On this basis, It is impossible to decide whether cholesterol has been broken down to small end-products or has merely been transformed to a non-preci- pitable steroid. Interpretations of data based upon the digitonin precipitation are further complicated by the non specificity of the reaction, for it is common to all sterols. In which there is a free hydroxyl group on position 3 and in cis relationship to the angular methyl group at (28). Similar limitations should be taken Into consideration if other methods of detecting cholesterol are employed. Balance experiments have proved that cholesterol can be destroyed as well as synthesized in the animal body. The first evidence of this type was presented in 1931 by Dam (29) who observed a loss of sterol in young chickens which could not be accounted for on the basis of excretion. Page and Menschick fed moderately large amounts of cholesterol to rabbits (30) and cats (31) and determined the difference 10 between the sterol fed and the sterol excreted, plus that found in the body. In both species there was a positive balance, showing that cholesterol breakdown had occurred. Cook (32) fed high-cholesterol diets to rats and discovered that 30$J of the sterol administered could not be accounted for in the feces and in the animals* bodies. Schoenheimer and Breusch (16), in the experiment previously referred to in connection with cholesterol syn thesis, showed that mice which, on low cholesterol diets, were able to synthesize their own cholesterol, were also able to destroy this substance when it was fed in large quantities. They concluded "that in the tissues cholesterol is continually being formed and destroyed. Either a positive or negative balance may be found depending upon experimental conditions, i.e. synthesis may be in excess of destruction, or vice versa." The site of cholesterol destruction has not yet been determined. The lung was studied as a possible site by French investigators, around 1924-1928. Remond, et al (33), observed that the blood of the right side of the heart was always richer in cholesterol than that of the left and inter preted this as evidence for cholesterol breakdown or storage by the lungs. Nitzescu (34), et al, found that the lung showed no cholesterolytic power in vitro. Asphyxia, caused by pinching the trachea,was found by Bouisset and Soula (35) 11 to cause a reversal of the effect noted by Remond, and these workers concluded that ventilation stimulates the ability of the lung to diminish cholesterol, while asphyxia inter feres with it. However, Bugnard (36) attributed this effect to a transference of cholesterol from the serum to the cor puscles during pulmonary transit, rather than to its des truction, and he suggested that regulation of the cholesterol content of blood is a function of the blood itself. Several Investigators have recorded increases in blood cholesterol following splenectomy. In 1921 Abelous and Soula (37) noted an increase in the cholesterol content of splenic pulp during aseptic autolysis and concluded that the spleen plays an important role In cholesterol metabolism. In 1937 Takagi (38) reported that when colloidal solutions of cholesterol were injected into rabbits, the blood choles terol alternately increased and decreased. If the animals were first splenectomized, or if the spleens were exposed to X-rays, the blood cholesterol remained high after the cholesterol was injected, suggesting to Takagi that the spleen functions in cholesterol breakdown. Takagifs observation that the injection of cholesterol preparations into the blood stream causes only a transient hypercholesterolemia has been repeated by other workers. Pitz and Bruger (39) obtained a similar reaction when dogs were injected intravenously with cholesterol in the form of 12 a colloidal suspension. Recently, Horlick and co-workers (40) prepared a physiologically emulsified form of choles terol from the plasma of chickens fed for several weeks on a cholesterol-rich diet. This plasma was given intravenously to dogs in varying doses, and the rate of disappearance of cholesterol from the blood stream was found to vary with its concentration above the normal value. It would be of interest to determine whether the cholesterol injected into the blood stream is destroyed in some manner or merely stored by an organ, such as the liver or lungs. The organ which seems at present to be the most logical site of cholesterol breakdown is the liver. In 1922 Artom (22, 23, 41) reported that the cholesterol con tent of perfused dog liver decreased If the organ was re moved during active digestion or from a fasting dog whose duodenum had been injected with hydrochloric acid. The same result was obtained during autolysis of liver, and in this case removal of the pancreas Increased the amount of destruction. Remond, et al (33), discovered that the sub- hepatic vein contains less cholesterol than the portal vein and attributed this to the ability of the liver to either store or partially destroy cholesterol. In 1948 Marx and Lipsett (42) observed a diminution in the cholesterol content of rat liver homogenates after incubation at 37°, Swell and Treadwell (43) recently reported that when rat liver homo- 13 genates were incubated with bile salt, there was an initial decrease in total cholesterol, followed by an increase until the concentration reached approximately the original value. Still longer incubation resulted in further increase. Val- deguie' and S^gu^las (44) incubated aqueous ground liver preparations with solutions of cholesterol, prepared with the aid of lecithin and bile salts, and demonstrated the production of significant quantities of acetone. Although they reported no measurements of cholesterol concentrations, it is possible that the acetone was derived from cholesterol. This ketogenic property was also exhibited by blood to a lesser degree. Further evidence that the liver is involved in the regulation of body cholesterol levels was provided by Schally (45) in 1936. He showed that parenterally administered liver extract increased to normal the blood cholesterol levels in pathological states characterized by hypocholesterolemia, while it reduced the hypercholesterolemia of patients with nephrosis, icterus, high blood pressure, and nephritis. This finding, along with evidence reviewed by Peters and Van Slyke (46), suggests that the liver plays a significant role in the metabolism of cholesterol— its synthesis, esteri- fication, storage, and destruction. Little is known about the products of cholesterol breakdown in the body. With the aid of deuterium as a tracer, 14 it has been shown to be a precursor of two structurally- related compounds. In 1943, Bloch, Berg, and Rittenberg (47) injected deuterio-cholesterol into dogs and isolated deuterium-labeled cholic acid from the bile, indicating a conversion of cholesterol to cholic acid. Later, Bloch (48) fed labeled cholesterol to a woman in the eighth month of pregnancy and subsequently isolated from the urine deuterium- labeled pregnanediol, an excretion product of the steroid hormone progesterone. The high content of chole sterol in the adrenal cortex and its structural similarity to the hormones of this gland have led to the theory that it may be utilized in hormone production. Although no direct evidence for this has been produced, Sayers, Sayers, White and Long (49) have shown that the cholesterol content of the adrenal cortex is markedly reduced by administration of adrenocorticotrophic hormone of the anterior pituitary. Evidence that acetone may possibly be a metabolic product of cholesterol has been mentioned previously (44). In summary, it has been shown that cholesterol is not an essential dietary constituent, but is synthesized in the adrenal glands and liver by the coupling of small mole cules. Excessive quantities, when ingested, are destroyed by a mechanism of unknown nature. Thus, the total amount of cholesterol in the animal is maintained at a constant level 15 by the interaction of absorption, storage, excretion, syn thesis, and destruction. CHAPTER III PLAN OP EXPERIMENT The purpose of these experiments was to continue the work of Marx and Lipsett (42), of this laboratory, who found that rat liver riomogenates may contain a system for breaking down cholesterol. In their preliminary experiments the cholesterol content of liver homogenates frequently diminished markedly during incubation. The results varied widely, however, and a decrease was not demonstrated in every experiment. Further work was aimed at (a) determining whether the system was of enzymic nature and (b) establishing conditions favorable for the occurrence of cholesterol des truction, as defined on page 9. The effect of heat was considered valuable in estab lishing the first point, since high temperatures would be expected to inactivate the system if it were enzymatic. Lipsett began to investigate this factor but did not collect sufficient data to draw any conclusions. \The optimum pH of the reaction was the factor chosen to study the second point.! ' ThOTrats used were fed a diet high in cholesterol, since other workers have shown that destruction of choles terol can be demonstrated in vivo only when there is an ab normally high cholesterol intake. Consequently, it was felt 17 that if a destructive mechanism were present in rat liver, it should be activated by feeding excess cholesterol. The experimental technique used at the beginning of this work was that previously established by Marx and Lip- sett (50), Briefly, the liver was homogenized and then in cubated with the solution, the effect of which was under investigation. The cholesterol content of the tissue was determined before and after incubation. It was later found necessary to modify the procedure for extracting cholesterol from the homogenate, and a new method was devised. CHAPTER IV EXPERIMENTAL PROCEDURES Treatment of animals. Male and female albino rats of the University of Southern California colony were plaeed on a high cholesterol diet at ages from three weeks to three months. The composition of the diet is given in Table I. Even distribution of the cholesterol throughout the mixture was effected by dissolving it in the cottonseed oil prior to mixing. The animals received this diet ad libitum until they were sacrificed, the feeding period varying in different cases from thirty days to several months. No correlation between the length of the period of high cholesterol intake and alteration of cholesterol concentration of the liver during incubation is evident, thus far. Preparation of liver homogenates. The procedure follows technique developed by Marx and Lipsett (50). The animals were anesthetized by intraperitoneal injection of 0.15 ml. of nembutal (60 mg. per ml.) per 100 grams body weight. The livers, which invariably exhibited a high degree of fatty infiltration, were promptly removed, rinsed, weighed, and chilled. The tissue was minced with scissors, and por tions weighing around 4-5 grams were homogenized with exactly twice their weight of liquid, which will be referred to as the homogenization fluid. The apparatus employed was con- TABLE I 19 COMPOSITION OF DIET Ingredients Per cent Whole wheat flour 33.75 Oats, ground 33.75 Skim milk powder 15.00 Alfalfa meal 4.00 Yeast, Strain ‘ ’ G’ 1 ^ 2.00 Cottonseed oil 7.00 Fortified oil2 2.00 3 Cholesterol 1.00 Bile salt^ 0.50'. Sodium chloride 0.50 Calcium carbonate 0.50 1 Brewer*s Yeast, Strain 1 1 GM, Anheuser-Busch Inc., St. Louis, Missouri. 2 Fortified oil contained 1600 I.TJ* of Vitamin A and 160 I.TJ. of Vitamin D per gram of oil. 3 Cholesterin, C.P., Merck and Co., Inc., Rahway, New Jersey. 4 Sodium glycocholate, City Chemical Corporation, New York, New York. 20 structed on a principle similar to that originated by Potter (51), This'consisted of a stainless steel, motor driven, pestle, which was later replaced by one made of lucite, and closely fitting 25 mm by 200 mm pyrex tubes. The homogenization was continued until the suspension appeared uniform and no large particles of connective tissue could be seen. This period usually amounted to around seven minutes with the steel pestle, while four minutes were suf ficient with the lucite pestle and ground tubes. All appara tus and tissue were maintained at a low temperature through out these operations by the use of ice-water baths. The homogenate was centrifuged five minutes at 5,000 RPM and filtered through gauze to remove bits of connective tissue. Different steps of the method were varied, in col laboration with Adams and Schotz, in an attempt to determine the conditions most favorable for the production of homo- genates exhibiting significant decreases in cholesterol during incubation. The medium of homogenization was studied by Schotz (52). He compared the change in cholesterol levels during incubation of homogenates prepared with distilled water and with a solution containing 0.9 M KC1 and 0.88 M sucrose. Apparent breakdown in amounts as large as 60$ were observed in homogenates made with the latter solution. An attempt was made to investigate the influence of 21 the extent of cellular disintegration by varying the period of homogenization. The results were inconclusive with res pect to the ability of the preparations to destroy choles terol. It was of interest that greater periods of grinding produced homogenates containing larger quantities of choles terol, showing that much of the endogenous substrate of coarser suspensions is lost during centrifugation. Temperatures at which the operations were carried out were also varied. A few of the experiments were conducted in a room maintained within a few degrees of 0° C, while in others, the tissue and solutions were chilled by means of ice baths, the method employed by Marx and Lipsett (50). In general, the greatest amounts of cholesterol destruction were observed in homogenates prepared in the cold room. However it can not be definitely stated that this method is preferable, since the results of a few control experiments using ice baths were inconclusive. The influence of duration and temperature of centri fugation of the crude homogenate was studied and reported by Adams (53) • Incubation of the homogenate. Following centrifugation, the homogenate was thoroughly mixed with an equal volume of the solution to be tested, and 1 ml portions were placed in either 15 ml test tubes or 25 ml volumetric flasks, depending upon the means of extraction to be used later. Volumes were 22 measured with extreme care, since small errors in measure ment in these steps would he magnified several times during l the assay for cholesterol. In experiments in which the object being investigated was some factor in the preparation of the homogenate, the suspension was merely Incubated with the liquid used in its preparation. The vessels containing the mixture to be incubated were placed in a large glass container of the type commonly used as a dessicator, A high humidity was maintained by filling the bottom section with water, to prevent alterations in the volume of homogenate during incubation. The chamber was filled with oxygen'*' and placed in a 37° incubator for 16 to 20 hours. The control samples were extracted at the beginning of this period, stoppered, and stored in a refri gerator in order to avoid losses due to evaporation until the Incubated samples were ready for assay. Extraction of cholesterol. The extraction method used In the early experiments of this series was that des cribed by Nieft and Deuel (54) and also employed by Marx and Lipsett (50). The procedure was to add a 3;2 mixture of alcohol-ether forcibly with a calibrated syringe, with 10,0 ml of solvent used for each 0,5 ml of tissue extract 1 Preliminary experiments of Marx and Lipsett (50) indicated that the system was active in an atmosphere of 0g, 23 or 1.0 ml of homogenate diluted with incubation solution* (This was approximately equal to a 60 jl ratio of solvent to fresh tissue*) The tubes were stoppered and allowed to stand for 10 minutes to complete the extraction. This procedure, which will be designated as Hextrac- tion method A,” was used with apparently satisfactory results in the first set of experiments which were concerned with pH effects. In subsequent studies on the effect of heating the homogenate prior to incubation, the amount of cholesterol recovered from boiled samples, particularly after incubation, was frequently less than that extracted from untreated homo genates. The possibility was considered that the coagulation of protein brought about by heating the tissue interfered with the extraction of cholesterol. The further reduction in extractable cholesterol of samples boiled and then in cubated was so untypical of an enzymatic reaction that an explanation was sought, and it was considered possible that incubation, per se, renders cholesterol less extractable either through trapping cholesterol in coagulated protein or through the production of cholesterol-protein complexes during incubation which are not readily broken down. It is possible that previous observations of cholesterol "break- down, ! by Lipsett and Marx (42) might be attributed to this effect. For these reasons it was felt that the efficiency of the extraction method should be thoroughly investigated, 24 for untreated as well as for boiled homogenates, and that a new method should be devised, if necessary. There are a few reports in the literature of tech niques for extracting cholesterol from tissues. Most of these require several lengthy periods of refluxing with var ious solvents, usually alcohol or ether, or a mixture of the two. Another method frequently used when total cholesterol values are desired involves the digestion of the tissue with alkaline solutions, followed by extraction of cholesterol with solvents. However, in the majority of cases reported by other workers, it was necessary to extract large quantities of tissue, while the experiments reported here required the extraction of only 150 mg samples of tissue, usually in a finely-divided state. Moreover, the large number of assays involved in each experiment made it desirable to devise, if possible, a simpler, less time-consuming method than has heretofore been reported. Prom a study of extraction procedures used by other workers, it became evident that four factors of extraction method A required modification: 1) the temperature of extraction, 2) the proportion of solvent to tissue, 3) ratio of solvent to water, and 4) the duration of extraction. The effect of temperature was studied first, while maintaining the 60:1 ratio of solvent to tissue. Different samples of homogenate prepared from the same liver were 25 extracted before and after incubation using method A, as previously described, and by refluxing for 10-15 minutes with 3:2 alcohol-ether as the solvent. The results are compared in Table II. In all but two instances, the amount of cholesterol extracted by refluxing was greater than that removed at room temperature. The influence of ratio of solvent to tissue was studied at room temperature and with refluxing. The ratios compared were 60:1 (or 10 volumes of alcohol:ether to 1 vol ume of diluted homogenate) and 150:1 (25 volumes of solvent to 1 volume of diluted homogenate). The latter proportion was chosen since the extraction could conveniently be carried out in 25 ml volumetric flasks fitted with reflux condensers. Table III shows that considerably larger quantities of cholesterol can be extracted from untreated as well as from boiled samples when larger quantities of solvent are used. The effectiveness of method A in removing cholesterol from both boiled and untreated homogenate was tested by re- extracting the residues by a method including refluxing for approximately 30 minutes and a solvent: tissue ratio of 150:1. This procedure will be designated "extraction method B." In executing these experiments, the extract from method A was decanted from the centrifuged residue and saved for analysis, and the residue was transferred quantitatively from the centrifuge tube to a 25 ml volumetric flask. It 26 was refluxed with 15-20 ml of alcohol: ether, cooled, and the flask filled to the mark. This extract was then assayed for cholesterol to determine the completeness of the first extraction. The results are shown in Table IV, with the amount of cholesterol reextracted expressed as milligrams and as per cent. In all cases the amount reextracted from the residues of homogenates not boiled previous to extraction was below 8$. Since the residues were not washed after decanting the first extract, some of the reextracted cholesterol can be accounted for as remaining from the original extract. If, for example, 0.5 ml of the first extract from method A remained on the residue, 5% of the quantity of cholesterol extracted would appear to be reextracted from the residue. Therefore reextraction of less than 5% of the amount of cholesterol removed at the first extraction is insignificant* With this interpretation in view, it may be concluded that in experiments 25 and 29, method A was sufficiently complete, while in experiment 26, significant amounts of cholesterol remained in the residue. Method A was quite ineffective in recovering cholesterol from boiled homogenates after incuba tion in all three experiments and from boiled samples before incubation in experiment 26. Contrary to the assumption that cholesterol becomes less extractable during incubation, there was no appreciable difference between the quantities 27 reextracted before and after incubation, in the case of the untreated homogenates. However, there was a significant effect of incubation on the extractability of cholesterol in boiled homogenates, particularly in homogenates prepared with sucrose-KCl solution. From the data presented thus far, it was obvious that method A was not reliable as a means of removing choles terol from tissues previously coagulated by heat, and that earlier data concerning the effect of heat on the system were of little significance. Furthermore, this procedure was not consistently effective for extraction of samples which had not been boiled. Consequently, its use was dis continued entirely in favor of the technique already desig nated as "method B," that is, refluxing with 150 volumes of solvent for 30 minutes. In Table V the quantities of cholesterol extracted from identical liver preparations by the two methods, A and B, are compared. In every case, more cholesterol was re covered with method B than with A. In heat-treated homo genates method B removed an average of 40*6$ more choles terol from unincubated tissue and 78$ more from incubated samples. Contrastingly, it was only 3.9$ more effective for control and 1.6$ more effective for incubated samples of an untreated homogenate. Although there are insufficient data here to be given much weight, the results bear out the 28 conclusion drawn from Table IV, that is, that the effect of incubation in rendering the cholesterol less easily extract- able is of far greater importance in boiled homogenates. It should also be noted that, at least in this ex periment, the apparent diminution of the cholesterol content of untreated homogenate during incubation was no greater with method A than with method B. This result was encour aging, since it might indicate that perhaps the ‘ ‘breakdown” observed in early experiments employing method A was perhaps a real effect and not an artifact of extraction* The effectiveness of method B was tested in a simi lar manner to that already described for the other method* After the first extraction, the residue was reextracted. Tk*om Table VI, it is evident that the first extraction was sufficient for untreated tissue, since, as in the experiments on method A, the residue was not washed prior to reextraction, and it has been calculated that any amount of cholesterol removed from the residue not exceeding 5$ was insignificant* In one instance (experiment 29), the first treatment of boiled samples was Incomplete. However it should be noted that the liver in this case contained a higher concentration of cholesterol than usual, and it is possible that the volume of solvent used was Insufficient to dissolve this quantity. (For this reason, animals should be used in these experiments which have been fed cholesterol for shorter 29 periods and consequently have a smaller concentration of cholesterol deposited in the livers.) On the strength of these results, thi3 procedure was adopted for extraction in all experiments not involving heating of the homogenate, while it was still felt to be of doubtful value in effecting complete removal of cholesterol in cases of boiled homogenates of livers containing unusually large amounts of cholesterol. One further type of treatment was investigated in this study of extraction methods. This involved digestion of the tissue with alcoholic potassium hydroxide prior to extraction* It was felt that by dissolving the coagulated tissue in this manner, the possibility of any cholesterol still remaining enclosed or trapped by coagulated protein would be eliminated. A further advantage would be the simul taneous hydrolysis of cholesterol esters, making this step unnecessary in the assay procedure which follows. The result of an experiment in which this treatment was compared with methods A and B is given in Table VII. The quantities of cholesterol recovered from untreated homogenates were prac tically identical to those obtained with method B, but the alkaline digestion was inferior for boiled samples. Further work should be done in refining a method of this type, since the time saved by eliminating the hydrolysis step in the cholesterol determination would be worthwhile. 30 TABLE II EXTRACTION Of CHOLESTEROL EFFECT OF TS23PERATOB! Experiment No. Homogenization Fluid Treatment of Homogenate and Medium of Incubation Mg, Total Cholesterol Extracted per Oram Fresh Liver Room Temperature Reflux (Method A) 6 Distilled water Saline Control 7.80 2.22 O.O5 H Succinate and 0.05 M Borate Incubated b.5 6 6.5^ 0.2 N Acetic acid and 0.2 N Sodium acetate Incubated 6.30 7.02 9 Distilled water Saline Control 19. bo 20.50 0.05 M Succinate and 0.05 M Borate Incubated 1M 0 ll &600 0.2 N Acetic acid and 0.2 N Sodium.acetate Incubated 16.20 15.20 26 Sucrose-KCl Boiled b minutes Control 9.5^ 10.20 Boiled b minutes Incubated Average 9.12 111.00 2.52 11.50 31 TABLE III EXTRACTION OF CHOLESTEROL EFFECT OF VOLUME OF SOLVENT Extraction Temperature Treatment of Homogenate* Mg. Total Cholesterol per Gram Fresh Liver SO Volumes 150 Volumes Al cohol:Ether(3:2) A1cohol:Ether(3:2) Room Temperature Untreated Control Incubated 10. SO 9.5^ 12.9 11.6 Reflux Boiled 4 min. Control Incubated 10.20 s. 52 12.3 13.7 * Homogenization fluid was Sucrose-KCl. 32 TABLE IV EXTRA.CTIQI OF CHOLESTEROL METHOD A Experiment Homogenization Treatment of Homogenate Total Cholesterol Extracted per Gram Eresh Liver ^°* Eluid First Extraction Reextraction of Residue Total Method ' ‘ A” mg. Method mg. 1 1 3 If fo mg. 25 Distilled Untreated Control 17.2 0.96 5.58 IS.2 water Incubated 15.0 0.84 5.60 15.S Boiled 4 min. Control 16.9 1.3S 8.16 18.3 Incubated 11. 8 3.12 26.40 lh.9 26 Sucrose-KCl Untreated Control 10.8 0.81 7.50 11.6 Incubated 9.5^ 0.74 7*75 10.3 Boiled 4 min. Control 9.5U 3.95 4i.4o 13.5 Incubated 9.1S M 2 47.20 13.5 29 Sucrose-KCl Untreated Control 11.6 0.32 2.76 11.9 Incubated ll.l 0.32 2.S9 11.4 Boiled 4 min. Control S. 53 0.54 6.29 9.12 Incubated 4.16 3. 9H 9H.70 ' 8.10 33 TABLE V EXTRACTION OF CHOLESTEROL COMPARISON OF METHODS A AND B Treatment of Homogenate Homogeni zat ion fluid Experiment No. Mg. Total Cholesterol per Gram Fresh Liver Method A Method B Control Incubated Control Incubated Untreated Sucrose-KCl Distilled water 29 30 Averages 11.6 JL2ii 12.3 ll.l l4.0 12.5 12.2 13-3 11.3 l4.l 12.7 Boiled 4 min. Sucrose-KCl Distilled water 26 29 Averages 9.5^ 8.53 “9.06 9.18 4.16 6.67 12.3 12.8 13.7 10.0 11.9 * TABLE VI EXTRACTION OP CHOLESTEROL METHOD B Experiment Ho. Homogenization Pluid Treatment of Homogenate Total Cholesterol Pirst Extraction mg. Extracted per Gram fresh Liver Second Extraction Total mg. $ mg. 2Sa Sucrose-201 Untreated Control 9.23 0.21 2.3 9. 1& Incubated 8.85 0.38 9.23 Boiled b min. Control 9.00 0.20 2.2 9.20 Incubated 8.70 0.15 1.7 8.85 28b Sucrose-KCl Untreated Control 10.90 0.21 1.8 11.10 Incubated 9.60 0.39 9.99 Boiled b min. Control 10.10 0.30 3.0 10. bo Incubated 9.23 O .36 3.9 9.59 29 Sucrose-KCl Untreated Control 12.20 0.^7 3*9 12.70 Incubated 11.30 0.33 2.9 11.60 Boiled ^ min. Control 11.30 1.H3 13*0 12.70 Incubated 10.00 2.16 22.0 12.20 35 TABLE 711 EXTRACTION OF CHOLESTEROL EFFECT OF ALKALINE DIGESTION Experiment 30 Treatment of Homogenate* Mg. Cholesterol Extracted per Gram Liver Room Temperature Reflux Digest with 2$ £6:1 150:1 alcoholic KOH Untreated Control Incubated 13-9 lU.O lU.H 1U.1 1U.3 1U.1 Boiled Control Incubated - ik.h ik.i 13.7 13.S * Homogenization fluid was distilled water. 36 Determination of cholesterol* The method employed was based upon a modification of the Schoenheimer and Sperry method developed by Nieft and Deuel (54), Duplicate aliquots of the alcohol-ether extract were measured into 15 ml centrifuge tubes and evaporated in alu minum test tube blocks provided with 50 watt cartridge heaters and a thermostat set for 60°* The evaporation was hastened by means of an air stream directed into the tubes through capillaries attached to an air-distributing manifold. The residue was redissolved in 1 ml of a 1:1 alcohol-acetone mixture* Two drops of 30$ aqueous KOH were added and the mixture heated at 60° for 30 minutes with frequent lateral shaleing to hydrolyze the esters present. (It was later shown that evaporation of the aliquot and redissolving it in acetone-alcohol was unnecessary, and the hydrolysis can be carried out equally well on the alcohol-ether extract. It is desireable, however, to bring the volume of the aliquot to 1 ml or less, since thorough mixture of the KOH is more easily accomplished In this 3mall volume.) At the end of this period, the solution was made acid to phenolphthalein with 15$ acetic acid. The volume was brought to approximately 3 ml with alcohol-acetone and 1 ml of 0.5$ digitonin in 50$ alcohol was added. After thorough mixing the tubes were allowed to stand at 37° for 3 hours. The cholesterol-digitonide was well packed by cent- 37 rifugation at 3,000 RPM for at least ten minutes, preferably longer, and the supernatant carefully decanted. The preci pitate was washed with 3 ml of ether, centrifuged, and the ether decanted. The tubes were placed in an aluminum block at 60° and the excess liquid evaporated off with a stream of air. Thorough drying at this point was necessary, since moisture has been found to interfere with the Liebermann- Burchard color reaction. The digitonide was dissolved by heating at 60° with 0.5 ml of glacial acetic acid. Exactly 3 ml of chloroform were added and the stoppered tubes trans ferred to a 35° block for color development. The color reagent was prepared just before using by addition of one part of concentrated sulfuric acid to 9 parts of acetic anhydride cooled in an ice bath. One ml of this solution was added to each assay tube, and at the end of exactly ten minutes, each tube was transferred to an ice bath and cooled for another 10 minute period. The tubes were read in the Klett colorimeter against a reagent blank with a 660 ngi filter within forty minutes after addition of the color reagent. CHAPTER V RESULTS Effeot of pH. The influence of different buffers upon the change in cholesterol content of liver homogenates during incubation is shown in Table VIII. The “Universal*1 buffer used in experiments 10 and 23 consists of a solution 0.02857 M with respect to diethyl barbituric acid, citric acid, potassium di-phosphate, boric acid, and hydrochloric acid which is titrated with an appropriate quantity of 0.2 N sodium hydroxide to obtain any desired pH within.the range of 2.4-12.0 (55). A pH of 5 was superior to a pH of 8 in promoting apparent breakdown of cholesterol in all experiments except #23. In all of these experiments, cholesterol was extrac ted from the homogenate by method A.. Later studies on ex traction procedures Indicated the possibility that the ex traction may not always have been complete using this method. It is not possible, therefore, to draw any conclusions from these results, unless they can be confirmed by using a technique giving complete extraction. Effect of succinate. The striking effect of succi nate buffer in experiments 2 and 4 (shown in Table IX) led to the consideration that perhaps the factor responsible 39 TABLE VIII EFFECT OF pH OH THE CHMGE Iff TOTAL CHOLESTEROL OF BAT LIVER HOMOGEHATET DURING INCUBATION Experiment Ho. pH 5 p H 2 Buffer Change in Cholesterol Mg/Gm Liver $ Buffer Change in Cholesterol Mg/Gm Liver $ 2 0.05 M succinates 0.05 M borate -h.h -64.2 O.O5 M borax s 0.1 K phosphate -1 .2 -17.6 k 1 1 -5.8 -U3.6 ti -0.7 - 5.3 9 it -5*2 -26.7 1 1 -h. 3 -22.0 10 "Universal" buffer* -'•6.7 -to.3 "Universal" buffer -4.6 -27.7 23 1 1 -3.1 -18.0 1 1 -5*0 -29.0 Average -5.0 -38.7 Average -3.2 -20.3 $ Homogenization fluid— distilled water. * 100 cc of solution of HC1, KHpPOij., citric acid, “ boric acid, and veronal (0.02257 M with respect to each constituent)titrated with appropriate volume of 0.2 H HaOH. 40 for the large decreases in cholesterol during incubation was not only the pH of the homogenate but the presence of suc cinate, Therefore a comparison was made of the change in cholesterol content of homogenates incubated in succinate buffer and in other buffers at a pH of 5, The results are summarized in Table X, In three experiments the decrease in cholesterol was greater when succinate was present, while in an equal number, it was greater without succinate. The average diminution was essentially the same In the two cases. In Table IX, the changes in cholesterol of homogenates incubated In succinate buffer and in unbuffered solutions are compared. Although the succinate solution is markedly superior, in experiments #2 and #4, this effect was not ob served in any of the other experiments. In connection with the study of succinate, as a pos sible agent for stimulating the system for cholesterol "destruction,” the action of malonate as an inhibitor was also investigated. The effect of incubation on the choles terol content of homogenates was compared in various solutions In the presence and absence of malonate. Table XI shows that addition of malonate produced an apparent Inhibition of cholesterol "destruction” of up to 70$. Effect of boiling the homogenate. A summary of the data concerning the influence of heat on the homogenate is given In Table XII. In experiments in which a significant 4i TABLE XX COMPARISON OP TEE EFFECTS OF SUCCINATE BUFFER AND SALINE ON THE CHANGE IN TOTAL CHOLESTEROL OF RAT LIVER HOMOGENATE DURING INCUBATION Experiment Incubated, in Saline Incubated in Succinate Buffer-pH 5 Ho. ------------------------------------ ------------------------------------ Change in Cholesterol Change.in Cholesterol Mg/ Gm Liver Mg/ Gm Liver $ 2 -0.5 -7.4 -4.4 -64.8 4 -1.1 -8.3 -5.8 -43.6 l4 -2.5 -16.4 -2.2 -14.5. 19 -2.2 -14.8 +0.2 negligible 23 -2.8 -16.3 -2.5 -14.5 average -1.8 -12.6 -2.9 -27.5 kz TABLE X EEEECT QP SUCCINATE OH THE CHANGE IN TOTAL CHOLESTEROL OE SAT LIVER HOMOGENATES* DURING INCUBATION Experiment No. pH Without Succinate With Succinate Buffer Change in Cholesterol Buffer Change in Cholesterol Mg/ Gm Liver $ Mg/ Gm Liver i. 0.05 M Borate - + ■ 0.05 M Borate + 1 + 7*5 0.05 M phosphate -0.7 - 5.5 0.05 M phosphate -1.6 -11.9 0.2 M Na acetate -t 0.05 M Succinate+ 5 5*0 0.2 M acetic acid -11.6 -1+1.2 0.05 M Borate -10.5 -37.2 6 5*0 1 1 1 1 -1.2 -lH.5 1 1 1 1 -1.7 -21,0 9 5*0 1 1 u -23.1 f -5-9 -2S.7 23 5*0 1 1 1 1 -3.0 1 1 1 1 -2.5 -ll+,6 li+ 5*5 Universal Buffer “ • 2.2 -11+.6 1 1 1 1 -2.2 -ll+,6 23 5.0 n 1 1 -3rl -1S.1 1 1 t i -2.5 -11+.6 Average -19.2 - P - -20.^+ * Homogenization fluid was distilled water 1+3 TABLE XI EFFECT OF MALONATE QN THE CHANGE IN TOTAL CHOLESTEROL OF LIVER HOMOGENATES DURING INCUBATION AT 350 Medium of Incubation Change in Cholesterol (-) Malonate (+) Malonate Mg/ Gm Liver $ Mg/ Gm Liver $ 0.9$ NaCl -2.5 -16.H -0.7 VO . J- 1 O.O5 M succinate + 0*05 M horate (pH 5) -2.2 -lH>6 -1.0 -6.6 Universal buffer (pH 5) -2.2 -l*u6 -1.1 -7.2 vo 44 decrease in cholesterol was observed upon incubation of untreated samples, boiling of the homogenated eliminated" this in one experiment (26), decreased it in two (28b arid 29), and increased it in one experiment (25)* In the re mainder, no significant change in cholesterol content was observed upon incubation of either boiled or untreated homo genates. It has already been pointed out, in the discussion of extraction studies, that during incubation of boiled homo genates some alteration takes place which renders the choles terol less readily extractable. The possibility has to be considered, especially in cases where the total cholesterol concentration was high, that perhaps extraction was still incomplete in the incubated samples. If this were true, the apparent decrease in cholesterol observed during incuba tion of boiled homogenates in several of the experiments could be accounted for on this basis. Additional work is necessary for a clarification of the effect of boiling on the decrease in cholesterol concen tration during incubation. > * 5 TABLE XII EFFECT OF BOILING AND INCUBATION ON THE CHANGE IN TOTAL CHOLESTEROL OF RAT LIVER HOMOGENATES DURING INCUBATION Homogenizing Experiment Medium No. Homogenate Untreated Homogenate Boiled H Minutes Mg. Total Cholesterol Extracted per Cm. Fresh Liver I Change Mg. Total Extracted per Cholesterol Gm. Fresh Liver i Change (1) Control (2) Incubated (1-2) (1) Control (2) Incubated (1-2) Distilled 25 18.2 15.8 2.1+ -13.2 18.3 11+.9 3 . H -18.6 Water 30a 14.1+ l*+.l 0.3 - 2.1 ik.b ll+.l 0.3 - 2.1 Sucrose- 26 11.6 10.3 1-3 -11.2 13.5 13.5 0.0 ' 0 :Y KCl 28a 9.^ 9.2 0.2 - 2.1 9.2 8.9 0.3 - 3.3 28b 1 1 . 1 10.0 1.1 - 9.9 10.1 + 9*6 0.8 - .7.7 29 12.7 11.7 1.0 - 7.9 12.8 12.1 0.7 - 5.5 CHAPTER VI DISCUSSION Interpretation of the results of the early experiments on pH and succinate effects is difficult, since later studies showed that the procedure employed for extraction of choles terol in these early experiments may not have been complete in some instances. Although this applied primarily to homo- genates boiled prior to incubation, it was also true to some extent of unheated preparations. With the adoption of more stringent methods of extraction, it was no longer possible to demonstrate the significant decreases in cholesterol con tent which had been observed previously. In an attempt to reconcile this with the early data, the possibility could not be excluded that the apparent diminution in cholesterol content of liver homogenates during incubation, which had also been observed by Marx §nd Lipsett (42), was actually an artifact* This might have been the result of some change occurlng in the homogenate during incubation which made the extraction of cholesterol more difficult than before incuba tion. This reasoning was shown to be correct in the case of homogenates which had been subjected to high temperatures prior to incubation; there was no doubt that in this case cholesterol was more difficult to extract after incubation. However, there was no indication that this was true 47 of incubated (but otherwise untreated) homogenates, since the quantity of cholesterol recovered from residues which had first been extracted by method A was essentially the same in incubated and control samples* Evidence that early observations of ’ ’breakdown” might not be attributed entirely to an artifact of extrac tion was provided by an experiment in which the decrease in cholesterol during incubation was observed to be almost identical regardless of whether extraction method A or B was used. The latter method had been shown to effect com plete extraction of cholesterol under the conditions of this experiment. Tests of the effectiveness of extraction method A showed that this procedure extracted at least 92$ of the total cholesterol from homogenates not boiled prior to ex traction, while in many of the early experiments, the amount of cholesterol recovered after incubation was only 40-80$ of that in the controls. In view of these findings, it seems improbable that the change in cholesterol concentration during incubation was due entirely to an artifact of extraction. Therefore, it was concluded that the early results may be significant if they can be confirmed with the new extraction technique. Attempts to accomplish this have thus far been unsuccessful* In the investigation of pH effects it was demonstrated 48 that incubation in a slightly acid medium was, in the major ity of experiments, more conducive to cholesterol decrease than incubation in a basic medium. The possibility was considered that the causative factor might have been not the pH but the presence of succinate in the pH 5 buffer. However, it was found that in solutions of comparable pH, the average change in cholesterol concentration during in cubation was the same whether succinate was present or not* It was concluded that greater decreases in cholesterol can be observed if the homogenate is incubated at a pH of 5 than at a pH of 8. However the possibility should be in vestigated that the protein of the homogenate is coagulated to a greater degree in the acidic solution, rendering the cholesterol more difficult to extract (than in neutral or slightly basic solutions). The action of malonic acid as an inhibitor of this system was clearly demonstrated, but in only one experiment. This might be interpreted as an indication that the succinic dehydrogenase system is involved and therefore, that the cholesterol "destruction" mechanism is of an enzymatic nature. Ho conclusion can be drawn until more evidence is collected. The effect of boiling on the system is still not clear. In the majority of experiments boiling the homogenate resulted in a decrease in the apparent "destruction*1 of cholesterol during incubation, but complete "inhibition" was 49 observed in only one instance. The demonstration that cholesterol is less readily extracted from boiled than from untreated homogenates in dicates that boiling altered the physical or chemical nature of the homogenate. The decrease in extractability might be an indication that during boiling cholesterol becomes mec hanically trapped within the coagulated protein. It is also possible that cholesterol in the tissue is bound to protein and that boiling coagulates the cholesterol-protein complex, rendering the cholesterol less accessible to the solvent. Although in recent experiments it has not been possible to demonstrate decreases in the cholesterol content of in cubated liver homogenates, the possibility that breakdown does occur in this system can not yet be discarded. The probability has been largely excluded that incomplete ex traction of incubated samples was responsible for the marked decrease in cholesterol content noted in many experiments on homogenates not heated prior to incubation. There remains only the explanation that some other factor in the procedure essential to the occurrence of breakdown has been altered unintentionally, for in recent attempts to exactly duplicate the techniques used in early experiments it was not possible to demonstrate breakdown. CHAPTER VII SUMMARY 1. The extraction method employed by Marx and Lipsett (42) in their experiments demonstrating destruction of cholesterol during incubation of rat liver homogenate was shown to effect only partial extraction of boiled homo genates* Extraction of untreated homogenates by this method was at least 92$ complete* 2. A new extraction method was devised which invol ved refluxing the tissue for 30 minutes with a solvent: tissue ratio of 150:1. This procedure was shown to be 98$ effective for untreated homogenates and equally effective for boiled samples of livers in which the cholesterol con centration did not exceed around 11 mg/ gm fresh tissue. 3. Extraction of cholesterol was shown to be more difficult after incubation of previously boiled homogenates than in the controls* This was not true of homogenates not boiled prior to incubation* 4. Incubation at a pH of 5 was more conducive to a decrease in cholesterol concentration than was incubation at a pH of 8* This effect was not attributable to the presence of succinate in the acidic buffer. 5. In boiled homogenate the cholesterol concentration decreased less during incubation than in untreated homogenates. 51 6* In early experiments significant decreases in cholesterol content of homogenates were observed as a con sequence of incubation. 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Asset Metadata

Creator Rice, Leslie Irene (author)

Core Title The effect of incubation on the cholesterol content of rat liver homogenates

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Biochemistry

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-779303

Unique identifier UC11347723

Identifier EP41309.pdf (filename),usctheses-c17-779303 (legacy record id)

Legacy Identifier EP41309.pdf

Dmrecord 779303

Document Type Thesis

Rights Rice, Leslie Irene

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA