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A study on the comparison of the cholesterol content of rat liver homogenates before and after incubation

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A study on the comparison of the cholesterol content of rat liver homogenates before and after incubation

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Content A STUDY ON THE COMPARISON OP THE CHOLESTEROL CONTENT OP RAT LIVER HOMOGENATES BEFORE AND AFTER INCUBATION A Thesis Presented to the Faculty of the Department of Biochemistry The University of Southern California In Partial Fulfillment of the Requirements for the Degree Master of Science by Myron Collens Schotz January 1950 UMI Number: EP41310 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 EP41310 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 Myron Pollens Schotz . under the guidance of A . . d L s . F acu lty Com m ittee, and approved by a ll its members, has been presented to and accepted by the Council on G raduate Study and Research in p a rtial fu lfill ment of the requirements fo r the degree of MASTER OF SCIENCE Date. Faculty Committee TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION ................................ 1 II. HISTORICAL REVIEW .......................... 2 Studies on cholesterol synthesis ..... 3 Studies on conversion of cholesterol to other steroids........................ 5 Studies on cholesterol breakdown ......... 6 In . vivo......... 6 b. In vitro ......... 6 III. METHODS .............................. 9 Treatment of the animals................. 9 Treatment of tissues .................... 10 Determination of cholesterol . ........... 10 IV. EXPERIMENTAL RESULTS ........................ 13 Series I .................................. 13 Effect of homogenization method ......... 13 Experiment A ........................... 14 Experiment B ............................ IT ( Series I I ................................ IT Time-rate study of change in cholesterol concentration ............... IT Series III................................ 18 iii CHAPTER PAGE Variation in extraction procedure .... 18 Series I V ............................... 20 Studies on the effect of supplementary factors on cholesterol "breakdownM . . 20 Experiment A .................... 20 Experiment B .................... 22 V. DISCUSSION OP RESULTS...................... 26 VI. SUMMARY . ...................... 29 BIBLIOGRAPHY .................................... 30 LIST OF TABLES TABLE PAGE I. . Effeet of Different Homogenizing and Incubating Medium on the Change in Cholesterol Concentration in Rat Liver Homogenates upon Incubation,........... . . 16 II. Results of Incubations Using Improved Extraction Technique ... ................ 21 III. Effect of Choline, Inositol and Insulin when Added to Incubation Medium............. 23 IV. Effeet of Lipotropic Factors when Added to the Diet of the R a t .................... 25 LIST OF FIGURES FIGURE PAGE 1. Time-Rate Curve.......................... 19 CHAPTER I INTRODUCTION A preliminary report by Marx and Lipsett (l) indicated that liver homogenates of rats fed a high cholesterol diet might contain a mechanism that causes, upon incubation, a decrease in cholesterol concentration as determined by a modified Sperry-Schoenheimer method. Furthermore, the liver has been shown to contain a factor which can break off the isopropyl group of cholesterdl. Further information about a mechanism for the bio logical breakdown or conversion of cholesterol to other products was considered desirable, not only to obtain a better understanding of the pathways of cholesterol meta bolism, but also in order to lead possibly to practical applications in regard to the problem of disposal of choles terol in cases of hypercholesteremia. Therefore, the effect of incubation of rat liver homogenates on cholesterol concentration was studied in this report. CHAPTER II HISTORICAL REVIEW For many years a relationship between cholesterol metabolism and atherosclerosis has been proposed by a number of investigators (2, 3). The well established ob servation that feeding high cholesterol diets to rabbits, guinea pigs and chickens produces experimental athero sclerosis substantiates this relationship. Recently, Steiner and Kendall (4-) have found that lesions closely resembling those seen in human arteriosclerosis develop in the dog following the ingestion of cholesterol and thiouracil over a period of time. Further evidence in dicating that cholesterol metabolism and atherosclerosis are interrelated is apparent from the fact that large amounts of cholesterol and its esters are found in ather- sclerotic plaques (5). Also, clinically, it has been found that in cases of uncontrolled diabetes mellitus and chronic glomerulonephritis— which are conditions wherein hyper cholesterolemia are common— the tendency to develop athero sclerosis is high (6). In contrast to the rabbit and the chicken; the rat is more resistant to developing hypercholesterolemia and atherosclerotic lesions on prolonged cholesterol feeding (7). 3 It would appear therefore that the rat somehow destroys or excretes the excess cholesterol more efficiently than the rabbit or the chicken, or that the rate of cholesterol turn over may be significantly different. Studies on cholesterol synthesis. That cholesterol is synthesized by the animal body has been recognized for, many years (8). Animals maintained on a cholesterol free diet continue to maintain relatively constant serum choles terol levels and continue to excrete sterols in the feces. This was demonstrated by Schoenheimer (9) by carefully con ducted balance experiments. Rittenberg and Schoenheimer (10) injected mice with sufficient D2O to raise the deuterium content of the body fluids to 1.5 atom per cent and this concentration was main tained for long periods by administering D2O in the drinking water. Groups of the animals were sacrificed and the deu terium content of the body cholesterol was measured. The Isolated cholesterol contained a proportion of deuterium that indicated 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 cyclization of a long-chain fatty acid; the authors therefore concluded that in mice cholesterol 4 is synthesized by the coupling of a large number of small molecules. Sonderhoff and Thomas (11) recovered so much deuterium from the unsaponifiable fraction of yeast grown on a medium containing deuterioaeetate that they concluded that acetic acid was directly converted to sterols. In 1942 Bloch and Rittenberg (12) fed sodium acetate containing deuterium to rats and mice. The quantities of deuterium in the sterols definitely established the fact that acetic acid can serve as one of the building blocks of cholesterol. More recently, Bloch and Rittenberg (13) have shown that all compounds which yield acetate in the course of their metabolism contribute to the formation of cholesterol. Although acetate is not an important dietary component, fatty acids and ketogenic amino acids are convertible to acetate, and acetate appears to be an intermediate for fat, carbohydrate and protein meta bolism. Therefore most dietary foodstuffs are possible pre cursors of cholesterol. Bloch, Borek and Rittenberg (14) have studied the site of cholesterol synthesis in the animal body by incu bating slices of different organs with heavy water, deute- rioacetate and doubly labeled acetate (containing deuterium in the methyl group and C1^ in the carbonyl group). Synthesis of cholesterol could only be obtained in liver slices. However, Chaikoff and his coworkers (15) have found recently that adrenal cortical tissue is also capable of synthesizing cholesterol from acetate, in vitro. Studies on conversion' of cholesterol to other steroids. Tracer studies with deuterium have shown that- cholesterol is the precursor of at least two related steroids. Intravenous administration of deuteriocholesterol to bile fistula dogs, by Bloch, Berg and Rittenberg (16) have shown that choles terol is converted to cholic acid. Feeding deuteriocholes terol to a pregnant woman resulted in the excretion of deuterio-pregnandiol in the urine. Since pregnandiol is a reduction product of progesterone, it can be postulated that progesterone is derived from cholesterol in the human body. Sterols have been found also in animal feces. The main steroids being coprosterol and dihydrocholesterol, which are saturated isomers of cholesterol. Schoenheimer (17) has shown that neither of these sterols can be absorbed from the intestines. Coprosterol appears to be formed in the colon, probably as a result of putrefactive bacteria (18). It has-been postulated by Schoenheimer (19) that the following series of reactions take-place: Cholesterol*— -f cholestenone---► coprostanone } 6 coprosterol. Studies on cholesterol breakdown. a. In vivo. Many workers have shown by means of balance experiments that cholesterol is broken down by the animal ill vivo. Dam (20) in 1931 showed by means of balance experiments, that the. loss of sterols in young chickens could not be accounted for on the basis of excretion. Page and Menschick (21) in similar experiments on rabbits and cats showed that cholesterol can be converted to a substance not precipitable by digitonin. Schoenheimer (22) established cholesterol destruction by humans in a case of xanthomatosis, a disease in which the patient was unable to excrete cholesterol. Schoenheimer and Breusch (23), in their carefully conducted balance experiments with mice, concluded, . . .our experiments lead us to the assumption that in the tissues cholesterol is continually being formed and destroyed. Either a positive or negative balance may be found depending on the experimental conditions, i.e., synthesis may be in excess of destruction or vice versa. In vitro. Bloch and Rittenberg (24) in their studies on surviving rat liver tissue found that when liver slices were Incubated with deuterium-acetate in the medium the deuterium was incorporated into the cholesterol. How ever, the total cholesterol before and after incubation 7 remained the same. Therefore, it can be concluded that "destruction" of the cholesterol is taking place simul taneously as the synthesis. Artom (25) in 1923 found that surviving dog liver during autolysis either increased or decreased in choles terol content contingent upon whether; the.dog was fed or starved just before it was sacrificed, and also upon the length of incubation. Takagi (26) in 1936 reported that serum from cholesterol injected rabbits when incubated with a cholesterol hydrosol at 37°C for twenty-four hours showed a decrease in cholesterol content. He also found that injections of cholesterol in normal rabbits did not appreciably raise the serum cholesterol, but if the spleen was removed or the reticulo-endothelial system was blocked, the serum cholesterol was comparatively high. In 1946 Valdiguie and Seguelas (27) found that ground liver preparations added to diluted colloidal solutions of cholesterol produced acetone in amounts up to 90 per cent (based on the conversion of the isopropyl group in the side chain of the cholesterol molecule) upon incubation at 37°C for eight hours. The authors also found this cholestero- r lytic power in the serum but to a much smaller degree. They concluded that the liver possesses a biochemical mechanism * . for the direct production of acetone from the isopropyl 8 group In the side chain. Swell and Treadwell (28) in 1949 also indicated that in rat liver homogenates the cholesterol content decreased within the first two hours but increased during further incubation. It was therefore decided to investigate further rat liver homogenates for a possible explanation of cholesterol "destruction." CHAPTER III METHODS Treatment of the animals. Male and female rats of the University of Southern California strain were used. The animals were obtained at the ages of from three weeks to three months and placed on a cholesterol diet. . This diet was prepared as follows: Forty grams of cholesterol'1 ' were dissolved in 280 grams of cottonseed oil by gently ; • heating the mixture. This is mixed with 1350 grams of ground oats and 1350 grams of wheat. To this mixture 80 grams of A-D oil,2 600 grams of skim milk, 80 grams of yeast “Gw,^ 160 grams of alfalfa, 20 grams of ii bile salt, 20 grams of sodium chloride and 20 grams of calcium carbonate were added. (The bile salt was included in this diet to facilitate the absorption of the cholesterol.) The animals received this diet ad libutum until they were sacrificed. The feeding periods varied from thirty days 1 Cholesterin C.P., Merck and Co., Rahway, N.J. 2 Fortified oii containing 1600 I.U. of Vitamin A and 160 I.U. of Vitamin D per gram of oil. . 3 Brewer*s Yeast, strain ”Gm, Anheuser-Busch Inc.,, St. Louis, Missouri. ^ Sodium glycocholate (pure), City Chemical Corp., New York, N.Y. . 10 to several months. Treatment of tissues. The animals were sacrificed by cervical fracture or injection of nembutal as anaesthetic. The livers were immediately removed and homogenized in a suitable medium. After homogenizing the suspension was centrifuged to remove debris. Samples were then removed. Controls were set aside for extraction with ethyl alcohol-, ether mixture and samples for incubation were placed in a humidity chamber and put in an incubator at 3T°C. At the termination of the incubation period, the samples were re moved and extracted with ethyl alcohol-ether mixture in the same way as the control samples. More specific information will be given in the description of the experiments. Determination of cholesterol. The method used is a modification of the Schoenheimer-Sperry method developed by Nieft and Deuel (29). An aliquot of ethyl alcohol-ether (3t2) extract was taken containing approximately 0.1 to 0.5 mg of cholesterol. This aliquot was evaporated to dryness by.means of a stream of air and a constant temperature apparatus at 6o°C. One ml of ethyl alcohol-acetone (1:1)solution and two drops of 33 per cent (by weight) potassium hydroxide were added, the tubes were corked and the samples were kept at 60°C for one 11 half to one hour with frequent lateral shaking. When the sample is more concentrated a longer hydrolysis time is preferred. The solution was then neutralized to the phenolph- thalein end point with 15 per cent acetic acid. Then 2 ml of ethyl alcohol-acetone (1:1) solution were added. The cholesterol digitonidewas precipitated by the addition of 1 ml of 0.5 per cent digitonin solution (in 50 « per cent ethyl alcohol) and incubated at 35°C for three hours. The precipitate was then centrifuged at a speed of approxi mately 3,000 R.P.M. for about ten minutes. The supernatant liquid was then carefully poured off and the precipitate was washed with 3 ml of anhydrous ether added down the side of the tube. This was then centrifuged at approximately 3,000 R.P.M. for eight to, ten minutes and the supernatant ether was discarded. The precipate was dried with air at 60°C, 0.5 ml of glacial acetic acid was added and the mixture was heated at 60°C until the precipitate was completely dis solved (this latter step takes from one to one and one half hours). Exactly 3 ml of chloroform were added and the tubes were brought to a temperature of 35°C. The color reagent, previously prepared by adding 1 ml of concentrated sulfuric acid to each 9 ml of chilled acetic anhydride, was then added. • ♦ The color was then read in a Klett-Summerson 12 photo-electric colorimeter which had been previously stand ardized with known samples. The filter was #42. A reagent blank consisting of acetic acid, chloroform and color reagent was always read. CHAPTER IV EXPERIMENTAL RESULTS SERIES I Effect of homogenization method. Hogeboom, Claude and Hotchkiss (30) observed that the cytochrome oxidase which is responsible for the oxygen consumption of succinicoxidase, systems is associated with the mitochondria. Hogeboom, Schneider and Pallade (31) later reported that rat liver homogenates prepared in hypotonic water or saline had dis torted mitochondria as observed under the microscope; in saline an appreciable amount of agglutination- of the mito chondria was recognized. In 0.88 molar sucrose these authors obtained rod shaped mitochondria which stained vitally, with Janus Green. Examination of the succinoxidase activity of all these preparations showed the greatest amount of activity in the sucrose preparation which contained morphologically intact mitochondria. It seemed possible that the "destruction1 * of cholesterol was due to an e n z y m e system associated with the mitochondria. Therefore the liver homogenates were prepared in a sucrose potassium chloride medium.1 1 The sucrose-potassium chloride solution was prepared as follows: 30 g sucrose and 0.9 S potassium chloride was dis- ' solved In 100 cc of freshly boiled distilled water. 14 Experiments A and B were designed to show the effect of homogenization in sucrose-potassium chloride solution as compared to distilled water, on the ability of rat liver homogenates to decrease the concentration of cholesterol upon incubation at 37°C for eighteen hours. Experiment A. Two rats which had been fed a high cholesterol diet (described previously) for forty-seven' days were injected intraperitoneally with 0.5 g each of nembutal in the cold room (temperature of 4oa). The livers were quickly extirpated and divided approximately in half. One half of each liver was immediately put into a bath of cold distilled water while the other half was placed in a cold solution of sucrose-potassium chloride. A portion of the liver (approximately 5 g) was re moved from the sucrose solution, blotted dry, and weighed on a torsion balance. It was then cut into small pieces and homogenized with the sucrose solution— 2 ml of solution to 1 g of tissue. Each sample was homogenized for five minutes. Homogenization was carried out in a tube surrounded by an ice bath and then poured into a centrifuge tube sur rounded by ice. After the homogenate was centrifuged for five minutes at 3,000 R.P.M. the supernatant was poured through four layers of cheese cloth. The filtered homogenate 15 was then mixed with an equal volume of incubation solution. The homogenization with distilled water was carried out in the same manner as with the sucrose-potassium chloride solution. One ml of the final mixture was transferred to a test tube by means of a graduated syringe. Ethyl alcohol-ether (3:2) mixture was added immediately to the controls in order to stop any enzymatic action. The tubes to be incubated were filled and put in a humidity chamber through which oxygen from a tank was passed for about fifteen minutes; the chamber was then put into an incubating oven at 37°C for eighteen hours. The incubates were then extracted in the same manner as the controls. A 1 ml aliquot of the controls and the in cubates were taken with a graduated syringe and evaporated to dryness. The cholesterol determination was then run as described previously. Three incubation media were used. The liver homogen ized with sucrose-potassium chloride solution was incubated both in a sucrose-potassium chloride solution and also in a succinate-borate buffer at a ph of 5. Tbe liver homogenized with water was incubated both in a 0.9 per cent sodium chloride medium and in the succinate-borate buffer. The results of Experiment A are given in Table I. 16 TABLE I EFFECT OF DIFFERENT HOMOGENIZING AND INCUBATING MEDIUM _ „ON.THE CHANGE IN CHOLESTEROL CONCENTRATION IN RAT LIVER HOMOGENATES UPON INCUBATION Temperature Mg cholesterol* per gram fresh- liver tissue Number genization ticn Medium . Medium Control Incubated Series I Experi-. ment A 4oc Sucrose-KCL Sucrose- Borate Buffer 10.36** 7.33 \ Sucrose-KCL Sucrose- KCL 11.29 7.06 HgO Succinate- Borate 15.18 . 15.38 H20 Saline 14.85 12.67 Series I Experi-c ment B 25°C Room tempe rature hut equipment precooled Suerose-KCL H20 Sucrose-KCL H20 Sucrose- KCL Saline 8.84 9.66 20.13 25.7^ 6.07 9.60 14.59 26.04 * All calculations are based on wet weight of tissue. ** All determinations were done in duplicate. 17 Experiment B. Two male rats which were on high cholesterol diet for 169 days were killed. The livers were treated in the same manner as in Experiment A except that the work was done at room temperature (25°C). The sucrose-potassium chloride homogenate was incu bated in sucrose-potassium chloride medium while the dis tilled water homogenate was incubated in 0.9 per cent sodium chloride solution. The results are shown in Table I. SERIES II Time-rate study of change in cholesterol concentra tion. Previously Marx and Lipsett (32) had done experiments on rat liver homogenates to determine the change in choles terol concentration after various lengths of time. The authors concluded that there was no change in cholesterol concentration until the incubation had proceeded at least five hours. These workers had run samples only after the first hour. It was thought that perhaps some change In cholesterol concentration had occurred during the first few minutes of incubation. Therefore the following time study was done. A male rat which had been fed 1 per cent cholesterol diet for fifty-seven days was killed in the cold room with 18 an intraperitoneal injection of nembutol. The liver was immediately removed, placed in a cold sucrose-potassium chloride solution, and cut into small pieces for homogeni zation. Homogenization, centrifugation, and filtration were done as described in Series I. The filtrate was mixed with an equal volume of sucrose-potassium chloride solution and 1 ml aliquots were taken and put into test tubes. In preparing the’aliquots all the tubes were im mersed in an ice bath at all times. ► - * The tubes were then put in a constant water bath at 37°C and at definite intervals of time the various tubes were removed and extracted with ethyl aicohol-ether mixture, as described above. The results are shown in Figure I. These results corroborate the previous findings of Marx and Lipsett (33). SERIES III Variation in extraction procedure. Rice (3*0 con cluded that the extraction process used in previous experi ments was 90 per cent accurate but that a more complete extraction could be obtained if 25 ml of ethyl alcohol- ether mixture were added to 1 ml of the homogenate and the mixture was refluxed for one half hour at 60°C. When © L w \ 3 H ; pfcl — f t j - A 9 = H 3 G ► - H r r 4 — 4. p r - 3 3 - r $ T t H — * O f i ■j > x j a • t T t $ * • t J * C ! I t . K [ r « V - j ( T ) *• ( i .l ) f r * c r 3 t l 1 f r G d ) h c > f. u - 1 3 f r t - >1 y *? # f c r j <r> r 4 t- - i j J > « £ 3 7 H 4 r 1 > * ✓ / » ✓ S s / \ / / IV) / © No. 6103—U n iv e rs ity B ook S to re , L o s A n g eles 20 this extraction technique was tried on rat liver horaogenates no noticeable difference was found between the control and incubated samples. These results are shown in Table II. SERIES IV Studies on the effect of supplementary factors on cholesterol “breakdown." In an attempt to stimulate the in vitro change in concentration of cholesterol the addition of the lipotropic factors choline and inositol, and the co- factor insulin were tried. The feeding of choline and inositol to rats on a high cholesterol diet also was tried. Experiment A. A male rat on the 1 per cent cholesterol diet for twenty-eight days was killed by cervical fracture; the.liver was quickly extirpated and placed in cold sucrose- potassium chloride solution. Although the work was done at room temperature all the equipment was kept as cold as possible by prefreezing. A piece of liver was then taken and homogenized for three minutes, using 2 ml of sucrose-potassium chloride solution for every gram of tissue. The. homogenate was then centrifuged for five minutes at 3.>000 R.P.M. and the super natant under the fat layer was removed with a syringe. 21 TABLE II RESULTS OP INCUBATIONS USING IMPROVED EXTRACTION.TECHNIQUE . Experiment Number " 'V Homogeni zat i on ■Fluid „ Mg total choles terol* per gram fresh liver Control Incubated 29** Sucrose-KCL 12.2 11.3 30 Distilled water 14.4 14.1 26 Sucrose-KCL 11.6 10.3 28a Sucrose-KCL 9.4 9.2 28b Sucrose-KCL 11.1 10.0 29 Sucrose-KCL 12.7 11.7 * All calculations are based on wet weight of tissue. ** Each determination was done in duplicate. 2 2 One half milliliter of the incubation medium was added with a graduated syringe to a 25 ml volumetric flask together with 1/2 ml of homogenate. The controls were extracted immediately with ethyl alcohol-ether (3:2) mixture. The other tubes were put in a humidity chamber filled with oxygen and incubated for eighteen hours at 37°C. The 25 ml volumetric flasks were filled to approxi mately 3/^ of their volume with ethyl alcohol-ether (3:2) mixture and refluxed for thirty minutes at 60°G. They were allowed to cool and then filled to volume and 2 ml aliquots were taken for the cholesterol determination. The results are shown in Table III. Experiment B. A female rat on the high cholesterol diet for two days was given an additional 1 per cent choline for seven days. The rat was killed by cervical fracture and the liver was extirpated and put in cold sucrose-potassium chloride solution. The homogenization was carried out for four rather than five minutes but all other steps were carried out as described in part A. c .'. .V The incubation was carried out for twenty-two hours and after extraction 3 ml aliquots were used. 23 TABUS III EFFECT OF CHOLINE, INOSITOL AND INSULIN .. WHEN ADDED.TO INCUBATION MEDIUM ... Cofactor added Concentration of . substance in 1 ml of homogenate- medium mixture - Mg cholesterol* per gram fresh- liver tissue Control Incubated Choline .125 mg** 7.5 7.5 Choline .006 mg 7.5 7.5 Inositol .125 rag 7.5 7.5 Inositol .006 mg 7.5 7.5 Insulin 2 units 7.5 7.5 Insulin 2 units 7.5 7.5 * All calculations are based on wet weight of tissue. ** Each determination was done in duplicate. 24 An identical experiment was also run on a female rat which had been on a high cholesterol diet for two days and was given an additional 1 per cent inositol for seven days. The volumetric flasks were incubated for seventeen hours. After extraction 3 ml aliquots were used for the cholesterol determination. The results are shown in Table IV. 25 TABLE IV EFFECT OF LIPOTROPIC FACTORS WHEN ADDED TO .THE DIET OF. THEf RAT. . Mg cholesterol* per Lipotropic gram fresh liver-tissue . factor Control Incubated Choline 12.5** 12.5 inositol IjA 16.8 * All calculations are based on wet weight of tissue. ** Each determination was done in duplicate. CHAPTER V DISCUSSION OF RESULTS The results of Series I shown in Table I seem to indicate that sucrose-potassium chloride solution is a better homogenizing medium than water, since a greater change in cholesterol concentration (as shown by the modi fied Sperry-Schoenheimer procedure) is obtained in the case of the sugar containing medium. The time-rate study undertaken in Series II (Table ’ II) showed that no change in cholesterol concentration occurred in the liver homogenate until after five hours of incubation. Thereafter there was a significant change in cholesterol concentration. This observation had been previously noted by Marx and Lipsett (3*0 using homogenates of rat liver tissue. The conclusion of Rice (35) that the extraction pro cess used in these experiments was only 90 per cent complete led to the adoption of a modified extraction procedure. When this modified extraction procedure was used on the rat liver homogenates, no significant difference could be observed between control and Incubated samples. It is possible that cholesterol exists in the tissue as a cholesterol- protein complex and that the process of incubation causes 27 denaturation and coagulation of the protein. Therefore a possible explanation for the results obtained in Series I and II is that the cholesterol became more difficult to extract from the denatured protein and that therefore the earlier procedure used for the extraction was inadequate. The method of extraction used in Series IV using a larger volume and which included refluxing at 60°C for thirty- minutes gave a more complete extraction and no change in cholesterol concentration was obtained. It was attempted in Series IV to stimulate a decrease in cholesterol concentration in rat liver homogenates by- adding the lipotropic factors choline and inositol, and the cofactor insulin iii vitro. As has been shown in Table III no significant change in cholesterol concentration was ob tained before and after incubation. In vivo attempts to stimulate the change in cholesterol concentration by adding to the diet respectively the liptropic factors choline and inositol also failed to show a change in cholesterol con centration when the liver was later homogenized and incu bated (see Table IV). Under the conditions reported here no change in cholesterol concentration was obtained. Earlier results may have been due to an artifact of extraction. It is not possible to draw any definite conclusion until all the work 28 presented has been repeated using both extraction procedures discussed in this thesis. CHAPTER VI SUMMARY An attempt has been made to confirm the results ob tained by Marx and Lipsett (36) that a cholesterol destroy ing enzyme is present in the liver. Early results indicated that sucrose-potassium chloride homogenizing medium was to be preferred over water and that no change in cholesterol concentration occurred until at least after five hours of incubation at 37°C had taken place. An improvement in the extraction procedure seems to invalidate these results since no subsequent change in cholesterol concentration was found on incubation of liver tissue alone or with the addition of the lipotropic factors choline and inositol, and the cofactor insulin in vitro and the lipotropic substances choline and inositol in vivo. A possible explanation for the early re sults has been suggested. A more extensive study is indi cated before definite conclusions can be drawn. 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Creator Schotz, Myron Collens (author)

Core Title A study on the comparison of the cholesterol content of rat liver homogenates before and after incubation

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

Contributor Digitized by ProQuest (provenance)

Advisor Marx, Walter (committee chair), [illegible] (committee member), Visser, Donald H. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-779344

Unique identifier UC11348932

Identifier EP41310.pdf (filename),usctheses-c17-779344 (legacy record id)

Legacy Identifier EP41310.pdf

Dmrecord 779344

Document Type Thesis

Rights Schotz, Myron Collens

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