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A method for the analysis of deuterium-labeled tissue cholesterol: Attempts to study the relation of the thyroid to the turnover of cholesterol in rats

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A method for the analysis of deuterium-labeled tissue cholesterol: Attempts to study the relation of the thyroid to the turnover of cholesterol in rats

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Content A METHOD FOR THE ANALYSIS OF DEUTERIUM-LABELED TISSUE CHOLESTEROL. ATTEMPTS TO STUDY THE RELATION OF THE THYROID TO THE TURNOVER OF CHOLESTEROL IN RATS 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 Ralph Werner Alexander January 19U9 UMI Number: EP41289 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 EP41289 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 Bis Wf. A 377 T/m thesis, w ritte n by .......................RALPH. lE M M ..4 ? fE M N D E R ...................... under the guidance of h.As.. F a cu lty Comm ittee, and approved by a ll its members, has been presented to and accepted by the C ouncil on Graduate Study and Research in p a rtia l f u lf ill ment o f the requirements fo r the degree of Master of Science ........ - ........E...S—Bogardye-..... Dean ........ Faculty Committee Chairman ACKNOWLEDGMENTS I want to express my sincerest gratitude to all members of my Committee for their helpful advice and direction. Especially I want to thank Doctor Harry J. Deuel, Jr. and Doctor Walter Marx for their very kind consideration and understanding. Furthermore, I am indebted to Doctor Roslyn B. Alfin for her cooperation in the cali bration of the mass-spectrometer at the Los Angeles County General Hospital. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION ..................................... 1 II. HISTORICAL REVIEW............................... 2 III. EXPERIMENTAL PROCEDURES.......................... 5 Analytical Procedures .................. 5 Methods for splitting of cholesterol digitonide . $ Refluxing with acetic anhydride ........ 6 Xylol extraction .................. 6 Dissolving with pyridine .................. 7 Preparation of hydrogen-deuterium gas mixture from deuterium labeled cholesterol............ . 7 Method of combustion of deutero-cholesterol . . 11 Method for reducing H2O-D2O mixtures to hydrogen and deuterium 1. ............. . 1 3 Calibration of the mass-spectrometer for the determination of. deuterium................... 17 Treatment of Animals .......................... 26 Experimental groups.......................... 26 Injections ............................ 27 Isolation and determination of tissue cholesterol 30 IV. RESULTS ........; .............. 31 V. DISCUSSION AND CONCLUSIONS .............. 3I 4. VI. SUMMARY . ................................. 3 6 BIBLIOGRAPHY.................................... 38 LIST OF TABLES TABLE PAGE I, Recovery of Cholesterol Using Xylol for Splitting the Digitonide . . . . . . . ....................... ♦ . 9 II. Recovery of Cholesterol Using Pyridine for Splitting the Digitonide ..».•#•••••••••..•• 10 III. Mass-Spectrometer Readings for the Theoretical Mass 3/Mass 2 Ratios as a Function of Deuterium Concentration ......... 25> IV. Modified Stock Diet . .. .................. 28 V. Average Body and Liver Weights................ 32 VI. Cholesterol Concentrations in Tissues ........... . . 33 LIST OF FIGURES FIGURE PAGE 1. Calibration Curve for Colorimetric Determination of Cholesterol . ......................... . 8 2. Combustion Train.............. 12 3. ' Water Reduction Apparatus ' . . . . 15 1 * . Calibration of Mass-spectrometer for Deuterium Deter mination Using Tank Hydrogen and 0.01 per cent D2O . 20 5. Calibration of Mass-spectrometer for. Deuterium Deter mination Using 0.1 per cent DgO ........... 21 6. Calibration of Mass-spectrometer for Deuterium Deter mination Using 1 per cent D2O .................... 22 7. Calibration of Mass-spectrometer for Deuterium Deter mination Using 10 per cent D 2O ............... 23 CHAPTER I INTRODUCTION It has been known for many years that the level of blood cholesterol is influenced by the thyroid hormone, but the mechanism of this phenomenon is not understood as yet. In particular, it is not known whether the change in the concentration of the blood cholesterol is a consequence of a shift between blood and tissues, or a modification in the rate of cholesterol synthesis and break down. In order to elucidate, especially the latter problem, it was decided to investigate the effect of the thyroid on cholesterol turnover using deuterium as a tracer. For this project, methods had to be developed by which deuterium-tagged cholesterol could be isolated from body tissues and its deuterium content determined quantitatively by means of a mass-spectrometer. CHAPTER II HISTORICAL REVIEW As far back as the latter part of the nineteenth century, many investigators have demonstrated the apparent relationship be tween the thyroid gland and the concentrations of cholesterol in the blood. Since that time various attempts have been made to correlate the clinical and experimental findings. Myxedema and other diseases producing a hypofunction of the thyroid gland were associated with a marked rise in the level of blood-cholesterol as shown by Hurxthal (193k) and Gildea, Man, and Peters (1939)• On the other hand, Schally (1935) and Man, Gildea, and Peters (191*0) demonstrated a definite hypocholesteremia in their hyperthyroid patients. These findings were reproduced by other authors in animals, but here a species difference was noted. Steiner and Kendall (191*6) enhanced the hypercholesteremia produced by a high cholesterol intake in dogs with a simultaneous feeding of thiouracil. Rats were relative ly resistant and developed only a slight, although well defined, hypercholesteremia after thyroidectomy, as was shown by Fleischmann and Schumacker (191*2). The findings on the rabbit are still quite contradictory. Although this species is very susceptible to hyper cholesteremia by mere cholesterol feeding, it is disputed whether the thyroid has any effect on this change. In a series of papers, Turner, et al (1933a, 1933b, and 1938) have reported that thyroidectomy has 3 no effect upon dietary hypercholesteremia in the rabbit, since it •* occurs to the same extent with or without the thyroid gland. On the other hand, Fleischmann, Schumacker, and Wilkins (191*0) reported a hypercholesteremia after thyroidectomy of rabbits. Because of this and for reasons of convenience, the rat was chosen as an experimental animal in this work. Practically all previous research on the effect of the thyroid on cholesterol metabolism was made on blood. Almost no work has been done regarding this relationship as far as the cholesterol contents of other tissues are concerned. Fleischmann and Schumacker (19i*2) examined the total body cholesterol of thyroxin-fed rats and found no correlation between serum and total carcass cholesterol. Since Aschoff proved the fundamental chemical constituent of athero- and arteriosclerotic lesions to be cholesterol, the rela tion of blood cholesterol to tissue cholesterol is especially important for understanding the mechanism involved in the development of atherosclerosis from experimental hypercholesteremia. The general assumption is that in hypercholesteremia* the tissue cholesterol con centration rises accordingly and that thus atherosclerosis may be produced. However, Fleischmann and Schumacker (19U2) showed that the tissue cholesterol content does not always decrease with the serum cholesterol under these conditions. The important question is, therefore: What happens to the cholesterol which disappears from the blood in hyperthyroidism? There sure obviously two possibilities: destruction or deposition. This h problem can be solved most conveniently by studying cholesterol turn over with tracer methods. CHAPTER III EXPERIMENTAL PROCEDURES Before animal experiments could be started, it was necessary to develop methods for the isolation of cholesterol from animal tissues, for the preparation of hydrogen-deuterium gas samples from deuterium-tagged cholesterol, and for analysis of the deuterium con tent of these hydrogen-deuterium gas mixtures using the mass-spectro meter. I. ANALYTICAL PROCEDURES A. Methods for splitting of cholesterol digitonide. The lipids from tissues were best extracted by the Soxhlet method for eight hours, using 3'2 alcohol-ether as the solvent. From this solvent the cholesterol was precipitated most conveniently with digi- tonin which is specific for cholesterol as determined by Windaus (1910). Since only minute amounts of cholesterol were available for analysis, the cholesterol digitonide had to be split before combustion to water; otherwise it would have diluted the deuterium in the final product so much that it could not be analyzed mass-spectrometrically because digitonin has more than three times the molecular weight of cholesterol. Since, therefore, cholesterol was isolated from animal tissue extracts in the form of its digitonide, it was necessary to develop a satisfac tory procedure for separating the cholesterol from this addition compound. All cholesterol digitonide used for the following procedures was prepared by the Windaus method (1909) and the purity'of the final 6 product was tested by melting point. No definite melting point was obtained, but a gradual decomposition occurred over 2U0°C., which is a characteristic of cholesterol digitonide. 1* Refluxing with acetic anhydride. It was attempted several times to convert about 200 mgs. of cholesterol digitonide to cholesterol acetate by refluxing in acetic anhydride for half an hour, according to Rittenberg and Schoenheimer (1937). At the end of this time, a heavy black precipitate was found in a blue-green solution. Upon the addition of 50 per cent ethyl alcohol, a white cloudy precipitate formed, but the heavy black precipitate remained so as to prevent separation and colorimetric readings. 2. Xylol extraction. Several portions of cholesterol-digi- tonide were extracted in Soxhlet extraction cups with xylol for ten hours as described by Windaus (1918). Aliquots of the xylol were evaporated, first in a water bath, then in a 60°C. aluminum block to dryness. The residue was dissolved with one half ml. of glacial acetic acid after one half hour heating in a 60°C. aluminum block. Exactly three ml. of chloroform were added, according to the Schoenheimer-Sperry method (193li), as modified by Chaney (unpub lished) , and the solution mixed, the tubes stoppered, and the temper ature brought to 37°C. in another aluminum block. In the meantime the color reagent was prepared as follows: One volume of concentrated sulphuric acid was added to nine volumes of ice-cold acetic-anhydride. One ml. of this ice-cold color reagent was then added to each tube. 7 The tubes were mixed and heated to 37°C. for exactly ten minutes. They were then transferred to an ice-bath and again left for ten minutes, after which time they were read in a Klett-Summerson colori meter, using a red filter (620 millimicrons). The blank for the zero * reading was prepared in the same manner.. The standard curve obtained by this method is shown in Figure .1. The results for the xylol extrac tion as calculated from this curve are given in Table I. Since in the animal experiments only those small amounts of cholesterol shown in Table I could be expected, this method, although it is the simplest, was discarded due to its low recovery results. 3. Bissolving with pyridine. Several samples of cholesterol- digitonide were dissolved in one ml. of hot pyridine per mg. cholesterol expected (Schoenheimer and Bam, 1933)• Insoluble fractions were neg lected. The digitonin was precipitated with ten volumes of ether per volume of pyridine used. A white, cloudy, colloidal type of precipitate formed which could not be centrifuged down at 2,500 R.P.M. This solu tion was filtered twice through No. h2 ¥i/hatman filter paper, until the filtrate was perfectly clear with no trace of cloudiness. Aliquots of this filtrate were tested colorimetrically according to the procedure given on page 6. The results are presented in Table II. The excellent recoveries obtained made this method the most suitable for small samples of cholesterol. B. Preparation of hydrogen-deuterium gas mixture from deuterium labeled cholesterol. Klett-Summerson Colorimeter Readings 8 220 200 180 160 1 2 * 0 120 100 60 20 * Cholesterol Mgs FIGURE 1 CALIBRATION CURVE FOR COLORIMETRIC DETERMINATION OF CHOLESTEROL i ‘ r Ay M .v r; , Lc:: TABLE I RECOVERY OF CHOLESTEROL USING XYLOL FOR SPLITTING THE DIGITONIDE Mgs. cholesterol- digitonide used Mgs. cholesterol calculated Mgs. cholesterol recovered Per cent recovery 26.2 6.1* 0.6 9.1* 33*3 8.1 0.8 9.9 69.0 16.7 1.2 7.2 1*6.3 11.2 0.9 8.0 TABLE II . RECOVERY OF CHOLESTEROL USING PYRIDINE FOR SPLITTING. THE. DIGITONIDE Mgs. cholesterol- digitonide used Mgs. cholesterol calculated Mgs. .cholesterol recovered Per cent recovery 19.9 ii.8 U.7 97.9 22.3 $.3 5.2 98.1 27.6 6.7 6.3 9U.0 25.1 6.1 5.9 96.6 II 1. Method of combustion of deutero-cholesterol. The combus tion procedure used was adapted from that described by Rittenberg and Schoenheimer (1935) • The deutero-cholesterol was burned with the aid of copper oxide (wire form) at 600°C. to a mixture of H2O and D2O. In order to transfer the cholesterol obtained from the pyridine-ether procedure into small combustion boats, the ether was distilled off and the pyridine evaporated at 60°C. until the cholesterol residue was dry. This was then redissolved with a minimum of 1:1 alcohol- acetone and the solution carefully transferred into a combustion boat. The boat was then placed into a vacuum oven, indicating a vacuum of thirty pounds, at 50°C. for about one half hour. If the volume of the solution exceeded the capacity of the boat, only a portion was used the first time, and the procedure was repeated with the remaining solution until all cholesterol from one sample was dried in one boat. The boat was then transferred into the combustion train shown in Figure 2. The apparatus was previously swept out for at least two hours with oxygen coming through at a rate of about thirty bubbles per minute until no more water collected in the dry-ice trap #2. Since electrolytic oxygen was used, it was first conducted through a copper oxide combustion tube heated to 600°C. to give any available hydrogen a chance to combine with oxygen. Any. water formed from the oxygen source could then be trapped out in dry-ice trap #1, so that it did not dilute the water obtained from the sample in the boat. The boat was placed into the combustion tube, which was immediately closed, and left outside of the furnace to dry for at least one hour. The Copper oxide- Combustion boat Furnace at 600°C. f44$l<Water trap #1 Dry ice bath Collecting tube --- Tank oxygen I Water—[ trap#2 Dry ice bath . FIGURE 2 COMBUSTION TRA.IN 13 dry-ice bath was then transferred from trap #2 underneath the collect ing tube, and the furnace was slowly moved over thevboat at a rate not faster than half an inch every five minutes. When the boat was finally completely inside the furnace, it was left there for at least one hour or until the last traces of cholesterol were burned to water* 2. Method for reducing H2O-D2O mixtures to hydrogen and deuterium. The amount of water obtained by the above combustion for the final gas analysis will obviously be extremely small. Reduction by electrolysis of less than .1 ml. of water would be very difficult and erroneous, especially since the difference of electrode affinity for hydrogen and deuterium is quite appreciable (Karaen, 19U7b). Thus it is best to let this water evaporate over some reducing substance. The difference in evaporation rates between H2O and D2O is negligible. At first, zinc mesh #10 was used as the reducing substance in a Pyrex combustion tube in a furnace of liOO°C, but water could be trapped out on the other end.of the reduction train when mixtures were evaporated. It was concluded that zinc at 1*00°C. was not active enough to reduce very small amounts of water vapor, and that its low efficiency would therefore produce great errors. Greater temperatures could not be used, since zinc melts at 1*20°C. Because of its higher melting point, magnesium was tried next (Henriques and Margnetti, 191*6, and Kamen, 19l*7a) for the water reduc tion. It was used in the form of turnings, in order to increase the active surface area, placed in a pyrex combustion tube at 520°C. I2i Under these conditions no magnesium hydride is formed. Although only a very small quantity of water could be trapped out after redaction, it was soon found that the hydrogen combines with Pyrex at such high temperatures, evidenced by the discoloration of the glass. When magnesium turnings were used in a quartz tube at 600°C., no water was trapped out indicating that the reduction was complete. Care should be taken that, if the furnace is left on, the magnesium is changed once a week, since its activity is quickly decreased when the turnings become coated with the oxide. The apparatus used for the collection of the gases is shown in Figure 3- The reduction of the H2O-D2O mixture was carried out as follows: The U-tube containing the water sample from the copper oxide combustion train was left in the dry ice bath and attached onto the reduction tube. The whole system was then evacuated by the vacuum pump with stopcocks 1, 2, 3> U, and 5 open, 6 and 7 closed, 8 open to air, and 9 closed to air. When the manometer indicated vacuum, stopcock 5 was closed, the dry ice bath was transferred from the sample containing U-tube to the water trap, and evaporation could proceed. The speed of evaporation could be controlled by stopcock 1, but for the small samples used in this experiment it was left com pletely open. The volume of the sample container usually used was about 20 ml., whereas that of Part C was about 250 ml. It was found that when the pressure was 50 mm. of mercury or greater, the Manometer Magnesium turnings Quartz tube Furnace 600°C. Collecting tube with frozen mixture Dry ice bath ■s Water trap FIGURE 3 WATER REDUCTION APPARATUS S ' Sample container Magnesium perchlorate drying tube Air inlet Mercury vacuum pump 16 hydrogen-deuterium gas mixture could be compressed into sample con tainer D to a pressure of 1 atmosphere by raising the mercury from the reservoir into container C. If the sample was too small and this pressure of 1 atmosphere could not be obtained after about three fourths of an hour evaporation, the gases in C were completely trans ferred into container X) and the entire operation repeated with the fraction of the gas sample left in A-B. If the volume of C and D was, for instance, 10 times as great as that from points A to B, 90 per cent of the gases were in C and D upon the first evacuation, 10 per cent remaining in A-B. After the second evacuation, 9 per cent additional gases could be collected, and upon the third time i — .9 per cent more. By this method practically all of the gas ob tained from extremely small quantities of water mixtures could -be collected in sample container D. In order to compress the gases into container D by the mercury contained in the reservoir, stopcocks 1, 2, and 5 were closed, and stopcock 6 gently opened. If equalization of the mercury level between the atmospheric air, coming through stopcock 8, and the gas mixture occurred before the mercury rose to stopcock 3, the latter was closed and the gases in I) were at a pressure of 1 atmosphere. When dealing with lesser pressures, the mercury had to be stopped just before entering stopcock 3 by closing stopcock 6. To return the mercury into the reservoir, stopcocks 3 and 8 were closed, and the reservoir was evacuated with the pump by opening stopcock 7* Stopcock 6 was opened next, but since there was a vacuum on both 17 sides of the mercury, the mercury could only fall until both columns were of equal height. Hence atmospheric air had to come into Cs Stopcock 7 was closed, the vacuum pump shut off, stopcock 9 opened to air, and stopcock 5 gently opened. When the mercury had been returned into the reservoir, stopcock 6 was closed and stopcock 8 was opened to air. When a second run on the same sample was neces sary, C of course had to be evacuated again: Stopcock 9 was closed to air, stopcock 2 was opened, and the vacuum pump started. When vacuum was indicated by the manometer, stopcock $ was closed again, and stopcock 1 was opened. The amount of gas produced was again indicated by the manometric pressure. Once more stopcocks 1 and 2 were closed, stopcock 3 opened, and the same procedure for driving up the gases was followed. This was repeated until the sample container was filled or until all the water had been reduced. Throughout the procedure, no water should be collected in the dry ice trap which would denote either a too rapid evaporation or an oxide coating of the magnesium. C. Calibration of the mass-spectrometer for the determina tion of deuterium. The quantitative determination of an isotope of hydrogen with the mass-spectrometer is more difficult than the deter mination of the other isotopes generally used in tracer studies. Hydrogen occurs in six masses, some composed of several ions which may therefore mask any individual ion considerably. Thus, the small reading for deuterium, (D)+, is completely overshadowed by the large 18 reading of normal hydrogen, (HH)+, which has the same mass. In a series of articles, Bleakney, et al (1932, 1933) have demonstrated mass 3, (HD)+, to be most indicative for deuterium readings since it can be masked only by (HHH)+, which not only exists in very small quantities but is, moreover, best read at a different pressure. Normal hydrogen can be determined as mass 2, (HH)+, since only (D)+ can mask it, and that fraction is negligible. Thus, the ratio of deuterium to hydrogen in the sample can be determined by the ratio of (HD)*/(HH)+. Both ions, of course, are read constantly at the same respective magnetic field which for the mass-spectrometer used here, was 3k gauss for mass 2 and 9£ gauss for mass 3 . In order to calibrate the instrument, different dilutions of D2O in H2O are reduced to hydrogen-deuterium gas mixtures. The voltages or pressures obtained for the peak of mass 2 are propor tional to the quantity of (HH)+ produced. Therefore different pressure- or voltage-points for the ionization of (HH)+ can be plotted against the ratio of masses 3/2- The abscissa P represents the peak of (HH)+ in millivolts, and the ordinate i/P represents fHD) + ♦ (HHH)+. --- Using these standard curves, the unknown samples can be determined. If we can assume that the value for (HD)+/(HH)+ is exactly twice as much as for D/H (Bleakney, 1932), then, accord ing to the approximate formula, that -!PP x (ratio 3/2) equals atom percent excess deuterium, it can be calculated that at no time could the actual ratio of 1 0 0 per cent D2O containing 1 0 0 atom per cent excess deuterium be higher than 2. On the other hand, ordinary tank 19 hydrogen in this laboratory did not give a ratio lower than J j . x 1 0”k although Bleakney (1932) was able to obtain a ratio of .67 x 10“^ for ordinary commercial electrolytic hydrogen. Therefore, between these ratios a series of curves has to be plotted on a graph such as shown, in Figures U, f>> 6 , and 7. Since the optimum ionization for (HHH)+ is at the square of the pressure used for (HD)’ * ’ , the 3/2 ratio can be expected to rise with higher pressure or voltage readings. For the same reason, the quantity of (HHH)+ produced, is negligible at zero pressure. Every standard curve should therefore be constructed from at least three different pressure readings so that the curve can be extrapolated to zero pressure, at which point the ordinate reads the actual (HD)+/(HH)+ ratio of the sample. All curves should be straight lines since the rise of both the pressure and the voltage for the peak of mass 2 is directly proportional to the rise in the 3 / 2 ratio of a given dilution. With greater D2 0 concentrations, (HHH)* can be large enough to cause a very steep curve, and it is therefore ad visable to determine the peak of mass U, if higher D2O concentrations (above 1 0 per cent ) , are used, and the curve becomes too steep for accuracy. Rain water, which may be assumed to contain the same deuterium content as body water or any- compound synthesized there from, has a ratio of B/H = 1/5,000 (Bleakney and Gould, 1933) > or a 3 / 2 ratio of 1/2,500 - I 4 x 1 0“^. This ratio is theoretically obtained by a . 0 2 per cent D2O sample containing . 0 2 atoms per cent excess deuterium. Obviously, therefore, with lesser D2O concentrations, I * icrl* i l l lilO 130 120 110 100 90 0 . 0 1 per cent DgO I 1 0 Tank hydrogen 12 Hi 16 Millivolts P = (HH) FIGURE U CALIBRATION OF MASS-fSPECTRCMETER FOR DEUTERIUM DETERMINATION USING TANK HIDROGEN AND 0.01 PER CENT D20 21 x 10' 3 0 0 290 280 270 260 2 ^ 0 X 2k0 i 230 220 210 200 190 180 170 160 150 Millivolts F = (HH) I FIGURE 5 CALIBRATION OF MASS-SPECTROMETER FOR DEUTERIUM DETERMINATION USING 0 .1 PER- CENT DoO • \ u . U 3 1 , I ' t m c r ^ i t j r B o o a S t o r e , Lou A n f t d c * 22 lik0 1*30 Uoo 390 3^0 370 360 M 350 330 320 310 300 Millivolts P » (HH) FIGURE 6 I CALIBRATION OF MASS-SPECTROMETER FOR DEUTERIUM DETERMINATION USING 1 PER CENT pop... _j . . . . No, rljj i, I'n K c r-.-n y Boolv S tc re , hot, A n ^ c lc it 2 0 3 0 2020 2010 2000 1990 i 1 9 8 0 1970 1 1 9 2 0 1 9 1 0 1 9 0 0 1890 Millivolts P = (HH) FIGURE 7 CALIBRATION OF MASS-SPECTROMETER FOR DEUTERIUM DETERMINATION USING 10 PER . CENT DoO _ I 1 t «-i i L !_ _ _ — j _ _ _ i 1.1 : A i . . . j N u w JIl, r n i\c r ; -.it y B o o k S to le , Loii A n ^ elc:; 2h the normal deuterium concentration for ordinary water has to be subtracted from the final results. For instance, the theoretical ratio of . 0 1 per cent D2O at zero pressure would be 6 x 1 0“^, but when the ratio for ’ ordinary water is subtracted, the actual ratio is only 2 x 1 0“^. It can be seen that once the instrument is calibrated this way, one reading of an unknown sample at any pressure suffices. The point obtained can then be extrapolated to zero pressure with reference to the standard curves between which it falls. It is, nevertheless, preferable to determine the ratio by two or more pressure readings to be able to check on the accuracy of the result. The theoretical 3/2 ratio at zero pressure can be calculated. Thus for 10 per cent D20: .( I 0,.* x jff) „ 1 8.2, average mole cular weight of water mixture. Therefore: 1 8 . 2 - 1 . 8 = 1 6 . h parts of hydrogen. D/H = 1.8/16.1* = .11 HD/HH = .22 (3/2 ratio) --322— = -322. - 9 . 9 atoms per cent excess deuterium in 1 0 —— ■ + i 1 0 . 1 .22 * X per cent D20. The theoretical 3/2 ratios of D 2O dilutions in purified H20 are given in Table III. Mass 3/mass 2 ratios greater than one cannot be determined unless the peak of mass i t is read. For calculations of atoms per cent excess deuterium, two formulas can be used: TABLE III MASS-SPECTROMETER READINGS FOR THE THEORETICAL MASS 3/MASS 2 RATIOS AS A FUNCTION OF DEUTERIUM CONCENTRATION Per cent dilutions of D 2O Ratios Atoms per cent excess deuterium 33.3 1.0 33-3 10 0.22 9-9 1 0.022 1.09 0.5 0.011 0.^5 0.1 0.0020 0.10 0.0$ 0.0010 0.05 0.01 0.0002 0.01 26 Atoms per cent excess deuterium ■ . . . 100 (exact formula) 2 + (3 / 2 ratio) _ 1 0 0 (3 / 2 ratio) (3 / 2 ratio) + ^ _ 1 0 0 x (3 / 2 ratio) (approximation formula) With concentrations of D2O greater than 1 per cent, the exact formula has to be used, but the results for lower dilutions can also be calculated from the approximation formula, since they do not differ from those obtained by the exact formula. Some curves obtained by Doctor Roslyn B. Alfin and the author are given in Figures U, 5> 6 , and ?• Ordinary electrolytic tank hydrogen was determined in addition to 1 0 per cent DgO, 1 per cent D2O, 0.1 per cent D2O, and 0.01 per cent D2O in purified water. How ever, the values obtained were higher than the theoretical 3 / 2 ratios and duplicate runs did not give satisfactory agreements. The ratio obtained for the electrolytic hydrogen standard was twice as high as it should have been. On the other hand, the values for the 1 per cent DgO and 1 0 per cent DgO dilutions were close to the theoretical values. Thus the calibration,has as yet not been completed. However, it can well be assumed that the final curves obtained by these different D2O dilutions will have the same shape as those shown in Figures, ii, 5> 6, and 7, although at a lower scale. II. TREATMENT OF ANHA.I5 A. Experimental groups. Thirty day old male, white rats of the colony at the University of Southern California were divided into 2? three groups as follows, and fed for one month a modified stock diet as given in Table IV: Group A. Three rats averaging 6 6 grams each were used as controls. Group B. Four rats were thyroidectomi zed. One thyroidec tomy was incomplete as revealed later by autopsy, leaving three rats in this group averaging 8 0 grams. Group C. Three rats received a supplement of .15 per cent dried thyroid powder with their diet. The animals averaged 73 grams. At the end of the one month feeding period all groups were weighed again. B. Injections. Sodium deutero-acetate was prepared through an exchange reaction between an(* malonic acid by a method adapted from Wynne-Jones (1935) and Halford-Anderson (1936). It was attempted to obtain a 50 atom per cent excess deuterium concentration in the sodium acetate. It has been shown that at least one half of the cholesterol-hydrogen comes from body fluids, and that 1 3 per cent of the newly formed cholesterol-hydrogen arises from acetic acid (Bloch and Rittenberg, 1 9U2). For .this reason the two substances were used together in order to cut down the inoculation time to four days, which was successful with the acetate alone for an eight day period (Bloch and Rittenberg, 19l;2), and in order to make use of all possible mechanisms for the incorporation of deuterium into cholesterol to reduce possible errors due to differences of deuterium sources. The amount of acetate together v/ith the dilution of D2 0 to be injected 28 TABLE IV MODIFIED STOCK DIET Ingredients Per cent Whole wheat 32.5 Oats 32.5 Skim milk 10.0 Alfalfa li.o Yeast "G" (1) 9.5 Fortified oil (2) 2.0 Cottonseed oil 8.0 NaCl 0.5 CaCO^ 1.0 (1) Brewer's Yeast, Strain "G", Anheuser-Busch Inc., St. Louis, Missouri. (2) Fortified oil contained 1600 I.U. of Vitamin A and 160 I.tJ. of Vitamin D per gram of oil. 29 intraperitoneally into white rats were determined as follows: The maximum non-pathological dose of D2O according to Barbour and Trace (1936) for mice seems to be about one half ml. of 15 per cent D2O per 10 grams body weight per day. When rats and mice were fed 1 . 6 mM of sodium acetate (containing 7 7 atoms per cent excess deuterium) per 1 0 0 grams of body weight per day, they showed no ill effects at all (Rittenberg and Bloch, 193k and 1935)• This acetate would have the approximate formula of (^H-COONa. The amount used would therefore be 85 x 1.6 * ■ 136 mg./lOO grams body weight. Both the acetate and D2O were fed below the pathological dosages at a level resulting in lower than 2 to 5 per cent D2O dilution in the circulating plasma. Above this dilution, D2O acts as a respiratory poison since the dehydrogenases cannot transport deuterium as fast as hydrogen (Kamen, 19k7b). Twelve grams of the sodium deutero-acetate were dissolved in 5 8 0 ml. of purified water and 2 0 ml. of 1 0 0 per cent D2O, giving a dilution of 20 mg. acetate per ml. in 3*3 per cent D2O. The solution was made as nearly isotonic as possible, neutralized, and before each injection heated to body temperature. For exactly four days following the one month feeding period each rat was given twice daily an intraperitoneal injection of 6 ml. of this solution per 1 0 0 grams body weight, the injections being at least 10 hours apart. Hence, 2i|0 mg. of deutero-acetate dissolved in 1 2 ml. of 3 * 3 per cent D2O per 1 0 0 grams body weight were given daily to each rat for four days. C. Isolation and determination of tissue cholesterol* After four days all rats were injected with 0 . 1 ml. nembutal per 1 0 0 grams body weight. As soon as no reflexes could be obtained, the blood was taken from the left ventricle with a syringe. It was then centrifuged and the serum volume measured. All of the blood and tissues of the rats in each respective group were pooled. The wet weights of livers, lungs, and adrenals were determined. The remaining carcasses were ground up and the body fluids distilled in vacuo for forty-eight hours cooling the receiver in a dry ice bath. The livers, lungs, and adrenals were ground and the lipids extracted in Soxhlets with 3:2 alcohol-ether for eight hours. One volume of the serum was extracted with fourteen volumes of 1 : 1 alcohol-acetone, the precipitated protein sedimented by centrifugation, and the supernatant fluid collected. Small aliquots were taken from all tissues and tested for cholesterol colorimetrically by the Schoenheimer-Sperry method (193U) as modified by Sobel and Mayer ( 19h$) and Chaney (unpublished). The solvents of all tissue extracts were then distilled off and the extracted residue dissolved with a minimum of 1 : 1 alcohol-acetone and transferred into 2*>0 ml. centrifuge tubes. The cholesterol was precipitated as digitonide by the Schoen heimer-Sperry method (193U)• The digitonide was split with pyridine and ether, and the deuterium-hydrogen gas mixtures from ,the isolated cholesterol and the body fluids were prepared for deuterium analysis as outlined under the Analytical Procedures. CHAPTER IV RESULTS The results for the quantitative cholesterol determinations were calculated from the curve shown in Figure 1, and are tabulated in Tables V and VI. The mass-spectrometric results for the deuterium analysis of the tissue cholesterols can only be obtained after proper calibra tion of the instrument which is still in progress. TABLE V AVERAGE BGDI AND LIVER WEIGHTS Group Number of rats Weight before feeding diet (grams) Weight after feeding diet (grams) Weight gain (grams) Weight of liver per 100 g. body wt, (grams) Normal 3 66 95 29 5 .1 Thyroidectoraized. 3 80 97 17 U.2 0.15 per cent thyroid fed 3 73 132 59 5 .9 TABLE 71 CHOLESTEROL CONCENTRATIONS IN TISSUES Group Number of rats Cholesterol in serum (mg./lOO ml.) Cholesterol in adrenals (per cent) Cholesterol in lungs (per cent) Cholesterol in liver (per cent) Normal 3 I*U 2.7 .1*8 .2 2 Thyroidectomized 3 86 2.1 . 1 * 5 - .2 0 0 . 1 5 per cent thyroid fed 3 37 1.7 .3 9 .19 K x > V *> CHAPTER V DISCUSSION AND CONCLUSIONS The experimental data in Table V show that the thyroid fed rats gained more than three times as much weight as did the thyroid- ectomizedj this was to be expected due to the higher metabolic rate in hyperthyroidism. Moreover, there was a significant difference in liver weights on a percentage basis between the three groups. The hyperthyroid animals presented a marked increase in liver weight, in agreement with observations reported by H. Fraenkel-Conrat, et al (191+2). The thyroidectomized rats showed correspondingly a decrease in liver weight. Pronounced changes were found in the serum cholesterol (Table VI). Thyroidectomy caused an increase of approximately 100 per cent in the concentration of the latter. It is evident, therefore, that extirpation of the thyroid gland resulted in a well defined hypercholesteremia. The serum cholesterol content in the hyperthyroid group, on the other hand, showed very little difference from the normal; if anything, it appeared slightly lowered. These results agreed with previous findings which stated that the change in serum cholesterol in hypothyroidism is usually far greater than in hyper thyroidism (Hurxthal, 193h and Man, et al, 1939, 191+0). The results obtained for liver, lung, and adrenal cholesterol did not present marked changes (Table VI). The thyroidectomized rats showed slightly lower cholesterol concentrations in their livers, 35 lungs, and adrenals than the normal controls, in spite of the fact that the blood cholesterol level was approximately twice as high in the former group. It is realized that it is difficult to interpret the change in adrenal cholesterol by itself since the latter varies widely with excitement, pain, temperature, or other environmental factors which may induce an "alarm reaction" (Selye, 191*3) • However, in spite of variability, the changes in adrenal cholesterol concentration corre lated with those of the livers and lungs (Table VI). These findings might indicate therefore that the thyroid inhibits a shift of cholesterol from the tissues into the blood rather than that it aids in the breakdown of cholesterol, and that hypothyroidism most likely does not result in an increased cholesterol synthesis as suggested by Foldes and Murphy (19U6). If the hypothesis is correct that in hypothyroidism all body tissues release cholesterol into the blood, the relative rise in the blood cholesterol level is expected to be much more drastic than the decrease in any other indi vidual tissue. CHAPTER VI SUMMARY VI. Various methods for the splitting of cholesterol digi tonide were compared. The greatest recovery of cholesterol was obtained by, treating the digitonide with pyridine and ether. 2. A method for combustion of samples of deutero-cholesterol, as small as 10 mgs., to H2O and D2O was described. Several procedures for the reduction of these ^Q-I^O mixtures to hydrogen and deuterium gases were compared. The best results were obtained by evaporating the mixtures over magnesium heated to 600°C. A special apparatus for the collection of the gases for mass- spectrometric analysis was designed and constructed, and its operation outlined. 3. Attempts were made to calibrate a mass-spectrometer with 1 0 per cent, 1 per cent, 0 . 1 per cent, and 0 . 0 1 per cent dilutions of DjJO. U. Thyroidectomized rats and rats fed O.lj? per cent thyroid powder were compared after a feeding period of four weeks with a group of normal animals regarding their cholesterol concentration in sera-, livers, lungs, and adrenals. In the thyroidectomized animals, the serum cholesterol level was twice as high as in the normal .groups. In spite of this change, the other tissue cholesterol levels were very similar in these two groups. Thyroid feeding did not induce a marked change from the normal cholesterol concentrations. 37 5. The rats in all groups were fed deutero-acetate and deuterium oxide over a period of four days. At the end of this time the serum, liver, lung, and adrenal cholesterols and the carcass fluids were prepared for analysis in a mass-spectrometer. The results can be obtained when the instrument has been calibrated. ■ BIBLIOGRAPHY 38 Barbour, H. and J. Trace, "Pharmacological Action of Deuterium- Qxide," J. Pharm., 58: 1:60-1:82, 1936. Bleakney, ¥., "A Search for Isotopes of hydrogen and Helium,” Physical Rev., hi: 32-38, 1932. Bleakney, W. and A. Gould, ’ ’ The Relative Abundance of hydrogen Isotopes," Physical Rev., Uh: 265-268, 1933* Bloch, K. and D. Rittenberg, "On the Utilization of Acetic Acid for Cholesterol Formation,”'J. Biol. Chem., 1U5: 625-636, 19U2. Chaney, A . L . (u n p u b lis h e d ) F leisch m an n , ¥ . and H . Schum acker, J r . , "The R e la tio n s h ip Between Serum C h o le s te ro l and T o ta l Body G h o le s te ro l in E x p e rim e n ta l h y p e r- and h y p o th y ro id is m ," Johns Hopkins Hosp. B u ll. , 71: 175-183, 19U2. Fleischm ann , ¥ . and H . Schum acker, J r . and L . W ilk in s , "The E f f e c t o f Thyro idecto m y on Serum C h o le s te ro l and B a s a l M e ta b o lic R ate in th e R a b b it," Am. J . P h y s io l. , 131: 317-327, 191:0. F o ld e s , F . and A . M urphy, " D is tr ib u tio n o f C h o le s te ro l and C h o le s te ro l- E s te rs and P h o s p h o lip id Phosphorus in B lood in T h y ro id D is e a s e ," P ro c . Soc. E x p e r. B io l, and M e d ., 62: 218-223, 19U6. Fraenkel-Conrat, H. L., M. E. Simpson, and H. M. Evans, "Effect of Purified Pituitary Preparation on Liver Weight of Hypophysec- tomized Rats," Am. J. Physiol., 135: 398-1:03, 19U2. Gildea, E. F., E. B. Man, and J. P. Peters, "Serum Lipoids and Proteins in hypothyroidism," J. Clin. Invest., 18: 739-755, 1939* Halford, J. 0., and L. C. Anderson, "Organic Deuterium Compounds, Acetic, Malonic, and Succinic Acids,” J. Am. Chem. Soc., 58, Part I: 736-7UO, 1936. Henriques, F. C.,r.Jr. and C. Marguetti, "Analytical Procedure for Measurement of Radioactive hydrogen (Tritium)," Ind. and Eng. Chem., Anal. Ed., 18: h20-1*22, 19U6. Hurxthal, L . M., "Blood Cholesterol and Thyroid Disease. III. Myxedema and Hypercholesteremia," Arch. Int. Med., 53: 762-781, 193U* Kamen, M., Radioactive Tracers in Biology. New York: New York Academic Press, 19U7a. Chapter VI, pp. 130-132. Kamen, M., "Use of Isotopes in Biochemical Research," Ann. Rev, of Biochem., 16: 631-65U, 191:7b. 39 Man, E. B., E. F. Gildea, and J. P. Peters, "Serum Lipoids and Pro teins in Hyperthyroidism," J. Clin. Invest., 19: 1*3-59, 191*0. Rittenberg, D. and K. Bloch, "Utilization of Acetic Acid for Fatty Acid Synthesis," J. Biol. Chem., If?!*: 311-312, 1931* • Rittenberg, D. and K. Bloch, "Utilization of Acetic Acid for the Synthesis of Fatty Acids," J. Biol. Chem., 160: 1*17-1*21*, 19l*5» • Rittenberg, D. and R. Schoenheimer, "Deuterium as an Indicator in the Study of Intermediary Metabolism," J. Biol. Chem., 111: 169- 171*, 1935. Rittenberg, D. and R. Schoenheimer, "Deuterium as an Indicator in the Study of Intermediary Metabolism," J. Biol. Chem., 121: 235- 253, 1937. Schally, A. 0., "Schilddruese und Cholesterinstoffwechsel, ” Zeit. f. klin. Med., 128: 376-385, 1935. Schoenheimer, R. and H. Dam, "Spaltbarkeit und loeslichkeit von Sterin-Digitoniden," Hoppe-Seyler's Zeit. f. phys. Ch., 215: 59- 63, 1933. Schoenheimer, R. and W. Sperry, "A Micromethod for the Determination of Free and Combined Cholesterol," J. Biol. Chem., 106: 71*5-760, 193U. Selye, H., Encyclopaedia of Endocrinology. Section I, Steroids. Montreal: Franks, 19U3. 1 * volumes. Sobel, A. and M. Mayer, "Improvements in the Schoenheimer-Sperry Method for the Determination of Free Cholesterol," J. Biol. Chem., 157, 255-261*, 191*5. “ Steiner, A. and F. Kendall, "Atherosclerosis and Arteriosclerosis in Dogs Following Ingestion of Cholesterol and Thiouracil," Arch. Path., 1:2: 1*33-1*1*1*, 191*6. Turner, K., "Studies on the Prevention of Cholesterol Atherosclerosis in Rabbits," J. Exp. Med., 58: 115-125, 1933a. Turner, K. and G. Khayat, "Studies on the Prevention of Cholesterol Atherosclerosis in Rabbits," J. Exp. Med., 58: 127-135, 1933b. Turner, K., C. Present, and E. Bidwell, "The Role of the Thyroid in the Regulation of Blood Cholesterol of Rabbits," J. Exp. Med., 67: 111-127, 1938. “ W indaus, A ., "Ueber d ie E n tg iftu n g d e r Saponine durch C h o le s te rin ,M B e r. chem. G e s ., JU2s 238-2U6 , 1909. W indaus, A ., "U eber d ie Q u a n tita tiv e Bestimnmng des G h o le s te rin s und d e r C h o le s te rin -E s te r in e in ig e n norm alen und p a th o lo g is c h e n N ie r e n ," H o p p e -S e y le r1s Z e i t . f . phys. C h ., 65: 110-117, 1910. W indaus, A ., " N o tiz u eb er d ie A u fs p a ltu n g des D ig ito n in -C h o le s te r id s ," H o p p e -S e y le r1s Z e i t . f . phys. C h ., 101: 276-277, 1918. W ynne-Jones, W. F . K ., ”The Use o f Is o to p e s in A c id and B a sic C a ta ly s is , Chem. R e v ., 17: 115-123, 1935. of Southern California U braiy

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Creator Alexander, Ralph Werner (author)

Core Title A method for the analysis of deuterium-labeled tissue cholesterol: Attempts to study the relation of the thyroid to the turnover of cholesterol in rats

Degree Master of Science

Degree Program Biochemistry

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

Tag health sciences, nutrition,OAI-PMH Harvest

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Advisor Marx, Walter (committee chair), [illegible] (committee member), Morehouse, Margaret G. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-775970

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