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Biological Fractionation of the Isotopes of Potassium

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Biological Fractionation of the Isotopes of Potassium r ria. / ^ KENSINGTON Permission has been granted by the Head of the School in which this thesis v/as submitted for it to be consulted s^mr and c opied ^^^ .... ^ This permission is contained in the Administration file "Availability of H.D. Theses" and applies only to those theses lodged with the University before the use of Disposition Declaration f orms. BIOLOGICAL FRACTIONATION OF THE ISOTOPES OF POTASSIUM. A thesis submitted as partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy in the Faculty of Science in the University of New South Wales. Submitted by S. H» Chorlton, v-flKg^ This thesis embodies the results of experimental work carried out in the laboratories of the Department of Nuclear and Radiation Chemistry, the University of Few South Wales and the Special Unit, Prince of Wales Hospital. Except where acknowledged, the work was perfomed solely by the author and has not been submitted to any other University or Institution for the award of a higher degree. S, H. Chorlton, August, 1964, ABSTRACT. The aim of this investigation -was to determine whether potassiiim isotopes were subjected to fractionation in living tissue - more specifically, to determine whether there was any difference for isotope ratios for normal, malignant and embryonic human tissues, and plant material. Mean determinations for these samples on potassium separated as the nitrate, after precipitation as the tetraphenyl-boron salt, did not differ significantly from ratios determined on A.R. potassium nitrate, for which a figure of 14.09 - ,06 (s. d.) was obtained for K^^/K'^^. Similar ratios were also found for impure (ashed) tissue samples. This differs from the results presented by Reutersward (1956) and Kendall (i960 a) v/ho found a higher ratio for samples in which the potassium was not chemically isolated. The differences in ratio reported by Lasnitzki and Brewer (I94I a, b and 1942) for cancerous tissue most probably were due to some similar impurity effect. The ratios for K^^/K^^ presented in this thesis are some A^io higher than the accepted value of Nier (l950) of 13.48 i .07, Attempts, however, to determine whether this was due to instrumental mass discrimination, by the use of synthetic isotope mixtures, were not conclusive, 41 / 40 K /K ratios were determined on A.R. potassium nitrate and potassium, separated as before, from samples of human and pig foetal tissue. The accuracy of these determinations was lov/, a mean figure for the pig samples of 569 i 32 (s. d.) being obtained (Nier's Value - 578 i 6.) K^^ specific activities were found for these pig foetal samples. Values were essentially the same as for A.R, potassium nitrate - 1.499 cnt./min./mg.K. The possible role of rubidium in these samples affecting the count rate, is discussed. G 0 N T E N T S. Page Section I. IMTRODUCTIOIT AMD BEVIES I 1. General Introduction 1 2. Biological Fractionation of Isotopes 2 3. Potassium Isotope Measurements 4 3. I. Potassixjm Ion Production by Thermionic Ion 6 Source (Theoretical Considerations and Ratio Determinations,) 3. 2. Isotope Analysis by Electron Bombardment 13 Miscellaneous Isotope Studies 14 3. 3. Determination of Natural Radio-active Isotope 14 of Potassium and Isotope Separation. 4. Outline of Project 18 Section II. INSTRI3MENTATI0N. 19 I. Isotope Ratio Studies - Mass Spectrometer 19 I. I. General Description 19 I. 2. Vacuum System 20 I. 3. Ion Source 23 I. 4. Electronic Units 27 1. 5. Ion Current Collection, Amplification and 30 Recording. 2. Potassium - 40 Radioactivity Measurements 32 3. Total Potassium Determinations. 32 Section III EXPSRII^nMAL 34 1, Sample Preparation 34 2, Source Preparation and Operation 39 2, I. Determination of Potassium Compound for 39 Ratio Studies 2. 2. Source Preparation and Operation 43 3. Analysis of Data 47 4. Calibration Determination and Investigation 54 of Errors. 4. I. Use of Internal Standard 54 4. 2, Synthetic Isotope Mixture in the Study of 57 Mass Discrimination Effects. 5. Instrument Performance 64 5. I. I/B2 - S G Mass Spectrometer 65 5. 2, Liquid Scintillation Counter 65 5, 3. Atomic Absorption Spectrometer 68 Section IV RESULTS 70 I. K^^/K'^^ Ratio Determinations ' 70 I. I. A.R. Potassium Nitrate Standards 70 1* 2. Cancer Samples 73 I. 3, Normal Tissue from Persons with Cancer 73 I, 4, Normal Tissue from Persons Free of Cancer. 73 I. 5. Human Embryonic Tissue 76 I. 6. Plant Samples 76 I. 7. Red Blood Cell Potassium 78 1. 8, Ashed Tissue Samples 78 2. Potassium 41/40 Ratios and Determination of 81 K^^ Levels. 2. I. A.R. Potassium Nitrate K^^/K^^ Ratios. 81 41/40 2. 2. K /K Ratios for Poetal Samples. 84 40 2. 3. K Specific Activity Measurements. 84 3. Calibration Determinations. 86 4. Total Potassium Levels - Tissue Sajnples. 88 Section V. DISCUSSION OF RESULTS. 90 ^CKNa'/LED®0!]NTS. 99 lOI APPENDIX. (a) Some Theoretical Considerations of i. Isotope Separation, (b) k^Vk^^ Ratio Determination. (c) Peak Shape and Resolution Determination vii. LIST OF TABLES. Table Number, Pa^e, List of Tissue Samples Collected for Isotope 35 Analysis. 2. K^^/K^^ Ratio Determinations on A.R. laO^; 42 RheniiHD Filament. 3. Checking of Range Factors. 51 4. (a) K^^/K^^ Ratios for Two Samples on the One 58 Filament Bead. 4. (b) K^^/K^^ Ratios for Two Samples of A.R. IQiO^ 58 on the One Filament Bead. 5. Effect of Heat on Li'^/Li^ Ratio 61 6. A.R. KNOjj 71 7. Cancer Samples 73 a 8. Normal Tissue from Persons with Cancer. 74 9. Normal Tissue from Persons Free of Cancer. 75 10. Human Embryonic Tissue 77 11. Plant Samples 77 12. Red Blood Cells 79 13. (a) Ashed Impure Samples 80 13. (b) A.R. OOg with Added Minerals 80 Table Number. Page K^^/K^^ Ratios for; 14. A.R. lOTO^ 82 o 15. (a) Hiiman Embryonic Tissue 82 15. (b) Pig Foetal Samples. 83 40 16. K Specific Activity for Pig Foetal Samples 85 17. K^VK"^^ ^"tios 87 IB. Total Potassium Determinations 89 LIST OF FIGURES. Figure Ntanber. Page 1. Diagrammatic Representation of the Vacuum 21 System of the ISS2 - S.G. for Thermal lonisation. 2. Photograph Showing the Pneumatic Valve (V.I.), 22 Used During Source Changing. 3. Ion Source of 1»IS2 - S.G. for Thermal lonisation 24 4. Exploded View of 1132 - S.G. Thermal Ionization 25 S oxarce. 5. Triple Filament Bead of Type Used in this Study 26 6. Circuit Diagram of Original Triple Filament 29 Power Supply. Key to Figure 6. 29 a 7. Part of K^^/k^^ Recorder Tracing for A.R. KNO^ 48 8. Linearity of Recorder and Amplifier 53 4T / 40 9. Part of K /K Recorder Tracing for A.R. mO^ 55 10. Effect of Varying Filament Current on K^^/K^^ 60 Ratios for A.R, KNO^ II. (a) K^^ Peak Shape from Recorder Tracing 66 II. (b) K^^ Peak Shape Graphed from Meter Readings 67 OF NEW SECTION I. ™RQ3)UCTI0IT AMD REVIM, General Introdiiotlon, Potassim is a "biologically important element, one of its main functions being to maintain osmotic equilibrium. An average 70 Kg, man has been estimated to have 140 gms. of potassim (determined from K4 0 and K4 2 measurements - Remenchik and Sliller, I96I.\} -with 9 gms. in the blood and 0,6 gms» in the plasma. The concentration of potassim in the cells is high (up to 400 mg./ lOOml.) whereas plasma levels are maintained at a low level of 17 mg./lOO ml. The mechanisms by which these different levels are maintained are not clear. In the developing embryo, however, extra- cellTilar potassium levels are raised, being almost three times as high as in an adult. There is an accmulation of potassium in the cells during growth, and in active tissue growth such as in cancer tissue the level of potassium is high. Shear (1933), in a review of the mineral content of cancer tissue, gives values for potassium levels in cancer of as against 1.4^0 for normal tissue. De Long et al. (I950) also reported a 60^ increase in potassium content of the mucosa in cases of intestinal cancer in humans; the increase in potassium is dtie in part to increased celltilar activity. Because of this association of increased potassium levels with cancer, and also because of the report by Lasnitzki and Brewer (I94I b; 1942) of the fractionation of the isotopes of potassium by cancerous tissue, Starr (1956) expressed an interest in the possibility of biological fractionation as an aid in cancer diagnosis. Subsequently, tissue samples were sent to Kendall (i960 a,) to be analysed on a mass spectrometer. Because of some doubt as to the reported isotope effects for "impure" samples in Kendall's results, it was decided to extend the investigation. If an isotope difference could be regularly found in partly purified potassium isolated from cancer patients, when compared with similarly partly purified potassium from normal patients, this could be of practical importance medically, 2, Biological Fractionation of Isotopes. Physical methods have been used for the separation of nattirally occurring isotopes, ever since the experimental work of Thomson (I9I3) Aston (I9I9, I92I and 1927) and Dempster (l922). To-day electro- magnetic separation is the commercial means of production of a great many stable isotopes. Separation by this method depends on differences in the mass/charge (m/e) ratio of gaseous ions. Since isotopes have essentially the same electronic con- figuration, they should exhibit the same chemical behaviotir. However, because of the zero point energy differences, slight chemical differences exist. Urey and Greiff (1935) produced enriched through chemical exchange of gaseous ammonia and ammonium salts.
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