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.

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.

Biological systems, which are dependent on chemical and physical reactions could conceivably give rise to fractionation.

Bowen (1959) has recently reviewed isotope fractionation by biological systems. The best conditions for fractionation, small percent uptake and excretion, or chemical changes which have large fraction-

ation factors occur in single cells. Much of the work on biological

fractionation has therefore been done on single cell organisms. As

might be expected, hydrogen, with its 2:1 mass difference for its

stable isotopes has been found to exhibit fractionation (Cloud et al

1958; Pratt and Curry 1937; Taylor and Harvey 1934.) Isotope effects, however, are not confined to this element; carbon (Urey, 1948),

oxygen (Peldon et al 1959) and sulphur (Thode et al 1954) have all been found to be similarly affected. A mineral effect has also been reported for lithium (Bowen 1956).

Although Tinicellular organisms provide the best soxarces for isotope separation, effects have been found with higher plants and animals. Plants have three metabolic pathways capable of fractionation- photosynthesis, respiration, and tiranslocation. There are reports of fractionation of the isotopes of carbon (Van Norman and Brown 1952), and oxygen (Vinogradov and Teis I94l) during photosynthesis. Mineral effects with potassium and lithim are claimed by Brewer and Baudisch

1937) and Jacques (l940), but these findings have not been verified.

The animal kingdom with its complex metabolism and excretion

should be capable of fractionating isotopes. Bowen states that, when looking for fractionation of isotopes in urine, it is not the readily absorbed elements such as sodium and potassitmi that should be studied, but elements such as calcium and iron. Some preliminary studies at the Special Unit (Wynne and Chorlton - impublished data) using 45 47 simultaneous adminiBtrations of the radio- isotopes Ca and Ca have indicated a discrimination by the body in handling these isotopes. An investigation to see whether the stable isotopes are similarly 45 47 affected or not, is to be imdertaken. If Ca and Ca can be differentiated by the body, and K^^ fractionation is also conceivable. Theoretical considerations of the feasability of fractionation of these isotopes by physiological processes is discussed in the Appendix Section of this thesis. Previous measurements in the animal kirjgdom have been confined to molluscs and vertebrates. Fractionation effects with carbon in molluscs (Craig 1953) and hydrogen in rats (Thompson and Ballou 1953, 1954) have been reported. Although Bowen made a very extensive review of biological fractionation, he did not refer to the papers of Brewer (1937 a), Lasnitzki and Brewer (1938, 1940, I94I a, b, 1942), or Penn, Bale and Mullins (l942), all reporting fractionation of the isotopes of potassium by htonan tissues. There are also 4report2 s of differences in renal and urine specific activities of K in humans (Blake and Emery 1957) which could be due to isotope discrimination. The measurement of potassiimi isotopes, however, will be discussed more fully in the next section. 3. Potassium Isotope Measurements. Potassium has three natural isotopes of mass niambers 39, 40 and 41, occiarring with the following abimdances (After Nier, 1950 — "Handbook of Chemistry and Physics, Table of Isotopes.") + 93.o4 • 04 + 40 0.0II9 + .0001 41 6.91 .04

Corresponding ratios are k"^^ - 13.48 .07

- 578 6.

Potaesi-um - 40 is a naturally occurring radioactive isotope, which has the following "breakdown pattern (Vinogradov, 1957)

Half - life - 1.33 x 10^ years.

40 K

E.G. II.25b p (8Q.&fo)

Em » 1.36 MeV. E

40 K is responsible for most of the natiiral radio acti"KdLty in man. This activity has been used as a measure for determining total body potassium (Anderson and Langham, 1959). Since Aston's (I92l) discovery of the isotopes of potassim, investigations to determine isotope ratios for potassium obtained from a wide variety of sources have been made. Most studies have been made lay mass spectrometry althoiAgh Manley (1936) used nuclear magnetic resonance for his measurements. Determinations by mass spectrometry may be made either by electron bombardment of potassium vapour

(mainly from distilled potassium metal) or by thermal ionis^tion of potassium salts from a metal siirface. A review of the development of mass spectrometry would be too lengthy, and woiald serve no usefiil purpose in this thesis; the subject is adequately covered in many good text books. However, details of previous studies on the isotopes of potassim will be given, discussing firstly results obtained for

K^^/ K^^ and K^® or K^^/ K^^ ratios from thermionic ion sources.

3. I. Potassium Ion Production by Thermionic Ion Sources.

Theoretical Consideration.

Thermal-emission sources work on the principle that when a material of ionisation potential I is heated on a starface of work function ^ there is a finite probability that some of the material will evaporate as a positive ion according to the Kingdom-Langmuir

Law + e( I - gi ) OC exp, KT where n"^ and n° are the number of positive ions and neutral atoms evaporated, e the electronic charge, K Boltzmann's constant and T the temperature in degrees absolute. For materials where ( I - ^ ) is positive, the process is highly efficient, and where negative, ionisation can be very low. The efficiency varies critically with temperature. For single filament ionisation (surface ionisation) the material being placed directly on the ionising filament, the choice of filament material is important. It should have a high work function so that ( I - ^ ) remains a positive quantity and should "be a refractory material. Tungsten, tantalm or rhenium are commonly used for filaments, since they all have melting points in the region of

3,00G° C. and work functions are of the order of 4 - 5 V. For a surface ionisation, the Kingdom-Langmuir equation is only a rou^ approximation of the degree of ionisation obtained in practice, because reaction may take place between the sample material and the filament, altering the work function. Phipps et al (1935, 1937, 1939) found they were unable to fit results of ionisation of alkali halides on tungsten filaments to the Langmuir equation. They found a variation in work function with temperature and in order to account for this they postulated the formation of a tungsten halide, which dissociates at high temperatures. Zemel (1957) however, has siiggested that patches of different work function occur on the surface of tungsten filaments, and that the ratio of ions to atoms emitted from the total surface, may be computed from the sum of Langmuir equations for each patch.

For materials of relatively high ionisation potentials ( 6 - 8 V), however, the efficiency of ionisation from single filaments is very low. This may be increased by raising the temperature of the filament, but in so doing, the sample life-time is reduced, and variation in isotope ratios may occur. A multi-filamented sotirce, however, enables materials of high ionisation potential to be ionised without any of these difficulties. Palmer (1956) says that ionisation by such sotirces, should be free of ionisation-induced fractionation. The

source used was of the type shown in Figs. 3 and 4, where the

filament which is raised to a high temperature for ionisation

(usually the centre filament) does not support the sample, Inghram

and Chupka (1953) describe the use of such sources, reporting

increases in efficiency which eliminate the use of electron

multiplier detection. The increased efficiency of ionisation may be

predicted by the equation;

+ - I Tj = exp. m m + KT I ^^

where T| and T^ are the temperatures for single filament and multiple filament ionisation. Multi-filamented sources were used in all sample analysis made in the present study.

Ratio Determinations.

The earliest report of potassium isotope ratio measurements was by Dempster (1922) who gave the ratio of K^^/ K^^ as approximately

18. Baingridge (I93l), working with leucite gave a ratio of 12,5 for

the main ratio stating that if existed, it was with a ratio of

less than 1/300.

V/ith the development of better vacuum techniques, enabling higher vacua to be obtained, and with improvements in electronics, greater accuracy and repeatability of determination was possible.

Many analyses in this period were made by Brewer and co-workers. Brewer and Kuesch (l934) using artificially produced aluminium

silicates in a Dempster type mass spectrometer obtained a ratio of

13.88 0.4, Brewer (1935), with potassium phosphate from basalt

impregnated in a platium disc over a tungsten filament (which was

said to give more reproducible results than a coating of the sample)

gave a K^^/ K^^ ratio of 14,25 and a K^^/ K^^ ratio of 6,300 ^ 100

Brewer then began a series of experiments to determine

the isotope ratios of potassium from a number of natiiral sources.

For Pacific Ocean sea water Brewer (1936 a) could find no difference

in the 39/41 ratio of samples down to a depth of 2,500 metres and obtained a ratio of 14.20 0.0035, Not all the sodium present

in the sample, was removed before the mass spectrometric study.

Brewer (l936 b) determined isotope ratios in mineral and plant material, again without complete separation of the potassium.

Mneral samples he found to vary slightly in ratio; a value of

14.25 was found. For ashes from plant material however, there was a 41 ^ marked variation, the percentage of K varying by as much as

Kelp varied the most in this regard, a figure of 12.6 0.2 being found for one sample. Brewer and Baudisch (1937) found an apparent increase in K^^ in underground water (13.85 O.l) and a small concentration of this isotope in Cryptozoon fossils (13.95 - 14.00

.01). Brewer (1939), using base exchanges of potassium and zeolite, altered the K^^/ k'^^ ratio from 14.20 - 13.60; but measurements carried out on potassium from phosphate rocks and soils showed no difference in isotope abundances. The analyses in the report by Jacques (I940) showing an increase in k"^^ in certain marine plants

(Valeria and Nitella) were carried out by Brewer, Ratios of

13,85 ,05 — 14,00 ^ ,03 are quoted. In an article describing the separation of the isotopes of potassium, Bradt, Parham and

Brewer (1947) report on isotope abundance ratios and find constant in nature (I4,20 0,02) "except ?/here acid-base exchanges take place," (This is a surprising statement in light of the anomalous ratios reported by Brewer and co-workers for samples of mineral, plant and animal origin.

Brewer also focused his attention on potassium in animal tissue. Potassium - 41 in various parts of the heart was reported to have an increased abundance (Brewer, 1937 b). Ashes from other animal tissues impregnated on a platinum disc, gave a constant ratio —

(14,20), Bone marrow however, had a high content of K^^, a ratio of

13,68 - 13,92 being quoted.

Lasnitzki and Brewer (1938) reported ratios obtained on ashes from Jensen rat sarcoma, red blood cell, spleen, lymph, heart, 41 bone marrow and bone. Bone marrow and bone were increased in K by

1,7^, skeletal muscle was reduced in this isotope by 0,4^o and the sarcoma by The rest of the tissues gave "normal" ratios, i,e, normal for Brewer's studies (I4.20), These same authors (Lasnitzki and Brewer, I940) reported that the K^^/ ratios in blood plasma of normal rats was increased by an average of 2,5^, again relative to their "normal" ratio. Later studies (Lasnitzki and Brewer, I94I a) confirmed these ratio differences in blood plasma and bone marrow. The increased ratio associated with tumovirs was also

verified (Lasnitzki and Brewer, I94I h), the unaffected muscles of

tumour-'bearing animals in this study also showing an increase of

0.9 - I.3fo in the isotope ratio. Increases in the same order were

also found for human cancer tissue (Lasnitzki and Brewer 1942)

although the non-cancerous tissue had a "normal" ratio of approx-

imately 14.20 in this instance.

Many measurements on K have "been carried out by

other workers, although mainly on mineral samples.

Bondy, Johannsen and Popper (1935) gave the K^^/ K^^

ratio as 16,2 2.2 while Bondy and Vanicek (1939) in later work

using surface ionisation techniques, obtained 14.1 O.I for this

ratio. Taylor and TJrey (1938) in experiments on chemical fraction-

ation of potassium isotopes, gave a normal ratio of 14,10 0,09.

Cook (1943) using a Dempster double focusing mass

spectrometer, accurately determined the

K"^^ ratios in certain

Pacific kelps, fossils and rocks of different geological ages. He

obtained an average ratio of 14.12 0.28 and could find no

significant differences for any of his samples, although some of

them were reported by Brewer and Baudisch (1937) to yield anomalous ratios. Cook noted fluctuations in isotope ratios, which he attributed

to fractionation effects of the hot filament source; samples left in

the instrument over night varied by as much as 2,5^o.

Paul and Pahl (l944) gave a value of 13,96 O.I for

K^^/ K^^ ratios. Rik and Shukoliukov (1954) found the K^V ^^^ ratio for terrestrial and meteoritic samples to be the same, 14,4.

Reutersward (l952) remeasiared the isotope ratios for potassium,

obtaining 14,32 ^ 0.05 for K^V ^^^^ ^^^^ "helxis

attributed to mass discrimination effects of the instrment. In a

later paper (Reutersward 1956) made a canprehensive study of errors

and fractionation effects of the thermal ionisation technique in

isotope analysis. He says that Erewer^s (1935 c) method of impreg-

nating his sample into platinum probably introduces a mass

discrimination effect, and that a coated filament is to be preferred.

Studies on powdered leucite gave results similar to those of Brewer

(1936) — ratios of 14.20. Dilute solutions of piire potassium salts,

however, gave ratios of 13.88 .07 ( p. e.). A sample of sea water,

after chemical separation, gave 14.06 ^ .04 while a kelp specimen

gave a ratio of 13.67 .08 ( determined from ash material.)

Chemical purification of this sample gave a ratio of 13.94 .03,

the lower figure obtained in the impure state being attributed to an

admixture of chemicals affecting ionisation. In this same paper,

Reutersward discusses the effect of sample size on the resulting

isotope ratio. If the isotope ratio is not to be disturbed by source

fractionation during measurement, thin sample coatings are essential. 41 The enrichment of K in thicker coatings varies irregularly

throughout the sample analysis. After correction for such variations,

Reutersward gives a K^^/ K^^ ratio of 13.57 ^ .09. VMte, Collins

and Rourke (1956) using a two stage mass spectrometer of improved

accuracy, searched for isotopes of low abundance. For K^^/ K^^ (from potassim nitrate) they obtained a value of 13.79 ,10

and 573 ^ 10 ( K^V a ^^ correction being made for

discrimination by the electron multiplier used^ A ratio of I3,96

.05 for K^^/ k"^^ was reported by Omura and Morito (1958),

Kendall (i960 a, b.) studied the potassium isotope

ratios for mineral, plant and animal tissue samples in the hope

of finding an explanation for ancmalous resoilts of Potassium-iirgon

dating of rocks and the isotope effects of cancerous tissue reported

by Lasnitzki and Brewer (1942). He foimd no isotope ratio variations

to confirm either of these two anomalies and obtained corrected

ratios of 13.77 0.03 ( T^^/ K^^ ) and 576 3 ( K^^/ K^^ ) for all

samples. On partly purified samples, however, he did find differences,

differences which were no longer evident after complete chemical

isolation of the potassium. These results are similar to the findings

of Reutersward (1956).

3. 2. Isotope Analysis by Electron bombardment«

No reference ^11 be made here to theoretical

considerations of ion production by electron bombardment. Results will be given only for analyses performed on potassium samples.

Mer (1935, 1936) determined the isotope ratios

from potassium metal, distilled into the ion source. For the ratio he obtained 13.96 ^ O.IO and, for K^^/ K^^, l/8,600 ^ 10 and calcialated an atomic weight for potassium of 39.096 ( accepted

present day value - 39.100). In a redetermination of the ratios,

the same author (Mer, I950), using two mass spectrometers of the o 60 sector type, calibrated by a synthetic argon isotope mixtiire,

obtained the values for relative abundances now accepted as being

most probable - 13.48 ^ 0.07 ( K^V ^^^ ) and 578 ^ 6 ( K^^ K^^).

Potassium metal was again distilled into the soiarce as a vapour.

White and Cameron (1948) obtained potassium ions from a

sample of potassium aluminium chloride, distilled into a 60^ mass

spectrometer of the Mer type (Nier 1947). The K^^/ k"^^ ratio

obtained was 13.66 O.I.

Miscellaneous Isotope Studies.

Differences in chemical atomic weight determinations can

give an indication of the isotope composition but the accuracy is

low. Loring and I>ruce (I930) claimed that the potassium from the ash

of potato stalk had a high atomic weight (40.5), which would imply an increased abundance of potassium - 41.

I5ahley (l936) measured K^^/ K^^ ratios by nuclear .j. magnetic resonance and obtained 13.4 ^ 0.5, a value very close to

the accepted value of 13.48 ^ 0.07.

3. 3. Determination of the natural Radioactive Isotope

of Potassium and Isotope Separe,tion.

Potassium had been shown to have a natural radio- activity (Biltz and Marcus I9I3) and von Hevesy (1927) working with partly separated potassium isotopes, came to the conclusion that

K'^^ was responsible for this activity. Bainbridge (I93l) had stated

that if an isotope of potassium existed at mass 40, it was present in a concentration of less than I in 300. Klemperer (1935) and Ne-wman and Walke (1935) independently stiggested 40 that evidence pointed to an as then unknown K isotope being responsible for the radio-activity of potassium. Firstly Nier (1935) and then Brewer (1935) presented mass spectrometric evidence for the presence of k"^^ obtaining / K^^ ratios of 8,600 ^ 10 and 8,300 ^

100 respectively. Kier (1936), from mass spectrometric studies, calculated that the4 0radio-activit y of potassium could be best explained by assuming that K was undergoing decay. S my the and Hemmendinger

(1937) by means of a high intensity mass spectrometer separated the 40 three isotopes of potassium and identified K as the radio-active isotope. Beta ray bands for potassium chloride and the separated isotope were the same. However, investigation of the constancy or otherwise, of the radio-activity of potassium in nature, had begun long before this time. Loring and Druce (I930) from their work on potato stalk ash previously mentioned, claimed that there was an abnormally high radio-activity for this sample, as measured photo- graphically. Ernst (1934), again by a photographic determination, claimed that the radio-activity of potassium from animal tissues was greater than that from mineral origins. However, chemically purified samples of potassium, isolated fxcm animal tissues, were shown by

Lasnitzki and Oeser (1937) to have the same activity as Analytical

Reagent (A. R.) potassium chloride ( within 2fo), Brewer (1938) confirming Smythe and Hemmendinger• s (1937) findings, suggested the use of potassium-argon ratios for determining geological age of rocks.

Lasnitzki (1939) with an accuracy higher than in his earlier work, reported on the activity of inusole, t-umour tissue, and a standard of mineral origin. He again found no significant differences. Brewer,

(1939) found that the ^ activity of k"^^ was the same for fresh

Vesuvius lava, clay soils, Saratoga Spring Water and commercial potassim. The activity of potassivim from deeply bxaried granite and surface layers was the same and it was concluded that cosmic ray activity had little effect in the production of Similar constancy for potassium specific activity was reported "by Schumb et al, (I94l) for meteoritic and terrestrial samples. Fenn et al. (1942) working with 40 / human crematorium ashes found K levels as much as I - lower than laboratory potassim chloride and suggested that such a difference could be due to the cell membranes favouring the lighter isotopes.

This, however, would mean that plasma isotope levels of

should be low, a finding inconsistent with those of Lasnitzki and Brewer (l940).

Mullins and Zerahn (1948) found the K^^ content for animal, vegetable and mineral sources constant vd.thin - Despite the reports that cosmic ray effect in potassium - 40 production is unlikely, and that meteoritic and terrestrial potassium activity is the same, anomalous

K"^^ contents have been found. Voshage and Hintenberger (1959 a, b.) and Honda (1959) report on anomalous contents of K^^ in the surface / 40 layer of iron meteorites, about 5fo of the potassium being K . This is apparently the result of cosmic radiation. 40 Attempts have been made to discover whether K is responsible for some features of the biological importance of potassium. Rahn (1936) found that k"^^ had no specific effect on the growth of micro-organisms. Crockett and Hiiff (1958) however, working with leiakaemic white cells, found that a reduction of the 40 / K content of media to 1/5 of the normal abundance, with constant total potassium content, gave rise to cells with larger nuclei, and cells of more imiform diameter. Degeneration was less as evidenced

"by fewer numbers of cells in the media. On the other hand Vinogradov

(1957) found that concentrations both above and below the natural abundance of did not affect the growth of Aspergillus nigricans.

Welte and Mechsner (1959) confirmed that K^^ is not responsible for 40 the biological importance of potassium, K increased by 46.6 times having no effect on the growth of the yeast Torula utilis.

There has been some degree of success in the separation of the isotopes of potassium by chemical means, mainly by the use of synthetic resins. Taylor and TJrey (1938) fractionated the isotopes of potassium by chemical exchange on zeolite colxamns, producing a change in K^^ ratios from 14.10 to 13.4. Brewer

(1939) also artificially fractionated the potassium isotopes in the laboratory by natural zeolites and suggested that the process might well occur in nature. Brev/er and co-workers (1947) concentrated the 39 isotopes of potassium by counter current techniques, the K concen- 39 / 41 trating at the cathode, where a K / K ratio of 24 was obtained after 500 hours; the ratio at the anode was 9.1. Experiments with chemical exchange on zeolite and counter current techniques were reported by Douglas et al (l950) in Y/estern Australia. They achieved an 6fo separation of the isotopes of potassiiam on zeolite, but could obtain no separation "by connter current techniques. Hevesy (1927)

partly separated the isotopes of potassium simply by repeated

distillation of the metal. He produced "heavy" potassium with an

atomic weight 0,005 units greater than normal,

4, Outline of Pro.iect.

The main aims of this study were to find out whether

the work of La,snitzki and Brewer (1942) on the isotope ratio of

potassium with human cancer could be verified or not. The evidence

suggested that these reported isotope effects were of instrumental

origin. However, even if the differences reported by Kendall (i960 a)

existed in impure potassium samples from cancerous tissues, it might be of some practical benefit in diagnosis of the disease. Consequently,

the project was planned in the following stages:

I. The determination of K ratios for A, R, potassium salts

(the most suitable one to be determined experimentally.)

II. The determination of K^^/ K^^ ratios for samples of normal and

malignant human tissue and plant samples as outlined in the

Experimental Section.

III. The determination of K^^/ K^^ ratios on ashed tissue samples

i.e. on an impure sample. r5Q / 4.T 4.T / An IV, The determination of K^^/ K and K^V k"^^ ratios for foetal

samples, 40 V. The determination of K radio-activity on these foetal samples,

VI. The calibration of the mass spectrometer by the use of a

synthetic isotope mixture for the determination of errors and

for the determination of true abundance ratios. SECTION II.

imTRTJMEmkTlQ-N,

I. Isotope Ratio Studies - Mass Spectrometer. I. I, General Description. The Mass Spectrometer used for the study of the isotope ratios reported in this thesis, was a conanercial instrument, a Metropolitan Vickers M S 2 - S.G. mass spectrometer capable of analysis of both gaseous and solid samples. The instrument is of the 9CP sector type with an all metal analyser tube of 6" radius. Ions, after being accelerated by a voltage of 300 - 2,000 volts, are sorted by a magnetic field according to their m/e ratios. Different ions could be collected by varying either the ion-accelerating voltage or the magnetic field strength (variable from 600 - 7,500 gauss) when direct current reading from a milliammeter was used. ?i/hen ion intensities were to be recorded for comparison purposes, magnetic scanning was the method used. (The magnet current may be set at any value between 10 and 250 m A and is constant to better than I part in 20,000 / minute with a general drift of less than I part in 5,000 / hour.) All isotope ratios in this study were derived from recorder determinations, the magnet current being varied in a cyclic manner by means of a sweep potenti 0-meter.

Although few changes Y/ere made in the instrument from as supplied, a more detailed description will be given here of some of the Units, with partictilar reference to the analysis of solid samples. I, 2. Vacmrni System.

This is shown diagrammatically in Pig. I , that part

of the system concerned in solid sample analysis only "being shown.

An essential feature, for solid source operation, is the provision

of a pneumatically operated valve (Vj) at the source end of the tube v/hich enables the source to be removed without breaking the main vacuum. ¥/hen the source is removed for sampling, or any other reason,

the source region above Yj may be evacuated along another vacuum line

through a two stage rotary pump (Metrovac type DRI - Pj) to less than -3 lO" mm.Hg within I minute. The valve V^ may be opened to the main pumping line after isolating Pj , and the system pressure can be reduced to better than lO'^mm.Hg in 5 minutes. Pig. 2 shows the position of this valve, with the ion source in position.

The tube is evacuated by the main pumping line through a stainless steel cold trap and two oil diffusion pumps

(OPj , OPg) in series. These piamps are backed by another rotary pump (Pg)' Tl^is backing line has a Pirani gauge (G^) which is inter- locked with the heaters of the diffusion pumps and switches the pumps off if the backing pressure is too high. A valve in this line

(Vg) enables a • vacuum to be maintained in the tube if the pumps are to be switched off. The cold trap refrigerant used throughout the present study was liquid nitrogen, which for the latter part of the study was dispensed by an automatic cold trap filler. This ensured that the cold trap refrigerant level never fell below half capacity.

A Phillips cold cathode ionisation gauge (G-^) mounted at the end of G?

Pig. I. Diagrammatic Representation of the Vacmcn System of the MS2 - S.G. for Thermal lonisation. Pig. 2. Photograph Showing the Pnematic Valve (V.I.) Used

'During Source Changing.Souroe Magnet is also shoim,

althou^ not xised in this study. the cold trap nearest the diffusion pmp measiared the pressiare

levels in the tube. This gauge was on a protective circuit which

ensured that neither the tuhe filament, nor the 2,000 volt power

supply could be switched on unless the pressure was less than 10 -5 inm.Hg and 10 nnn.Hg respectively. The sensitivity of this gauge is

such that grid resistances from 10,000 ohms to 100 megohms correspond

to pressures of 10 to 10 mm.Hg (full scale).

A water relay was also included in the diffusion pump protection circuit, which consisted of a microswitch being

operated by bellows. Since the Jilass Spectrometer was in an air- conditioned room on the top floor of a three storied building, it was found necessary to insert a piimp in the water supply to increase the pressure to operate these bellows,

I* 3, Ion Source.

A photograph of the ion source for solid samples is shown in Fig, 3 and an exploded view is shown in Fig, 4, A multiple filamented bead (fig, 5) was used for results quoted in this

study (unless otherwise stated,)

The ion source is basically that developed by Craig

(1959), Essentially the source consists of a stainless steel block

(see Fig. 4) into which the filament is inserted by means of its locating pins. The focus plates on this block perform the function of withdrawing the ions from the block, and of focusing the ion beam.

One of the plates is fixed at a voltage of 1,600 V relative to earth and the other is variable between 1,988 and 1,700 Y relative to earth. Pig. 3. Ion Source of MS2 - SG for Thermal lonisation

pyrophillite block shown. 32 ^lUi IiehroM Rut. IIBA llehroM fMh«r. lo.l nat« (LlMltlng slit plmU) .080- sllt^

A^ (W 5/32" O.D X .969*lff.Ilchrcm a Spacer.

.004" slit llBi z 2 7/l6"l«.Ilohrca« Pillar. Io«.2 k 3 FUtaa (SaflBlnc slit plat*) IIBA z 1 5/l6"lc.IlohraM Pillar. .062" allt IIBA llchroM lut and laaher. IIBA X ^V'io^ca* lUlar.

IIBA IlehroM Vut Jt Waahar Quarta Spacer. »o.4 Plate .040" allt^S^^ —3hb ^artc Spacer. ("D" or Focus Plataij)

Block Ian. 2 OB CJ)x .7J]g-Quartz Slew*. (HlchrcBa) ^-Ibb Ceramic Spacer.

ISB Ceraalc Spacer. IIBA Hlchrom* Hut & Waaher.

Triple rilaMnt Bead.

Fyrophjrlllte Block.

Source Support.

Pig. 4» Exploded View of the liSSZ ^ SO Thermal lonisation

Source* Locating Pine

Side FilsBsnte Centre Pilar^nt

ug •TP i iN I- -Glesn Baee. .'1 I ! I nIr

Fig. 5. Triple Filament Bead of Type Used in this Study. Control for this focusing is situated in the Sweep Control chassis*

By placing a voltage of 300 V on one of the plates, the beam may be

deflected, so that it does not emerge from the source. These focus

plates are .040" apart. From the block the beam is accelerated down

the ion source, throijgh the defining slit, set at 0.004" and out

through the limiting slit plate (0.080"). The beam then passes

through the analysing tube where magnetic sorting of the ions takes

place, and thence through the collector slit on to the collector,

where the ions release their charge.

In the original soiirce described by Craig (1959), an

image of 0.3 - 0.6 X the filament was produced at the collector.

Observation of the ion bvirns on the collector of the MS2 - S.G.

indicated values nearer the upper limit.

A source magnet of 150 gauss which is provided, is

essential for collimating the ion beam in gas analysis. 55his was

found to have no beneficial effect in solid source analysis and so

it was removed for K^^/ K^^ ratio determinations.

Some of the problems associated with this original

source, and the slight modifications made, will be discussed in later

sections. The filament control unit and sweep control imit are different from those used with the gas source and they will also be described later.

I. 4. Electronic Units.

Except for the units mentioned already these are similar to those used for gas analysis. The units concerned are: (a) The 250 Volt stabilised Power Unit which supplies the power to

the critical valves in the electrometer amplifier, the H, T. for

the electrometer amplifier, and filament supply to the first

stage of the magnet power supply. Output is constant to better

than I part in 50,000 per minute and drifts less than I part in

5,000 per hour. Low frequency output ripple is approximately

20 m. Volts.

(b) 2,000 Volt stabilised Power Supply and Potential Dividers. The

2,000 V output is held constant to better than I part in

20,000 and is fed to the potential divider on the sweep control

panel, which may give a voltage of 300 - 2,000 V to the source.

scanning magnetically for solid source analysis, a poten-

tiometer was adjusted so that any preselected mass range could

be scanned. For potassim this was generally 38,5 to 41.5. The

charging and discharging of a condenser provided for the mass

range to be scanned in a cyclic manner as many times as required.

When recording, the automatic range changer, operated by the

recorder, enabled the scan rate to be slowed down to ensvire that

the peak amplitude was drawn correctly.

(d) Triple Filament control. (See Pig. 6 for circuit details.) This

provides the power to heat the centre and/or the side filaments

(one at a time.) Originally a constant voltage transformer

supplying the power to the Variacs was incliided in this circuit.

However, this was found unsuitable as also was the constant /

ni J I Tl

—f h R1 i t I TT-'-y-r T1

bS \ 5)2 m RG p / m » ^^ VW-^ ) G2 K: VFf i RB T3 - f 0

Fig. 6. Cipciiit Diagram of Original Triple Filament Power Supply^

T. I, was not used in this study. 29 a

Key to Figure 6. Triple Filament Power Supply.

Resistors.

HI 1.2 K 100 W 50^

2-3 3.3 K 5 W

4-9 10 K f W IC^o

Transformers.

T I Constant Voltage Transformer.

(Not used for this study.)

2-3 Yariacs. (V 5 H)

4 - 5 230/5V 6A. (Parmeto Type 600/57.)

Meters.

M I - 2 0.6A (Sangamo Weston S5I.)

Switches.

S I - 2 S. P. S. T.

S 3 Rotary Switch. (Santon.)

Output.

Numhers 44, 45, 46, 55, 62, 63 and 64 - to ion sotirce.

44, 45 - Centre Filament.

55, 62 - One Side Filament.

63, 64 - Other Side Filament.

46 - Cage. voltage transformer supplying the power to all electronic iinits,

A voltage stabiliser was therefore used, and the filament power supply was fed frc»n this stabiliser. The power to the filaments was controlled by Variacs fed through a transformer. This form of filament control has its faults and is thought to be inferior to a constant current supply, as described by Reutersward (1952) and

Kendall (i960). Faults, similar to those found dtiring this study, were also reported by Errock (i960) for a filament power supply of this type,

I. 5, Ion Current Collection, Amplification and

Recording,

Provision is now made for double collection of isotopes, mainly for work on isotope ratios. However, it was only when practically all the K^^/ K^^ ratios had been determined that a double collector was made available and since this form of 41 / 40 collection was not stiitable for the K / K study, it was decided to carry out all determination on a single collector. The collector slit width for all K^^/ K^^ determinations was set at 0,025" and for k'^V k"^^ ratios at 0,018", The mass spectrometer has provision for electron multiplier collection, but this was not found to be very satisfactory and so all results wei^e obtained by plate collection.

(A further source of mass discrimination was thus avoided.)

The electrometer valves (954 - grid current less than 10-1 4amps ) are housed in the electrometer box at the collector end -4 of the tube. This box may be evacmted to 10 mm,Hg. A negative feed back amplifier measures the ion current* With the collector plate, eleven sensitivity ranges are provided covering cijrrents of 2,5 X lO"^^ amps to 5,0 X lo"^^ amps for full scale deflection with an input grid leak resistor of 20,000 megohms (Welwyn T/i,) For some of the early work, a 40,000 megohm resistor w&s used, and this was also used 41 / 40 for the K /K determinations giving a corresponding increase in -15 sensitivity. Foise level is 3 X 10 amps.

The Recorder is a Simvic R.S.P. 2 potentiometer type, requiring 25 mV to give full scale deflection in one second. Linear accuracy is "better than 0,2fo of f .s.d. and resolution O.I^, The chart speed may be varied but was set at I" per minute for all determinations.

When scanning, the automatic range changer affects the amplifier sensitivity so that any peak is drawn as near as full scale as possible on the recorder. When recording, there are only eight sensitivity ranges varying in sensitivity from I to 200. As has already been said, when recording a slow down device comes into operation slowing down the magnet scan and thus ens\iring that the correct peak height is recorded. A move of 2fo f.s.d. from the base line operates the switch for this mechanism. Range values are printed automatically.

Linearity of both the amplifier and the recorded were checked.

Details of this are given in the Experimental Section.

This is only a brief outline of the various units in the Metropolitan - Vickers, 1!S2 - S.G. mass spectrometer, but as has been said, modifications have been very few and of minor importance only. For a more complete description, reference is given to the

Instruction lianual for this instrument. 2, Potassium - 40 Radioactivity Measurement.

The p activity due to potassium - 40 in foetal samples was measured on an Ekco Liquid Scintillation Coimter Type H664A at

the Special Unit, Prince of Vifales Hospital. This is a bench sta.nding

shielded instrument capable of in vitro counting of millimlcrociirie

quantities of beta and gama emitters. Beta emitters were counted by

liquid scintillation counting using the jdiosphor described in the

Experimental Section, The counter was housed in a deep freeze unit at - 20° C. The counter comprises a thirteen stage photo-multiplier

tube in a lead shielded container with special light-proofing safety devices. A liquid coupling medium is used between the sample container

(pyrex glass for all determinations) and the tube face, to improve efficiency of light collection. The pulses arriving at the photo-tube may be amplified by factors of 25 , 50, 100, 250, 500, 1,000. A gain 40 of 25 was used for K counting. This counter was used in conjunction with an Ecko Automatic Scaler Type N6IQA. which is a combined scaler and timer, incorporating a pulse-height analyser40, a wide band linear amplifier and a stabilised H. V. supply. For K counting, all pulses above the threshold level were counted. Details of the circuitry are available in the appropriate Instruction Ivlanual.

3. Total Potassium Determinations.

Early potassium levels in sane of the biological material were determined by flame photometry on a Beckman D U

Spectrophotometer with photo-multiplier attachment. Later results,

including checks of potassium levels in foetal samples for potassium- 40 coimting, were obtained by atomic absorption spectrometry as described by Walsh (l936), and Gatehouse and Willis (l96l).

The amplifier and H.T, voltage supply had been specially developed for maximum sensitivity. An air-acetylene flame (I2 lb. in.*" :

3,5 lb. in.~ ) was used. A Phillips Type 93103 E Potassium lamp operated at a c\irrent of 850 mA, was the light source. A mechanical light chopper was provided. The monochromator was a Hilger D290 in which the grating had been replaced by a quartz prism. Instability of the potassium lamp made it necessary to carry out quantitative analyses at the 404 m^ line. SECTION III.

EXFERHvEM'AL.

I. Sample Preparation,

Tissue samples representative of the following five

groups were collected and isotope ratio determinations were made:

I, Cancer tissue.

II. Noinnal tissue from persons with cancer.

III. Normal tissue from persons free of diagnosahle cancer.

IV. Emhryonic tissue.

V, Plant tissue.

Samples included in the various groups appear in Table I.

Most of the tissue samples were obtained from operations

performed at the Special Unit, Prince of Wales Hospital. Some of the

tissues were obtained at post-mortem, immediately after death (see results of total potassium determination) and show a slightly lower potassium level, indicating possible post-mortem shifts. Human foetal

tissue came mainly from the Crown Street V7omen's Hospital, samples being frozen until treatment began. The kelp samples were from

Southport (Tasmania) and were obtained from the School of Biological

Sciences, University of New South VYales. These samples had been previously dried and so had only to be finely ground before being

chemically treated.

It was decided to determine the radio-activity of the 40 K isotope on foetal tissue, and to compare this determination with Table I. List of Tissue Samples Collected for Isotope Analysis.

Normal Tissue Normal Tissue H\iman Cancer Tissue. from from Non-Cancer Embryonic Plant Tissue. Cancer Patients. Patients. Tissue

I II Ill IV V Sarcoma Bone Breast Brain Kelp Bladders (soft tissue)

Wilm^s Tumour Liver Skin (2) Muscle Kelp Fronds.

Metastases Kidney 1" Granulation Liver Kelp Stipes. (Sub. Cut.) Tissue 3

Bone Sarcoma Muscle Rectum Kidney lliole Plant Lymphoma Pancreas (2) Ulcer Heart Tobacco^ Lymphosarcoma Lung Edge of Ulcer Lung

Ca. Rectum Cervix Skin around Bone Ulcer

Ca. Pancreas Spleen S taphyl oc oc cal Intestine (3) Pus. Ca. Lung (3) Ovary Granulation Tissue Ca, Breast Uterus Red Blood Cell ''^Samples ashed, not digested. (5) Wilm's Tumour Red Blood Cell Saliva (5) Ca. Ovary Saliva Samples for which both ashed and digested results are Fibrosarcoma available. Me dulloblast oma

Unless otherwise indicated, all samples were subjected to the chemical treatment outlined in the text. the K^V ratio. It was thus hoped to get some pattern of the 40 K concentration in different tissues throu^out foetal development and, because of their availability, pig foetuses were used. These were obtained from the State Abbatoirs, Homebush, and were frozen tintil treated. Total weight and cro;^ - rump measurements were taken to get some idea of the foetal age (Patten 1948; Lowrey I9Il). Organs of all litters of a similar age were pooled. All samples were freed of excess blood by ;vashing with water, oven dried at IIO°C and fat extracted with CHClj^ for 18 - 24 hours. Samples, once dried, were ground in a small hammer-mill and a sub-sample taken for total potassium determinations, the remainder being digested by an HNO^^ - HCIO^ mixture (after the method of Gieseking et aL, 1935). The residue was dissolved in dilute HGl and the potassium Yias precipitated as the tetraphenyl boron salt, from a slightly alkaline solution. This precipitate was washed with ether and alcohol, dissolved in acetone, and re-precipitated with glass- distilled water. Conversion to the nitrate was achieved by double decomposition with AgiW^)^ , excess silver being removed by drop- wise addition of dilute HCl. Samples were taken at all stages of the purification to determine possible losses of potassium. (Note: The final form of potassium for analysis, namely KNO^ , was not decided upon until tests had been made on A.R. salts by the mass spectrometer to determine which gave the most satisfactory aresults.) All the above samples were purified by this chemical process, and mass spectrometric tests have failed to show the presence of any ionisable elements other than potassium in the products.

A sample of A.R, KNO^ iwas put through the chemical purification process and the isotope ratio determined. All samples were made up

in solution to a concentration of about potassium (W/l7). Sampling

on to the filament was from a micrometer syringe, I pg/mm samples being loaded for analysis.

This chemical purification was similar to that used in

Kendall's (i960 a) work, but was developed independently. The tetraphenyl boron purification was decided upon following reports

of its use by Plaum and Howich (1956); conversion to the nitrate by silver nitrate was used after Findeis and De Vries (1956) had used silver nitrate titration as a method for determining total potassium.

Total Potassium Determination.

Total K determinations on the sub-samples were made by

Flame Photometry on a Beckman DTJ. Spectrometer at 768 mp. wave length.

Determination of potassiiim losses in the chemical purification were made on an atomic absorption spectrophotometer, the instrument previously described. For the qualitative measurements, the more sensitive wave length 766 n^ was used; however, because of instability at this wave length, the less sensitive region of 404 m^ was adopted for the potassiian determinations on the pig foetal samples subject to

K4 0 analysis.

40 K Specific Activity. 40 KCTOg samples for K determinations were first analysed 39 / 41 41 / 40 {for K /K and K /K ratios. These samples were then re-crystallised, taken into solntion, filtered through V/hatman No 41 filter paper and evaporated to dryness. Weights of samples were determined on a balance correct to 0,1 mg. Samples were then counted on the liquid scintillator counter (instrumentation Section.)

Phosphors used for the counting (after Stiglitz,

1962) were of the following composition:

400 ml Toluene, 8g/l PPO; 50 mg/l POPOP,

300 ml Triton X - 100.

20 ml Methanol.

(Methanol (A.R,) was redistilled from furfural and ifo

KaOH after Vogel (l956) to remove acetone and then redistilled from sodium metal and methyl benzoate. Toluene (A.R.) was redistilled from sodium metal after drying with M^O^). Samples were dissolved in

I ml of distilled water and added to 7 ml of phosphor for determination of B activity. For counting an amplifier gain of 25 was used and a discriminator setting of I5V (threshold) was adopted. A plateau for k'^^ was found using A.R. KNO^ (lOO mg of K) and a voltage setting of

1450 V was used for all activity determinations. Calculations of specific activities were based on the weight of sample taken.

Potassium concentrations in the phosphor mixture were determined, after counting, so that a check on the potassium content might be obtained. Standards of A.R, KNO^, corresponding to the weight-range 40 of the foetal samples, were used for K coimting and potassim concentration determinations. These enabled corrections to be made with some degree of confidence,

2, Soiorce Pre-paration and Operation,

2, I, Determination of Potassiaam Compound for Ratio

Studies.

The chemical composition of potassitim samples used for

isotope ratio determinations by thermal ionisation has varied widely.

Bainbridge (I93l) used leucite as the sample for his isotope studies;

Blummell and Jones (1936) state that ionisation of alkali metals

is very efficient when they are present as the alumini-um silicates.

Brewer and Kuesch (1934) carried out their potassium studies on such

compounds and Cook's (1943) extensive study of plant and mineral

samples also was made with the same chemical compounds. However, for

many of the biological studies of Brewer and co-workers, the form of

the sample is not quite clear. For mineral samples from rocks,

(Brewer, 1935), the compound used was potassium phosphate as it was

also for his later study on phosphate rocks (Brewer, 1939). Although

in this case no chemical preparation was made. Bradt et al (1947) in

counter current experiments also used the phosphate, stating that

there was difficulty in impregnating the filaments with chloride and

sulphates. For sea water. Brewer (1936 a) used the perchlorate but

for work on plant, human and animal tissues (Brewer, 1936, 1937 a;

Lasnitzki and Brewer, 1938, I94I a, I94I b, 1942) the form of

potassium used is not always indicated. It appears to have been ashed material mainly, without any attempt at chemical purification,

Hendricks et al (1937) in studies of thermal ionisation properties of tungsten filaments used potassium halides. Smythe and Hemmen- f dinger (1937) and Smythe (1939) attempting to identify the radioactive isotopes of K, used potassium chloride. Later studies on isotope ratios, carried out with thermal ionisation sources, have been on purified chemical compounds. V.Mte et al (1956) used potassium nitrate. Reutersward's (1956) exhaustive study was made on a number of different forms of IC, in an attempt to duplicate the results of previous workers; powdered leucite, potassim phosphate, potassium chlorate and ashed tissues were all tested. Kendall (1936 a) found potassium nitrate gave the best results.

In the present study, a number of chemical compounds were tested. Those tested were: KNO^ , KCl, K^SO^ , K^CO^ , (C^H^)^ BK.

Of these salts KITO^ again proved most satisfactory. Difficulty was experienced with KCl and K^CO^^ in retaining the sample on the filament, while the organic form (a compound produced in the purification process) gave short runs only, although with a surprising constancy of £«.tio. Potassium sulphate did not appear to give a very satisfactory ion current. Potassium nitrate on tungsten appeared to give an ample and steady ion current without having to use a high filament current (about 2.5 amps) and so was the compound decided upon.

A difficulty arose in reducing the potassium contamination in the filament to a satisfactory working level in tungsten filaments. (Before sampling, the ion current was reduced to

Multiple-filament technique overcame this difficulty. A multiple- filamented bead as shoMi in Fig. 5 was used. The sample ?/as placed on either one or both side filaments and the centre filament used for ionisation, using a higher cvirrent. In some of the earlier studies, the centre filament carried the sample, and the side filaments did the ionising. Apart from the expected change in focus, little difference was observed in the ion current, no difference being foimd in the K ratio. This method gave fairly satisfactory results, as can be seen from Table 2, There is no clear reason for the change in the ratios from values of about 13,85 to values of 14,06, but the change is similar to the effect reported by Cook (1943) on samples left in the mass spectrometer over night. Subsequent ratios obtained for all samples gave results in the region of 14,00, At this stage, technical difficulties arose due to two main causes;

(a) Poor qixality in filament bead supply (this was a fault

found by others using this type of solid source filament

- Tushingham (i960).).

(b) Unsuitable nature of the pyrophillite block to maintain

constant electrical contact (Russell, I960),

Unstable ion currents resulted from these faults, giving rise to variations in the isotope ratios. Arcing in the 2KV power supply due to poor connections in the pyrophillite blocks. Table 2.

K Ratio Determinations on A.R. KNO,

Rhenium Filament - Double Filament lonisation.

-7 Cuirent (amps) Pressure XIO Ratio + s. d7

Sample I. 2.25 4.6 13.85 + 01

II 4.4 13.83 + 01 + It 4.3 13.84 01

2.70 4.4 13.88 + 01

Sample 2, 2.02 3.0 13.86 + 01 + u 3.0 13.85 01

«» 2.8 13.85 01

+ Sample 2.35 2.7 13.86 01 ti 2.6 13.85 + 01

2.36 2.5 13.86 + 01

Ratios are means of eleven determinations. caused rapid rises in filament ciirrent, with subsequent increases in ion current. It was not until these blocks had been replaced by- quartz blocks with stainless steel screw connections, and a new supply of filament beads obtained, that stability was regained. The centre timgsten filaments of these beads were substituted by tantalum, since it was found far easier to remove the contaminating potassium from these filaments; in order to ensure good electrical contact, the tantalum was double-welded with an orthodontic spot welder,

2, 2, Source Preparation and Operation.

In this thesis, the source refers to the source block

(see Pig. 4.) the filamented bead and ion collimator. As has just been stated, beads with tantalum centre and tungsten side-filaments were used for all the ratios reported in the Result Section. These filament beads were cleaned in trichlorethylene, boiled three times in distilled water and dried for 30 - 60 minutes under an infra-red lamp. Before sampling, each filament v/as de-contaminated by heating in the mass spectrometer until the level of potassium referred to previously (<.0,08% of K at 3 amps) was obtained. V/ith tantalum filaments so treated this took no longer tlian 30 minutes, fractionation of the isotopes being observed during this time. (Rs.tios of

13,10 were not uncommon during this procedure,) If a filament had been used previously for potassium analysis, the sample was first removed by scraping with a razor blade before being subjected to the above treatment. Compsjfcon (1963 personal commimication) uses a different method of

decontamination. He states that by raising the filament current to

a high value in order to reduce the potassium level to some pre-

determined value, he can cause the source block and other parts of

the source assembly to give off potassium. He therefore heats his

filaments for a fixed time. No effect vjas found in the present study which could be due to potassium emission from other parts of the

source than the filament, but this could be due to the relatively

short time and low temperature required for de-contamination.

After reduction of background potassium, the sample was placed on the filament by means of a micrometer syringe. Early difficulty was experienced with the filament becoming re-contaminated with potassim after it was removed from the mass spectrometer. The atmosphere appeared to be the source of this contamination. This effect was reduced by allo^ving the centre filament to cool in the mass spectrometer before removal of the source, and to use the filament power supply of the mass spectrometer itself to supply the filament current for sampling. This meant that the filament had to be handled as little as possible. Samples fran the syringe were placed

on the side tungsten filaments, using a current of up to 1.5 amps to dry the sample. The source was then replaced in the mass spectrometer ready for ratio determinations. The pneumatically operated valve described previously (instiumentation Section) enabled a working vacuum (of the order of

was found to reduce any background potassium that may have "been

picked up during sampling. The potassium ion current originating

from this source, and from the potassium sample, were found to have

slightly different focusing properties. The working current was

then gradually increased until a satisfactory ion current ms pro-

duced. Increasing the filament current too quickly caused out-gassing

and sometimes loss of the sample. A filament current of 2,4 - 2.8 39 amps was usually found satisfactory to produce a K ion current

of the order of 3 X lO""^^ amps. A current of this magnitude was aimed

at for all analyses so that the 39 and 41 ion currents could be on

the same two amplifier sensitivity ranges, and could therefore be

compared relatively. (Problems associated with the range changing

device will be discussed later.) Por all 39/41 measurements, a

collector slit width of .025 in. (measured electrically) was used.

All ratios were determined from recorder tracings. 41 / 40 For K / K ratio determinations, much the same

techniques were used. Sample and source preparation were the same as

for K^V

analyses. As a matter of fact, values v/ere first

determined for this ratio using the collector slit width previously mentioned. This slit width was then reduced .018 in. and the filament

current increased to about 3 amps (sometimes a little higher).

Decreasing the slit width reduced the effect of the larger 39 beam on

the K^^ current. Both 39/41 and 4l/40 scans were obtained by a variable magnet scan. As has been said the mass reinge scanned for k'^^ ratio was from about mss 38.5 to mass 4I,5. For the / 39 41/AO work the ran^e was reduced to 39,5 to 41.5 so that K 40 would cause as little contribution to K as possible. Even so it 39 41 40 T^as impossible to remove the tails of K and K from K and 40 accuracy of measurements of the K ion current ms^ limited by this difficulty.

Earlier tests using the electronmultiplier and peak switching techniques had given unsatisfactory results. Filament current instability, similar to that reported by Errock (i960) Tiras observed. This considerably reduced the accuracy of the isotope

2?atio runs. The problem seemed to be partly due to dirty glass-to- metal seals in the ion source. Therefore, whenever this developed, these glass to metal seals at the sample end of the source, were cleaned with trichlorethylene. Between a series of determinations

(usually 5-10 different samples for any one ion source) the source was completely dismantled. Any ion burns on sample being removed with 0000 emery paper, and all metal parts cleaned firstly in trichlorethylene and then by boiling three times in distilled water.

Ceramic spacers were first boiled in nitric acid and quartz sleeves in hydrochloric acid, before boiling in mter. All parts were then dried under an infra-red lamp for up to 2 hours. The ion source was then re-assembled using jigs for the source block and the collimating system. The ion block became contaminated more quickly and v/as, therefore, cleansed more often, mostly after each sample determination, although in these instances, the block was not always dismantled. 5. yjialysls of Data.

Fig, 7 shows part of a typical recorder tracing of K^^/

The method of calculation of a complete scan for an A, R. potassium nitrate is shown in the Appendix, Briefly, it involves recording twelve 39 and 41 ion currents by magnetic scanning, the ion beams appearing in the following order:

39, 41, 41, 39, 41 and so on.

This is done by the charging and discharging of a condenser, as mentioned in the Instrumentation Section. A mean ratio is then obtained by meaning the first and second 39 peak heights, and dividing the result by the mean height of the first and second 41 recordings.

Mean ratios are then found from the second and third tracing, and so on for the ccanplete scan. This means that eleven K ratios are obtained from twelve 39 and 41 ion current measurements. An overall mean and standard error is then calcxilated for these eleven ratios. Three series of eleven such ratios were determined for each sample. This method of ratio determination could produce the "bunching effect" mentioned by Kendall (i960 a) and measurements of the standard errors quoted in this report could support this claim. However, the average standard deviation quoted for determinations is 0.06, a value not significantly different from the standard deviation of repeated ratio determinations on all samples. Inclusion of this ratio and its accompanying standard deviation gives an idea of the precision of ratio determinations, therefore, and should not be construed as giving an overall repeatability measurement. All peak height measure- r

R r c

Pig. 7. Part of K^Vk:^^ Recorder Tracing for A.R. KNO_. fD iinents were made with the same rule, taking each measurement accurately to 0,2 mm , This method of determination of ion current, therefore, introduced a limitation on the accuracy of ratio determinations, but was found to "be more accurate than amplifier meter readings. Tests were carried out to determine whether peak height measurements were repeatable or operator dependent. Although individual measurements may vary by 0.4 mm , the ratio so determined varied no more than the previously determined error. All ratios quoted in these results, however, were calculated fran measurements made by the author alone.

Now has an ion current approximately 10 times the 41 magnitude of K . This means that the 41 ion current recording would be observed on a range 10 times more sensitive than 39, by means of the automatic range changing device. For most of the 39/41 recordings, the recorder ranges used were 0 and 3 for 39 and 41 resijectively. If the sensitivity factor from range 0 to range 3 were 9.9.. instead of 10, absolute ratio determinations would not be possible. However, relative ratio determination could be made if these two ranges only were used. For some samples, however, it was impossible to get an ion current of the order of 3 X amps due to small sample size. Kow ratio determinations which had been observed on different sensitivity ranges had been found to vary. Consequently the linearity of the amplifier and recorder responses were checked.

?/hen recording, there are ^ight sensitivity ranges on which an ion current may be registered, depending on its magnitude.

The most sensitive of these ranges, range 7, is 200 times more sensitive than range 0. The varicois ranges, with the increase in sensitivity are:

Recorder Range: 0 1 2 3 4 5 6 7

Increase in Sensitivity: I 2 5 10 20 50 100 200

If these range factors are not quite correct, an error may he introduced when the recorder automatically changes range; i.e. a

39/41 ratio obtained from ion current measurements on ranges 0 and 3, may "be different from one determined from ranges I and 4. It was therefore decided to test the correctness of these range factors. In order to obtain ion ciirrents on the different ranges with a sample it is necessary to vary the filament current (and ionising temperature) thus introducing another variable which could confuse the issue. A signal was therefore placed on the amplifier by varying the gain for the zero potentiometer of number two amplifier. A "reading" was set on range 7, at approximately the value at which the recorder would automatically change to range 6, except that in these tests, the range change mechanism was manually operated. This was repeated for all pairs of ranges 7/6, 6/5, 5/4 l/O and the "ion currents" measured. Range-factor differences were found by multiplying the less sensitive range value by the range-change factor and comparing with the direct reading on the more sensitive range. The results of these tests, shown in Table 3, do tend to indicate that the range factors quoted are not quite lOOfo correct, ¥hat this testing has done in fact, is to check the feed-back resistor valves in the automatic range changer. A check was also carried out on the linearity of the amplifier and recorder, but this proved to be quite satisfactory as the Table 3.

^CHECKING OP RAUGE FACTORS.

I Ranges O/I 1/2 2/3 ; , 3/4 6/7 1 , 4/5 > 5/6 \ i » Recorder 62.3 154.2 159 172.8 176,, 3 172.4 176 170.3 175.3 173.8 176 172.8 175.3 "Values in 60.2 mm. 62.8 154.6 158.9 172.8 176 172.8 176 170.3 175.3 174 176 174 175.3 60.6 60.8 62.6 154.8 158.9 172.6 176 172.6 176 170.5 175.4 174 176.I 174 176.2

60.8 62.4 155.2 159 172.6 176 172.8 176 170.5 175.8 174 176.2 174 176

60.4 62.3 154.6 159 172.6 176 172.8 176 170.5 175.8 174 176.3 174.4 176.3

60.2 62.3 154.8 159 172.6 176 172.8 176 170.5 175.8 174.2 176.8 174 176

60.2 62.4 154.8 159 172.6 176 172.8 176 170.8 .175.8 174.4 177 174 176.1

60.2 62.4 155.2 159 172.6 176 172.8 175.9 170.8 175.8 174.2 177.I 174.2 176.2

60.4 62.1 155.2 159 172.6 176., 2 172.6 176 170.8 175.8 174.4 177.2 174.4 I76.I

60.0 62.1 155.4 159.1 172.6 176 172.6 176 171 175.9 174.4 I77.I 174.4 176.6

60.4 62.5 154.8 159 172.6 175,, 8 172.8 176 170.5 175.8 174 176.3 174.4 176.3

Mean Difference s I.,6 2 4.4 3,,4 3., 3 5.. 1 2.4 2.. 0 i Meter i Readings 1.4 2.78 2.79 6.99 3.9 7.72 3.9 7.74 3.1 7.70 3.9 7.74 3.9 7.73

The first figure in each case TTas obtained by multiplying the measurement on the less sensitive

range by the stated range factor. graph in Pig. 8 shows.

The effect of a difference in range factors will be

two-fold. Firstly, it vdll affect the absolute ratio determinations , 41 since, in obtaining the 39/41 ratio the K value is obtained on a 39 range ten times as sensitive as that for K , If the factor is not

ten, then the ratio will not be correct. Secondly, if the difference for the range factors is not the same for all ranges, then the ratio will alter whenever the recorder range changes. This appeared to be the case for some of the sample determinations. For ratios not

obtained on ranges 0 and 3, therefore, a correction must be adopted

so that all values are relative. All that has been said fo4r1 / 40 ratio determinations, also applies for determination of K / K ratios. The electronmultiplier offers increased sensitivity (Leland,

1956) over plate collection for ion current detection. The electronmultiplier of the KS2 - S.G, •vi'as tested, but, because of poor activity

of the dynodes, proved unsatisfactory. (On the other hand, discrimination effects were avoided). Plate collection with a 40 Kl-I grid leak detector was used and ion currents of 1.25 X to 2.5 X could thus be measured. The procedure for the determination of 41 40 / \ K / K ratios (only made on foetal and A.R, samples) was as follows;

(l) Using a .025" collector slit width, a single scan of II

ratio determinations for K^^ was made. (II) The slit width was adjusted to .018" and the filament 41 current raised so that a K ion current of the order of

amps was obtained. Scans similar to those for Nuflibero Q-.7 refer 22 L to sensitlvi-ly rsssge

20 I

X8 I

14 I

10 L

6 ^

4 L

EIECIHOMEE •m.TiXm

Pig. 8, Linearity of Recorder and Amplifier, ratios were run, two of eleven each,

(III) The slit width was reset at .025" and eleven K^^/K^^ ratio

determinations again made. 41 / 40 An example of a K /K tracing is shown in Pig, 9,

As can be seen both and K peaks "tailed" badly, making accurate measurement of the K 40 peak virtmlly impossible. The procedure adopted was to allow the minimum value to be recorded on either side 40 of the K peak before manually changing the amplifier range. When measuring the

peak height its base line was taken as being half way between the "base" measurements on the 39 and 41 sides. Ratio determinations were then the same as for The problems associated with range-change differences still remain, and are magnif- rangied ei f 0(4anythingI peak), . foBecausr range e o7(4f al0 l peakthes)e idifficultiess 200 times ,mor measuremente sensitives than 41 / 40 40 of K /K ratios were of low accuracy. The K activity of the 41 / 40 foetal samples was canpared with their K /K ratios, determined in this manner.

4. Calibration Determination and Investigation of

Errors.

4. I. Use of Internal Standards.

The use of internal standards for precise measoire- ments of abundance ratios has been developed in the last few years, in an attempt to improve accuracy in thermal ionisation mass spectrometry. In this way, it had been hoped to be able to approach the accuracy and convenience of sample and standard comparison in gas U u J

Fig. 9. Part of K^^/ K^^ Recorder Tracing for A.R. KHO^. analyses. Bietz et al (1962) described a method using an internal standard to give a five fold improvement in the precision of

\3ranium analysis. The method calls for the use of two isotope spikes of TJ and U which are not naturally occurring isotopes of uranium. The observed ratios of the known mixture is then used to correct the ratio for the

Correction can then be made for the mass discrimination effects for the lighter isotope. Patterson and Tvllson (1962) also working with uranium, use a somewhat similar method for determining their abundance ratios. Their comparison was with an enriched 235/238 sample, and they report a 3-fold increase in accuracy. These workers used a slightly modified triple filament, all three filaments being parallel. The mathematical considerations of precise isotope analysis by s\irface ionisation using internal standards are discussed by Dodson (l963).

The method of analysis for uranium, used by Patterson and Wilson would not be successful for compounds such as lithium nitrate which evaporate by radiant heat from the centre filament.

The same should also hold for potassium nitrate. Nevertheless, an attempt was made to see if this method could be adopted for potassium nitrate, so that accuracy of determination could be improved.

After experimenting with a number of different modifications to the filament layout it was decided to test the feasibility of placing samples on the back of the side filament, and use the side filament itself as a shield for the sample. Qualitative tests were carried out using lithium nitrate and potassium nitrate as the two different samples, each placed on the back of a side filament, A current was passed throiigh each side filament in t\im, to evaporate the sample, while keeping the centre filament at a higher current.

Ylhen one sample was being evaporated and ionised, no ion current could be found for the other sample. This would probably be an inefficient form of ion production, but this may not be of great con- cern for samples such as potassium and lithium, v/hich are so readily ionised. Tests carried out with A.R, potassium chloride and A.R, 39 potassium chloride to which had been added tracer K , also indicated that this may be a practical possibility. (See Table 4 a.) Compston, however, claims to find O»lfo differences in K?^/ k"^^ ratios of the same sample, when ionised from samples on different side filaments,

(Personal data), and this might be the factor limiting the accuracy obtainable. The results of tests carried out in this survey on A.R. potassium nitrate ionised from alternate side filaments are shown in

Table 4 (b).

This method of standardisation was not used, however, since samples of separated isotope in sufficient quantities were not available and so other methods were adopted.

4. 2. Synthetic Isotope Mixtures in the Study of Mass

Discrimination Effects.

Mass discriminations in a thermal ionisation source have been shown to be dependent on the presence of a mixture of cations in the sample, and the weight of the sample used (Kendall,

I960 a; Reutersward, 1956.) In a multiple filamented source, mass Table 4 (a)

K^^/ K^^ Ratios for Tvro Samples on the One Pilament Bead.

+ Sample Ratio S. D.

+ KCI 14.20 .06 + A.R. i™^ 14.05 .03 + KCl"^ 14.22 .03 + A.R. KCI 14.03 .07 + KCl"^ 14.56 .04 + A.R. KCI 14.21 .04 + KCl"^ 14.29 .09 + A.R. KCI 14.10 .03

KCI - K^^ Enriched Sample.

Table 4 (b)

K^^ Ratios for Two Similar Samples of A.R. KNO^ on the One

Filament Bead.

Left Side Filament. Right Side Filament.

I. 14.18 i .04 14.14 t .09

2. 14.08 - .05 14.08 t .03

3. 14.08 - .05 14.10 i .02

4. 14.05 i .03 14.04 - .05

5. 14.12 i .03 14.07 - .06 discrimination effects may be less than those for surface ionisation,

but vaporisation of the sample before ionisation could give rise to

discriminations. All purified samples used in this study were found

to be of a fairly high degree of purity - contamination from the

filament most probably providing the greatest source of error. For

ratio determinations on A.R. salts, a slightly larger sample size

(about 5 ug.mm," ) was used than for samples of biological origin

(of the order of I ug.mm." ).

Boerboom (I960) had found that varying temperatures, as the result of using different filament currents, affected the V 6 Li / Li ratios he obtained. His results are shown in Table 5. They

indicate a discrimination of about for an 800° C rise in temperature. During this study it v;as possible to obseirve the K^^/ ratios as a fimction of filament current. The actual temperature

involved was not measured, but should be of the same order as that

in Boerboom's study. The results (see Fig. lO) of these observations

do not appear to show any definite relationship between ratio and filament current, although the current was varied from 2 to 3 amps.

This should correspond to a temperature range of 1,100 to 1,800° C. according to the results of Boerboom. In sample analyses, however,

the variation in filament current was much less than this. Mass discrimination from variation in the ionising temperature during the

K^^/ k"^^ ratio determinations discussed in this thesis does not

therefore appear to be of any great importance. The pressure during all ratio determinations was 3 X 10 mm.Eg or better and so gas 14o20

M ^ 14olO « 9

« 99 0 o« e 0 ® 0

d o« © @ 14^00 -T 2,00 2o25 2.75 S«00

Filanient Current (anipo)

Oi Pig. 10. Effect of Varying Filament Cmrent on K^^/ k"^^ Ratice for A.R, KNO^ O Table 5,

V fn Effect of Heat on Li / Li Ratio. (Reference A. Boerboom. )

I Centre I Side Temperature Ratio hl^/ Li^ (amp.) (amp.)

1.675 4.00 780 11.723 t .017

1.70 3.75 815 12.027 i .027

1.80 3.65 958 12.328 t .014

2.00 3.40 II70 12.346 t .007

2.20 3.20 1345 12.373 i .006

2.40 3.00 1460 12.417 i .009

2.60 2.35 1555 12.412 i .018

2.70 0.00 I58I 12.436 i .036

Sample on Side Filament. scatter sshould have been considerably reduced,

Brev/er (1936 c) stated that abtmdance ratios of

potassiiim are constant for a single sample and are not affected until of the potassium has evaporated. Reutersward (l956) on the

other hand says that isotope ratios and total emission vary erratic-

ally with time, a finding which is supported by Cook (l943). In the

present study samples were not run to exhaustion, and no fall in ratio was observed, although there was a slight variation (see results of ratio determinations in later Sections,) Nevertheless the ratios

obtained (about 14,08) showed a discrimination when compared with

the accepted value of Nier (I950) of 13,48. As has been mentioned previously, earlier determinations reported in this investigation

gave values of 13.85. For no reason that could be found, there v/as a

change to the higher ratios found during the remainder of the survey,

A synthetic isotope mixture ms therefore used to see v/hether correction factors could be adopted for mass discrimination and sb enable an "absolute" ratio for K^^/ K^^ to be obtained, 39 41 Samples of the separated isotopes K and K v/ere obtained from

Oak Ridge, U.S.A., 50 mg and 5 mg of potassium respectively as the

chloride. Percentages of the isotopes were: Potassium 39 Potassium 41.

K^^ 99.85 ^ 0.02 1.4 ^ 0.05

K^^ 0.02 ^ O.OI 0.03

K^^ 0.14 O.OI 98.60 0.05 The isotopes were prepared as follows, on the advice of Dr W, Ganpston. (Personal communication,)

To ensure that the isotopes were stoichemically true, they were fused "by heating in a muffle furnace to 776° C. The temperature was measured by a Cambridge thermocouple, recently re-calibrated and checked by a mercury thermometer before use up to 200° C. No corrections were found necessary. Unfortunately, the potassium - 41 sample was accidently contaminated in the muffle furnace and most of it had to be discarded. This made it difficult to prepare an accurate standard, since a l/l mixture of 39/41 had been suggested by Compston.

Milligram quantities of the isotopes had to be weighed accurately. This was done on a Kahn electron balance. The final isotope mixture consisted of 0.6853 mg 0.4212 mg converted to the nitrate. A mixture with the ratio of approximately unity was suggested because range change factors and amplifier differences could thus be eliminated.

It was also decided to get some idea of the percent fractionation that the mass spectrometer could detect. Consequently, a sample of potassium 39 was accurately weighed and made into a solution (again as the nitrate) of concentration I mg K per ml. Volumes of

O.IO, 0.20, 0.50, 0.70 and 1.00 mis of this solution were added to

I ml. samples of a solution containing 100 mg. K/ ml. Assuming a figure of 14.06 as a mean figure for Y?^f

determination by the mass spectrometer, these volumes represent a percent fractionation of 0.07,

0.14, 0.35, 0.49 and 0.70 respectively. determined in the usual manner. Y^^ ratios were Since the ratio for K^^/ k"^^ obtained on the I®2 - SG was about higher than the accepted value, I ml of the K^V l/l mixture ms added to a I ml sample of the 100 mg K/ ml solution.

This should therefore give a sample which has a content higher than normal.

The results of all of these tests are shown in the

Results Section (IV. 3.)

Some of the other errors which mi^t affect ratio determinations have already been mentioned. These include the range- change factor, and poor resolution of peaks resulting in "tailing". 41 / 40 This second problem affected the K / K ratios more than the

ratios, the resolution in the latter case being satisfactory.

The effect of chemical impurity was tested on samples which had been ashed only. The source magnet was removed for all ratio determinations, and so errors caused by the presence of magnetic fields in the source region were not possible. Repeated cleaning of the source components kept any memory effect to a minimum. Also, tests on samples with anomalous isotope ratios in no v/ay affected subsequent ratio determinations on "normal" samples, indicating that the memory effects were non-existent* 5. Instrument Performance.

Checks were carried out on the performances of the

Mass Spectrometer, liquid scintillation counter and atomic absorption spectrometer. 5. I» MS2 ~ SG Mass Spectrometer.

The main criterion for performance of the mass spectrometer was peak shape, and thus, resolution. Ebcceedingly hi^ temperatiures were not used, and so vacuum conditions were fairly 41 / 40 reproducible and resolution adequate, except for the K / K determinations. Resolution is a function of the tuhe radius, and collector and slit vridths; with the 1132 - SG, it was found to be about 150 for

39/41 analysis. Peak shape was determined by recording the peak shape automatically, by varying the percentage scan of the magnet so that one mass number only was recorded during the magnet scan. A tracing so obtained is shown in Pig II a. A peak shape was also determined from meter readings for 0,2 volt steps and the results graphed (Pig. II b).

The method of determining resolution from these peak shapes is shown in the Appendix. Routinely however, resolution was simply checked by observing the number of half volts between the O.I^ deflection on either side of the peak. Slit width vras checked by determining the number of half volts for the half peak height. Voltage was varied for these tests by the accelerating voltage divider. Provision was made for moving the tube in the magnet if these checks indicated that it was necessary.

5. 2. Liquid Scintillation Counter.

The Ekco Liquid Scintillation Counter used for

determinations v/as used for counting C and H samples. It had not been used for potassium or any emitter of higher energy ^ particles. Tests were carried out with concentrations of potassium Fig. II (a). K^^ Peak Shape from Recorder Tracing. ACCELERAIUJa VOUTAGE

Pig. II (b/ )N K3 9' Peak Shape graphed from Meter Readings. nitrate varying from 50 to lOCF/o saturation. Saturated solutions

precipitated immediately the phosphor solution was added, the 95%

solution fdien adapting to - 20° C, and the 90^o solution only after

a considerable time at - 20° C. Therefore, sample concentrations of

potassium nitrate of up to SCffo saturation should he possible for

count rate determinations. With a solution of 100 mg. K/ml., a plateau

was determined for the counter, using lOV and 20Y discrimination levels,

with a threshold range of 5 - 50 V. Voltage settings from 1,250 to

1,650 were tested, in 50 volt steps. For all counts the multiplier

gain v/as set at X 25 because of the higher energy (1.35 meV) for the 40 4 p particles from K . 10 counts were determined for all foetal

samples and for concentrations of A.R. potassium nitrate covering the

sample range. Sample weights for potassitmi nitrate varied from 7 to

135 mg.

5. 3. Atomic Absorption Spectrometer.

During routine determination of potassium on

clinical samples at the Special Unit, instability at 766 m^ had been

observed. Errors so introduced at this sensitive wave length would be greater than those for the counting of potassium- 40 and so would

limit the accuracy of specific activity determinations. The other wave

length for potassium, 404 mu was almost 200 times less sensitive. This

loss in sensitivity would not be important for the foetal sample

determinations, because a fairly high concentration of potassium

could be obtained. Tests were carried out at this wave length and a

straight line trend was found for aqueous standards (lO - 50 mE.K/l.). potassiim determinations on the foetal samples (in the phosphor solution) were made at this wave length. The A.R, potassi-um nitrate samples on which activity had "been determined, were used to construct a standard curve. SECTION IV.

RESULTS.

39 / 41 41 / 40 Results for both K /K and K /K ratio determinations are given in tabular form and as such are fairly self-explanatory.

The ratio is given as a number (corrected for rajige change v;-here necessary) and not as a permil deviation from a standard as in Kendall's

(i960 a) thesis. It must be realised that the numbers tabulated are not meant to represent absolute ratio determinations. They are listed solely for comparing relative ratio differences.

I. K^^/K^^ Ratio Determinations.

The results are grouped according to the four class- ifications of samples given in the Introduction. Results for ratio determinations on A.R. KNO^^ have been included. All samples were analysed as Kl'^O^ o^i tantalum/tungsten triple filamented beads. Space does not permit the inclusion of all ratio determinations. The figures in the tables are the m.eans of three runs each of eleven scans for different samples. The standard deviations quoted for these means give an indication of the precision of the measurements. The group determinations indicate the reproducibility of the ratios for different samples.

I. I. A.R. Potassium Nitrate Standards.

A considerable amount of time was spent on these ratio determinations in an attempt to improve the accuracy of 39/41 readings. The results (Table 6) would be the yardstick for the accuracy of ratio determinations. Approximately half of these results were iTable 6.

K^^/ K^^ Ratios for A.R. Potassium Nitrate,

39/41 14.23^ 14.22 14.18 14.16 14.13 14.09 14.08 14. II S.D. .07 .05 .11 .04 .05 .07 .08 .07

39/41 14.12 14.02 14. II 13.98 14.04 14.04 14.04 14.01 ~ S.D. • .04 .06 .06 .04 .03 .04 .05 .10

39/41 14.05 14.08 14.08 14.06 14.07 14.19 I4.II 13.82 S.D. .04 .06 .06 .08 .03 .03 .09 .02

•vl 39/41 14. II 14.03 14.20 I4.II 14.04 14.08 14.15 14.01 M S.D. .07 .06 .10 .05 .07 .06 .07 .04 1

j 1 39/41 14.14 14.05 14.08 14.08 14.01 14.10 14.07 14.14 1 S.D. .04 .03 .03 .05 .06 .06 .02 .02 1

Mean 39/41 Ratio 14.09 i .06

Values quoted are the mean of 33 Ratio Determinations on sub-samples of

A.R. KNOg. determined before any biological samples had been analysed. The remainder of the ratios were calculated from samples of A.R, KII0„ ' 3 which were included in the series of analyses on test samples, as a means of checking the instrument. Approximately one standard was included for each four unknown determinations. A more desirable method of checking would be the inclusion of a standard with each test sample.

The method outlined in the Experimental Section, involving the ionisation of two separate samples from the one filament bead, could offer a big improvement in this regard. It was not used routinely for the sample analysis in this thesis because sample variations were no greater than those for the A.R. lOfO^. It will be noted that one A.R. sample gave a mean determination for K^^ of 13,82. This reading was similar to those obtained in earlier determination of K^^/ ratios on rhenium filaments. No reason can be given for this sudden change in ratio, which would represent approximately discrimination 39 against K on the values obtained in these results. Because of this chance variation, any difference occiirring in the biological samples was not taken as a true difference unless it was found for a number of sample determinations.

The mean K^^/ k"^^ ratio found for A.R. KKOg samples therefore, was

14.09 - .06 (s.d.), this error representing day to day variations such as filament current, vacuum conditions, instrument performance and changes in filament geometry. No corrections, other than for range change differences, have been made. The figure of

13,82 was not included (on statistical grounds) in obtaining the group piean and standard deviation quoted. The standard deviation ( ~ 0.4/^

expressed as a percentage of the mean) gives the accuracy of the method

for A.R. potassium nitrate.

I. 2, Cancer Samples.

The samples analysed in this section are shown in

Table 7. Samples showing two results were analysed on two separate

occasions.

The results (Table 7) shov/ that there is no significant

difference from the ratios obtained for A.R. KlIO^ , a mean figure of

I4»II i .08 (s. d.) being obtained. Again the only correction was for

range change factors. This correction is the only one that has been

made for all the ratios obtained for the samples in this Section IV.

I. 3. Normal Tissue from Persons with Cancer.

Again the 39/41 ratio (Table 8) obtained is

similar to that for the other two groups, except that the variation is

g-reater (14.08 ^ 0.14). The ratio obtained for one pancreas sample

varied considerably. However, ratios obtained on another pancreas gave

a constant and "normal" ratio (I4.I2). The samples differed slightly

in that the earlier sample had secondary cancer in the pancreas

(although not present in the sample) whereas the second sampl^-evwas free of secondaries.

I. 4. Normal Tissue from Persons Free of Cancer.

The K^^ ratios for this group are shown in

Table 9. Values shown are similar to those of A.R. samples, but again 73a

^able 7.

K^^ Ratios for Cancer Samples,

Sample Ratio - S. D.

Metastases (Sub-cutaneous) 14.15 i .04 ; 14.21 t .09

Soft Tissue Sarcoma 14.26 I .10

Bone Sarcoma 14.24 i .04 ; 14.00 t .03

Y/ilm's Tim our 14.13 i .06

Lymphoma 14.14 i .06

Lymph o-sarc oma 14.07 i .04

Rectum 14.II t .05

Pancreas 14.08 t .06

Lung 14.07 t .06

Lung 13.99 t .10

Lung 14.09 i .07

Medulloblastoma 14.05 i .05

Mean K^V ^^atio 14.II t .08 „ Table 8.

Ratios for Normal Tissues from Persons with Cancer,

1 1 Sample Ratio i S. D.

+ + + Bone 13.95 .06 14.31 .17 14.10 + .04 14.17 .06 + Limg 13.95 .14 + + + Pancreas 13.78 .09 14.05 .03 13.81 .03 + Pancreas 14.12 .05 + Muscle 14.27 .13 + + Kidney- 14. II .02 14.00 .03 + + Cervix 14.03 .17 14.02 .05 + Spleen 14.17 .06 + Ovary 14.03 .06 + Uterus 14.09 .05 + + + Liver 14.18 .05 14.16 .06 14.23 .04 + Kidney 14.00 .03 + Saliva 14.29 .06

Mean K^V^^^ Ratio 14.08 i .14 Table 9.

Ratios for Normal Tissue from Persons Free of Cancer,

Sample Ratio i S. D.

+ + Breast 14.26 .02 14.II .03 13.87 t .08 + + Skin 14.19 .04 14.18 .09 + Skin 14.09 .02 + Granulation Tissue 14.20 .03 + Granulation Tissue 14.16 .04 + Granulation Tissue 14.19 .04 + Rectum 14.05 .05 + Ulcer Edge 14.23 .03 + Skin Arotmd Ulcer 14.20 .06 + + Staphylococcal Pus 14.29 .15 14.41 .08 14.40 t .04 + + Staphylococcal Pus 14.05 .03 14.00 .03 + Saliva 14.02 .04

Mean K^^/K^^ Ratio 14.12 - .10 have a slightly greater variation. The staphylococcal pus sample gave

a significantly higher 39/41 ratio on each occasion it Twas analysed.

However, another sample chemically purified "by the method described

(Experimental Section) gave a figure which did not differ from the

standard. The sample which gave the anomalous ratio must he classed as "being doubtful. The doncentration of potassium nitrate, as indicated by amounts of sample present when sampling on the filament was higher

than ms expected. Therefore it may be possible that this sample v/as

contaminated subsequent to its chemical separation. However, even when

the sample vias piirified the high 39/41 ratio persisted.

I. 5. Human Embryonic Tissue.

39 41 The results for K / K ratio determination

on pig foetal samples are not included here, but are given with the 41 / 40 results for K / K ratios. The samples were from pooled tissues of various organs of foetuses of ages varying from 2 to 3 months. Ratios 41 / 40 obtained are shown in Table 10. K / K ratios for these samples appear later.

I, 6. Plant Samples. Analyses for this group were on kelp (llacrosytis

pyrifera) only, samples being representative of the various parts of the

plants such as stipes, fronds, bladders and ivhole plant. The ratios

obtained (Table II) were very constant, the accuracy being as good as for the A.R. KITO^^ samples. No differences were found for this plant

(compare with Brewer's findings) and no differences were found between

the different parts of the plant. Table 10. HL K Ratios for Human fiabr^ TIBSUG + Sample Ratio S. D.

+ Liver 14.12 .06 + Bone 14.24 .06 + Muscle 14.08 .03 + Intestine 14.15 .05 + Brain 14. II .06 + Kidney 14.19 .04 + Lung 14.15 .03 + Heart 13.99 .03

+ Mean Ratio 14.13 - .07

Table II.

K*^^/ Y^"^ Ratios for Plant Samples,

Sample Ratio - S. D.

+ + Kelp Stipes 14.07 .06 14.II i .06 14.05 .07 + + Kelp Fronds 14.04 + .03 14.14 i .04 13.97 .07 14.18 .05 + + Kelp Bladders 14.06 .03 14.05 t .06 14.01 .05 + Kelp Whole Plant 14.14 .03 14.12 i .08 + Tobacco 14.16 .07

Mean K^^K^^ ^^tio 14.08 i .06 I. 7, Red Blood Cell Potassium.

The red blood cell is a site of high concentration of potassim in the human body (315 mg/lOOcc ; Winton and Bayliss 1948) and a site where potassium exchange occurs. Five blood samples, matched for blood group, were collected from cancerous and non-cancerous persons.

The analyses (Table 12) show that no differences exist either between the two groups or between the groups and A.R, KITO^.

I. 8. Ashed Tissue Samples.

Both Kendall (i960 a) and Reutersward (l956) reported K^^/ k"^^ ratio anomalies for impure samples, Kendall reporting differences on ashed cancer samples. In the present investigation, ashed samples prepared as described in Sample Preparation were subjected to K^^/ k"^^ analysis. Not one of these samples (Table 13 a) gave an anomalous ratio* The only other ion present was sodiim, and, in most cases, gave a lower ion current than potassium. This, of course, does not exclude the presence of other elements in the sample. Studies with samples of calcium compounds, for example, have shown that it is very difficult to produce calcium ions whenever there is a background of contaminating potassium present in the filament.

Tests were carried out on samples of A.R, KUO^ to which unmeasured amounts of different elements had been added. These elements (sodium, magnesiiom, calcium and iron as chlorides) were ones that could be expected to occur in human tissue samples. The results

(Table 13 b) show that the K^^/ k'^^ ratio is disturbed but that the pattern is not consistent. TaWe 12.

K^^/K^^ Ratios for Red Blood Cells,

Control Group. Cancer Group.

Ratio is. D. Ratio i S. D.

+ + 14.03 .04 14.01 .04 + + 14.07 .03 14.08 .03 + 14.14 .04 14.16 .03 + + 14. II .05 14.08 .04 4- 14.08 + .04 14.12 .07

+ + .n 14.09 .04 Mean 14.09 .06 Table 13 a. 80

Ratios for Ashed Impiare Samples.

+ Sample Ratio S. D. + Cancer Breast 14. II .05 + Wilm's Ttffliour 14.04 .05 + Cancer Ovary 13.97 .05 + Fibrosarcoma 14.05 .02 + Normal Bone 14.02 .05 + Normal Liver 14.18 .04 + Normal Muscle 14.07 .03 + Normal Kidney 14. CO .03 + Granulation Tissue 14.14 .07 + Kelp (Whole Plant) 14.05 .05 + Tobacco 14.15 .05

.41 Mean 14.07 - .07

Table 13 b.

Ratios for A.E. KITO^ with Added Minerals,

+ Added Mineral. Ratio S. D.

Nil - A.R. imO^ 14.10 .05 + NaCl 14.36 .06 + NaCl, FeClj^ 14.21 .08 + NaCl, FeCl„, CaCl^ 14.24 .07 + NaCl, FeCljj, CaCl^, MgCl^ 14.29 .14 • . 2. Potassium 41/40 Ratio and Determination of

K Levels.

The pig foetal tissues were the only samples on 41 / 40 40 which K / K and K measurements were made. The samples analysed are shown in Tahle 15 h. Organs of different litters, of comparable foetal age were pooled. The only three organs analysed were liver, brain and muscle, each of which has a peak period of development at different stages during the gestation period. The period of development would be associated with a high concentration of potassium4,0 an d it was hoped to find out whether there was any involvement v/ith K levels.

2. I. A.R. KNO^ K'^V Ratios.

Ratio determinations were made on sam.ples of A.R. KITO^. As has already been said, the measurements of actual peak heights for K4 0 were not very accurate, neither for the standard nor the tissue samples. For both A.R, and foetal samples, 39/41 ratios 41 / 40 were determined before and after the K / K ratios. Slit widths were

.025" for 39/41 and .018" for 41/40 determinations. The reason for determining the 39/41 ratio after the 41/40 ratios, was to see whether increasing the filament current to get measiirable levels of K4 0 had any effect on the ratios. Results obtained are shown in Table 14. The mean 39/41 ratio does not differ significantly from the value previously found, but the 41/40 ratio is somewhat lov/er than other authors quote, and the variation between runs is high. Table 14.

K^^/K^^, K^^/K^^ Ratios for A. R. KNO^

K^V^ i S. D.

+ + 14.12 .06 540 6 + + 14.12 .07 564 6 + + 14.04 .07 488 4 + + 14.04 .02 496 4 + + 13.98 .04 578 8 + + 14.10 .06 478 14

+ + Mean 14.07 .06 524 42

Table 15 a. AT # 4-0 K /K Ratios for Himan Embryonic Tissue.

^9/^41 + ^41/^40 + Sample S. B.

+ Liver 14.12 .06 396 i 2 + Bone 14.24 .06 562 t 7 + Intestine 14.15 .05 474 - 5 + Muscle 14.08 .03 484 t 4 + Heart 13.99 .03 476 i 5 + Kidney 14.19 .04 590 i 4 + Lung 14.15 .03 488 i 10 + Brain 14.II .06 582 t 9

+ Mean 14.13 .07 Mean 507 i 82 Table 15 h

r5Q , AT 4.T . 4.n ir^'/K -^. K -^/K Ratios for Pi^ Foetal Samples,

Sample 39/41 Ratio t S. D. 41/40 Ratio ^ S. D.

+ + Muscle 14.06 .01 592 6 + + Brain 14.13 .01 574 7 + + Liver 14.12 .04 572 12 + + Liver 14.17 .03 556 12 + + Brain 14.23 .01 588 10 + + Muscle 14. II .04 642 18 + + Brain 14.II .03 564 6 + + Muscle 14.08 .04 594 6 + + Liver 14.09 .03 586 6 + + Brain 14.03 .03 576 8 + + Liver 14.20 .04 590 7 + + Muscle 14.03 .04 574 II + + Muscle 14,14 .09 600 6 + + Liver 14.12 .05 606 7 + Brain 14.12 .11 590 + + Muscle 14.10 .05 500 4 + + Liver 14.16 .04 568 4 + + Brain 14.12 .05 554 8 + Liver 14.10 .04 514 10 + + Brain 14.18 .03 564 10 + + Liver 14. II .05 528 6 + + Liver 14.15 .05 546 8 + + Liver 14.16 .02 532 6 + + Liver 14.14 .03 538 6 Mean 14.12 •f .05 Mean 569 + 32 41 / 40 2. 2. K / IC Ratios for Foetal Samples.

Results for these determinations, shown in Table

(I5 a, h) vrere obtained in a similar manner to those for A.R. KNOg.

The K ratios for the human foetal samples have already been given, and these were found to give values similar to the "normal" 41 / 40 ratio. The K / K ratios on these samples, however, were generally even lower than those found for A.R, KNO^ with the variations between runs being even Y/orse, V/ith pig foetal samples (Table 15 b) Y^'^/ k"^^ ratio determinations gave somev/hat more reliable results. The mean ratio

(569 I 32) Y/as closer to the accepted value and variability was considerably less. No relationship between the ratio and foetal age

?/as found. The A.R, KNO^ no fractionatioK ratin beino wags agaievidenn tsimila whichr mighto tht e bevalu causee fod r by the higher temperatures involved in obtaining a measurable K 40 peak. 40 2.3. K Specific Activity Measurements.

Pig foetal samples on which Y^^/ K^^ ratios had 40 been determined, were recrystallised twice, and the K was counted by liqiiid scintillation using the phosphors previously described.

Samples were weighed before adding them to the phosphor solutions, and

A.R, KWO^, covering this sample range, was similarly treated. After counting, potassium levels in the samples were determined by atomic absorption spectroscopy. Potassium specific activity levels were calculated both from the weights of samples and the levels as determined by atomic absorption. Results are shown in Table 16. A.R, KNO^^ Table 16. K Specific Activity for Pig Foetal Sajnples.

Specific Activity Cnt/min/mgK.

t.41 /„40 -n . . Sample Weight of Sample K Determination K /K Ratio.

80 Days Liver 1.790"^ 1.967"^ 586

Brain 1.913 1.516 564

Muscle 1.649 1.321 594

85 Days Liver 1.292 1.297 536

Brain 2,504 1.798 588

I5uscle 1.341 1.531 642 CO •H 92 Days

Brain 1.357 1.325 576 o 0 Muscle 1.508 1.380 574

•H Brain 1.156 1.123 574

Muscle 1.478 1.444 592

104 Days Liver 1.564 1.533 606 •H 03 •P ra Brain 1.887 1.756 590 0) Muscle 1.597 1.320 600 * 108 Days Liver I. 541 1.559 550

Brain 1.553 1.502 559

Muscle 1.428 1.433 500 Standards 1.499 standards included for the K^^ counting and atomic absorption determinations allowed for corrections to "be made to the results.

All count rates, even for the lighter samples, were significantly higher than the background (31.98 cnt/min.) The specific activities as determined from sample weights were influenced by the purity and form of the preparation. The specific activities as found from the amount of potassium determined by atomic absorption and weight of sample varied, some being higher than those for A.R. KJFO^. The results of the determinations on foetal samples will be dealt with more fully in the Discussion Section. 41 / 40 K / K ratios are also included in Table 16 thus enabling comparisons to be made. 3. Calibration Determinations.

The results of these tests appear in Table 17. If 39 one considers the ratios found for the separated isotope of K , there appears to be a very high preference for this isotope. However, a reliable estimate of discrimination would not be gained from ratio determinations on potassium which has an isotope abundance so different from the normal. Similar objections would, therefore, apply to the ratios determined on the K^^/ k"^^ isotope 1:1 mixture. In this case, contamination with "normal" potassium, and v/eighing errors could also contribute to the "higher than expected" ratio obtained. The best estimates of discrimination would, therefore, probably be obtained from tests on potassium in v/hich the ratio was only slightly differen39 t from the normal. Potassium nitrate to which varying amounts of K and Table 17.

Cali"bration, Ratios.

Sample Ratio Found Ratio Expected

+ A.R. KNO3 - 100 mg.nml I3.96 ,04 14.09 - ,06

A.R. OO3 Iml + O.I mg.K^^ 14,10 ,04 14,10

+ A,R. KKO3 1ml +0.2 mg.K^^ 13.95 .04 14,11

+ A.R. 1ml + 0.5 14.07 .05 14,14

+ A.R. 1ml + 0.7 mg.K?^ 14.12 .04 14,18

+ A.R. KNO3 1ml + I.O 14,25 .05 14,23

+ A.R. IQI03 1ml + 1.0 ml K^^/K^^ mixt. 13.91 .02 13,74

-f K^^ EOJOg 1[99.85^ 0 I ,462 15 7X3,2

13103 (0,6853 K^^ t 0.4212 K"^^) 2.905 1 28 1,627 K- had been added, gave such results. The results are a little indefinite, but appear to indicate that the instrument does not 39 preferentially detect the K isotope. Differences between the found and expected ratios are, in the main, within statistical variation. 4. Total Potassium Levels — Tissue Samples.

Total potassium levels were determined for the tissue samples shown in Table 18. The potassium determinations were made by flame photometry on the Beckman D U Spectrophotometer. The levels of potassium in the cancer tissues are elevated, but are not as high as those found for the kelp samples. The high level found for the bone sample is most probably due to the presence of bone marrow and therefore reflects this tissue potassium concentration. The same would not hold completely for the bone sarcoma. Wilm's tumour (a tumour of the kidney) has an elevated level when compared ?fith the normal kidney value. ,Table 18. Total Potassiiam Determination,

Sample (Group No.) MgK/gm D. Wt. Normal Concentration. Ivlg/gm D. Wt.

Wilm's Tmour (I) 23.45 Skin (3) 2.65 2.86 3.96 Sarcoma (I) 3.77

Lung (2) 12.22 11.22 + 6.82

Kidney (2) 7.97 8.75

Metastases (Sub. Cut.) (I) 11.59

Pancreas (2) 6.10 -h 11.60

Liver (2) 8.83 6.94 10.27

Bone (2) 10.66 18.17 -H 1.09

I&iscle (2) 5.82 5.62 + 17.14 Kelp Stem (4) 38.08 Kelp Frond (4) 36.60 Kelp Whols Plant (4) 27.18 Normal Rectum (3) 18,77 19.33 Bone Sarcoma (I) 25.90 Normal Uteirus (3) 9.77 7.35

Adapted from A. T. Shohl - "lylineral Metabolism". (I939)

Reinhold, II.Y.

-f- Post-mortem Samples, SECTION V.

DISCUSSION OF RESULTS.

The significance of the differences in potassim isotope ratios reported by Brewer and co-workers is not easy to ascertain. If one looks at some of the errors quoted, it would appear that the accuracy of the determinations was exceptional; hut subsequent ratio determinations using improved techniques and instrumentation have shown greater variations than indicated by Brewer's results,

A clear distinction between the precision of ratio determinations on a single sample, and the repeatability of determinations on a number of different samp!J.es, is not made by Brewer, For sea-water he gives a percent, error of ~ .025^ whereas for other samples he quotes errors of - 0.14 - 1.59^ , the higher values being on samples for which he claimed an isotope ratio difference. Cook (1943), however, has shown variations of up to in ratios may occur in the mass spectrometer during repeated sample analysis, and gives his deviation as i I^, a far more realistic value.

Subsequent experiments by Reutersward (1956) and

Kendall (i960 a) have indicated that the ratio differences reported by Brewer and his fellow workers were most probably due to impurities in the samples. Kendall found no difference in the ratio of potassium purified from cancer tissue, normal tissue or mineral sources. The present study verifies these findings, although it does not find any isotope anomalies for partly purified potassium, as reported by Kendall. The reason for this is not clear. That the J/IS2 - S. G. Mass Spectrometer is able to detect small differences

in isotope ratios, has been shOTm by the tests carried out during

calibration and on samples that had their ratios deliberately-

altered. Also, the ratio determinations made on samples to which

known "impijrities" had been added, indicated that anomalies in the

detected ratio could be produced. Therefore, it is unlikely that the

ion source T/as impurity-insensitive. A distinct possibility for the

failure of the present study to find any impurity effect could be

that the levels of impurity present were too low to upset the ratios.

Kendall, (i960 a) found that addition of sodium as the chloride,

did not appreciably affect the ratio obtained, and said the effect must be due to some other impurity. The levels of sodium present in

the ashed sample of this survey were not high (as indicated by ion current production, ) and it is possible that other elements were

there if only in small amounts.

The ratios reported for the tissues and Analytical

Reagent cannot be claimed to be "integral ratios", as reported in

Reutersward's (1956) study, for no samples were run to exhaustion.

However, Kendall (i960 a) reports that variations in the isotope ratio obtained by his single filament vapour ionisation technique were almost non-existent after sixty minutes operation. Ratios quoted in this thesis were obtained from samples which had been run in the mass spectrometer for a similar period of time. The ion current ratios then varied only within the limits quoted in the Results, showing no tendency to fall, although observed for several hoin's. The sample size used for ion production T^as, on the

-2

average, 5 jog.mm, for A.R, samples. However, because the sample

was dried out by passing a current through the filament during

application, the concentration at the centre of the filament would

be less than this. Also, most of the biological tissues gave low sample weights and the amount present per mm would be less. (About -2

I pg.mm." ). Nevertheless, the regions of higher concentration

could be responsible for erratic ion production, similar to that

reported by Reutersward (1956). For this reason, an integral ratio

could not be determined.

Most of what has been said, applies equally to

^^^ k^I/k^O ^.atio determinations. However, K^^/K^^

determinations on the ¥32 - S.G. mass spectrometer, have not been very successful. The reasons for this have been enumerated. The

problem, is not one of an inability to determine peak heights from

the recorded results, but one of resolution of the potassium 40 ion

current. The values obtained during sample runs were quite good, but

reproducibility was poor, both for foetal samples and A.R. potassium

nitrate. The mean ratio determinations for the two groups were lower

than the accepted value, although the pig foetal samples were slightly down (569 V. 578).

In an attempt to obtain true abundance ratios, a number of different corrections have been used by various authors.

Mer (1950) in arriving at the K^^/K^^ ratio of 13.48 - .07, now accepted as the true abundance ratio, calibrated his mass spectro- 36 40 meters with a synthetic mixture of isotope A and A of argon,

lonisation was produced "by electron bomhardment of potassium vapour.

Scane doubts have been cast on the justification of the use of

isotopes separated by 4 a.m.u, as calibration of samples separated

by only 2 a. m. u, Nevertheless it does allow for corrections to be made with a fair degree of confidence. Kendall (i960 a) advocates

the calibration of a mass spectrometer by the use of a synthetic

standard of isotopes of potassium of known abimdances. A working

standard could then be calibrated and this could be used to calibrate further mass spectrometers. The form of ion production in this

instance could be by thermal ionisation.

Despite this, most stated "true abundance ratios", have been arrived at by the use of sometimes doubtful correction factors. V/hite et al (l956) adopted a 3/i discrimination correction

for the electronmultiplier used, in quoting their 39/41 ratio.

Reutersward (1952, 1956) gives figures for mass discrimination effects during thermal ionisation, in the second paper quoting results of tests to support his figures. On thinly coated samples, he obtained a iTatio for of 13.87, for which he measured an

observed discrimination (OC ) of 2.70fa. The higher initial ratio found on thick sample coatings he postulates as being due to diffusion within the sample srystal structure. He proposes a similar explanation for the effect of the presence of other elements in the sample, resulting in crystal structural differences. Ion production, he says,

is affected by these differences and isotope ratio differences result.

Kendall (i960 a) in quoting his "true isotope ratios" gives a list of corrections made. It is his "correction for fractionation" which greatly reduced the variations in his Uncorrected ratios. He quotes corrections from - to + 0.56^0 for this "error",

"but does not discuss methods used for its calculation in the correction and errors section of his thesis. The only reference made to the derivation of this factor, is that it is assumed to be responsible for the fractional differences between the "integral ratio" obtained and the ratio obtained from the "box comparison" readings. No reasons are given why the fractionation should vary so much. The uncorrected readings given for

ratios in this thesis, have less variation than the uncorrected ratios quoted by Kendall. Nevertheless, they represent a slightly more than higher value than the "true" abundance ratio of Nier. This is in agreement with results found by

Compston, using a similar mass spectrometer. Compston suggested the method adopted for the calibration of the mass spectrometer used for this study. These tests were not as conclusive as had been hoped, because of the small sample size involved.

Use of the sample with a very high 39/41 ratio might have meant that the collector an4d1 associated systems were not able to accurately detect the small K ion current in the presence of 39 such a large K ion current. The problem is thus similar to the accurate determination of K^^/K"^^ ratios. However, this test did indicate that contamination with "normal" potassim should be fairly low under the sampling conditions used, since the presenc41 e of any "normal" potassim should increase the abundance of X . Therefore, it seems unlikely that "normal" potassium should be responsible for the "high" ratio found for the calibrating mixtiire

(2,905 instead of 1.627)• Determination of the potassiiim content

of this sample gave the expected value, and hence the low 41 figure

cannot be due to loss of potassium as such. Ratio determinations

on samples in which the ratio as found in this study was altered only

slightly by the addition of and indicated no preference by 39 the mass spectrometer, above statistical variation, for K . However,

more accurate determinations of the mass discrimination effects of the

MS2 - S.G, mass spectrometer (which is probably greater than Apfo)

could be made by the use of larger amounts of separated isotopes.

If such quantities of isotopes were available, calibration could be

made by using a synthetic mixture giving approximately the expected

ratio, and one with a ratio of K^^/K^^ of approximately l/l. Only then could a"true" abundance ratio be given. No attempts were made to

correct the K 41/ /K 40 ratio by the use of similar is»tope mixtures, 40 because the supplies of the K isotope of a stiitable degree of

pircity were not available. The determination of the K4 0 specific activities

for foetal tissue demands special consideration, following the

ratio determinations it was thought that there would be no 40 variation in K specific activity levels. By determining the actixal weight of samples taken, and the potassium levels by atomic

absorption, it was hoped to obtain a double check. The results

obtained, however, did not completely agree, differences in specific

activities being found. How the weight of sample counted could be influenced "by a nuEiber of factors. The form of potassium may be in

doubt (but this is not very likely due to the method of preparation);

the compound may be present as the hydrate, or at least have some water present; or there could be foreign matter, in the form of

other chemicals. Most of these possibilities would, however, be expected to lower the specific activity. Total potassium determination on the samples would overcane these problems. To allow for the

slight possibility of a difference in efficiency during counting, because of the different weight of the samples, five samples of

A.R. KNOjj were included, covering the range of weights of the samples.

Count rates for all these standards were constant within 1.2% and indicated an efficiency of better than 90/o. Some of the foetal samples, however, gave somewhat higher counts/minute/mg, than these standards, when based on ?;eights of samples. As has just been said, the specific activity would be expected to be slightly lower, if any- thing. The specific activities, of course, would vary, depending on whether the potassium nitrate was present as the mono-hydrate or not. liihen the specific activities were based on the weights of potassium as detemiined by atomic absorption, the count s/minute/mg. were lower.

The specific activities as detennined in this manner would be dependent on the ability to determine potassium accurately in the phosphor solution. However, the A.R. potassium nitrate samples were used for calibrating the standard curve, so any interference should be thus allowed for. It was then thought that rubidiimi could be responsible for some of';these inconsistencies in activities. It had not been seriously considered as a source of contamination originally because it is not thcoight to be an important "biological element. Sodium tetraphenylboron will precipitate this element, and because of the fairly high abimdance of its natural radio isotope (Rb 8?) this might explain the anomaloijs activities. The settings used on the liquid scintillation counter (particularly the 25 X multiplier gain) were thought to eliminate rubidim JB particles. However, a sample of rubidium chloride was found to give 11.56 counts/minute/mg Rb, for the instiTument settings used for counting. Subsequent determinations by atomic absorption spectrometry indicated the presence of rubidium in the phosphor solutions, in varying amounts. The percentage content of rubidium in the samples ranged from 0.38 - with a mean of The weights of rubidium per potassiijm samples were from .023 - ,390 mg. ( mean .160 mg). For the mean weight of rubidiim present, 1.85 counts/minute would thus be incorrectly attributed to potassium. For the mean weight of potassium sample - 17.620 mg. (as determined by atomic absorption) a count rate of 26,41 counts/minute would be expected. This represents an average rubidium potassium concentration of 7^ which would account for an increase in count rate of 0,104 counts/minute. The differences in K^^ specific activities above statistical variation, could therefore be explained, in part at least, by the presence of rubidium in the samples. This element was also most probably responsible for ion currents found (of the order of -14 \ 10 amps.) in blank filaments. It would be interesting, because of this association with actively growing tissue (viz. foetal tissue) to find out whether cancer tissue is similarly affected. If this were foiind to be involved also, it might then be conceivable that the ratio differences reported previously for partly purified cancer tissue v/ere due to the presence of this element in the samples. Threshold levels could exist for the amounts of rubidium present to affect the potassium isotope ratios and this could explain why the foetal

K^^/K^^ ratios were not upset in this study. However, a study of these possibilities would be extensive and beyond the scope of this thesis, 40

K specific activities for the foetal tissues, therefore, are probably not any higher than those for A,R, potassium nitrate. One sample (a brain sample of earlier foetal age) gave a higher activity even after allowing for rubidium content. This, however, wa,s a light sample (7 mg. KtlO^) and specific activities determined both from the weights and potassium determination were of low accuracy.

Low values found for some samples are not so easily explained; but there does not appear to be any consistent pattern with regard to either foetal age or organ site. Pee and Yletl (1963) find total potassium levels in human foetuses constant throughout the gestation period, and this may be the same for the differen40 t organs. It is therefore unlikely that concentration of K should vary if no increase in potassium metabolism is indicated. ACKlOTLEDGilENTS.

The New South ¥ales State Cancer Council is thanked for granting permission to present this work for a higher degree*

This study was first suggested "by Dr K. Y/. Starr, Honorary Director

of the Special IMit for Investigation and Treatment of Cancer,

Prince of Wales Hospital.

The Staff of the Hospitals and Institutions which provided the samples, and in particular, the staff of the Special Unit, are thanked for their co-operation.

Ivtrs S. Salasoo helped with tra.nslations of some of the references, and also assisted with the potassium radio-activity measurements.

Mr K, N, Wynne has shown a continued interest in this project, and was responsible for many helpful suggestions.

My CO-supervisors, Professor D. P. Mellor and

Professor J. H. Green have been most helpful throiaghout this extended survey, and many stimulating discussions have been had with both.

The co-operation of the Department of Nuclear and

Radiation Chemistry, University of N. S. W., is acknowledged.

Commander J. Mason was responsible for the continued operation of the

Mass Spectrometer and his technical assistance in this regard is gratefully appreciated. Mr J. Bell is thanked for his assistance on problems of an electronic nature.

Dr W. Compston, of the Australian National

University, suggested the method adopted for the calibration of the mass spectrometer.

The help of Miss M. Gilfillan and I\Ir J. D. Tolhurst in the compilation of this thesis is gratefully acknowledged* lOI

References*

Anderson, E. C. & Langham, W. H. (1959) Science 130 713.

Aston, F, W. (I9I9) Phil, l-iag. ^ 707•

Aston, P. W» (I92I) Phil. Mag. ^ 436.

Aston, P. W. (1927) Proc. Rpy. Soc. (Lend.)

A 115 487.

Bainbridge, K. T. (I93l) J. Prank. Inst. 317.

Biltz, W. & Maxcus, E. (I9I3) Zeit. Anorg. Chem. M 369. Black, D, A, K. & Emery, E. W. (1957) Brit. Med. Bull. 13 7.

Bliimell, J. P. & Jones, E. J. (1936) Phys. Rev. ^ 464.

Boerbooci, A. J. H, (i960) Proc. User's Gonf.

Thermal lonisation

(A. E, I, Manchester) p.38

Bondy, H. Johannsen, G. & Popper, K. (1935) Zeit. F. Physik. 95 46,

Bendy, H. & Vanicek, V. (1939) Zeit. P. Physik. lOT 186.

Bowen, H. J. M. (1956) J. Nucl. Energ. 2 255.

Bowen, H. J. M. (1959) Int. J. Appl. Rad. & Iso.

7 261.

Bradt, P. Parhan, 0. & Brewer, A. K. J. Res. Nat. Boar. Stand.

(1947) ^ 162.

Brewer, A. K. (1935) Phys. Rev. ^ 640.

Brewer, A. K. (1936 a) J. Amer. Chem. Soc. ^ 365.

Brewer, A. K. <1936 h) J. Amer. Chem. Soc. ^ 370.

Brewer, A. K. (1936 c) J. Chem. Phys. 4 350.

Brewer, A. K. (1937 a) J. Amer. Chem. Soc. ^ 869.

Brewer, A. K. (1937 b) Science. 86 198. Brewer, A. K. (1938) Ind. Eng, Chem, 30 893•

Brewer, A. K« (1939 a) J» Amer. Chem. Soo. 61 1597

Brewer, A. K» (1939 b) Phys. Rev. ^ 669,

Brewer, A.K» & Baudisch, 0. (1937) J. Amer, Chem. Soo* ^ 1578

Brewer, A. K. & Kuesch, P. D. (1934) Phys. Rev. ^ 894.

Brewer, A, K,, Madorsky, S,. Taylor, J. J. Res. Nat. Bur. Stand.

Bibeler, V., Bradt, P., Parhan, 0., 38 137.

Britten, & Reld, J. (1947)

Cloud, P,, Friedman, I. & Sisler, P. Science. 127 1394.

(1958)

Compston, ¥. (1963) Personal Communication.

Cook, K. L. (1943) Phys. Rev. ^ 960.

Craig, H, (1953) J. Geol. &2 115.

Craig, E. D. (1959) J. Sci. Instr. ^ 38.

Crockett, W. A. & Huff, R. L. (1958) Ann. Soo. Clin. Invest.

Dempster, A. J. (1922) Phys. Rev. ^ 631.

Be Long, R., Coman, D. & Zeidman, I. Cancer. 3 7i8.

(1950)

Bietz, L, A,, Pachucki, C. P. & Land, G. Anal. Chem. 34 709.

A. (1962)

Bodson, M. H. (I963) J. Sci. Instr. 289.

Bouglas, D. M., Elliott, G. A., Proc. Conf. on Applic. of

Ellis, R. & Lee, R. H. (I950) Iso. in Sci. Res. (Melb.)

P. 125. Ernst, E. (1934) Katurwiss* ^ 479,

Errock, G. A. (i960) i^oc. User's Conf. Thermal

lonisation (A. E, I.,

Manchester) p» 43•

Pee, B. A. & Weil, W, B. (1963) Ann. N. Y. Acad, Sc. IIO 869.

Feldman, D. E., Yost, H. T. & Science. I^ 146.

Benscai, B. B. (I959)

Fenn, Bale, V/. & Mullins, L, (1942) J. Gen. Physiol. ^ 345.

Pindeis, A. P. & Be Vries, T. (1956) Anal. Chem. 28 1899.

Gatehouse, B, M. & Willis, J. B. (I96l) Spectrochim. Acta. 17 710.

Gieseking, J, E,, Snider, H. J, Ind. Eng. Chem. 7 185.

Getz, C. A. (1935)

Honda, M. (1959) Geochim. Cosmochim. Acta.

^ 148.

In^am, M, G. & Chupka, W. A. (1953) Rev. Sci. Instr. ^ 518.

Jacques, A« (1940) J. Gen. Physiol. ^ 741.

Kendall, B. R. P. (i960 a) PhD Thesis, Uni. W. Aust.

Kendall, B, R. P. (I960 b) nature. 166 225.

Klemperer, 0. (1935) Proc. Roy. Soc. (Lond.)A

148 638.

Lasnitzki, A. (1939) Amer. J. Cancer. ^ 225.

Lasnitzki, A. & Brewer, A. K. (1936) Nature. 142 538.

Lasnitzki, A« & Bi^wer, A. K. (I940) Nature. 146 807.

Lasnitzki, A. & Brewer, A. K. (I94I a) Biochem. J. 35 144.

Lasnitzki, A, & Brewer, A, K. (I94I b) Cancer Res. I 776. Lasnitzkl, & Brewer, A. K* (1942) Cancer Res« 2 494, Lasnitzki, A. & Oeser, E. (1937) J. Chem» See. II 1090. Leland, W. T. (1956) Phys, Rev. 77 634. Lowrey, L. (I9II) Ajner. J. Anat. 12 III. Loring, P. H. & Druoe, J. G. F. (I930) Chem, News. 140 34. Mauley, J. S. (1936) Phys. Rev. 4^ 921. Meade, E, C. & Stiglitz, R. A. (I962) Int. J. Appl, Rad. & Iso. 13 II. Mullins, L. & Zerahn, K. (1948) J. Biol. Chem. 174 107. Newman, F. H. & Waike, H. J. (1935) Phil. Mag. 12 767. Nier, A. 0. (1935) Phys. Rev. ^ 283, Nier, A, 0. (1936) Phys. Rev. ^ I04I. Nier, A. 0. (I950) Phys. Rev. 77 789. Ofinara, I. & Morito, N. (i958) J. Phys. Soc. Japan. 13 659.

Palmer, G. H. (1956) Electrcmagnetically Enriched Isotopes & Mass Spectrometry, (Butterworth) p. 156. Patten, B. M. (1948) Embryology of the Pig 3rd Edition. Blakiston Co. Philadelphia,

Patterson, H. & Wilson, H. W. (1962) J. Sci. Instr. ^ 84. Paul, W. & Pahl, M. (1944) Naturwiss. 32 228. Pflaxm, R. T. & Howick, L. C, (1956) Anal. Chem. ^ 1542. Phipps et al

Copley, M. & Phipps, T. (1935) Phys. Rev. ^ 960. Hendricks, D,, Phipps, T. & J. Chem. Phys. 868,

Copley, M. (1937)

Johnson, A, & Phipps, T. (I939) J. Chem. Phys. 7 1039,

Pratt, R. & Curry, J. (1937) ^er. J. Bot. ^ 4X2.

Eahn, 0. (1936) J. Bact. ^ 393.

Remenchik, A. P. & Miller, C. E. (I96l) Proc. Sym. on ?/hole Body

Goimting. (Vienna) p. 331.

Reutersward, C. (1952 a) Ack. P. Pysik. 4 203.

Reutersward, C. (1952 b) J. Sci. Instr. ^ 184.

Reutersward, C. (1956) Ark. P. Pysik. n 37.

Rik, G. R. & Shukoliukov, Y. A. (1954) Ddkl. Akad. Hauk. S.S.S.R.

Russell, R. B. (i960) Proc. User's Conf. Thermal

lonisation. (A.E.I., Man-

chester.) p. 57.

Schumb, IT., Evans, R. & Leaders, J. Amer. Chem. Soc. 63 1203

(I94I).

Shear, M. J. (I933) Amer. J. Cancer. K 924.

Shohl, A. T. (1939) Mineral Metabolism Reinhold

Pittsberg Corp. (N.Y.) p. 19

Smythe, W, R. (1939) Phys. Rev. ^ 316.

Smythe, W. R. & Henmendinger, A. (1937) Phys. Rev. ^ 178.

Starr, K. W. (1956) Second Report on Projected

Cancer Research Programme -

N.S.W. State Cancer Coimcil.

p. 64. Taylor, G, W. & Eairvey, E. N. (1934) Proo. Soc. Exper. Biol.

(N.y.) 31 954.

Taylor, T. & Urey, H. (1938) J. Chem. Phys. 6 429.

Thode, H., Wanless, R, & Wallouch, R. Geochim. Goemochim. Acta.

(1954.) 5 286.

Thompson, R. & Ballou, J. (1953.) Arch. Biochem. Biophys.

^ 219.

Thompson, R & Ballou, J. (19540 J. Biol. Chem. 206 lOI.

Thompson, J, J. (I9I3) Rays of Positive Electricity

and their Application to Chem.

Analysis. Longmanns, Green. (Lond)

Tushingham, R, (i960) Proc. User*s Conf. Thermal

lonisation. (A.E.I. Manchester)

p. 53.

Urey, H. C. (1948.) Science. 108 489.

Urey, H. C. & Greiff, L. (1953.) J. Amer. Chem. Soc. ^ 321.

Van Norman, R. W. & Brown, A. H, (1952) Plant Physiol. ^ 691.

Vinogradov, A. P. (1957.) Biochem. ^ 12.

Vinogradov, A. P. & Teis, R. V. (I94l) Ddkl. Alcad. Nauk. S.S.S.R.

^ 490.

Vogel, A. I. (1956) Practical Organic Chemistry.

Longmanns, Green & Co. (Lond.)

p. 170.

Von Hevesy, G. (1927) Nature. I^ 838.

Voehage, H. & Hintenberger, H. (1959 a) Zeit. P. Naturforsch. I4a 194. Voshage, H, & Hintenberger, H. (1959 e) Zeit. P. Natixrforsch.

M a 828.

Walsh, A, (1962) Proc, Int. Conf. on Spectr.

p. 127.

Welte, E, & Mechsner, K. (1959) Nature. 184 1958.

White, J. R. & Cameron, A, E. (1948) Phys. Rev. 74 991.

White, P. A. & Collins, T, L. & Phys. Rev. lOI. 1786.

Rourke, P. M. (1956)

Winton, P. R. & Bayliss, L. E. (1948) Human Physiology. 3rd Edit.

Churchill. (Land.) p. 96.

Wynne, K. N. & Chorlton, S. H. (i960) Unpublished data.

Zemel, J. (1957) J. Chem. Phys. ^ 410. APPEUDDC.

(a) Some Theoretical Consideratione of I sot ope Seijaration.

Isotope separation may be achieved by two types of

processes - rate processes and eqtiilibrium processes. To the first

class belong barrier diffusion, thermal diffusion, electrolysis, etc.; and to the second, distillation and chemical exchange. Rate processes are irreversible, while equilibrium processes may be conducted in an almost completely reversible manner. Separation factojps for the two methods vary; for light atom and molecules, reversible chemical processes show separation factors of the same magnitude as rate processes. However, for heavier atoms or molecules, irreversible rate

process separation factors greatly exceed those for reversible pro-

cesses. For example, the separation parameter (£ ) for A^^/A^^ ^ axe

0.006 (distillation) and 0.055 (diffusion). Therefore, for the isotopes

of potassiiim, diffusion processes would offer greater scope for separation.

The mechanism in biological systems, involving uptake of

organic and inorganic substances by the cells, is not clearly under- stood. Simple diffusion through a membrane may take place, or chelation may be necessary for the passage across the membrane. Clearly, then, both types of processes discussed previously, could be operating, and could be responsible for isotope fractionation. Prom theoretical considerations, due to differences in ionic mobilities, one would expect the lighter isotope to move into and out of the cell, at a

^ Glueckauf, E. (I96l) - Endeavour ^ 42. faster rate than that of the heavier isotope. The best conditions for fractionation require a small percent, uptake or excretion or chemical reaction processes with large fractionation factors (Bowren,

1959).

Now, as has been said, for elements such as potassium, diffusion should offer the best conditions for isotope separation. 2 Senftle and Bracken discuss this form of separation, in geological processes, although the results are applicable to any system involving diffusion from one phase to another. These authors find the concentration C at a distance x inside a solid from the equation

C = Co (l-erf where Co is the concentration at the interface, k is a constant for any pair of phases, m is the mass of the diffusing species and t the time.

From this expression, the percentage isotope enrichment may be calculated as a function of the distance from

100 ( CiGz,o/ OtOiyO - I ) for two isotopes I and 2. Enrichment increases as the distance the element penetrates. For cellular masses, however, there is a limit to the distance of penetration. Theoretically, it is possible to obtain large enrichments by diffusion; however, in practice, the amount of

p * Senftle, F. E. and Bracken, J. T. (I956) Geochim. et Cosmochim.

Acta. 7 61. enriched material is vanishingly small.

Brewer et al. (1947) in their experiments on potassium

isotope separation by counter-current techniques, estimate separation factors. They find a maximiffii separation coefficient - l) of -2 0.385 X 10 , giving details of the method of calculation. For ionic mobility, they suggest a possible hydration of K. 'J'HgO, which gives a of 1,006, P5Q coefficient (( ) for K7 7H2O

Although probably of little importance for biological

fractionation of the isotopes of potassium, some of the results of

Brewer's counter-current experiments vrlll be given;

Ratio 39/41 Separation Factor (s) (£ - l) X 10^

14.70 1.035 0.229

14.95 1.052 0.II2

15.20 1.070 0.227

Brewer's normal K^^/k"^^ ratio is 14.20. Therefore, for an

increase in the separation factor (calculated from Ro/Rr)of 0.035, he gives an increase in the isotope ratio of 0.50. The ratios reported in this thesis give abnormal" value of 14.09 - .06. A difference of 2 S. D. would alter the ratio by 0.12, and a 3 S. B. difference, by 0.18. From Brewer's experiments, these differences would represent separation factors of approximately 1.008 and 1.013 respectively. The value given earlier by Glueckauf for the separation

of A^ by diffusion is higher than this, as also is the value given by Bigeleisen ^ for Ca^^/Ca^^ (1.08).

Prom these considerations, therefore, if biological fractionation of the isotopes of potassiiJin occurs it could take place at such levels which could be detected by ratio differences as determined by mass spectrometry.

* Bigeleisen, J. (1949). Science IIO 14. (b) K^^/k^^ Ratio Determination,

These were the measurements taken from the recording of a series of three of eleven ratio determinations on A,R. KNO^. For convenience, the recording itself is not included, only peak height measiirements. Scan I, ,

Measurements (mm) Mean Measiarements

41 39 41 165.1 117.1

162.9 117.0 164.0 117.05 14.01 162.4 115.1 162.65 116.05 14.02 160.3 114. 9 161.35 115.0 14.03

160.0 113.3 160.15 114.1 14.04 158.0 113.0 159.0 113.15 14.05 157.3 II2.0 158.15 112.5 14.01 156.9 III.7 157.1 III.65 14.00

155.2 II0.4 156.05 110.55 14.01 153.5 109.9 154.35 II0.I5 14.01

I53.I 109.0 153.3 109.45 14.01 152.0 108.2 152.55 108.6 14.05

Mean Ratio 14.02^ .

\sfhere ^ ( X " X )' 1. D. n ( n - I ) Scan 2. Scan 3.

Measurements (mm) Measurements (mm)

39 41 39 41

147.8 104.9 128.8 91.9

146.8 104.7 128.2 91.8

146.4 104.0 128.8 91.2

145.8 103.8 128.0 91.2

145.6 103.3 128.0 91.0

144.1 103.0 127.4 91.4

143.8 I0I.9 128.0 91.0

143.8 I0I.8 127.3 91.0

142.8 I0I.3 127.4 90.7

142.2 lOI.I 126.8 90.7

142.0 100.9 126.8 90.2

141.0 100.6 126.3 90.2

Mean ^^atio 14.06 t .02 Mean Ratio 14,03 - .02

The standard deviation gives a measure of the precision

of ratio determination. The means quoted in the tables are estimated from all three scans. Overall group means and their standard deviations give an indication of the repeatability of ratio determination. 41 / 40

K /K ratio determinations were made in a similar manner to those for K 7K ratios. Vll

Refer to Pig. II a and II b.

Collector slit width and resolution may be checked from the peak shape. Straight lines are drawn through the sides of the peak (as in Fig. II b) cutting the tops of the peak at C and D and the axis at A and B. The width of the beam at the collector slit may be determined from the intervals (AC' and d' B) cut off on the axis measured in half volts.

The collector slit width is obtained from the measurement XY (again in half volts.)

Slit width then equals XY in.

The resolution is determined from the value of EF, the distance between the points where the peak height falls to less than 0»Tfo, The resolution is then obtained from

Resolution « 3950/ E F

In practice, however, peak shapes were rarely recorded or plotted, the collector slit width and resolution being determined by observing the values of X Y and E F from the accelerating voltage divider. >006983812