THE pomssmm, swsum Am cassum CONTENT E o: ANIMALS AND THE RELATIONSHIP 0s ctmposmon. Thesis far the Degree a? Ph. .D. MICHIGAN STATE UNNERSJTY Man Henry Kitten “£962 f This is to certify that the thesis entitled THE POTASSIUM, SODIUM AND CESIUM CONTENT OF ANIMALS AND THE RELATIONSHIP TO COMPOSITION presented by Alan Henry Kirton has been accepted towards fulfillment of the requirements for Ph D degree in Mace ‘Majgrv professor Date November 20L 1962 0-169 LIBRARY Michigan State University ABSTRACT THE POTASSIUM, SODIUM AND CESIUM CONTENT OF ANIMALS AND THE RELATIONSHIP TO COMPOSITION By Alan Henry Kirton The potassium, sodium and cesium content of animals and their parts were determined and related to their composition. Live lambs and pigs were studied as well as 38 lb. samples of ground pork and lamb. The samples of ground pork and lamb were chosen to cover a wide range in chemical composition. The samples were uniform in size in order to make calibration of the scintillation counting system easier. Potassium-40 and cesium-137 were investigated as non-destructive methods for measuring animal composition. Flame photometry was used for measuring the sodium content and as an alternative method for measuring potassium content. A comparison was made of four methods of extracting potassium and sodium from muscle samples for flame photometry. The methods included homogenization in 2% TCA, oven ashing, acid ashing or boiling in water followed by acidification of the solution. Results suggested that oven ashing was inaccurate as an extraction procedure. Extraction by homogen- ization in a 2% TCA solution was found to be reliable and readily adapt- able to the equipment available, so it was adopted for use in this study. Potassium-40 analyses showed that live lambs weighing an average of 88 lb. contained 0.18% potassiun. Their carcasses averaged 48 1b. and contained 0.23% potassium. An average of 37 gm. of potassium was removed from the skin and wool by washing the lambs, although they had been shorn rather recently. Flame photometry and potassium-40 measurements were in essential agreement as to the potassium content of the fatty tissues and 'muscular tissues from lamb carcasses. Similar results were obtained for the ground pork and lamb. For lambs, bone contained approximately one Alan Henry Kirton half and fatty tissue approximately one quarter as much potassium as the ‘muscle tissue (0.30%). Pigs averaging 198 1b. in live weight had 0.20% potassium in their empty bodies (G.I. contents excluded) and 0.21% potassium in their car- casses. The potassium content of the remaining body compartments was determined individually and the data are presented. Where data were available for comparison, it was shown the composi- tion of the animals, their carcasses and ground meat samples could be ‘more accurately predicted from flame photometrically determined potassium than from potassium-40. The relationships between composition and potass- ium content were closer for pork than for lamb. In general, the standard errors of the regression equations for predicting composition from potass- ium content were too large to suggest that the method based on potassium- 40 is likely to have any wideSpread application. Possible reasons for the magnitude of the standard errors have been fully discussed, as well as some possible non-destructive alternative methods for determining com- position. The sodium content of various tissues was also determined by flame photometry. In contrast to potassium, the levels of sodium in the carcass of the pig were higher than in the non-carcass compartments. Sodium was found to be less closely related to composition than potassium. The cesium-137 levels in the lambs were found to agree with other data from North American sources. These levels were lower than some pub- lished from Scandanavia following nuclear weapons tests. The cesium con- tent of the lambs was found to be unrelated to their composition. THE POTASSIUM, SODIUM AND CESIUM CONTENT OF ANIMALS AND THE RELATIONSHIP TO COMPOSITION By Alan Henry Kirton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1962 ACKNOWLEDGEMENTS The author wishes to acknowledge with gratitude the constant inter- est and assistance of his committee chairman, Dr. A. M. Pearson, in the selection of course work and guidance of research project. Gratitude is also eXpressed to the other members of the author's guidance committee, Dr. B. S. Schweigert, Chairman of the Department of Food Science, Dr. E. P. Reineke, Professor of Physiology, and Dr. J. L. Fairley, Professor of Biochemistry. Part of the research project would not have been possible without the permission and assistance of Dr. W. H. Langham, Director of the Los Alamos Scientific Laboratory, Los Alamos, New Mexico, Dr. E. C. Anderson and Mr. R. L. Schuch of the same address for making available the Los Alamos human counter, providing instruction on its use, and furnishing laboratory facilities. The author gratefully acknowledges the assistance of Mr. and Mrs. Frank Mbntoya of the Los AlamOs Scientific Laboratory for their helpfulness throughout the experiment. The author also wishes to eXpress his thanks to Mr. Leslie 8. Porter, County Agricultural Agent at La Jara, Colorado, and to Mr. J. R. Chavez, County Agricultural Agent at Santa Fe, New Mexico, for assistance in locating and purchasing the animals used in the Los Alamos experiment. The author wishes to acknowledge the cooperation of Radiation Counter Laboratories, Inc., Skokie, Illinois, who provided the counting equipment used in Experiment II and advice on its use. In particular the help of Dr. James W; Haffner, Mr. M. Green, Mr. J. Rzechzkowski, and Mr. Arthur G. Murphy is gratefully acknowledged. 'Mr. Roy W. Porter assisted with tranSportation of materials to and from Chicago. ii iii Dr. R. H. Gnaedinger provided samples from 24 pigs for sodium and potassium analyses and the data on the gross chemical analyses of the samples. Dr. W. T. Magee and Dr. R. H. Nelson provided statistical ad- vice. The author wishes to acknowledge the financial support of the Michi- gan Agricultural Experiment Station and funds provided by research grant No. AMD 4172-03 provided by the National Institutes of Health for vari- ous phases of the experimental work. Travel of the author to and from the United States was made possible by a Fulbright Travel Grant provided through the United States Educational Foundation in New Zealand. The author was assisted financially by a Graduate Research Assistantship from the Departments of Animal Husbandry and Food Science of Michigan State university and the Macmillan Brown Agriculture Research Scholarship pro- vided by the University of New Zealand. The author wishes to express his thanks to Mrs. Beatrice Eichelberger for her typing of this thesis. TABLE OF CONTENTS INTROD UCT ION . C O O O O O O O O O O I O O O O O O O 0 Experimental Objectives . . . . . . . . . . . REVIEW OF LITEMTURE . O O O O C O O O O I I O O O 0 Potassium and Animal Composition . . . . . . . . Theoretical Basis . . . . . . . . . . . . . Measurement of Potassium Content from the Radioactivity 0f POtflSSiUfll-‘lfl o o o o o o o o o o o o e o o o o o 0 Measurement of Potassium Content by Flame Photometry . Relationships between Potassium Content and composj-tion O O O O I O I O O I O O O O O O O 0 O O 0 Sodium and Animal Composition . . . . . . . . . . Theoretical Basis . . . . . . . . . . . . . Relationship of Sodium Content to Composition Cesium-137 and Animal Composition . . . . . . . . Theoretical Basis . . . . . . . . . . . . . Relationship of Cesium-137 Content to Composition DEVELOPMENT OF FLAME PHOTOMETRIC PROCEDURES . . . . . ‘Materials and Methods . . . . . . . . . . . . . . Apparatus . . . . . . . . . . . . . . . . . Sample preparation . . . . . . . . . . . . . Standard solutions . . . . . . . . . . . . . Samples . . . . . . . . . . . . . . . . . . Calculations . . . . . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . iv 11 ll 12 12 12 13 14 14 l4 l4 l7 l8 l8 19 21 EXPERIMENT I. LOS ALAMOS LAMBS . . . . . . Experimental . . . . . . . . . . . . . Counter . . . . . . . . . . . . . Animals . . . . . . . . . . . . . Counting and Sampling . . . . . . Chemical Analyses . . . . . . . . Potassium and Sodiun Estimations . Cesium-137 Estimations . . . . . . Results and Discussion . . . . . . . . Animal Composition . . . . . . . . Potassium Content and Composition Sodium Content and Composition . . Cesium Content and Composition . . Summary and Conclusions . . . . . . . . EXPERIMENT II. GROUND PORK AND LAMB . . . . Experimental . . . . . . . . . . . . . Pork and Lamb Samples . . . . . . Chemical Analyses . . . . . . . . Scintillation Counter . . . . . . Counting Methods . . . . . . . . . Potassium Standards . . . . . . . Results and Discussion . . . . . . . . Potassium Standards . . . . . . . Gross Meat Composition . . . . . . Potassium and Sodium Content of the Ground and Relationships with Composition . . . . . . . . . . Summary and Conclusions . . . . . . . . Meat Samples Page 22 22 22 29 29 29 30 3O 31 31 31 42 44 48 49 49 49 50 50 51 51 51 51 52 53 61 vi Page EXPERDMENT III. TWENTY FOUR PIG BODIES . . . . . . . . . . . . 62 Introduction . . . . . . . . . . . . . . . . . . . . . . . 62 Experimental . . . . . . . . . . . . . . . . . . . . . . . 62 Animals and Their Gross Analyses . . . . . . . . . . . 62 Potassium and Sodium Analyses . . . . . . . . . . . . 64 Results and Discussion . . . . . . . . . . . . . . . . . . 65 smary and CODCIUSi-ons O O O O O O O C O O O O O O O O O O 74 81mm 0 O O O O O O O O O O O O I O O O O O O I O O O O O O I 83 LITE MTUm CITED 0 O O O O O O 0 O O O O O O O O O O O O O O O O 84 APPENDIX 0 O O O C O C O O O O O O O O O O O O O O O O O O I O O 9 0 ll. 12. l3. 14. 15. 16. LIST OF TABLES Page Summary of data taken from the literature on potassium content of various tissues . . . . . . . . . . . . . . . . 6 A comparison of different methods of extraction of potaSSi‘m and sodim O O O O O O O O I O I O O O O O O O O 20 Weight and composition of the lambs . . . . . . . . . . . 31 Potassium content of 10 live lambs and their components as estimated from potassium-40 counts and from flame photome- try 0 O O O O O O O I O O O O O I O O O O O O O O O O O O 32 Correlations between the potassium content of live lambs and carcasses and carcass composition . . . . . . . . . . 37 Regression equations for predicting carcass composition . 41 Sodium content of the carcasses and some separable compon- ents of ten lambs as measured by flame photometry . . . . 43 Correlations between the percent sodium in the edible carcasses of ten lambs and other carcass variables . . . . 44 Cesium-137 content of ten live lambs and their components 45 Correlations between the cesium content of live lambs (ppc cesiun-l37/Kg. tissue) and some carcass components . . . . 47 Gross chemical composition of the counted meat samples . . 53 Potassium and sodiun content of ground pork and lamb samples as measured by flame photometry or from potassium-40 . . . 54 Relationships between the percent potassiun in ground meat samples (X) as estimated from potassium-40 and other chemi- cal components . . . . . . . . . . . . . . . . . . . . . 56 Relationships between the percent potassium in ground meat samples (X) as measured by flame photometry and other chemical components . . . . . . . . . . . . . . . . . . . 57 Correlations between the percent sodium in ground meat samples and other chemical components . . . . . . . . . . 59 Body weight and components from 24 pigs (from Gnaedinger, 1962) O O I O O O O O O I O O O O I O O O O O I O O O O O 66 vii Table 17. 18. 19. 20. 21. 22. 23. 240 viii Composition of the empty bodies and frozen carcasses of 24 pigs (from Gnaedinger, 1962) . . . . . . . . . . . . . Potassium content of the empty bodies and their components frotn 24 pigs I I I I I I I I I I I I I I I I I I I I I I Sodium content of 24 pigs and their components . . . . . Correlations between the potassium content of the frozen carcasses and the empty bodies of 24 pigs and the compon- ents of the frozen carcasses . . . . . . . . . . . . . . Regression equations for predicting the composition of 24 frozen pig carcasses from the potassium content of the carcasses and of the empty bodies . . . . . . . . . . . . Relationships between the percentage potassium (X) in 24 empty pig bodies and other body components . . . . . . . Correlations between the sodium content of the frozen carcasses of 24 pigs and other carcass components . . . . Relationships between percentage sodium (X) in 24 pigs (empty bodies) and other body components . . . . . . . . Page 66 67 7O 71 72 72 73 74 Figure II. III. LIST OF FIGURES Page General View of Los Alamos human counter . . . . . . . 24 Method of restraining animals for counting . . . . . . 26 The Los Alamos human counter outside its lead shield showing the banks of photomultiplier tubes . . . . . . 28 ix LIST OF APPENDICES Comparison of different methods of extracting potassium and SOdimn I I I I I I I I I I I I I I I I Slaughter and separation data from Los Alamos lambs . . Chemical composition of separable fat and lean from Los Alamos lambs I I I I I I I I I I I I I I I Potassium content of Los Alamos lambs from potassium-40 co‘mts I I I I I I I I I I I I I I I I I I Sodium and potassium content of separable lean and fat I from Los Alamos lambs as measured by flame photometry . Cesiun-l37 content of Los Alamos lambs from gamma counts Composition of 38 lb. ground pork samples . Composition of 38 lb. ground lamb samples . Potassium content of 24 pigs . . . . . . . Sodium content of 24 pigs . . . . . . . . . Page 90 91 91 92 92 93 93 94 95 96 INTRODUCTION One of the most important deficiencies in research techniques avail- able to medical and biological investigators, is an accurate, non-destructive method that could be used to measure the gross composition of the animal body. It is an obvious prerequisite that such a measurement should not result in the death of the subject. Such a method would have many appli- cations in the animal industries as well as health related implications. A useful method could be utilized to predict composition either in physi- cal tenms (fatty tissue, muscle and bone) or in chemical terns (ether- extract, water, protein and ash). If selection for leanness is to be successfully employed in a breed- ing program for meat animals, it is obvious that the method must enable one to recognize "meatiness" or muscling prior to mating. In.many nutri- tional and physiological experiments, it would be desirable to measure the gross composition of the same experimental animals at the beginning and the end of an experiment in order to ascertain changes in composition. On a more practical level, there would be many advantages accruing from a method that would permit farmers to measure accurately when their meat animals were sufficiently fat for slaughter. It is, in fact, possible, that the day may come when farmers will be paid on the basis of the com- position of the animals they market. The present assessment of fatness is commonly made by "eye" or "hand" and is frequently inaccurate and wasteful. In the research laboratory, the only methods that can be used to obtain accurate body compositional data are direct analyses. In many laboratories, the expense, labor and physical difficulties involved in -2- direct analyses are sufficient to prevent such analyses from being used. This reduces the validity of the experimental work and indicates another of the areas in which a non-destructive method of measurement is urgently needed. Reviews of the many non-destructive methods that have been or are currently being investigated have been compiled by Keys and Brozek (1953), Harrington (1958) and by Brozek and Henschel (1961). Many of the methods described appear promising, but at present, no one method appears to have the qualities of sufficient accuracy and ease of Operation to be used on any widespread basis. A recent method which appears to have many advan- tages is based on the natural radioactivity from potassium-40, which is a normal component of all animal bodies. The method is easy to apply and causes a minimum of inconvenience to the subject being studied. Experimental Objectives As earlier reports did not establish the accuracy with which animal composition could be estimated from their potassium-40 content, it was decided to study the relationship between potassium content and actual composition along with the source and extent of the errors involved. It also appeared to be desirable to have an alternative to the potassium-40 method of estimating potassium. Thus flame photometry, which is a des- tructive technique, was also employed. Because it is a normal procedure in many laboratories studying potassium-40 to simultaneously measure the cesium-137 content (one of the products of nuclear weapon testing), these data were also obtained in one of the present experiments. As the liter- ature suggests the possibility of potentially useful relationships between sodium content and gross body composition, the samples that were prepared for potassium analyses by flame photometry were also analysed for sodium. REVIEW OF LITERATURE Potassium and Animal Composition Theoretical Basis - Evidence has accumulated showing that potassium is found mainly in the intracellular fluid of animals, and is present as a relatively constant prOportion of this fluid compartment for a given species (Manery, 1954; Conway, 1957; Wolstenholme and O'Connor, 1958; Robinson, 1960). The membranes of animal cells are capable of performing metabolic work, which establishes a concentration difference of ions on the two sides of the membrane (Guyton, 1956). Potassium is the main cation found in the intracellular fluid, and sodium is the main cation in the extracellular fluid. The level of potassium present in a given species has been shown to be influenced by age and sex (Spray and Widdowson, 1950; Wolstenholme and O'Connor, 1958; Anderson and Langham, 1959; Allen ggnal,, 1960). The potassium content increases quite rapidly early in life and then levels out and later slowly decreases. Anderson and Langham (1959) have shown that after 11 to 12 years of age, the potassium concentration in the bodies of human females is less than in males. Anderson (1959) has stated, "Since the concentration of potassium in living cells is held constant by homeostatic processes, a determina- tion of potassium content is equivalent to determination of cellular mass. There is no potassium in fat and very little in bone. ..... Applications to the meat industry are based on this proportionality between potassium and the mass of lean tissue." These arguments present the rationale for expecting relationships between potassitm content and composition. As most of the intracellular -4- fluid is present in the muscular tissue and organs of the body, it would be expected that the higher the proportion of muscular tissue in a sample, the higher the prOportion of potassium. 0n the other hand, the higher the prOportion of fatty tissue (containing little intracellular fluid in mature animals) the lower would be the preportion of potassium. A review of the literature showed that Anderson's statement concern- ing the potassium content of fat and bone are incorrect (table 1), unless they are elaborated to read "chemical fat" and "crystalline bone". In this connection, some confusion of terms exists in the animal and medical literature. When a medical or animal research worker refers to fat and bone, he is most often referring to the fatty tissue and green bone (con- taining marrow and in some cases a little flesh), which can be dissected or separated from the animal body. It is well known that bone marrow contains potassium (Archdeacon g£“§1,, 1961), while there is evidence from direct analyses in both the rat (Bergstrom and Wallace, 1954) and man (Casey and Zimmermann, 1960) that bone contains an appreciable amount of potassium, which cannot be explained on the basis of intracellular and extracellular fluid (table 1). However, Blaxter and Rook (1956) were unable to detect potassium in sections of the metacarpal bones of cattle. Data from the literature indicating the potassium content of muscle, fatty tissue and bone are presented in table 1. These data confirm that there is considerably more potassium in muscle than in fatty tissue or bone. Since bone forms a very much lower prOportion of an eviscerated carcass than muscle, it would seem reasonable to expect a relationship between potassium content and composition. 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The possibility of non-destructively measuring composition by means of a low level gamma ray counter capable of measuring potassium-40 activity appear to offer such an advantage. Measurement of Potassium Content from the Radioactivitygof Potass- ium-40 - Suttle and Libby (1955) have reported that potassium-40 comprises 0.0119 percent of the natural potassium isotOpes and has a half-life of about 1.25 x 109 years. Potassium-40 emits 10 beta particles for every gamma ray. The natural mixture of potassiun isotOpes emits 2.96 gamma rays per second per gram with an energy of 1.45 Mev. Kulwich gtflgl, (1960a) stated that, "Potassium from different sources does not vary by more than i 0.5% in its potassium-40 content (Vinogradov, 1957), so that a determination of the potassium-40 content of a biolo- gical sample should provide an excellent index of the total amount of potassium present. Potassium-40 is the principal naturally radioactive isotOpe present in all organisms. The data of Vinogradov (1957) indi- cates that, in the case of humans, there is 7 times as much radioactivity emitted by potassium-40 as there is by the next most prevakant naturally radioactive isotOpe, carbon-l4." Anderson (1958) described how the danger of counting the gamma radio- activity from cesium-137, one of the products of nuclear weapon testing, can be minimized by limiting the range of the gamma spectrum from which counts are recorded. As the gamma rays from cesium-137 have an energy of 0.66 Mev. it is possible to simultaneously record the activity from this source and the activity of potassium-40 (1.45 Mev.) on separate channel settings of the detection apparatus, with a small spill over of counts from the other channel. The natural radioactivity of radium and thorium contribute many more counts to the lower energy channel than does potassium, and the main fission products in fallout such as ruthenium-103 and rhodium-106 will be counted almost exclusively in the lower channel (Anderson, personal communication). It can therefore be seen that the measurement of the penetrating, high energy gamma rays of potassium-40 furnish a non-destructive method of measuring the potassium content of biological material. The first determinations of body potassium by means of its natural radioactivity were reported by Sievert (1951, 1956) and by Burch and Spiers (1953), using large high-pressure ionization chambers. Although the early work was mainly directed toward detection of small amounts of the other gamma emitters, Sievert noted a change in potassium content with age and differ- ences between the sexes. He explained the differences in terms of body composition. Burch and Spiers (1953) estimated the potassium content of 13 human subjects to be 0.21% of body weight. Measurement of Potassiuerontent by Flame Photometry - Flame photo- metry offered an alternative but destructive method of measuring the potassium content of animal tissues. A very thorough review of the field of flame photometry has been given in a recent book by Dean (1960). Since different methods of preparing the samples for flame photometry are reported in detail later in this thesis, the pertinent literature will be reviewed and discussed at that time. Relationships between Potassium Content and Composition - Cheek and West (1955) investigated the potassium content of 30 rats using flame D photometry. They plotted a regression equation which showed that lean body mass was related linearly to total body potassium. Woodward ggugl. (1956) demonstrated a linear relationship between the gamma activity and lean body weight of 13 human subjects. However, no error of estimate was given. Kulwich ggmgl. (1958) reported a highly significant correlation between the gamma activity per pound and the percent of fat-free lean from two pairs of hams at various stages of subdivision (the radioactivity was attributed to potassium-40). The gamma activity was also negatively correlated with percent fat. Zobrisky g£_§1. (1959) reported that the potassium-40 content of live hogs may possibly be useful as a rapid, non- destructive index for determining meatiness. Up to 1959 the results available showed that significant relationships occurred, but did not give any indication of the accuracy for predicting composition. In 1960 (a), Kulwich £5.31. used a destructive procedure to measure the beta radioactivity (from potassium-40) of 34 meat samples taken from pork hams. The standard errors for the regression equation using the beta radioactivity to predict the percentage of the chemical components and their correSponding correlations were as follows: ether-extract -- 2.2% (r = -.99); protein -- 0.64% (r = 0.99); moisture -- 1.7% (r = 0.99). Later Kulwich st 31. (1961a) related the potassium-40 gamma activity of 34 hams to their separable components which were expressed on both a weight and a percentage basis. Potassium-40 (net count per minute per pound of ham) was significantly correlated with percent separable lean (0.87) and percent separable fat (-.86) in the hams. The standard error of the regression equation for predicting percent separable lean was 2.5 in the hams studied, which had a range of 47-68% in separable lean. -10- Kulwich gt El, (1961b) carried out a similar experiment with 16 beef rounds. The potassium-40 content was correlated with percent separable lean (0.80), percent separable fat (-.87) and percent separable bone (0.06). The standard error of the regression equation for predicting lean from gamma activity was 2.1%. A group of German workers (Pfau 25 31., 1961) have shown a close relationship between the potassium-40 content and the amount of lean in hams (r = 0.90). Research has also been undertaken on human subjects, relating potass- ium content to composition. In order to make comparisons, the composition had to be determined non-destructively by some indirect method. Allen gtflgl. (1960) demonStrated a linear relationship between the potassiun content and M3 (residual mass of body after removal of bone mineral, fat and water from gross body mass) for a group of subjects, but no estimate of the accuracy of prediction was given. M3 was estimated from body water determinations. A new approach to the problem of estimating fat content of the living human being has been claimed by Forbes gtflal. (1961). They related the potassium-40 activity of children and young adults to their skinfold thickness measurements and weight:height ratio, both of which were re- garded as indices of fatness. Forbes 25.31, reported that the correlations between the potassium-40 measurement and average skinfold thickness was 0.80 for the males, and with weight/height was 0.56 (males only). The data were plotted but no regression equations were presented to show the accuracy of prediction. In ensuing publications, (Anderson and Langham, 1961; Forbes and Hursh, 1961) the validity of the claim to have develOped a new method for a non-destructive measurement of fat content was discussed. -11- It would appear in the light of the literature reviewed that the claim is not completely justified. Sodium and Animal Composition Theoretical Basis - There is some evidence to suggest that the sodium content of animals might be related to their composition. It is fairly well known that sodium ions form a relatively constant pr0portion of the extracellular fluid (Keys and Brozek, 1953; Manery, 1954), in fact, the dilution volume of radioactive sodium is often used as a measure of the extracellular fluid space. The sodium dilution volume is regarded as a measure of exchangable sodium. Exchangable sodium differs from total body sodium because of a sizable pool of slowly exchangable bone sodium (Edelman, 1945a, b; Bergstrom and wallace, 1954; Forbes and Perley, 1951; Casey and Zimmerman, 1960). Edelman (1961) explains the distribution of sodium in the body in the following words: "The distribution of body sodium is unusual because of the complex nature of bone sodium. There appear to be three distinct phases of bone sodium: (a) free extracellular sodium (presumably all ex- changable), (b) exchangable sodium adsorbed to the surface of bone crys- tals, and (c) nonexchangable bone sodium in the crystalline structure. ..... These estimates indicate that total exchangable sodium represents about 70% of total body sodium." Exchangable sodium in human beings varies little between sexes and with age when expressed on a per unit weight basis (Edelman, 1961). In healthy animals, the extracellular water comprises a relatively constant part of total body water, which is known to be related to body composi- tion (Keys and Brozek, 1953). Thus, it would seem reasonable to investi- gate the relationship between sodiun content and composition. -12- Relationship of Sodium Content to Composition - Edelman (1961) has stated, "Accordingly, one might expect a close dependence of plasma sodium concentration on the amounts of sodium, potassium and water in the body. This, in fact, is precisely what has been foun ." However, Edelman did not relate exchangable sodium to body water, which would have been the interesting relationship from the point of view of the present eXperiment. Blaxter and Rook (1956) present an equation from which the water content of cattle tissue may be estimated from the sodium and potassium content. The above experiments suggest that it could be of interest to relate the composition of tissue or of entire animals to their sodium content. Cesium-137 and Animal Composition. Cesium-137 was discovered in the late 1940's, and it is well known that this isotope is produced by fission of uranium-235 in atomic energy reactors (Thoraeus, 1961). Of greater concern is the fact that this iso- tOpe is one of the longer lived by-products of nuclear weapons testing (Langham and Anderson, 1959; Langham, 1961), with a half-life of approxi- mately 30 years. Theoretical Basis - In animals, cesium-137 is found in the same tissue sites as potassium (Langham, 1961). The level in animal tissues is being followed with interest in many countries, because of the potential danger of radiation damage to man and other animals if the concentration should become too high (Langham, 1961). The cesium-137 level gives an indication of the amount of radioactive fallout. Because the tissue distribution of cesium-137 is similar to that of potassium, it might be expected that there would be a relationship between cesium-137 content and composition. -13- .Rglationship of Cesium-137 Content to Composition - Kulwich ggugl. (1961a) reported a correlation of 0.47 between the weight of lean in pork ham and the cesium-137 content. Pfau §£_§1, (1961) noted a correlation of 0.78 between the percent lean in hams and their cesium content. In both cases, these correlations were lower than the equivalent correlation coefficients for potassium which were 0.96 and 0.90, reSpectively. Kul- wich 25 al. (1961b) have reported similar results for beef rounds. In this case, the correlation between the cesium-137 content of the round and the weight of lean was 0.81, which was lower than the equivalent po- tassium-40 correlation (r = 0.98). DEVELOPMENT OF FLAME PHOTOMETRY PROCEDURES MATERIALS AND METHODS Apparatus A Beckman Medel D.U. SpectrOphotometer with MOdel 9200 flame attach- ment was used. The flame attachment was fitted with an oxygen-hydrogen burner and its Operation has been described in Beckman Instrument In- struction Manual 334-A. The 768 mp.wave1ength setting was used for potassium determinations, and the 589 mp.setting was used for sodium. The burner was Operated at a pressure of 6 lb. per square inch of hydrogen and 13 lb. per square inch of oxygen. This gave the Optimum burner conditions according to the man- ufacturers recommendations. The photometer was Operated on phototube position 1 (with filter at in position) for potassium and phototube 2 (filter out position) for sodium. A slit width of 0.01 - 0.03 was used for sodium and 0.15 - 0.30 was used for potassium. A sensitivity setting of 5 was used on the power supply unit, while the selector switch was set at position 0.1. Sample Preparation Before flame photometry can be used to measure the concentration of an element in a sample, a method must be employed to extract the element and get it into a solution that is suitable for atomization. Such a so- lution must not contain any substance that will clog the very fine atomizer burner tube. Several methods have been successfully employed to extract potassium from animal tissues and fluids (Dean, 1961), and several methodology -14- -15- studies have been made in this area. Grove §£_§1, (1961) compared wet and dry ashing with particular reference to the temperature at which sam- ples may be ashed without getting losses. It was found that the maximum temperature at which animal tissues may be dry-ashed for a 24 hour ashing period without loss of sodium and potassium is 550°C for l - 5 gm. same ples. Stone and Shapiro (1948) boiled muscular tissue under reflux and compared the potassium content of the supernatant liquid with the potass- ium remaining in the tissues. They reported that the same concentration of potassium was found in the boiled tissues as in the supernatant liquid. Siebert 25 21. (1951) compared dry ashing of tissue with boiling under reflux, as methods of extracting the potassium from the tissue, and found that the two methods gave results that were in agreement to within about 4%. MOunib and Evans (1957) compared homogenization in 2% trichloracetic acid (TCA), boiling under reflux, and boiling under reflux plus acidifi- cation as methods of extracting sodium and potassium from tissue samples. Homogenization in 2% TCA gave results which appeared to be reliable, and in the case of the boiled tissue samples, it appeared that acidification was necessary to get complete ion release from some tissues. The method of homogenization in 2% TCA, which was subsequently adapted by Mounib and Evans (1960), was suited to the equipment available in this laboratory. However, it seemed advisable before using this method on a routine basis, to compare it with another method. The TCA method seas finally accepted as the routine method for potassium and sodium ex- ‘traction from the tissue, so it is described in detail. A 2% TCA solution (w/v) was made by dissolving 40 gm. of TCA in 2000 IIiL. of de-ionized distilled water. Between 1.5 and 3.5 gm. of previously -16- ground tissue was accurately weighed and transferred by washing into an aluminum blender. The sample was homogenized for 5 - 10 minutes with 150 ml. of 2% TCA (measured with pipettes). The solution was then transferred to a 250 m1. Erlenmeyer, which was stOppered and stored Overnight in a 38°C cooler. The length of storage following homogenization (0 - 48 hours) was found to be without effect on the results. The stored samples were filtered through Whatman No. 40 filter paper into polyethylene storage bottles, and 5 ml. of this solution was made up to 15 ml. in a test tube with 2% TCA solution. The test tube was capped with "Parafilm" and shaken to get complete mixing. In addition to the method adapted and outlined above three other ex- traction procedures were compared. Thz first of these involved oven ashing at less than 550°C for 24 hours in porcelain crucibles, after a preliminary rough ether-extraction (diethyl ether), if the samples were very fat. The ash was then dissolved in a few drOps of concentrated HCl and diluted with 150 m1. of 2% TCA solution. The second method of extraction utilized wet ashing with nitric and perchlotic acids after a preliminary rough ethervextraction. The proce- dure outlined by Benne and Lindon (1960) was followed after some minor modifications. After digestion was complete and the liquid evaporated, the sample was dissolved in 150 ml. of 2% TCA solution. The last of the three methods involved refluxing the sample for 30 minutes in 150 ml. of de-ionized distilled water, followed by acidification with a few drops of HNO3 as recommended by Mounib and Evans (1957). Following extraction the procedure for all methods was the same. Five ml. of the extracted sample was diluted to 15 ml. in a test tube -17- with a 2% TCA solution. The test tubes were always covered with "Para- film" and shaken to ensure complete mixing. Blank solutions were prepared and all samples were diluted using a 2% TCA solution. The photometer sample beakers were washed with the solution that they were to contain before being filled for a series of readings. Standard Solutions For the first two experiments which involved both muscular and fatty tissue, a liter of primary standard containing 1000 parts per million (ppm) potassium and 200 ppm sodium was prepared as suggested by Dean (1960). Analytical grade KCl was used as the source of potassium (K) and the same grade NaCl as a source of sodium (Na). De-ionized distilled water was used for making up the standard. The solution was then stored in a poly- ethylene bottle. Fifteen ml. of the primary standard was then.made up to 500 ml. (vol- tmetric flask) with 2% TCA solution. This gave a concentration of 30 ppm potassium and 6 ppm sodium. Then 100 ml., 75 ml., 50 ml., 30 m1. and 10 m1. of this solution was pipetted into polyethylene bottles (4 ounce capa- city) and made up to 100 ml. with 2% TCA solution by pipette. This gave the range of 30, 22.5, 15, 9, 3 and 0 (blank TCA solution) ppm of potass- ium and 6, 4.5, 3, 1.8, 0.6 and 0 ppm sodium. These points were used in plotting the standard curve. In experiment III, it was found that some of the body components of the pigs being analyzed had a higher Na:K ratio than was suitable for analysis using the original primary standard of 1:5. Thus, a new primary "Standard containing 1000 ppm potassiun and 1000 ppm sodiun was prepared 311d was diluted to give the desired strength for the standard curve. -18- Samples In order to compare methods of extraction of potassium and sodium, a homogeneous sample of ground pork was utilized. A.series Of analyses was made on the pork after it had been extracted by both TCA homogenization and the oven ashing methods. When the two methods of extraction failed to agree, a recovery trial was carried out by running 5 ml. of the pri- mary standard containing 1000 ppm potassium and 200 ppm sodiun completely through the two extraction methods. The potassium and sodium contents were computed on the basis that they were present in 1 ml. of the original solution (i.e. containing 5000 ppm potassiun and 1000 ppm sodiun). Using the TCA extraction method, it was possible that organic mater- ial could cause erroneous results by altering emission intensity (Dean, 1960). Thus, it was decided to use an acid-ashing procedure, which would destroy any organic material. A fourth accepted method, involving boil- ing of the sample was also used for comparative purposes. Ground beef and another ground pork sample were used for comparing all four methods of extraction. Calculations A standard curve was prepared by plotting percent transmittency against known concentrations of potassium and sodium. The potassium and sodium content of the tissues was computed from the concentration of these elements, which were estimated from the standard solution prepared for flame photometry. This concentration was multiplied by a dilution factor and finally corrected for the blank reading. The dilution factor as 150 + x x grams, and the factor of 3 allows for the dilution of 5 ml. of the homo- w x,3, where X is the weight of the tissue sample analyzed in genized, filtered sample to 15 m1. _19- RESULTS AND DISCUSSION The results of various methods of extraction upon potassium and sodium analyses are shown in table 2. On the tissue samples, the TCA homogeni- zation extraction procedure resulted in higher potassium estimations and lower sodium estimations than the oven ashing procedure. The lower stan- dard deviation of the TCA method showed that it was more reproducible than oven ashing. As these two extraction procedures gave different estimates, it seemed to be desirable to run known quantities of potassium and sodium completely through the procedures to see if any loss or gain of the ele- ments occurred due to faulty methodology. The results suggested that the TCA extraction was more accurate for potassium and that an apparent loss of sodium occurred in the ashing procedure. The recovered elemental levels were Opposite to what would have been predicted from the tissue analyses. NO explanation for this is known. There were several possible reasons for the different results observed on the tissue samples. A loss of potassium might have occurred in oven ashing if the oven controls were inaccurate. Grove 25 31. (1961) have noted that the maximum temperature at which animal tissue may be dry-ashed for a 24 hour ashing period without loss of sodium and potassium is 550°C. However, this would not explain the larger sodium content observed in the oven-ashed samples. Another possible explanation was that an exchange of sodium and potassium occurred between the ash and porcelain crucible sur- face at high temperatures. A third possibility was the enhancement of the emission intensity from the TCA extracted samples due to the possible IJIesence of organic components in the solution (Dean, 1960). .mcowum>HomnO mo Hones: n a .oaeewm some mo momaameo poumoeou scum weeuasmou Aaee n GOHHHHE you wanna efiv meowuma>op pudendum H momma mum pouaomoue mosam> . 0 n_ cm H «mm m eHs H Np H oon s a H Ham m xHom ems H see H so H awn H aH H mes a «mom - - mm H was H omH H Baa 0 Hz sea oooH - - mom H mmm a mo H awe a xHom asHpom ma H meas m Hmms H mma H ammm s mH H HHHH m HHom osma H Home H aHH H omea s mm H Home a prm - - mma H amen H mm H moon s a sea ooom - - mom H mmHm a HHH H oHos a xHom aaqmmmuom amoz a com: a and: a can: a oaeawm posed pau< Mmzm.+ poaaom ponmm co>o nowumwweowoeos <99 ueoaoam .asapOm pew anammmuoa mo coauomnuxo mo mposuoe u:ouommap mo aomwuomaoo < .N manna -21- Some of these suggested difficulties could be overcome by the use of chemical ashing as a method of comparison. In this method, high tempera- tures were unnecessary and the organic material was destroyed. Table 2 shows the results following chemical ashing. Chemical ashing and a fourth procedure involving boiling of the samples gave estimations Of the sodium and potassium content of the tissues that agreed with those Obtained from the TCA homogenization method, but disagreed with the results obtained after oven ashing. It is not known why the oven ashing procedure, which has apparently been successfully employed in many laboratories, gave erroneous results in the present eXperiments. Possibly, better results would have been Obtained if platinum crucibles had been used. The re- sults suggested that the TCA homogenization method was reliable. The trend for decreasing variability Of results with the increased use Of the method suggested that the estimations were increasing in accuracy as ex- perience was gained in using the procedure. SUMMARY AND CONCLUSIONS A comparison was made of four methods of extracting potassium and sodiun from muscle samples. The methods included homogenization in 2% TCA, Oven ashing, acid ashing or boiling in water followed by acidifi- cation of the solution. Results suggested that oven ashing was inaccur- ate as an extraction procedure. Extraction by homogenization in a 2% TCA solution was found to be reliable and readily adaptable to the equip- ment available. EXPERIMENT I. LOS ALAMOS LAMBS EXPERIMENTAL Counter The Los Alamos four Bi liquid scintillation counter (Fig. I, II) was used to measure gamma activity. The Los Alamos counter is a well-type counter which can accommodate sanples weighing up to 300 1b. Because the scintillation solution surrounds the sample, the geometrical efficiency approaches 100% and makes a very short counting interval possible. Anderson (1958) has described the counter as follows: "The counter itself is a cylindrical steel tank 6 ft long and 30 in. in diameter." (see Fig. III). "An axial well 18 in. in diameter accommodates the sub- ject or sample which is surrounded by a layer of liquid scintillation solution (terphenyl and POPOP in toluene) 6 in. thick. The counter is shielded by 5 in. of lead. Scintillations are detected by 108 photomul- tipliers (2 in. diameter cathodes), which Observe the solution through ports in the outer wall. The photomultipliers are connected in two banks of fifty-four tubes each, which are Operated in coincidence in the usual manner. The energy resolution of the system is not good, but is adequate to separate gamma-rays whose energies differ by a factor of 2 or more. Thus, the machine is usually operated for simultaneous counting in two energy channels: 1 - 2 MeV, giving ordinarily only the natural K40 (1.45 MeV gamma) activity, and the 0.5 - 0.8 MeV, giving some K40 activity (easily calculable from the upper channel count) and also any 03137 acti- ‘vity (0.66 MeV gamma) present in the sample." In the present experiment the potassium determinations from the upper channel were based on the gamma rays which deposit more than 0.8 -22- -23- Figure I. General view of Los Alamos human counter. -24- -25- Figure II. Method of restraining animals for counting. -27- Figure III. The Los Alamos human counter outside its lead shield showing the banks of photomultiplier tubes. -29- Mev. in the scintillator. Animals Ten recently shorn, blackfaced lambs with a mean liveweight of 88 lb. were purchased from a feedlot at LaJara in southern Colorado in March of 1960, and tran3ported to Los Alamos, New Mexico. Counting and Sampl ing Because of the possibility of potassium and other radioactive con- tamination on the wool and skin, counts were made on the live lambs both before and after they had been thoroughly washed with detergent and warm water. It was found that a second washing did not reduce the gamma acti- Vity below the level accomplished by one washing. The animals were res trained during counting in a cardboard drun in the same manner as the dog shown in Fig. 11. After being counted, the lambs were slaughtered and dressed in the us Dal manner. The carcasses and the non-carcass components of the animals we re counted for potassium-40 activity. The non-carcass components in- cluded the hide, feet, head, blood and also all of the internal organs and their contents. Three lOO-second counting periods were used in all ea~Ses, except for the carcasses which were counted for five lOO-second I)eili‘iods. The carcasses were physically separated into fat, lean and bone and each component was counted separately for potassium-40 activity. The fat and lean were then ground separately and sampled for chemical analy— ses. The combination of the separable fat plus lean tissue of each lamb Q0I‘responds to the edible or boneless carcass. Mm Analyses Three to 7 gm. samples of ground tissue were taken for analysis of -30_ water and ether-extract. Water was determined by difference after drying for 24 hours at 100°C (Benne _e_t_§_l_., 1956). Ether-extract was determined on the water-free sample, which was dried in a disposable aluminun dish and then extracted with diethyl ether in a Goldfisch apparatus (Hall, 1953). Protein was determined on 1-3 gm. samples of tissue by the Kjel- dahl method as described by Benne g; 3.1. (1956). Potassium and Sodium Estimations The total potassium content of the live lambs and their various com- Ponents was estimated from potassium-40 counts. The raw courting data and efficiency calibrations were entered on punched cards, and calcula- tions of total grams of potassium per kilogram of material were carried Out on an I.B.M. electronic computer, which tabulated and printed the results (Anderson 91331., 1959). It should be noted that l mu. of potass- iutn per kilogram of tissue is equal to 0.1% potassium or 1000 ppm potass- ium. The potassiun and sodium content of the separable lean and fat from the carcasses was determined by flame photometry on the samples that had Ibeen taken for gross chemical analyses. The method of extraction of the I)CDtassium and sodiun from the tissues by homogenization in 2% TCA and the conditions used for flame photometry have been described earlier in this thesis. Duplicate analyses were carried out on each tissue sample. %m-137 Estimations The raw counting data obtained from the lower channel setting were a”180 entered onto I.B.M. cards, and the computer tabulated and printed the results as micromicrocuries (FPO) of cesiun per gram of potassium. -31- RESULTS AND DISCUSSION Animal Composition The weights and composition of the lambs are presented in table 3. It may be noted that the variation in liveweight was not extreme. The range of separable components in the carcass amounted to about 18% in fat content and 10% in lean content. The data on the chemical composition of the boneless carcasses show that the range in percentage for both ether-extract and moisture was 20.1 and for protein 4.3. Table 3. Weight and composition of the lambs Item. ‘Mean S.D. Range Liveweight (1b.) 87.9 9.5 76.6 - 105.5 Hot carcass weight (lb.) 45.7 5.9 38.2 - 56.3 Non-carcass components (lb.) 42.3 3.9 35.6 - 48.5 Carcass composition Separable fat (1b.) 9.7 3.5 4.1 - 14.1 Separable fat % 20.8 5.9 9.7 - 27.8 Separable lean (1b.) 27.1 3.1 23.4 - 33.0 Separable lean % 59.4 3.2 55.3 - 65.5 Bone (1b.) 7.5 0.9 6.1 - 8.4 Bone % 16.5 2.1 14.4 - 20.4 Edible carcass composition Ether-extract % 22.7 6.9 9.1 - 32.5 Protein % 15.7 1.4 14.0 - 18.3 Water % 60.9 5.6 52.7 - 72.8 Potassium Content and Composition The total estimated potassium content of the live lambs and their components is presented in table 4. The mean potassium content was esti- mated to be 108 gm. in the unwashed lambs and 71 gm. in the washed lambs. Washing the lambs reduced the potassium content from 0.271 to 0.177% or -32- Table 4. Potassium content of 10 live lambs and their components as esti- 'mated from potassium-40 counts and from flame photometpy Counting Mean' errorsa Item % K S. D. Range S.D. (%) Potassium-40 Live unwashed lambs 0.271 0.030 0.229 - 0.316 2.29 Live washed lambs 0.177 0.020 0.147 - 0.209 3.56 Dressed carcass 0.225 0.033 0.171 - 0.262 3.68 Non-carcass components 0.178 0.039 0.094 - 0.233 6.56 Separable fat 0.070 0.044 0.013 - 0.154 66.41 Separable lean 0.298 0.020 0.267 - 0.319 5.35 Separable bone 0.141 0.048 0.071 - 0.206 34.85 Flame photometry Edible carcass 0.255 0.028 0.219 - 0.297 -- Separable lean, run 1 0.313 0.014 0.298 - 0.340 -- Separable lean, run 2 0.316 0.016 0.286 - 0.340 ~- Separable lean, run 2, fat-free basis 0.340 0.017 0.306 - 0.363 -- Separable fat 0.082 0.023 0.056 - 0.126 -— Separable fat, fat-free basis 0.239 0.031 0.209 - 0.310 -- aStandard deviation computed by the method described by Comar (1955) expressed as a percent of total counts. Based on mean sample count (10 observations) and mean background count. on average 37 gm. of potassium was removed from each animal by washing. Analysis of variance showed that the difference in potassium content be- tween the washed and unwashed lambs was highly significant. While the potassium activity dropped to 0.65 of its original value with a standard deviation Of 0.07, the apparent total amount of cesium-137 present re- mained at 0.99 of the unwashed level, with a standard deviation of 0.12 (Anderson, personal communication). This strongly suggests that the radioactivity removed was almost entirely potassium. The natural activi- ties of radium and thorium contribute many more counts to the lower energy channel than does potassium, and the main fission products in fallout such as ruthenium-103 and rhodium-106 will be counted almost exclusively -33.. in the lower channel. If any of these activities were being removed, the apparent cesium-137 activity would droP significantly. The presence of potassium in the wool is not difficult to account for, since the external secretion of the sheep are rich in potassium, much of which is trapped in the wool. In fact, Kulwich pg g1. (1960b) suggested that the radioactivi- ty of wool measured in the upper channel (potassium setting) of a low level gamma ray detector might be used as a non-destructive method for measuring the impurities present in unscoured wool. NO satisfactory explanation can be given for the fact that the total potassium content on adding the carcass and non-carcass components together was greater than the potassium content for the live washed lambs. Any self absorption of the gamma rays that occurred in the larger samples, such as for the live animals, should have been allowed for in the calibra- tion of the counter. A highly significant correlation of 0.84 was found between the potassium content of the carcass and the non-carcass compon- ents. Since much of the intracellular fluid, which contains potassium, is present in the non-carcass components, a positive relationship would necessarily exist between the potassium content of the carcass and non- carcass components in order to accurately predict carcass composition from the gamma counts of the live animal. When the potassium content of the carcass was computed from the weight of the component tissues and their potassium content as estimated from potassium-40, it was found that a level of 0.222% of potassium was present. This value agreed well with the figure of 0.225% potassium when the radioactivity of the intact carcass was counted. This suggests that the average values for the potassium content of the separable fat and -34- bone are approximately correct. The presence of potassium (or radioactive material) in fatty tissue and bone (table 4) has also been shown by Kul- wich £5.31. (1961a, b) and Pfau ggngl. (1961). The counting errors given in table 4 show that the estimated potassium content of the fatty tissue or bone from any particular animal is likely to be inaccurate. This is because the small sample size in relation to the size of the counter re- sulted in a small sample count in relation to the large background count, which has some sort of a normal level for a given counter. The variation of the means for separable lean and fat based on the 10 duplicate analyses (making up any of the flame photometry potassium means, (table 4) was separated by analysis of variance into components due to differences between individual lambs and an error term. The stan- dard error of the duplicate means was computed from the error mean square and found to range from 15 to 25 ppm of potassium, which suggested reason- able agreement between duplicate samples. .As shown in table 4, there was agreement in the average values ob- tained for the potassium content of the separable lean or of the fat as measured by flame photometry or by the natural gamma activity. For the lean, analysis of variance showed that although the difference between the flame photometry mean of run 2 and the potassium-40 mean was small, it was significant at the 5% level. Similar results were reported by Pfau 2E 31. (1961), although they did not report on the variation of the two methods or whether the means had been tested for significant differ- ences. The present results and those of Pfau ggngl, (1961) confirm the presence Of potassium in fatty tissue at levels that cannot be ignored. Thus, the results from direct chemical analyses show that the statement -35- by Anderson (1959) that, ”There is no potassium in fat", is incorrect un- less qualified. In the present experiment, the results for both lean and fatty tissues were found to have lower standard deviations for potassium content when.measured by the flame photometric method than from the potassium-40 method. Since the investigators suSpected that there were important day-to- day variations in the flame photometry results during the course of the first run, a second set of potassium determinations was carried out on the lean tissues. Thus, all determinations in run 2 were carried out on the same day and were calculated from the same standard curve. When the duplicate means of run 1 were correlated with the duplicate means of run 2, a highly significant correlation Of 0.88 was found. How- ever, the standard error Of the regression equation for predicting the potassium content of the lean in run 1 from run 2 was found to be 70 ppm, which indicates that run-to-run errors in measurement are an important source of variation. Factors contributing tothe differences will include sampling errors and slight differences which occur in the standard curves. The variation could also result from small differences in the rate of atomization of the solutions, and other factors apparently inherent in the photometer and which cannot be precisely controlled. When the potassium content of the separable fat as measured by flame photometry was correlated with the potassium content of the separable fat as measured by potassium-40, a non-significant correlation of 0.40 was found. The potassium content of the separable lean as measured from po- tassium-40 was correlated with the flame photometrically determined po- tassiun«content of the lean in the two runs. Non-significant correlations -36- Of 0.57 and 0.52 were found in run 1 and run 2, respectively. However, a great deal of weight should not be placed on the lack of agreement be- tween the individual values for the potassium content of the tissues as estimated by the two methods. The counting errors in table 4 show that the potassium-40 method would not be expected to give an accurate esti- mate of the potassium content of the separable fat or lean. A range of 0.27 to 0.34% of potassium was observed in the separable lean of the lambs on combining the data from both methods. These results compare favorably with a range of 0.20 to 0.30% for lamb muscle as re- ported by Toscani and Buniak (1947), 0.27 and 0.31% reported by Harris .g£.§l. (1952) for sheep muscle, a figure of 0.30% for sheep muscle re- ported by Blaxter and Rook (1956), and mean figures in the order of 0.43% for the fat-free, blood-free biceps femoris muscle of sheep reported by Mounib and Evans (1960). As the separable lean in the present experiment contained from 4 to 10% chemical fat, the correction of the potassium content to a fat-free basis increased the potassium figures on average by 0.025% to give a value of 0.34%. Further work is needed to establish the potassium levels of different muscles of different breeds and Species of animals before accurate comparisons can be made. In a similar manner, the conversion of the potassium content of the separable fat (which con- tained from 41.2 to 77.4% chemical fat) to a fat-free basis increased the calculated potassium content from 0.082% to 0.239%. It is interesting to note that on this basis the muscular tissue has a higher potassium content than the fatty tissue. The K/N ratio has been suggested as a measure of the constancy Of the potassium content of various tissues and organs. The mean K/N ratio -37- (wt./wt.) in the separable lean was 0.1045 (S.D. = 0.0047; range = 0.0936 to 0.1115) and in the separable fat was 0.0753 (S.D. = 0.0049; range = 0.0677 to 0.0818). These results present the same picture as when the potassium content was expressed on a fat-free basis with the lower potass- ium content being found in the fatty tissue. .Table 5 shows the relationships between the potassium content and other variables for the lambs. The potassium content of the edible car- casses (by flame photometry) was computed from the percentages of potass- ium in the separable fat and lean (run 2) and the weights of these tissues. Table 5. Correlations between the potassium content of live lambs and carcasses and carcass composition. Flame From potassium-40 photometry Live Live Dressed edible unwashed washed carcass carcass Item, % K % K % K % K Live washed lambs, % K (K-40) 0.66* - - - Dressed carcass, % K (K-40) 0.30 0.40 - - Separable fat, % of dressed . carcass -0.79** -0.73* -0.38 -0.92** Separable lean, % of dressed carcass 0.57 0.58 0.52 0.81** Separable lean, 3 of dressed carcass, on a fat-free basis 0.66* 0.63 0.57 - Separable bone, % of dressed carcass 0.86** 0.78** 0.14 0.81** Ether-extract, % of edible carcass -0.79** -0.71* -0.41 -0.87** Water, % of edible carcass 0.77** 0.65* 0.40 0.81** Protein, % of edible carcass 0.80** 0.83** 0.41 0.94** * Correlation significant at 5% level. **Correlation significant at 1% level. While most of the potassium in the dressed carcass was present in the muscular tissue (82%), the correlation between the estimated potassiun content of the live animal and the lean content of the carcass was not significant. If, however, the percentage lean in the carcass was corrected -38- to a fat-free basis, the correlation between the potassium content of the unwashed live animals and percentage lean was just significant. These correlations, which were very much smaller than would have been anticipated from results that have been published in the literature, can perhaps be explained in part by the small variability of the percentage lean in this group Of animals. The percent of separable lean of the 10 carcasses showed a standard deviation of 3.2% (table 3). However, the counting statistical error as given in table 4 is 5.4% for the lean and 3.7% for the carcass. Since the variability of the carcasses as deter- mined by dissection is less than the precision of the potassiun measure- ments, a significant correlation would not be expected. Percent separable fat in the live animal or carcass would be expected to show a negative correlation with potassium concentration, since fat contains comparatively little potassium. As the dissection data show a larger variability for fat than lean (S.D. of 5.9% versus 3.2%, table 3), it was not surprising that significant correlations were found between potassium in the live animal and separable fat in the carcass (table 5) in contrast to the results for lean. The fact that the standard devia- tion for separable fat (5.9%) was larger than the error in estimation of potassium content of the live animals and carcasses (table 4) is in con- trast to the data for separable lean. Although the correlations between separable fat in the carcass and the potassium content of the live animal were significant, they were not high enough for practical application. The reason for the positive correlation between percent potassium (from potassium-40) and percent bone is not clear. It is known that per- cent bone tends to follow muscle percent in growing animals, but the -39- variability Of bone in these animals was even less than the variability in separable lean (S.D. of 2.1% versus 3.2%, respectively). Thus the positive correlation between potassium in the live animal and percent bone needs further verification. Although it might be anticipated that skin and wool contamination of the unwashed lambs would reduce the relationship between the amount of potassium in the live animal and carcass composition, for some unknown reason the Opposite situation was found to be the case. In general, washing lowered the correlations between the potassium content of the live animal and the various components. The counts on both the live animals and carcasses have been related to carcass composition. In the case of live animals, it seemed likely that the potassium in the non-carcass components would lower the relation- ship with carcass composition. However, this was not found to be the case, as none of the correlations between carcass potassium content (as estimated from potassium-40) and the percent of the separable components of the carcasses were significant. This is in marked contrast with the comparable correlations with percent potassium in the edible (boneless) carcass as estimated from.flame photometry and the percent of separable components in the dressed carcass. For all separable components, these correlations were highly significant. It is not considered that the ex- clusion of bone potassium, which is a small proportion (11%) of carcass potassium, is the explanation for the significant correlations observed. Evidence already presented suggests that bone is positively correlated with the potassium content of these lambs, and so the presence of bone potassium when the potassium-40 method was used to measure dressed carcass -40- potassium content should not greatly alter these relationships. The ex- planation for the significant correlations when flame photometry was used to measure potassium content would appear to be due to the greater accur- acy for estimation of potassium than for the potassium-40 method. The correlations between the potassium content of the 10 lambs and the chemical composition Of the edible portion of their carcasses are also presented in table 5. As was the case for the separable tissues, significant relationships were found with the radioactivity counts for the live animals, but not for their carcasses. The highest relationships were found between the potassium content of the live animal and the per- cent protein in the edible tissue. This would be expected if most Of the potassium were present in the muscular tissue or lean. Since most of the body potassium is present in the intracellular fluid (Wolstenholme and O'Connor, 1958; Conway, 1957; Robinson, 1960) and most of the carcass intracellular fluid is present in.muscular tissue, it might be exPected that the highest relationships to potassium-40 would be with percent lean in the carcass and/or with percent protein in the edible carcass. The highly significant correlations between the percent potassium (flame pho- tometry) in the edible carcass and its composition were in contrast with the non-significant correlations between the potassium content (from po- tassium-40) for the dressed carcass and the composition of the edible carcass. This again suggests that the use of flame photometry resulted in more accurate estimations of the potassium content than the use of the potassium-40 method under the conditions of application. Table 6 gives the regression equations which could be used for pre- dicting carcass composition from the potassium content of the live animals -41- es.m mm.a- +.x~.~oH u w AooooHoo o-oHpo .oao-mv e a Hoop: a ew.m mm.- + x-.mHH u w Apoeooooo o>HH .os-ev e e Hoop: a so.m xm.-H - on.os u » Aooooooo o-oHpo .oaoHeV a a Hoe a NH.m xo.ooH - om.oo u w Aeoeoos ooHH .oe-ev a e Hoe a em.o xo.me - mm.~e u H Apoooosoo ooHH .oe-ev a a How a em.o mo.m +.x~.ko n w nooooHoo o-oHpo .otoHeV a a oHoHon a xw.o HH.m +.em.om u H Apoeooa o>HH .oe-ev e a aHoHon a so.H oe.m + xo.mm n w Aoonooooo ooHH .os-ev e a oHooon a m mmOku QHSH Um sm.- oH.H + xo.om u » AooooHoo oHoHeo Hoes-mo e e . oaom a e-.H o~.o +_xm.oo n e Aeoaooooo o>HH .oe-ev a a ooom a as.~ xH.HoH - mH.oa u H Aooooooo o-oHoo .osoHov a a How oHooHooom a so.q xo.mmH - om.~o n w Apoeooooo ooH- .os-ev e e Hoe o-ooHooom a em.H oa.mm + em.~o n » AooooHoo oHoHpo Hoaoamv a a noo- o-ooHooom a so.m oo.-m + xo.mw n w Apoaoozoo ooH- .oo-ev a e AoHooo . ooumuummV coma manoumeom N m mmOHwO GQQMGHQ x.%m nowumnvo eowmmouwom manuanm> unopcoeopeH o~nmaum> unopcooon nowuwmomeoo mmmuumo wdHOOHponm Mom meowumado nowmmoumom .o canny -427 and their carcasses. The standard errors of these equations give an indi- cation of the accuracy with which they could be applied to estimate the composition of lamb carcasses. When these standard errors are compared with the range of carcass components shown in table 3, it can be seen that the non-destructive potassium-40 method for estimation of carcass composition would not discriminate very well between animals. Although flame photometry apparently gave a more accurate estimation of potassium content, even this method did not result in an accurate prediction of carcass composition. For example, the total range of protein in the car- casses of these lambs was only 4.3%, although the most accurate equation for predicting carcass protein has a standard error of 0.5%. However, the range of composition of these lambs is one over which an experimenter may wish to determine treatment effects in a critical experiment. It must also be remembered that flame photometry is a destructive technique, which was used solely to investigate the relationships present and has no practical significance. 1 Results show that significant relationships exist between the po- tassiun content of the lambs and their carcass composition. However, the natural variation in the potassium content of the different tissues shown in table 4 is probably sufficient to prevent any marked reduction in the errors of prediction. This suggests that if the results found in this experiment are typical for the sheep, then the potassium-40 method is not likely to be very useful for predicting the composition of this Species. Sodium Content and Composition The data on the sodium content of the lamb carcasses are presented in table 7. The standard error of the duplicate means was found from -43- Table 7. Sodium content of the carcasses and some separable components of ten lambs as measured by flame photometryp Mean Item % Na 8. D. Ramge_» Edible carcass 0.073 0.006 0.063 - 0.083 Separable lean 0.075 0.005 0.069 - 0.081 Separable lean (fat-free basis) 0.080 0.0051 0.074 - 0.087 Separable fat 0.070 0.017 0.051 - 0.111 Separable fat (fat-free basis) 0.206 0.014 0.183 - 0.225 analysis of variance to be 9 and 10 ppm of sodium for the separable lean and separable fat, reapectively. It was interesting to note that when the chemical fat was removed (mathematically) from the separable fat, the remaining material (protein, water and ash) had a greatly increased sodium content. 0n correcting to a fat-free basis, there was more sodium in the separable fat than in the separable lean, whereas, for potassium the re- verse was true. A range of 0.069 - 0.081% of sodium was Observed in the separable lean of the lambs. These values are lower than the range of 0.079 - 0.140% for lamb muscle reported by Toscani and Buniak (1947), but agree with the figures of 0.073 and 0.074% for sheep muscle Observed by Blaxter and Rook (1956). However the values obtained for sodium in the present eXperiment are higher than the figures of 0.062 and 0.064% reported for sheep muscle by Harris et a1. (1952) and the mean figures of 0.050 and 0.045% sodium in the fat-free, blood-free biceps femoris muscles of sheep reported by MOunib and Evans (1960). These results suggest that differ- ences exist between.muscles or between breeds of sheep and perhaps between 'methods of analysis. Further work is needed to clarify the factors in- volved. -44- The relationships between the sodium content of the edible carcass and other carcass components are presented in table 8. It may be observed that with the exception of bone, all the correlations between the sodium content of the edible carcass and carcass composition were significant. Table 8. Correlations between the percent sodium in the edible carcasses of ten lambs and other carcass variables. Item r Edible carcass % water 0.82** % ether-extract - .80** % protein 0.70* Dressed carcass % separable lean 0.79** % separable fat - .78** % separable bone 0.48 * COrrelation significant at 5% level **Corre1ation significant at 1% level However, these correlations tended to be the same or lower than the equi- valent potassium correlation coefficient (cf. table 5). The sodium rela- tionships are likely to be of less interest in meat animals as radioactive sodium isotOpes are not naturally present in the tissues. Therefore, the addition of isotOpes would probably render the meat unsuitable for human consumption. Yet these relationships may be of interest in work with experimental material . Cesium Content and Composition The cesium-137 content of the lambs and their components is present- ed in table 9. As is customary in the literature, the cesium content has been expressed as the cesium/potassium ratio and as the cesium content of cl: -45- the tissue. It can be seen that washing the lambs increased the cesium/ potassium ratio. As was eXplained earlier, this occurred because washing removed potassium from the skin and wool without markedly affecting the cesium content of the lambs (see cesium/Kg. tissue, table 9). Table 9. Cesium-137 content of ten live lambs and their components Item Mean S.D. Range Live unwashed (ppc cesium-l37/gm. K) ~ 58.0 10.6 41.8 - 74.5 Live washed (ppc cesium-l37/gm. K) 84.8 .17.9 60.2 - 121.0 Dressed carcass (pnc cesiun-l37/gm. K) 81.5 23.0 43.0 - 122.4 Non-carcass components (ppc cesium-l37/ gm. K) 96.8(9)a 59.4 25.9 - 189.2 Separable lean (ppc cesium-l37/gm. K) 79.5 18.1 59.1 - 107.7 Live unwashed (ppc cesium-l37/Kg. tissue) 155.5 24.1 126.3 - 182.8 Live washed (ppc cesium-137/Kg. tissue) 148.7 21.4 118.8 - 183.3 Separable lean (ppc cesium-l37/Kg. tissue) 236.6 54.3 167.8 - 323.1 Counts for separable fat and bone on the lower channel were so close to background that several of the samples were estimated to have a negative cesium content and all results on these tissues were obviously inaccurate. For this reason they have not been reported. aMean is based on 9 observations as one negative value was discarded. In the section on Potassium Content and Composition it was stated that "the apparent total amount of cesium-137 present remained at 0.99 of the unwashed level, with a standard deviation of 0.12 (Anderson, personal communication)", whereas the figures in table 9 suggest that 0.96 would be a better estimate. The difference between these estimates is due to ' the fact that two sets of potassiun and cesiun figures were available on two of the washed lambs. These were washed twice in order to determine whether all the external potassium was removed by one washing. The fi- gures in table 9 include those obtained after one washing, which was .- v--JJ~ \ UHU -46- comparable to the situation on the remaining lambs. However, Anderson used the figures obtained after two washings, which for some unknown rea- son gave a somewhat higher estimate of cesiun. It is probable that the increase in cesium count was a chance occurrence. It can be seen from table 9 that the cesiun/potassium ratio in the washed lambs and their components was fairly constant which would be ex- pected as cesium is metabolically similar to potassium (Langham, 1961). This resulted in a higher concentration of cesium per unit of muscular tissue than in the live lambs, because the lean tissue also had a higher concentration of potassium (table 4). The levels of cesium reported are similar to those found in cattle tissue in 1957 - 1959 in the U.S.A. (Van Dilla §£H§1,, 1961), in pigs and calves prior to 1962 in Canada (Green g£“§1,, 1961; McNeill and Robinson, 1962) and in human beings, largely from the U.S.A. (Langham, 1961). How- ever, these values are very much lower than those observed in Norway and Sweden in beef, horse, mutton, pork, reindeer and to a lesser extent in man (Hvinden and Lillegraven, 1961a, b; Baarli 32 $1,, 1961; Liden, 1961). As all these observations were made prior to the recent nuclear weapons test series of the U.S.S.R. and the U.S.A. beginning in August of 1961, it would appear that these levels resulted from earlier weapons tests. Differences may be due to different world fallout patterns and different eating habits. For example, the cesium/potassium ratio in Norwegians (Baarli g£H§1., 1961) was very much lower on average than in Laplanders Liden, 1961). Differences in sheep, cattle and reindeer (Hvinden and Lillegraven, 1961b; Liden, 1961) were attributed to variation in grazing habits. f) -47- Some correlations between the cesium content of the live lambs and carcass composition are presented in table 10. Components were chosen which were highly correlated with potassiun content in the present exper- iment, and also lean content, which has been shown by other workers (Kul- wich ggugl., 1961a, b; Pfau g; 91., 1961) to be significantly related to Table 10. Correlations between the cesium content of live lambs (ppc cesium-137/Kg. tissue) and some carcass components Live unwashed Live washed Item cesium content cesium content % Protein (edible carcass) -.240 -.063 % Separable fat (dressed carcass) 0.042 -.183 % Separable lean (dressed carcass) -.094 0.257 cesium content. None of the correlations in table 10 were significant. However, Pfau ggugl, (1961) and Kulwich.g£“§1, (1961a, b) have shown that cesium-137 is less closely related to lean content than potassium, and the latter workers concluded that cesium could not provide a useful esti- mate of leanness. As none of the potassium relationships (from potassium -40) with carcass composition in the present experiment were very high, the lack of relationship with cesium-137 content is not too surprising. It has been shown that the individual estimates of potassium from potass- ium-40 were not very accurate in the present experiment and as the same conditions were used for cesium detenminations, a significant relation- ship would not be expected. The literature shows that the cesium content of animals may vary from laboratory to laboratory and will change with continuing nuclear weapons tests. For this reason cesium is potentially less useful than potassium-40, which is a constant proportion of all po- tassium. The differing levels of cesium would make a comparison of the results from different laboratories more difficult than is the case for potassium. -47- Some correlations between the cesium content of the live lambs and carcass composition are presented in table 10. Components were chosen which were highly correlated with potassium content in the present exper- iment, and also lean content, which has been shown by other workers (Kul- wich g; 31,, 1961a, b; Pfau g£“§1,, 1961) to be significantly related to Table 10. Correlations between the cesium content of live lambs (ppc cesium-137/Kg. tissue) and some carcass components Live unwashed Live washed Item cesium content cesium content % Protein (edible carcass) -.240 -.063 % Separable fat (dressed carcass) 0.042 -.183 % Separable lean (dressed carcass) -.094 0.257 cesium content. None of the correlations in table 10 were significant. However, Pfau §£_§l, (1961) and Kulwich g£_§13 (1961a, b) have shown that cesium-137 is less closely related to lean content than potassium, and the latter workers concluded that cesium could not provide a useful esti- mate of leanness. As none of the potassiumrelationships (from potassium ~40) with carcass composition in the present experiment were very high, the lack of relationship with cesium-137 content is not too surprising. It has been shown that the individual estimates of potassium from potass- ium-40 were not very accurate in the present experiment and as the same conditions were used for cesiun determinations, a significant relation- ship would not be expected. The literature shows that the cesium content of animals may vary from laboratory to laboratory and will change with continuing nuclear weapons tests. For this reason cesium is potentially less useful than potassium-40, which is a constant proportion of all po- tassium. The differing levels of cesium would make a comparison of the results from different laboratories more difficult than is the case for potassium. -48- SUMMARY AND CONCLUSIONS Ten lambs with a mean live weight of 88 lb. were used to determine the accuracy with which their carcass composition could be measured from their potassium-40 content. It was found that on average the live lambs contained 108 gm. of potassium which was reduced to 71 gm. by washing, which indicated the presence of appreciable amounts of potassium on the skin and wool. The potassium content of the live lambs when estimated from potassium-40 was significantly related to carcass composition. How- ever, carcass potassium as estimated by the potassium-40 method was not significantly related to physical or chemical composition. In contrast, the flame photometrically determined potassium content of the carcass was significantly related to carcass composition. This suggested that the carcass potassium content was more accurately determined from flame pho- tometry than from potassium-40. However, it should be noted in this experiment, that potassium did not give an accurate estimate of carcass composition, regardless of the method of measurement. It was shown that the sodium content of the carcasses was less closely related to carcass composition than was potassium. The sodium and potassium contents of fatty and muscular tissue were found to be in general agreement with the results of other workers. The cesiun-137 content of lamb and its possible use for predicting composition were dis- cussed. It was concluded that cesium was likely to be less useful than potassium for measuring the composition of live animals and the carcass part8. EXPERIMENT II. GROUND PORK AND LAMB The relationship between the potassium content and both physical and chemical composition in Experiment I was lower than was anticipated from the literature. It was decided to test the potassium-40 method again on homogeneous tissue samples of exactly the same weight and with a wider range in composition than was present in the lambs. Under these more ideal conditions, it was anticipated that the relationships would be closer than would normally be eXpected if the potassium-40 method was adopted on a routine basis for measuring composition. Flame photometry was used as an alternative method to check the accuracy of the potassium determinations, and in addition, to measure the sodium content of the samples. EXPERIMENTAL Pork and Lamb Samples Both pork and lamb samples were obtained from several carcasses, which were boned out and prepared at the Michigan State University Meat Laboratory. No attempt was made to keep the meat separate from different carcasses within each Species. The meat was ground into a homogeneous ‘mixture in a "silent cutter", which is used for making sausage emulsions. The range in chemical composition of the samples approximated that which may be found in sausage emulsions. Exactly 19.0 lb. of the meat emulsion were packed separately into waterproof, cardboard cartons, which were about 10 inches high and 9.5 inches in diameter. At the same time, a sample was taken for chemical analysis. Each carton of meat emulsion was capped to prevent evaporation and frozen for ease of handling and to pre- -49- -50- vent deterioration. Twenty cartons of pork and seventeen cartons of lamb were prepared. Chemical Analypes The samples taken for chemical analysis were frozen and stored in glass jars until analyzed for percent water, fat and protein by the methods described previously. The samples were then used for duplicate potassium and sodium analyses by flame photometry after being extracted by the TCA homogenization method described earlier. Scintillation Counter Radioactivity was measured with the Radiation Counter Laboratory Model 55400 Ratio Computer (Regas £5;él:: 1959). It is a large scintill- ation detector with a centrally positioned well 12 inches in diameter and 24 inches deep. The counting well was optimistically described as being able to contain 90 lb. of meat. With the exception of the end areas, the sample is completely surrounded by the scintillation material, thus giving a counting geometry approximating a four Bi configuration. The detector tank was surrounded by a 3 inch lead shield to reduce the radiation back- ground in the counter well. Eight photomultiplier tubes 5 inches in dia- ‘meter view the scintillation material from the end areas. At the time that the experiment was carried out, only five of the eight photomultiplier tubes were functional. Pulse heights were counted in the range of 1.2 - 1.6 Mev., which included the potassium-40 peak (1.45 Mev.). Limiting the range reduced the possibility of counting the radioactivity from other elements. -5]_- Counting.Methods Two cartons were always counted at the same time; thus, the gamma activity of the meat was determined on 38-lb. batches. Each of the 19- lb. cartons was counted in two different combinations, giving a total of 20 observations for pork and 15 for lamb. Cartons were measured in pairs to increase the precision of counting. All samples and backgrounds were counted for two consecutive 5~minute periods. With the first 10 pork combinations, background counts were taken after each pair of cartons had been counted. Since background remained fairly constant, two pairs of cartons were counted between each background observation for the remain- ing pork samples. With lamb, background counts were made only after counting every third pair of cartons, or at about 40~minute intervals. Potassium Standards Four l9-lb. lots of commercial sucrose, which were shown to have no gamma activity, were put in cardboard cartons identical to those used for the meat samples. Three lots of chemically pure KCl containing 33, 66 and 132 gm. of potassium were added to three of the sugar cartons to act as potassium standards as outlined by Anderson (1959). Then each stan- dard was counted with a carton of pure sugar giving 38-lb. standards. The potassium standards were counted for two 5-minute periods twice dur- ing the experiment. RESULTS AND DISCUSSION Potassium Standards The counts per second (cps) on the potassium standards were related to the grams of potassium in the standards by the following linear equa- tion: -52- K (gm.) = 2.6554 cps + 0.52 Since the potassium standards fulfilled the requirements discussed by Anderson (1959), the above equation was used to predict the potassium content of the meat samples. The predicted potassium content was then expressed as a percentage of the fresh meat sample. The scintillation detector recorded an average of 0.3716 cps per gram of potassium. Since the natural mixture of potassium isotOpes emits 2.96 gamma rays per second per gram.(Anderson, 1959), the efficiency of the detector was 12%. It is probable that a higher counting efficiency would have been achieved if all eight photomultiplier tubes had been functioning. Conceivably, this lowered efficiency could have reduced the accuracy Of the determin- ations. Gross Meat Composition The gross chemical composition of the meat samples is presented in table 11. The means, standard deviations and ranges are based on the 38- 1b. samples that were counted for gamma activity. Thus, each individual observation comprises the mean composition Of the contents of each pair of l9-lb. cartons that were counted together. It can be seen that there was quite a wide range in composition, with the variation for pork and lamb being about the same. The summation of the individual components does not total 100%, because the ash content of the samples was not deter- mined. -53- Table 11. Cross chemical composition of the counted meat samples Item. Mean S.D. Range Pork (20 observations) Water % 51.1 7.4 37.7 - 63.1 Fat % 33.5 9.5 17 5 - 50 7 Protein % 14.5 2 2 10 8 - 18 3 Lamb (15 observations) water % 53.7 7.7 36.6 - 63.1 Fat % 30.0 10.0 18.4 - 52 5 Protein % 15.8 1.8 11.5 - l8 0 Potassium and Sodium Content of the Ground Meat Samples and Relationships with Composition The mean counts per second observed on the pork samples were 15.7 (S.D. = 3.2; range = 9.7 - 20.8) and on the lamb samples were 16.5 (S.D. = 3.0; range = 10.9 - 20.8). With both pork and lamb, the counting error (S.D.) was approximately 3.6, when expressed as a percentage of the total number of counts. Since the background was determined more frequently with pork, the over-all accuracy may have been greater than with lamb. The counts were converted to potassium content of the meat samples as described earlier. The standard error Of the mean Of the duplicate potassium analyses by flame photometry was found from the error mean square of analysis of variance to be 23 ppm for the pork samples and 16 ppm for the lamb sam- ples, respectively. The standard error of the mean for the duplicate sodium analyses was found from the error mean square of analysis of vari- ance to be 5 ppm for the pork samples and 7 ppm for the lamb samples, respectively. It must be re-emphasized that these figures take no account of day-to-day variation in the Operation of the flame photometer, as the -54- duplicate analyses were always carried out on the same day. However, it should be noted that for potassium, the standard error would have to be 88 ppm to give a standard error of 3.6%, which could be considered equi- valent to a counting error of 3.6%. This suggests that under the condi- tions of use, the flame photometry estimations Of potassium were probably more accurate. The data on the potassium content of the ground pork and lamb as measured by flame photometry and as measured from the potassium-40 con- tent are presented in table 12. It may be observed that both methods Table 12. Potassium and sodium content of ground pork and lamb samples as measured by flame photometry or fromppotassium-40 Mean Species Method % n S. D. Range Potassium Pork Flame photometry 0.245 20 0.038 0.175 0.307 Pork Potassium-40 0.244 20 0.050 0.153 0.323 Pork Flame photometry, fat-free basis 0.367 20 0.008 0.352 0.382 Lamb Flame photometry 0.244 15 0.038 0.163 0.287 Lamb Potassium-40 0.256 15 0.046 0.171 0.323 Lamb Flame photometry, fat-free basis 0.348 15 0.008 0.337 0.361 Sodium Pork Flame photometry 0.042 20 0.003 0.035 0.047 Pork Flame photometry, fat-free basis 0.064 20 0.005 0.056 0.072 Lamb Flame photometry 0.062 15 0.006 0.049 0.070 Lamb Flame photometry, fat-free basis 0.089 15 0.006 0.081 0.104 estimated the same potassium content of pork. However, analysis of vari- ance showed that the difference between the means of the potassium content of lamb, although small (120 ppm), was significant (P'< .05). As was noted in the experiment with the live lambs and their components, the use -55- of flame photometry resulted in a smaller standard deviation for the potassium content of both Species, than when potassium-40 was used. Correction of the potassium content of the ground pork and lamb to a fat-free basis permitted a comparison to be made of potassium levels. It is interesting to note that on this basis the potassium content of the ground lamb (0.348%) was almost identical with the potassium content of separable lamb lean (0.340%, table 4) observed earlier. However, in contrast, it was shown by a "t” test that the difference in potassium content between the pork and lamb on a fat-free basis was highly signi- ficant (P«< .001). Further work is needed to investigate whether pork normally has a higher potassium content than lamb, and to determine if factors such as age and sex may influence potassium levels as has been found true for hunans (Anderson and Langham, 1959). All such factors would have to be considered before any potassium-based technique could achieve accuracy. The data on the sodium content of the ground meat samples are also presented in table 12. Application of a "t" test showed that the sodium content of ground lamb was greater than for pork on a fat-free basis (P-< .001). This was the opposite of the results for potassium. The sodium content of the ground lamb on a fat-free basis was higher than similar values for lamb muscular tissue observed in table 7. This can probably be eXplained by the high sodium content of the non-fatty mater- ial found in separable fat (table 7), as a considerable amount of separ- able fat was present in many of the ground lamb samples. Table 13 shows the correlation and regression equations used in pre- dicting the composition of the meat samples from their potassium content -56- Table 13. Relationships between the percent potassium in ground meat samples ()0 as estimated from potassium-40 and other chemical components Dependent variate Correlation1 Regression equation ' Sy.x Pork (20 observations)2 % Water 0.977 Y = 144.89X + 15.70 1.61% % Fat -0.975 Y = 79.36 - 187.60X 2.20% % Protein 0.962 Y = 42.14X + 4.20 0.61% Lamb (15 observations)2 % Water 0.917 Y = 153.13X + 14.39 3.20% % Fat -0.908 Y = 80.21 - 196.15X 4.35% % Protein 0.883 Y = 34.93X + 6.62 0.89% AIAll correlations significant at the 1% level Each observation was made on a pair of 19 lb. samples. The composition of these 38 lb. samples have been related to their potassium content. as estimated from their natural radioactivity. In pork, the accuracy with which the composition of the samples could be predicted looked pro- mising. The standard errors of the equations based on the gamma activity of the pork are almost exactly the same as those given by Kulwich it; 31., (1960a) from the beta activity of pork samples. In contrast, the error in estimating the composition of lambs was quite high. The magnitude was similar to that observed in regression equations for predicting the composition of lamb carcasses (table 6). The relationships between the potassium content of the ground meat as measured by flame photometry and some of the other chemical constitu- ents are presented in table 14. All correlations are larger than the correSponding relationships reported in table 13, when the potassium con- tent of the samples was estimated from natural radioactivity. It can be seen from table 14 that the standard errors of the regression equations for predicting meat composition were very much smaller when flame photo- metry was used than when the natural gamma activity was used. These re- -57- sults suggest that part of the explanation for the relatively high stan- dard errors resulting from the potassium-40 approach are due to errors involved in counting the gamma emissions. It is likely that better instru- mentation and longer counting times than those used in the present exper- iments would reduce the errors involved. The precision indicated in table 14, where the standard error of estimate of the fat and water con- tent of the pork was less than 1%, would be sufficiently accurate in a routine, non-destructive method of analysis. The results in tables 13 and 14 suggest that potassium is more closely related to the composition of pork than of lamb. Table 14. Relationships between the percent potassium in ground meat samples (X) as measured by flame photometry and other chemi- cal components Dependent variate (Y) Correlation Regression equation Sy.x (%) Pork (20 observations) water % 0.997 Y = 190.88X + 4.43 0.54 Fat % - .996 Y ==094.00 - 247.35X 0.90 Protein % 0.986 Y = 55.71X + 0.88 0.38 Potassium % (from K-40) 0.977 Y = 1.261X - 0.064 0.0109 Lamb (15 observations) Water % 0.993 Y = 204.17X-+ 3.83 0.97 Fat % - .990 Y - 94.22 - 263.36X 1.46 Protein % 0.975 Y - 47.52X + 3.98 0.42 Potassium % (from K-40) 0.936 Y - 1.153X - 0.0251 0.0168 The high correlation between the tWO‘mEthOdS of measuring potassium content shown in table 14 is in contrast to the lack of a significant re- lationship for the same comparison on the separable lean or fat for the Los Alamos lambs. It can be explained by two factors. First, the wider -58- range in chemical composition in the second experiment probably would increase the magnitude of any correlation. A second and more important explanation, is the fact that the use of larger samples in relation to the size of the counter may have increased the statistical precision of counting in the second experiment. As was mentioned earlier, the Los Alamos counter was not designed to count samples as small as the separ- able fat from the lamb carcasses, and so the poor results for separable fat cannot be regarded as a criticism of the technique. It was noted that the potassium content on a fat-free basis of the IgrOund pork when measured by flame photometry was Significantly related (r = -.675) to the chemically determined fat of the ground pork. In a similar manner, when the potassium content of the fat-free ground pork was estimated from the potassium-40 content, there was a negative correl- ation with percent fat of -.784. The potassium content of the ground lamb was also calculated to a fat-free basis from flame photometry and potassium-40 estimations. The potassium content on a fat-free basis was in both cases negatively and non-significantly correlated with the percent fat in the ground lamb. It can be seen from table 15 that the sodium content of the ground imeat was closely related to composition. However, the sodium content was in all cases less closely related to composition than the potassium con- tent (cf., table 14), when the same method was used for estimating both sodium and potassium. It was interesting to note that by expressing the sodium content on a fat-free basis, the signs of all correlation coeffi- cients were reversed. This was apparently due to the high sodium content of the non-fatty material (water, protein and ash) found in the separable -59- fat, which tends to increase in weight directly with the weight of chemi- t cal fat. As this non-fatty material in the case of lamb (table 7), has two and a half times the sodium content of the fat-free separable lean, the percentage of sodium in the fat-free ground tissues increased as the percentage of fat increases. Table 15. Correlations between the percent sodium in ground meat samples and other chemical components Item % sodium % sodium (fat-free basis) Pork (20 observations) water % 00 934 'o 931 Fat 70 -0928 0.934 Protein % 0.912 -.933 Lamb (15 observations) Water % 0.967 -.938 Fat % -.968 0.941 Protein % 0.972 -.920 It is noted that the relationships observed are likely to be much closer in a homogeneous material, such as ground tissue, than in live animals or their carcasses. As potassium is more closely related to composition than sodium and is at the same time naturally radioactive, it is considered that the use of potassium is likely to prove more fruit- ful than sodium analyses in the investigation of body and carcass composi- tion, unless the intracellular and extracellular fluid Spaces are to be investigated separately. A practical application of the potassium-40 counting method to the meat industry has been suggested by Anderson (1959). He pointed out that the counting method might have an outstanding advantage over chemi- cal analysis because Of its adaptability to continuous systems. He stated, "If the sample to be analysed (e.g., comminuted meat) can be -60- passed through the counter in a continuous stream of constant bulk den- sity, the counter will provide through a ratedmeter a continuous measure- ment of the lean content of the meat. The use of such a system for control of the ratio of lean/fat input to the grinders is obvious. Whether or not such a system is feasible depends on the precision and reaponse time requirements.’ Later in the same paper Anderson states "there must be sufficient mixing and grinding before the counter to ensure ' Anderson indicated some a homogenous material at the point of counting.’ of the problems that would have to be overcome before the potassium-40 method could be used. In the light of the present experiment, it would appear that the counting method would not be sufficiently accurate for lamb and may not be accurate enough for pork. Anderson (1959) did not mention how back- ground determinations would be fitted into a continuous system. It is known that the frequency Of background determinations greatly influence the accuracy of any radioactivity determinations where background is not constant. It should also be remembered that the statistical precision of the counting method which was discussed by Anderson, applies only to the accuracy with which potassium can be estimated, and takes no account of the errors in estimating meat composition, if the potassium content is known with complete accuracy. These errors would be due to the biolo- gical variability of the potassium content of meat. Finally, it should be noted that in the present experiment it took 10 minutes to obtain a relatively inaccurate estimate of the composition of homogeneous meat samples. -51- SUMMARY AND CONCLUSIONS The potassium content of 20 lots of ground pork and 15 lots of ground lamb, each weighing 38 1b. was estimated from the potassium-40 content and from flame photometry. A wide range of gross chemical composition was found in the 38-lb. lots of ground tissue from both species. The ground pork was found to contain significantly more potassium (0.37%) than the ground lamb (0.35%) on a fat-free basis. Both methods of potassium estimation gave approximately the same potassium content of the individual samples. However, the flame photo- metrically detemined potassiun content was more closely related to the chemical composition of the samples in terms of percentage water, fat and protein, than was potassium content as determined by the natural gamma activity. The data suggest that such relationships are closer for pork than for lamb. The sodium content of the samples as measured by flame photometry was also very closely related to the composition of the samples, but the relationship was not as good as that observed for potass- ium. Results of the potassium analyses using flame photometry demonstrated that a close relationship exists between potassium content and chemical composition. If a non-destructive method (such as potassium-40) is to be sufficiently accurate to be useful, then a degree of precision at least comparable to that obtained by flame photometry is needed. EXPERIMENT III. TWENTY FOUR PIG BODIES INTRODUCTION The potassium content of animal tissues was more closely related to the gross composition of the samples when estimated from flame photometry than from potassium-4O (Experiments I and II). The tissue samples uti- lized in this study came from the bodies and carcasses of 24 pigs for which the major chemical components had been detenmined (Gnaedinger, 1962). Although the size of the pigs would tend to make sampling for chemical analysis more difficult and less accurate than in previous ex- periments in which flame photometry was used to measure the potassium content, these samples were considered suitable for investigating the relationships between the gross composition and potassium content by flame photometry. Unfortunately, facilities were not available for mea- suring the potassium content of the live pigs from their natural gamma radioactivity. It was, however, considered that the relationships estab- lished by flame photometry would give an indication as to whether the non- destructive potassium-40 technique was likely to be useful in estimating the composition of live pigs and their carcasses. Sodium analyses by flame photometry were also conducted. EXPERIMENTAL Animals and Their Gross Analyses Twenty four pigs ranging in liveweight from 181 - 220 lb. were used in this experiment. A description of the slaughter procedure and methods of gross chemical analysis (fat, protein, water and ash) has been published -62- -63- by Gnaedinger (1962). At slaughter each animal was divided into 7 com- partments. These included the carcass (including the skin), hair (includ- ing scurf and toenails), head (including the tongue), blood, viscera (liver, lungs, eSOphagus, trachea, heart, kidney and Spleen), empty G.I. tract (stomach, intestines, caul and ruffle fat), and the contents of the G.I. tract. It should be noted that the eviscerated carcass contain- ing the skin is equivalent to a commercially dressed carcass with the exception of leaf (kidney) fat which is normally removed under commercial conditions. The commercial practice of scalding the carcasses in 142°F water prior to scraping and dehairing was followed. This would undoubted- ly remove some of the sodium and potassium from the exterior of the ani- m als and from the skin and hair. Each body fraction.was frozen, sawed into thin strips, ground until homogeneous and samples were taken for 'subsequent chemical analyses (Gnaedinger, 1962). The samples were stored in a frozen condition in glass jars until thawed for analyses. water, ether-extract and protein were determined by the methods described earlier. Ash content was determined on samples weighing approximately 5 gm. which were ashed for 24 hours at 525°C as described by Gnaedinger (1962). An anomaly was noted in the data on pig 1 from the point of view of the present experiment. This pig was skinned, whereas, the skins were left on the remaining 23 hogs as is the commercial practice. The skin (plus some subcutaneous fat) of pig 1 weighed 23.4 lb. and the dressed carcass weighed 139.6 lb. For purposes of analysis, Gnaedinger included the skin of pig 1 under the hair classification. This compartment aver- aged l.l lb. in weight on the remaining animals. This had no effect when total body composition was computed, but made some differences to carcass -64- composition in terms of both the gross chemical composition of carcass l and also its electrolyte content. As compared to the hair, scurf and toenails of the remaining pigs, the skin of pig 1 weighed a great deal ‘more and had more water and fat, and less protein and ash. Thus,two sets of calculations will be presented in many of the statistical analyses re- lated to the carcasses of these pigs. In one set, the frozen carcass composition figures presented by Gnaedinger (1962) which excluded the skin and hair of pig 1, will be used, and in the second set of calcula- tions, the skin components of pig 1 will be added to the frozen carcass components. As Gnaedinger (1962) was mainly interested in the body com- position of live pigs, the classification of the skin of pig 1 was not a matter of importance providing that it was included in the total body compartments. It may be noted that the original experiment of Gnaedinger was not planned from the standpoint of determining the potassium and sodium con- tent of the pigs. Blood was not available for all animals as some of the samples had been completely utilized in earlier analyses. As the blood samples were prevented from coagulating by the use of sodiun citrate, they were unsuitable for sodiun analyses. Three further blood samples were obtained from three mature hogs at the time of slaughter and these were analysed for sodium and potassium. Coagulation was prevented in these samples by the use of citric acid. Potassium and Sodium Analyses Potassium and sodium analyses were made after the samples had been extracted and prepared by homogenization in 2% TCA as described earlier. Unlike the two previous experiments, ground bone was also present in the -65- carcass and head samples. Dean (1960) has shown that over 100 ppm cal- cium can depress potassium readings and enhance sodium readings in the range that these elements were being determined in this experiment. As it was possible that the method of extraction may dissolve a little of the finely ground bone, a preliminary analysis was carried out for calcium on ground material from carcass 1. Extraction and dilution was carried out in the usual manner. Analysis indicated that about 12 ppm of calcium was present in the solution analysed, and so it was assumed that the cal- cium level would not seriously influence the readings. For samples other than those obtained from the carcasses, it was found necessary to prepare a new standard solution with a lower K:Na ratio than 5:1, thus, a ratio of 1:1 was chosen. This gave a range of O - 30 ppm potassium and sodium for the standard curves. The potassium and sodium content of the empty bodies of these pigs were computed from the weights of the body components and their potassium and sodium contents. It was realized that this method of determining the potassium and sodium content of the empty body would underestimate these elements, since some of these elements may be trapped by particles of bone and teeth from the head and carcass. RESULTS AND DISCUSSION The weights of the major components of the bodies of 24 pigs are pre- sented in table 16. It may be noted that the frozen carcasses comprised 76% of the liveweight of the animals. The value recorded for blood re- presents the difference in weight of the pig body before and after bleed- ing and is not a measure of the total blood volume of the animal. Dukes (1955) suggests the blood volume of swine is on average 7.4% of body weight. -66- Table 16. Body weight and components from 24 pigs (from Gnaedinger, 1962) Mean Item (1b.) % Rangey(lby) Live weight 198.4 100.0 181.0 - 220.0 Hot dressed carcass 156.2 78.7 140.2 - 173.6 Frozen dressed carcass 151.3 76.3 136.0 - 169.6 Head and tongue 10.2 5.1 8.3 - 13.0 Gastrointestinal tract + caul fat 10.5 5.3 8.7 - 12.9 Remaining visceraa 7.8 3.9 6.5 - 9.0 Hair, scurf and toenails 1.1 (23) 0.6 0.8 - 1.5 Blood 7.5 3.8 4.2 - 9.8 Gastrointestinal contents 3.4 1.7 1.7 - 5.4 aLiver, lungs, eSOphagus, trachea, heart, kidney and Spleen. However, the blood weight recorded represents the loss in body weight due to bleeding that might be expected in a commercial slaughter operation. The chemical composition of the frozen carcasses (excluding the skin of pig 1) and the empty bodies (the whole animal excluding the contents of the G.I. tract) of the 24 pigs are presented in table 17. It may be noted that the range in body composition is less than the range in carcass composition. Similarly, the range in protein and ash is quite small. Table 17. Composition of the empty bodies and frozen carcasses of 24 pigs (from Gnaedinger, 1962) Frozen carcass Empty body Item Mean Range Mean Range Water % 44.4 36.4 - 48.2 48.5 43. 6- 52.5 Ether-extract % 38.6 32.8 - 48.3 33.5 28.4 - 41.5 Protein % 13.6 12.3 - 14.8 13.8 12.5 - 14.8 Ash % 2.8 2.1 - 3.3 2.7 2.2 - 3.2 The potassium content of the empty bodies and their components are given in table 18. The empty bodies of these pigs contained 0.20% potass- ium, or 0.30% on a fat-free basis. Green gt El, (1961) have estimated 0.24% potassium in pigs of unSpecified age and weight, while Spray and Widdowson (1950) have estimated that the fat-free bodies of adult pigs -67- contained about 0.28% potassium. These values agree fairly well with the results of the present experiment, which may slightly underestimate po- tassiun content because of the potassium trapped by bone fragments. Table 18. Potassium content of the empty bodies and their components from 24 pigs Item n Mean % S.D. Range Frozen carcassa 24 0.2098 0.0186 0.1737 - 0.2455 Head and tongue 24 0.1470 0.0092 0.1253 - 0.1668 G.I. tract + caul fat 24 0.1873 0.0159 0.1617 - 0.2275 Remaining viscera 24 0.2571 0.0110 0.2343 - 0.2887 Hair etc.b 1 0.0978 -- -- -- Bloodc 14 0.2199 0.0090 0.2061 - 0.2368 Empty bodyd 24 0.2013 0.0146 0.1734 - 0.2316 aIf the skin etc. of pig 1 was included in the composition of this car- cass, the mean potassium content of the 24 hog carcasses was reduced to 0.2089%. A composite sample from several animals. CEleven samples from the 24 hogs (range 0.2061 - 0.2386) plus 3 additional samples from 3 more hogs (range 0.2092 - 0.2155) and the mean value was used in computing body composition. dIn computing empty body composition the loss of weight between the hot carcass and frozen carcass was assumed to be water containing no potass- ium or sodium. The contents of the G.I. tract have been excluded from the computations. The frozen carcasses contained 0.21% potassium, or 0.34% on a fat- free basis. It was shown in table 12 that ground pork (lean and fat) contained 0.37% potassium on a fat-free basis. It is possible that the bone present in the carcasses in this study may be sufficient to explain the lower value for the entire carcasses. Filer 22 El. (1960) presented some data from which it was possible to estimate the potassium content of 8 week old piglets. The animals were used in an experiment to study the influence of protein and fat con- tent of the diet on body composition. For experimental purposes Filer ~68- .gg‘gl. (1960) divided the animals into 8 groups, the lightest of which averaged 39 lb. and the heaviest 42 1b. liveweight. The average carcass potassium of 32 of the pigs which were fed on a high-fat diet was 0.18% and of 14 of the pigs fed on a low-fat diet was 0.21%. It appeared from the data that the difference in potassium content of the carcasses from the two feeding treatments was significant and that the lowered potassium content in the high-fat pigs was apparently due to the lower protein con- tent in their carcasses. However, the potassium content of the pigs on the low-fat diet was approximately the same as that found in the carcasses from the 198 1b. pigs in the present eXperiment. When the potassiun con- tent of the carcasses of the piglets (Filer gtvél,, 1960) was expressed on a fat-free basis, there was little difference between the animals on the high- and low-fat diets. The average potassium content was 0.26% on a fat-free basis. This is considerably lower than the 0.34% observed in the present experiment, and does not appear to be in agreement with the data of Spray and Widdowson (1950), which suggests that pigs reach chemi- cal maturity in terms of sodium and potassium content at 3 weeks of age. The low potassium content of the head (table 18) is probably the re- sult of the high bone content (7.6% ash; Gnaedinger, 1962), even though pig brain is quite high in potassium (Widdowson and Dickerson, 1960). The low fat content (Gnaedinger, 1962) and high potassium content (Widdow- son and Dickerson, 1960) of many organs explain the relatively high potassium content of the remaining viscera. The low potassium content of the hair, scurf and toenails may be at least partially due to the soaking that these components received during the scalding process. The observed potassium content of the blood (0.22%) is very much higher than the value of 0.09% reported by Green-gt.§1. (1960). In this ~69- connection, it should be noted that McCance and Widdowson (1958) found 0.47 - 0.48% potassium in the erythrocytes from mature pigs. Widdowson and McCance (1956) found 0.02 - 0.03% potassium in mature pig serum. Assuming a haematocrit of 41.5 (Dukes, 1955) and using the above data to calculate the potassium content, gives an estimate of about 0.21% po- tassium in the blood of the mature pig. This suggests that either Green 25 31. (1960) had a group of pigs with widely different blood potassium from those in the present eXperiment or else the potassium-40 method of analysis was inaccurate on blood. It should be noted that in the sheep, strains have been found with blood levels of potassium which differ as much as for the groups of pigs discussed here (Evans, 1954; Kidwell $331., 1959), but the presence of high and low potassium strains of pigs have not yet been reported as far as the author is aware. The sodium content of the 24 pigs and their various components is shown in table 19. It can be seen that the remainder of the body has a considerably higher sodium content than the carcass, with the head and the blood having the highest sodium content. When computed on a fat-free basis, the sodium content of the empty bodies was 0.124% and of the frozen carcasses was 0.116%. The former figure is less than 0.140% sodium in- dicated by Spray and Widdowson (1950) for the fat-free bodies of mature pigs. This latter figure agrees quite well with the results of Filer .ggugl, (1960) on the carcasses of 46 piglets. Calculation from their data showed that the mean sodium content of the piglets on a fat-free basis was 0.127%. All results indicate that the sodium content of the entire carcasses is almost double that of the soft tissues of the carcass (fat and lean; see ground pork, table 12), because it was shown earlier -70- that ground pork contained 0.064% sodium on a fat-free basis. The in- creased amount of sodium in the entire carcass probably comes from the bone, which has been estimated to contain 36.5% of the total body sodium in man (Edelman, 1961). It should be emphasized that it is not known what proportion of bone sodium would be removed by the method of extract- ion used in the present experiment. Table 19. Sodium content of 24 pigs and their components Item n Mean % S.D. Range Frozen carcassa 24 0.0715 0.0049 0.0611 - 0.0807 Head and tongue 24 0.1528 0.0161 0.1157 - 0.1852 G.I. tract and caul fat 24 0.1115 0.0122 0.0886 - 0.1439 Remaining viscera 24 0.1132 0.0044 0.1053 - 0.1205 Hair etc.b 1 0.0918 -- -- -- Bloodc 3 0.1825 -- 0.1760 - 0.1889 Empty bodyd 24 0.0825 0.0042 0.0730 - 0.0896 3If the skin of pig 1 was included in the composition of this carcass, the mean sodium content of the 24 hog carcasses was increased to 0.0717%. A composite sample from several animals. cFrom 3 additional hogs as samples from the original 24 hogs were unsuit- able for sodium analyses because coagulation was prevented by the use of sodium citrate. dSee footnote d to table 18. If potassium-40 is to be used to non-destructively predict composi- tion then several relationships are likely to be of interest. workers in the field of animal production may wish to predict the composition of live animals and/or of their dressed carcasses, by making potassium-40 counts on the live animal. Similarly, workers interested in carcass com- position may wish to estimate this from counts on the dressed carcass. The present data were used to investigate these relationships. Table 20 indicates the relationships between the potassium content of the empty body which is approximately equivalent to the potassium con- -71- tent in the live animal and the potassium content of the frozen carcasses in relation to the gross chemical composition of the frozen carcasses. Table 20. Correlations between the potassium content of the frozen car- casses and the empty bodies of 24 pigs and the components of the frozen carcasses Frozen carcass Empty body Frozen carcass composition % K_(l) % K (2) % K (3) % K (4) % water 0.822** 0.809** 0.810** 0.813** % ether-extract -.83l** -.821** -.818** -.819** % protein 0.712** 0.682** O.6W¢** O.669** % ash 0.469* 0.425* 0.417* 0.422* % K (carcass) - - 0.985** 0.989** (1) Skin of pig 1 omitted from both major components and potassium analyses of the carcass. (2) Skin of pig 1 included in both major components and potassium analy- ses of the carcass. (3) Skin of pig 1 omitted from major components of the carcass. (4) Skin of pig 1 included in major components of carcass. Two sets of data are presented, one ofvwhich includes the skin of pig 1 in the carcass compositional data and the other set omits the skin from the carcass. It can be seen in table 20 that this made very little difference to the relationships, even although the inclusion of the skin lowered the potassium content of carcass 1 by approximately 10%. It is interesting to note that the correlations between the potassium content of the empty body and carcass composition are almost as high as when carcass potassium content is related to carcass composition. This is not surprising, however, when it is noted that the empty body contained on average 176.8 gm. of potassium (S.D. = 14.4) and the frozen carcass con- tained 143.7 gm. (S.D. = 12.7), which is 81.3% of the total body potassium. The regression equations that could be used for predicting carcass composition are given in table 21. The standard errors indicate that there is little difference in accuracy between body or carcass potassium -72- content for predicting carcass composition. It should be noted that when carcass potassium is used to predict composition, plus or minus one stan- dard error includes 29.8% of the total range in carcass water content, 29.6% of the range of fat content and 37.6% of the range in protein con- tent. This suggests that the potassium content is not very accurate in discriminating between individual carcasses. Table 21. Regression equations for predicting the composition of 24 frozen pig carcasses from the potassium content of the carcasses and of the empty bodies Frozen carcass Independent variable component (Y)a (X) Regression equation Sy.x % water % K frozen carcassa Y = 133.9X + 16.1 1.76 % ether-extract % K frozen carcassa Y = 76.4 - 180.4X 2.30 % protein % K frozen carcassa Y = 25.3X + 8.3 0.47 % water % K empty body Y = 168.3X + 10.3 1.81 % ether-extract % K empty body Y = 84.2 - 226.6X 2.37 % protein % K empty body Y = 30.5X + 7.46 0.50 % potassium % K empty body Y = l.257X-0.0432 0.0032 8Skin of pig 1 omitted. Table 22 presents the relationships between the potassium content of the empty body and other body components. The correlations are similar to those observed on the carcass (table 21). Although the standard errors of the regression equations given in table 22 are lower than those in table 21, they do not discriminate between individual animals any better, because the range in body composition is lower than the range for the car- casses. Table 22. Relationships between the percentage potassium (X) in 24 empty ypig bodies and other body components Dependent variable (Y) r Regression equation Sy.x % water 0.819** Y = 145.8X + 19.2 1.52 % ether-extract -.794** Y = 71.3 - 187.9X 2.15 % protein 0.708** Y = 27.6X + 8.2 0.41 % ash 0.439* -73.. The correlations between the sodiun content of the carcasses of the pigs and their composition are presented in table 23. In this case, it Table 23. Correlations between the sodium content of the frozen carcasses of 24ypigs and other carcass components Frozen carcass component % Na (1) % Na (2) % water 0.458* 0.526** % ether-extract -.508* -.569** % protein 0.472* 0.465* % ash 0.349 0.468* (1) Skin from pig 1 omitted from both major components and sodiun analy- ses of the carcass. (2) Skin from pig 1 included in both major components and sodium analyses of the carcass. can be seen that the inclusion of the skin and some attached subcutaneous fat in the composition of the carcass of pig 1 increased the correlation, except in the case of protein where the correlation was essentially un- changed. It may also be noted that these sodium correlations were very much lower (except in the case of ash) than were their potassium counter- parts. This agrees with the results from the earlier experiments. The relationships between the sodium content of the empty bod- ies and the major chemical components of the empty bodies are given in table 24. The correlations for the empty body are higher than for the equivalent components on the carcass (cf. table 23). An explanation for this is not known unless the method of analysis removed some of the non- exchangable bone sodium, which would comprise a higher proportion of car- cass sodium than of body sodium. It might be expected that exchangable sodiun, which comprises a constant proportion of extracellular fluid, would be more closely related to body composition than total sodium. The sodium analyses indicated that the carcasses contained on average -74- Table 24. Relationships between percentage sodium (X) in 24 pigs (empty bodies) and other body components Dependent variable (Y) r Regression equation Sy.x % water 0.700** Y = 430.2X + 13.0 3.59 % ether-extract -.655** Y = 78.3 - 543.3X 2.64 % protein 0.597** Y = 80.3X + 7.2 0.47 % ash 0.428* - - 47.4 i 4.1 gm. of sodium and the bodies contained 72.5 i 5.0 gm. This meant that the carcass contained 65.4% of total sodium, although the frozen carcass comprised 76.3% of body weight. If the standard errors of the regression equations in table 24 are compared with those in table 22, it can be seen that sodium is considerably less accurate than potass- ium for estimating body composition. It should be remembered, however, that .a method that estimates exchangable sodiun might be more closely related to body composition than the method used in this experiment, which may include some non-exchangable sodium. SUMMARY AND CONCLUSIONS The chemical composition of 24 pigs ranging in live weight from 181 - 220 lb. was determined. The animals were divided into 7 compartments at slaughter. These included the carcass (including the skin), hair (in- cluding scurf and toenails), head (including tongue), blood, viscera (liver, lungs, eSOphagus, trachea, heart, kidney and Spleen), empty G.I. tract (stomach, intestines, caul and ruffle fat) and the contents of the G.I. tract. These compartments were frozen, sliced on a bandsaw, and ground until homogeneous. They were then sampled for analysis into water, protein, fat and aSh. These same samples were also used in the present experiment for sodium and potassium determinations by flame photometry. -75- The empty bodies of the pigs contained on average 0.20% Potassium (177 gm.) and their frozen carcasses contained 0.21% (144 gm.). When ex- pressed on a fat-free basis these figures were increased to 0.30% and 0.34% potassium, reSpectively. The sodium content of the empty bodies was 0.083% and of the frozen carcasses was 0.072%. All correlations between the gross chemical composition of the fro- zen carcasses and either the potassiun content of the empty bodies or frozen carcasses were significant. Similarly, all correlations between the gross chemical composition of the empty bodies and their potassium content were Significant. The magnitude of the standard errors from the resulting regression equations suggests that potassiun is of questionable value for predicting the chemical composition of pig bodies and carcasses, unless methods are found for reducing some of the errors in these rela- tionships. These errors include any inaccuracies in the measurement of the potassium content of the pig. Percent sodiun was less closely related to. the composition of the pigs than was potassium. It was shown that sodium was more closely rela- ted to the composition of the bodies than the carcasses. GENERAL DISCUSSION The usefulness of potassium-40 as a non-destructive method of esti- mating composition must be considered, firstly, in terms of its absolute accuracy, and secondly, in terms of possible alternative methods. Sources of Errors in the Potassium-40 Method In regard to accuracy, the Standard errors of the regression for predicting composition from potassium-40 in the experiments reported herein, have been rather high. Where comparisons are available, the present ex- periments appear to agree with the results in the literature (Kulwich ggugl., 1960a). The lack of precision may be due to errors in measuring potassium by natural radioactivity, or due to errors in measuring the actual composition from the true potassiun content because of biological variability in the animals or, finally, to errors in measuring animal com- position. Two experiments were performed in which it was possible to compare potassium-40 with flame photometry for estimating potassium content. The fact that the composition of animal carcasses or ground meat samples could be more accurately estimated from flame photometry than from natural gamma activity suggests that flame photometry measures potassiun more accurately. The accuracy of the potassium-40 method of estimating potassium can be improved by using longer counting times than those used in the pre- sent eXperiments. It is also possible that the use of improved electronic equipment and the utilization of more efficient liquid scintillation fluids may increase the efficiency of counting. Even when flame photometry was used to measure potassium, the accur- acy of estimating composition was generally unsatisfactory. The only -76- -77- exPeriment in which the standard errors of the regression equations were small enough to give the desired accuracy (standard error of 0.56% for water and 0.90% for fat) was when flame photometry was used to predict the composition of ground pork samples. The problem of biological variability will always be a final limit- ing factor in relating potassium content to composition. For example, it was shown that when the potassium content of the lamb muscle was ex- pressed on a fat-free basis, the standard deviation was 5% of the mean value, so that only approximately 66% of obServations would fall within a range of 10% of the mean value. Similar results were observed when the K/N ratio was investigated. This suggests that potassium comprises a slightly different proportion of the muscular tissue of the different animals in the sheep. Because of this, the muscular tissue and other components would not be closely related to potassium content. Evans (1954), Evans and King (1955) and Kidwell.ggnal. (1959) have shown that the red cells of individual sheep could be classified as either high potassium (approximately 83~m~equiv. per liter of erythrocytes) or low potassium (approximately 23~m-equiv. per liter of erythrocytes). These differences were reported to have a genetic basis. With blood com- prising approximately 8% of body weight in sheep (Dukes, 1955), the inclusion of both types of sheep in an .eXperiment would be sufficient to introduce some error. If the widely different potassium levels should occur in the muscles of sheep then even larger errors would occur unless the types were studied separately. However, Mounib and Evans (1960) have Shown that the potassium content of the skeletal muscle of sheep of the two different blood types was not significantly different, although it -78- tended to vary slightly in the same direction as blood potassium. Fur- ther work is needed to establish the potassium content of different ‘muscles in a given Species of animal, and also to ascertain whether differ- ences occur between various Species of animals. Several authors have shown that the potassiun content of different organs of sheep (Mbunib and Evans, 1960; Blaxter and Rook, 1956), cattle (Blaxter and Rook, 1956) and pigs (Widdowson and Dickerson, 1960; Green .EEHEL-y 1961; McNeill and Robinson, 1962) differ considerably between organs. As the organs vary in weight within a given species, this may also add to biological variability. For the potassium approach to be successful in predicting composition, the over-all effects of different organs and glands muSt be the same. Other sources of biological variabi- lity may include potassium contamination on the skin, or in the case of sheep, on the wool. Another source of variation may be the potassium in the contents of the digestive tract. For ruminants, this may be eSpecially important because of the relatively large proportion of the contents to body weight. Blaxter and Rook (1956) have shown that the prOportion of potassium in the G.I. tract of cattle may vary considerably. It has been shown in all species of mammals that have been investi- gated, that the potassium content initially increases until the animal reaches "chemical maturity" and then levels off and declines with advancing age. Spray and Widdowson, 1950; Anderson and Langham, 1959; Allen 25.3g3, 1960). The decline in potassium with age occurs even when the ratio of potassium to body composition is calculated after removing the effects of fat, water and bone mineral (Allen ggugl,, 1960). This indicates that the reduction of potassium is not due to increasing fatness with age. The -79- effect of age and maturity need to be further investigated in farm animals, so that corrections for these factors may be applied. The lower potassiun content in the fat-free carcasses (0.26%) of the 8 week old piglets of Filer_gt_§1. (1960) as compared to the higher values (0.34%) for the fat-free carcasses of the 198 1b. pigs in the present ex- periment may be due to age. However, the data of Spray and Widdowson (1950) do not support this hypothesis as chemical maturity in terms of electrolyte content appeared to be reached at about 3 weeks of age. Fur- ther work in this field is obviously needed. The variation in potassiun content due to sex may not be important from the point of view of prediztion, as Spray and Widdowson (1950) found that when the bodies of male and female rats and rabbits were adjusted to a fat-free basis, no differences could be detected. In other words, any differences due to sex in the potassium content of these species were ex- plicable on the basis of fat content. However, Allen ggugl., (1960) found that for human beings after 15 years of age, the ratio of potassium to body composition with the effects of fat, water and bone minerals removed, was less for females than males. If potassium content is influenced by sex in other Species then an adjustment would be needed in order to com- pare or combine data on males and females. Therefore, there may be a large number of factors, apart from the variation of potassium content in the tissues of animals of the same age and sex, which can effect the bio- logical relationships between potassium cou:ent and composition. Another problem involved in estimating the absolute accuracy of pre- diction by the potassium-40 method concerns the errors in measuring the composition of the carcasses or the intact animals. Apart from.weighing or other human errors, the most important source of inaccuracy in measuring -80- physical composition in terms of separable or dissectible components comes from evaporative water losses. This can, however, be minimized by the use of damp towels. In experiments which measure the composition of animals or carcasses in terms of gross chemical composition, sampling will likely be a source of error. It was shown by Kirton g£_§l., (1962) that the standard error of the composition of a lamb carcass (based on twelve 50 gm. samples per carcass) was 0.41% for water, 0.54% for ether-extract and 0.22% for pro- tein and ash. Taking duplicate samples from the carcass would not have greatly reduced the accuracy of the determinations of the chemical com- position. The lamb carcasses averaged 39 lb. in weight and were not ground as finely as the pig carcasses (averaging 151 lb.) studied in the present experiment. In the case of the lambs, however, very much larger samples were analyzed. Results suggest that some of the error involved in measuring the relationship between the chemical composition of the carcasses or bodies and their potassium content may be due to inaccuracy in measuring the gross chemical composition. Potassium-40 in Relation to some Alternative Non-Destructive Methods The other main factor involved in ascertaining the potential useful- ness of the potassium-40 method is the accuracy of possible alternative methods. Currently used methods for estimating composition include "eye' and "hand" appraisal and body weight measurement, which are known to be inaccurate. A few methods more recently suggested for indirectly deter- mining body composition will be discussed. In all eXperiments cited, the actual body composition was determined by chemical analysis and related to the non-destructive method being investigated. -81- p The technique used by Gnaedinger (1962) for determining live animal specific gravity from air diSplacement and helium dilution was not suffi- ciently accurate to permit the estimation of the body composition of pigs. In discussing the errors from the air diSplacement technique, Gnaedinger Stated that, ”The greatest source of error appeared to result from inaccur- acies in reading relative hunidity." He also reported, "The major diffi-' culties involved in the helium dilution technique were caused by the activity of the experimental animals inside the chamber." Similarly, Kay and Jones (1962) reported on the use of Specific gravity (underwater weighing with helium dilution to determine head and lung volume) to predict the body fat content of pigs. The results were tmsatisfactory when compared with the tritiun dilution method of estimat- ing body fat from the estimated body water. A correlation of 0.96 was reported between body water as determined by tritium dilution, and body fat. The mean difference between the predicted and actual body‘water content was 1.17%, which appears promising. Panaretto and Till (1962) have shown that tritiated water is more accurate than antipyrine and its amino derivative, N-acetyl-4-aminoanti- pyrine (NAAP), for measuring the chemically determined body water content of goats. The use of antipyrine and NAAP resulted in biassed estimates of body water. The standard error of the regression equation for estimat- ing body water from tritium dilution was 2.1% and for estimating body fat was 2.8%. Unfortunately, experiments relating potassium-40 content to complete body composition determinations have not been reported. Doornenbal ggugl, (1962) have made some just criticisms of the potass- ium-40 method and suggested the use of chromium-51 determined red cell ~82- ‘volume for predicting lean body mass in pigs. These workers defined lean body mass as carcass protein, which differs somewhat from the usual defi- nition. Doornenbal 2; a1, (1962) gave no indication of the accuracy for predicting carcass protein from red cell volume. On calculating the re- gression equation and standard error from the data presented by these workers, it was found that the standard error of the regression equation for predicting the weight of carcass protein was 1.23 lb. over a total range of 5.72 lb. Thus, one standard error included 22% of the total range, and discriminated little between animals. Nutritional and health- related factors could also greatly limit the application of this method. Definite recommendations cannot be made until more experiments and comparisons have been performed. However, on the basis of the experiments reported in this thesis, it does not appear that the potassium-40 method is sufficiently accurate for most experimental purposes. SUMMARY The potassium, Sodiun and cesiun content of animals and their parts were determined and related to their composition. Flame photometry and potassium-40 measurements were in essential agreement as to the potassium content of fatty tissue and muscular tissue from lamb carcasses. Similar results were found for ground pork and lamb samples. The composition of the animals and their tissues could be more accurately predicted from the flame photometrically determined potassium content than from potassium-40. Relationships between potassium content and composition were closer for pork than for lamb. In general, the standard errors of the regression equations for predicting composition from potassium content were too large to suggest that the potassium-40 method is likely to have useful applica- tion. Possible reasons for the magnitude of the standard errors have been fully discussed, as well as some possible non-destructive alternative methods for determining composition. The sodium content of the various tissues was also determined by flame photometry. In contrast to potassium, the levels of sodium were higher in the non-carcass compartments than in the carcass of the pig. Sodium was found to be less closely related to composition than potassium. The cesium-137 levels in lambs were found to agree with other data from North American sources. These levels were lower than some published from Scandanavia following nuclear weapons tests. The cesium content of the lambs was found to be unrelated to their composition. -33- LITERATURE CITED Allen, T. H., E. C. Anderson and W. H. Langham. 1960. Total body potass- ium and gross body composition in relation to age. J. Gerontol. 15: 348. Anderson, E. C. 1958. The Los Alamos human counter. p. 211. Liguid Scintillation Countipg. Permagon Press, New York - London. Anderson, E. C. 1959. Application of natural gamma activity measurements to meat. Food Res. 24:605. Anderson, E. C. and W. H. Langham. 1959. Average potassiun concentra- tion of the human body as a function of age. Science 130:713. 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APPENDIX .N manmfi mammnu cw wouaomoua maowumH>om fiumwsmum was mamoz Ema ca moaam> neon «mam H68 mmnq one. mamm 6mm Nana woo mans has mama neon oamm can Haws «no mans “as mmms Nam Hmnm can «mus N anon «mm “has mos Neon amm seem moa mnom an» anon was amaa New Some 8am Baas 62 Egg coon N66 «sum mama maom a gag coon . .6... 2m .82 3.. m2: . «on amma «as mean ems mam. mam new. «an eon. ems mmmn mos mmma see «man mas cams owe mama name 648 mwmm Nan Nwam 68w anon New «mos man main «mm emoq coo mmam was oaoq SHHH NSSN awe Hoas moan coca ans «mam mus «Hem mmq sacs H.306m gunmuOm gflmmwuom gfiflom gfimmmuom gfifiom gwmmduom SHUOW gflmmwuom gnaw moans uHo< mozm +stHHwom moans ao>o sowumuammwwson <03 wosuoe_sowuomuuxm ESHpOm was aaammmuom waauomuuxo mo mpoauoa_unouommap mo somHHmano .H manna -91- Table 2. Slaughter and separation data from Los Alamos lambs Hot Separable carcass Lamb Live carcass Non-carcass components No. weight weight components Lean Fat Bone 1 105.5 56.3 48.5 33.0 14.1 8.19 2 88.0 49.0 40.0 27.1 13.6 7.41 3 93.5 46.9 46.1 26.8 10.3 8.35 4 80.2 42.1 38.7 23.5 11.7 6.05 5 76.6 41.9 35.6 25.3 8.9 6.10 6 100.8 54.4 45.2 31.3 13.1 8.25 7 88.1 43.8 44.4 26.0 8.7 7.87 8 84.3 42.1 43.4 26.5 7.1 6.95 9 83.4 42.7 41.3 28.0 4.1 8.70 10 78.3 38.2 39.4 23.4 5.4 7.12 Weights in lb. Means and standard deviations presented in thesis table 3. Table 3. Chemical composition of separable fat and lean from Los Alamos lambs Lamb Separable lean Separable fat N9, Water Ether-extract Protein Water Ether-extract Protein 1 73.5 6.7 19.1 25.1 69.3 5.19 2 70.0 0.0 18.8 18.2 77.4 4.38 3 72.0 8.4 18.6 29.4 63.0 7.28 4 72.4 8.0 18.6 19.5 76.1 5.00 5 72.1 8.8 18.2 25.8 68.5 6.00 6 72.7 7.5 19.0 23.7 70.1 5.94 7 74.2 5.7 18.9 28.5 64.9 6.00 8 73.2 6.4 19.1 28.6 63.5 7.81 9 76.5 4.4 18.3 47.6 41.2 11.22 10 72.6 6.3 20.3 28.6 61.5 9.69 Composition values in %. Means and standard deviations for edible carcass (separable fat + lean) presented in thesis table 3. -92- Table 4. Potassium content of Los Alamos lambs from potassium-40 counts _" Separable carcass Lamb Live Non- components Live No. washed Carcass carcass Lean Fat Bone unwashed 1 0.177 0.262 0.233 0.297 0.056 0.135 p 0.250 2 0.174 0.194 0.151 0.287 0.014 0.123 0.229 3 0.179 0.171 0.094 0.267 0.042 0.071 0.270 4 0.151 0.230 0.175 0.308 0.074 0.199 0.267 5 0.147 0.220 0.171 0.271 0.013 0.094 0.237 6 0.162 0.181 0.166 0.314 0.096 0.147 0.268 7 0.183 0.249 0.214 0.284 0.089 0.194 0.293 8 0.194 0.256 0.171 0.319 0.112 0.155 0.261 9 0.196 0.235 0.188 0.316 0.154 0.206 0.316 10 0.209 0.250 0.216 0.318 0.052 0.087 0.314 Potassium content in %. Means and standard deviations presented in thesis table 4. Table 5. Sodium and potassium content of separable lean and fat from Los Alamos lambs as measured by flame photometry Potassium Sodium Lamb. Separable lean Separable Separable Separable No. Run 1 Run 2 fat lean fat 1 0.298 0.286 0.064 0.081 0.066 2 0.306 0.319 0.056 0.070 0.051 3 0.305 0.313 0.079 0.073 0.068 4 0.299 0.311 0.064 0.069 0.051 5 0.303 0.297 0.079 0.082 0.067 6 0.317 0.320 0.070 0.073 0.062 7 0.315 0.318 0.074 0.070 0.076 8 0.340 0.340 0.086 0.070 0.071 9 0.311 0.316 0.126 0.079 0.111 10 0.331 0.337 0.119 0.078 0.079 Sodium and potassium content in %. Values are the mean of duplicate analyses. Means and standard deviations for potassiun have been presented in thesis table 4 and for sodium in thesis table 7. -93- Table 6. Cesium-137 content of Los Alamos lambs from gamma counts Separable carcass Lamb Live Live Non- components No. unwashed washed Carcass carcass Lean Fat Bone 1 50.5 78.1 73.2 60.0 96.6 -51.5 124.9 2 62.4 86.8 84.4 49.2 107.7 727.9 400.6 3 67.7 83.1 122.4 145.6 80.7 909.4 401.5 4 68.2 89.8 81.4 175.2 104.9 176.3 234.5 5 74.5 121.0 75.2 -24.4 68.3 1.6 -69.9 6 61.2 99.6 92.6 189.2 72.1 39.3 75.2 7 47.7 64.9 50.9 55.3 59.1 62.5 153.3 8 48.8 70.6 96.9 101.7 64.0 -71.9 -74.6 9 57.2 93.5 95.1 69.0 80.9 74.4 76.4 10 41.8 60.2 43.0 25.9 60.3 510.6 14.1 Values presented are ppc cesiunfgm. potassium (potassium estimated from potassium-40). Results on separable fat and bone are very unreliable because of the small number of counts. . Means and standard deviations are presented in thesis table 9. Table 7. Composition of 38 1b. ground pork samples Potassium Sodium Ether- Flame flame Samples Water extract Protein K-40 photometry photometry 8 + 14 37.7 50.7 10.8 0.153 0.175 0.035 15 + 26 43.8 42.9 12.3 0.191 0.209 0.041 13 + 16 45.3 40.8 13.1 0.209 0.211 0.039 21 + 17 48.6 36.8 13.9 0.244 0.233 0.042 24 + 10 49.9 35.3 13.9 0.241 0.234 0.041 22 + 12 52.7 31.8 14.6 0.252 0.253 0.042 23 + 27 54.2 30.3 14.8 0.292 0.267 0.045 11 + 25 56.0 27.0 16.0 0.274 0.268 0.044 9 + 20 59.7 22.2 17.3 0.299 0.290 0.046 18 + 19 63.1 17.5 18.3 0.312 0.307 0.046 8 + 15 38.5 49.4 10.9 0.167 0.178 0.036 14 + 26 43.0 44.2 12.2 0.181 0.206 0.040 13 + 21 46.6 39.4 13.3 0.204 0.224 0.042 18 + 9 60.1 21.4 17.6 0.323 0.291 0.045 19 + 20 62.7 18.3 18.0 0.308 0.306 0.047 24 + 23 51.4 33.8 14.1 0.247 0.248 0.043 10 + 22 52.3 32.1 14.6 0.256 0.248 0.041 16 + 17 47.4 38.2 13.7 0.213 0.220 0.039 12 + 25 53.7 30.1 15.1 0.261 0.260 0.043 27 + 11 55.2 28.3 15.6 0.262 0.265 0.045 Composition figures in %. Sample values the mean of two 19 1b. cartons of ground pork. Means and standard deviations are presented in thesis tables 11 and 12. -94- Table 8. Composition of 38 lb. ground lamb samples Potassium Sodium Ether- Flame flame Samples Water extract Protein K—40 photometry photometry l + 2 36.6 52.5 11.5 0.171 0.163 0.049 3 + 31 49.0 35.5 14.6 0.217 0.218 0.059 29 + 34 54.1 30.1 15.5 0.277 0.249 0.064 32 + 35 63.1 18.4 17.6 0.323 0.287 0.066 4 + 28 58.8 23.2 16.7 0.310 0.277 0.064 6 + 36 57.3 25.2 16.8 0.271 0.262 0.064 7 + 33 55.5 26.8 16.1 0.259 0.247 0.064 5 + 30 55.3 27.5 16.1 0.270 0.257 0.063 28 + 37 58.9 23.1 16.8 0.318 0.270 0.067 1 + 3 40.4 46.7 12.4 0.180 0.182 0.051 2 + 31 45.2 41.3 13.7 0.209 0.198 0.057 32 + 37 62.2 18.6 18.0 0.270 0.283 0.070 29 + 30 54.2 29.5 15.3 0.263 0.254 0.061 33 + 34 54.7 28.1 16.0 0.236 0.242 0.066 35 + 36 59.6 22.9 16.6 0.272 0.272 0.066 Composition figures in %. Sample values the mean from two 19 lb. cartons of ground lamb. Means and Standard deviations are presented in thesis tables 11 and 12. -95- Table 9. Potassium content of 24 pigs Empty Pig Frozen G.I. Remaining Empty No. carcass tract viscera Head Blood1 Hair2 body 1 0.2285 0.2024 0.2887 0.1668 0.0669 0.2063 2 0.2214 0.2275 0.2591 0.1538 0.0978 0.2121 3 0.2276 0.1923 0.2527 0.1577 0.0978 0.2166 4 0.2135 0.1918 0.2704 0.1529 0.0978 0.2074 5 0.1752 0.1904 0.2750 0.1433 0.0978 0.1746 6 0.2455 0.1988 0.2510 0.1560 0.0978 0.2316 7 0.2008 0.1796 0.2515 0.1440 0.2247 0.0978 0.1936 8 0.2375 0.1617 0.2513 0.1583 0.2168 0.0978 0.2211 9 0.2297 0.1597 0.2343 0.1436 0.2216 0.0978 0.2145 10 0.1872 0.2006 0.2512 0.1253 0.2061 0.0978 0.1830 11 0.2060 0.1872 0.2595 0.1503 0.2386 0.0978 0.1974 12 0.2164 0.1866 0.2577 0.1369 0.2238 0.0978 0.2063 13 0.2163 0.1698 0.2511 0.1531 0.0978 0.2067 14 0.2112 0.1856 0.2517 0.1569 0.0978 0.2043 15 0.2045 0.1821 0.2515 0.1434 0.2192 0.0978 0.1967 16 0.2205 0.1996 0.2556 0.1364 0.2117 0.0978 0.2110 17 0.1737 0.1984 0.2674 0.1442 0.2270 0.0978 0.1734 18 0.2193 0.1907 0.2526 0.1497 0.2308 0.0978 0.2106 19 0.1901 0.1684 0.2453 0.1351 0.0978 0.1845 20 0.2103 0.2062 0.2539 0.1380 0.0978 0.2027 21 0.2186 0.1966 0.2680 0.1481 0.0978 0.2097 22 0.1980 0.1674 0.2505 0.1458 0.0978 0.1908 23 0.1947 0.1689 0.2555 0.1426 0.2218 0.0978 0.1893 24 0.1891 0.1839 0.2644 0.1467 0.0978 0.1868 All figures presented are % potassium. Mean of duplicate analyses. The blood from the remaining samples had been completely used in previous analyses. Samples were collected from three additional mature pigs at Slaughter and found to contain 0.2155, 0.2092 and 0.2111% potassium. values for the 14 pigs were averaged and the mean value was used in com- puting the body potassium content. The value for pig 1 differs from the remaining value because the skin and some subcutaneous fat is included. sample was analysed. The means and standard deviations are presented in thesis table 18. The weights and chemical composition of the 24 pig bodies and their com- partments have been presented in the appendix of Gnaedinger (1962). For the remaining animals a composite The -96- Table 10. Sodium content of 24 pigs Empty Pig Frozen G.I. Remaining Empty No. carcass tract viscera Head Blood1 Hair2 body 1 0.0648 0.1180 0.1115 0.1852 0.0995 0.0830 2 0.0727 0.1346 0.1112 0.1673 0.0918 0.0844 3 0.0728 0.1439 0.1150 0.1573 0.0918 0.0865 4 0.0637 0.1081 0.1053 0.1521 0.0918 0.0756 5 0.0611 0.0973 0.1166 0.1391 0.0918 0.0730 6 0.0734 0.1245 0.1078 0.1647 0.0918 0.0868 7 0.0707 0.0997 0.1076 0.1435 0.0918 0.0801 8 0.0807 0.0886 0.1133 0.1569 0.0918 0.0896 9 0.0657 0.0993 0.1091 0.1513 0.0918 0.0770 10 0.0709 0.1097 0.1125 0.1537 0.0918 0.0832 11 0.0746 0.1076 0.1141 0.1611 0.0918 0.0849 12 0.0746 0.1039 0.1118 0.1157 0.0918 0.0830 13 0.0751 0.1139 0.1168 0.1609 0.0918 0.0851 14 0.0715 0.1252 0.1202 0.1603 0.0918 0.0818 15 0.0672 0.1021 0.1148 0.1481 0.0918 0.0793 16 0.0762 0.1201 0.1205 0.1194 0.0918 0.0863 17 0.0690 0.1107 0.1064 0.1540 0.0918 0.0789 18 0.0675 0.1103 0.1152 0.1564 0.0918 0.0789 19 0.0753 0.1127 0.1099 0.1297 0.0918 0.0835 20 0.0798 0.1162 0.1180 0.1346 0.0918 0.0889 21 0.0774 0.1140 0.1152 0.1716 ' 0.0918 0.0876 22 0.0729 0.1047 0.1216 0.1666 0.0918 0.0830 23 0.0694 0.1033 0.1103 0.1612 0.0918 0.0793 24 0.0699 0.1076 0.1118 0.1566 0.0918 0.0804 All figures presented are % sodium. Mean of duplicate analyses. 1As the blood samples had been prevented from coagulating with sodium citrate, they were unsuitable for sodium analyses. Samples were collected from three additional mature pigs at slaughter and found to contain 0.1826, 0.1760 and 0.1889% sodium. The mean value was used in computing body sodium. See footnote 2 in appendix table 9. The means and standard deviations are presented in thesis table 19. The weights and chemical composition of the 24 pig bodies and their com- partments have been presented in the appendix of Gnaedinger (1962). a. v. 3142 5006 II»unmfiluwfinflmmum»unuuimiuiflnfiiim