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A Study of nitrogen metabolism, with special reference to mink Oldfield, James Edmund 1949

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A STUDY OF NITROGEN METABOLISM WITH SPECIAL REFERENCE TO MINK. James Edmund O l d f i e l d A thesis submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of Master of Science i n Agriculture. IN THE DEPARTMENT OF ANIMAL HUSBANDRY The University of B r i t i s h Columbia August, 1949. A STUDY OF NITROGEN METABOLISM WITH SPECIAL R2PERSNCE TO MINK. by James Edmund O l d f i e l d ABSTRACT: Experimental studies with mink at the University of B r i t i s h Columbia had t h e i r o r i g i n with the a c q u i s i t i o n by the University of a mink colony i n 1947. In September of that year various l o c a l mink ranchers donated some 60 animals to the University with a view to establishing an experimental unit on which research might be c a r r i e d out. The ultimate -object of such research was to be the formulation of d e f i n i t e feeding standards f o r mink, such as are already available f o r other species. This project was recognized as a long-term proposition, and i n i t i a l experiments were designed to inves-tigate the protein requirements, of mink. Preparatory to the experimental project, a survey of the l i t e r a t u r e concerning general nitrogen metabolism,'and more p a r t i c u l a r l y the concept of so-called "endogenous" nitrogen metabolism was c a r r i e d out. * This survey constitutes the opening portion of t h i s Thesis. The actual experimental work undertaken was divided into two phases: 1. Investigation of the endogenous nitrogen excretion of mature animals maintained i n a f a s t i n g condition, or on a nitrogen-free d i e t . ...ABSTRACT (2) 2 . Conventional nitrogen balance t r i a l s , involving the establishment of nitrogen equilibrium at the lowest possible l e v e l , using certain s p e c i f i e d sources of dietary protein. The method followed involved the c o l l e c t i o n and analysis of urine samples from adult animals maintained under d i f f e r e n t stated conditions of n u t r i t i o n . Total nitrogen was determin-ed by the Kjeldahl-Gunning procedure, and creatinine content was estimated by the F o l i n - J a f f e a l k a l i n e picrate reaction. Various supplementary procedures were instigated to guard . against possible interference by abnormal urinary constituents. The results obtained would appear to have extensive imp-l i c a t i o n s regarding future investigations into the n u t r i t i v e requirements of mink. F i r s t , at the expense of a great deal of time and e f f o r t , equipment has been b u i l t which has proven satisfactory for the laboratory Investigation of t h i s newly domesticated animal. Second, the close c o r r e l a t i o n of actual data with figures c i t e d i n the l i t e r a t u r e f o r other species of s i m i l a r bodily dimensions suggests that the mink i s not phys i o l o g i c a l l y abnormal, and that predictions as to i t s nut-r i t i v e behaviour may be made i n comparison with.other species with reasonable accuracy. Third, the experiments dealing with protein requirements suggest that considerable overfeeding of proteins may be common prac t i c e , especially i n cases of mere maintenance of mature animals. A very strong suggest-ion i s put forward f o r future studies into the b i o l o g i c a l values of d i f f e r e n t native proteins f o r mink. ...ABSTRACT (3) Detailed descriptions of a n a l y t i c a l procedures and ex-perimental equipment, and discussion of additional topics regarding mink n u t r i t i o n are appended'to the main body of the Thesis, i n the hope that they may serve as a useful reference f o r future investigations along t h i s theme. These l a t t e r include figures representative of time of passage and basal metabolism; reference to the natural diet of the mink i n the wild state; correlations between organ size and body weight i n mature animals, and weight changes exhibited by growing mink k i t s . Approved T H . M . King) Frofessozyand Head, Department of Animal Husbandry. ACKNOWLEDGEMENT The writer wishes to acknowledge h i s appreciation to Professor H.M. King, Head of the Department of Animal Husband-ry f o r putting at his disposal the resources of the Department and especially the University Mink Colony on which the exper-imental work contained herein was ca r r i e d out. Special thanks are tendered Dr. A.J. Wood, Associate Professor i n the Department of Animal Husbandry f o r the ben-e f i t 'of h i s teaching i n Animal Nutrition, his keen c r i t i c i s m , and his boundless encouragement and enthusiasm in'the conduct of t h i s project. The writer also wishes to acknowledge the co-operation of various organizations interested i n the advancement of science i n agr i c u l t u r e . A scholarship administered by the A g r i c u l t u r a l I n s t i t u t e of Canada made i t f i n a n c i a l l y possible f o r the writer to carry out t h i s program of research. A grant from the B r i t i s h Columbia Ind u s t r i a l and S c i e n t i f i c Research Council made possible the provision of the necessary apparatus and supplies. A STUDY OF NITROGEN METABOLISM WITH SPECIAL REFERENCE TO MINK. TABLE OF CONTENTS PAGE I INTRODUCTION 1 The general scope of nitrogen metabolism i n -vestigations and balance t r i a l s with a note re t h e i r p a r t i c u l a r a p p l i c a t i o n i n the case of mink. I I LITERATURE SURVEY 3 General Nitrogen Metabolism 3 Amino Acid Metabolism 3 Enzymatic Action 5 Absorption 6 Functions of Nitrogenous Compounds i n the Tissues 8 S p e c i f i c Dynamic Action 11 Protein Storage 13 Mi c r o b i a l Action 15 Endogenous Nitrogen Metabolism 17 Theories of Endogenous Nitrogen Metabolism 17 Measurement of Endogenous Metabolism 19 The Significance of Nitrogen Balance 23 D e f i n i t i o n 25 Calculation of the State of Nitrogen Balance 23 N u t r i t i v e Value of Proteins 25 II I EXPERIMENTAL 31 Some Considerations Involved i n Planning a N u t r i t i o n Experiment. Choice of F i e l d f o r Experimentation 33 Plan of Experiment 34 Method 35 Observations and Discussions 39 Endogenous Nitrogen Excretion 39, Nitrogen Balance Experiments 42 Creatinine Excretion ' 48 Summary 51 IV APPENDICES i Preparation of Reagents and Laboratory Tech- i niques. Animal Techniques v i Additional Data Re Mink N u t r i t i o n x A STUDY OF NITROGEN METABOLISM WITH SPECIAL REFERENCE TO MINK In both plants and animals a substance i s contained which i s produced within the former and imported through the food to the l a t t e r . I t i s one of the most complicated substances ... very changeable i n composi-ti o n . I t i s unquestionably the most impor-tant of a l l known substances i n the organic kingdom. Without i t no l i f e appears possible on our planet. Through i t s means the chief phenomena of l i f e are produced. - G. J . Mulder (1840) INTRODUCTION Possibly no' single phase of metabolism has been the sub-j e c t of so much controversy, or the object of more det a i l e d investigation than has the metabolism of proteins and of n i t -rogenous compounds generally. From the earliest days of the study of the science of n u t r i t i o n , investigators have believed proteins to be endowed with some v i t a l property not attributed to the other classes of nutrients. In the words of Rubner, (1920), one of the pioneers i n t h i s f i e l d , "Protein contains the magic of l i f e , ever newly created and then dying, a process continuous since the advent of l i f e upon the earth." Among the major foodstuffs, carbohydrates and fats contain b a s i c a l l y only carbon, hydrogen and oxygen i n various proportions. Proteins, i n addition to these elements, contain nitrogen and usually sulphur, and quite commonly other inorganic elements including phosphorous, i r o n and copper. Perhaps even more important than the mere presence of these elements, however, i s t h e i r organization into the complexity of form i n which they are f i n a l l y used by the organism. Through t h e i r complicat-ed structure, therefore, as well as t h e i r diverse d i s t r i b u t i o n , - 2 -("Protoplasm sans protein does not e x i s t " - C a h i l l (1944,a), proteins o f f e r to the student of n u t r i t i o n a f i e l d which i s at once absorbing and enlightening. Not a l l of the nitrogenous constituents of the animal body are proteins; however by v i r t u e of t h e i r vast quantita-t i v e s u p e r i o r i t y and t h e i r metabolic s i m i l a r i t y to the other nitrogenous components, proteins are commonly assessed as a v a l i d expression of nitrogen metabolism as a whole. In the following pages a survey of the l i t e r a t u r e i s presented i n an attempt to c l a s s i f y and explain, as f a r as i s presently possible, the various phases of nitrogen metabolism as inves-tigated i n various animals and f i n a l l y an experiment i s des-cribed wherein some of these data are applied i n the s p e c i a l case of the mink. Since the time of Sanctorius of Padua, (Lusk, 1931a), the value of so-called "balance t r i a l s " i n the in v e s t i g a t i o n of n u t r i t i v e requirements of animals has been recognized. Nitrogen balance, or the precise comparison of nitrogen i n the ingesta and excreta of an animal, serves as ah i n d i c a t i o n of the nitrogen metabolism of that animal. In addition, the importance of nitrogen balance studies i s enhanced by the f a c t that a supply of proteins or t h e i r components i s i n d i s -pensable to higher organisms as already mentioned (Jackson, 1945). A study of the theory of nitrogen metabolism comple-mented by an experimental nitrogen balance t r i a l i s p a r t i -c u l a r l y applicable i n the case of mink for several reasons. The incomplete domestication of these animals causes them to be extremely resentful of changes i n t h e i r environment, necessitating rather c a reful d e f i n i t i o n of experimental con-d i t i o n s . In view of t h i s f a c t , i n v e s t i g a t i o n of the nitrogen metabolism of mink which as t y p i c a l carnivores h a b i t u a l l y consume diets r i c h i n meat and f i s h would seem a l o g i c a l s t a r t i n g point i n the c a l c u l a t i o n of t h e i r complete n u t r i t i v e requirements. LITERATURE SURVEY General Nitrogen Metabolism The source of the great majority of the nitrogen meta-bolized by the animal body i s the dietary protein. Some food materials, notably green vegetables and roots, contain apprec-iable quantities of free amino acids; however i n p r a c t i c a l n u t r i t i o n i t i s doubtful whether these can be considered as important sources of nitrogen. Demonstration of anaphylactic shock i n sensitized animals i n cases of certain protein a l l e r -gies (Wilson, 1935), suggests the d i r e c t absorption of at lea s t small traces of proteins from the digestive t r a c t . This operation probably occurs i n minute quantities only, however, and the b i o l o g i c a l value of such absorption i s doubt-f u l . Amino Acid Metabolism The c l a s s i c a l view of nitrogen metabolism involved the hydrolytic breakdown of dietary protein into i t s constituent amino acids followed by the recombination of these same amino acids to y i e l d the proteins necessary to the animal body. I f , - 4 as i s often the case with "normal" diets f o r mature animals, more protein i s supplied than i s necessary to f u l f i l the several requirements f o r nitrogenous materials i n the body, the excess i s degraded and i t s nitrogen eliminated mostly i n the forms of urea, ammonia, or u r i c a c i d . (Baldwin, 1947a). I t i s also immediately evident that as proteins are complex conjugations of amino acids i n the higher animals at l e a s t i t i s these amino acids which are the ultimate l i m i t i n g f a c -t o r i n the entire scheme of nitrogen metabolism. Obviously, i t w i l l be impossible to b u i l d a protein within the animal body i f one of i t s constituent amino acids i s unavailable, therefore the necessary amino acids must eithe r be supplied i n the d i e t or synthesized from other substances by the animal. Preliminary studies on the biochemistry of proteins were contingent on the development of suitable methods f o r the i d e n t i f i c a t i o n and separation of amino acids. I t was d i s -covered quite early that proteins when boi l e d with strong mineral acids would break down into mixtures of amino acids but the i s o l a t i o n of these l a t t e r remained a perplexing prob-lem u n t i l the a p p l i c a tion of the low pressure f r a c t i o n a l d i s t i l l a t i o n technique by Emil Fischer i n 1901. This method involved the f r a c t i o n a l d i s t i l l a t i o n of the ethyl esters of amino acids under conditions of very low pressure, and i t resulted i n very rapid progress i n the knowledge of protein structure. Even the adoption of such improved techniques could not completely c l a r i f y the scheme of protein constitu-t i o n , however, due to the extreme complexity of the native substances. That i s to say, although p a r t i a l fragmentation of the protein molecules and i d e n t i f i c a t i o n of t h e i r compon-ents could be carried out, the remainders involved immense technical d i f f i c u l t i e s and our knowledge on the subject i s s t i l l f a r from complete. Enzymatic Action The l i b e r a t i o n of the i n d i v i d u a l amino acids from t h e i r mother proteins takes place i n the i n t e s t i n a l t r a c t and i s the culmination of many and varied enzymatic reactions. A complete description of the mechanisms of these breakdown processes i s a study i n i t s e l f and l i e s beyond the scope of t h i s paper; however, a b r i e f resume i s necessary f o r contin-u i t y of the theme. Enzymatic action upon proteins i s hydro-l y t i c i n nature - that i s , causing a cleavage with the addi-t i o n of the elements of water at the point of cleavage. (Mitchell, 1929a). One of the important features of such a breakdown procedure i s that i t e n t a i l s an almost n e g l i g i b l e loss of chemical energy thus r e s u l t i n g i n a high conservation of energy i n the digested products. Hydrolysis proceeds stepwise, r e s u l t i n g i n the successive formation of smaller and smaller fragments of the mother protein accompanied at each stage by the l i b e r a t i o n of c e r t a i n amino acids or amino acid groups. Digestion of dietary protein commences i n the stomach through the action of a p r o t e o l y t i c enzyme, pepsin, i n the strongly acid medium of the g a s t r i c j u i c e . Another stomach enzyme, rennin, converts milk casein to paracasein which - 6 -forms an insoluble calcium s a l t (curd) thus retarding the rate of passage of th i s food through the digestive t r a c t while increasing the absorptive surfaces by distension. Pepsin, of course, acts upon the protein of the c l o t i n i t s usual manner. Action of these enzymes c a r r i e s the fragmentation of the native proteins to the phases of metaproteins, pro-teoses and peptones. Following these i n i t i a l breakdown pro-cedures, the protein materials are transported into the i n -testine as a component of chyme, whereupon they are subjected to more vigorous action by the pancreatic enzyme, trypsin, and the i n t e s t i n a l enzymes, erepsin. The mode of action of these l a t t e r i n common with that of pepsin i s characterized by the formation of proteoses and peptones, however, the l i b e r a t i o n of increased quantities of amino acids indicates a somewhat d i f f e r e n t point of attack. Experiments conducted by Frankel (1916) would seem to point to a complementary ac-ti o n by the various p r o t e o l y t i c enzymes - that i s , the hydro-l y t i c effects of t r y p s i n and erepsin are more complete when preceded by peptic digestion. Absorption Considerable doubt s t i l l e x ists as to whether proteins are absorbed e n t i r e l y i n the amino acid form, however an overwhelming mass of evidence points to t h i s method of ab-sorption as the normal procedure. The presence of hy d r o l y t i c enzymes capable of cleaving the native proteins into t h e i r constituent amino acids i n the digestive t r a c t ; the occurr-ence of considerable quantities of free amino acids i n the - 7 -i n t e s t i n a l contents; and the apparently normal n u t r i t i v e con-d i t i o n of animals fed amino acids i n place of proteins a l l lend weight to t h i s concept. Absorption of the end products of protein digestion appears to be most active i n the duodenal region where an extensive surface i s presented through the involutions or v i l l i of the i n t e s t i n a l mucosa. SCHEMA OF INTESTINAL VILLI (DOG) ... a f t e r Maximow and Bloom (1947) I t w i l l be noticed from the diagram presented above that two pathways are open to the products of protein digestion absorbed through the v i l l i underlying the i n t e s t i n a l mucosa. They may enter the blood c i r c u l a t o r y system d i r e c t l y through the c a p i l l a r y network or i n d i r e c t l y by way of the l a c t e a l s , the lymph c i r c u l a t i o n and the jugular vein. I t i s generally - 8 -accepted that protein and carbohydrate products take the former path and fat s the l a t t e r although the separation of the nutrients does not appear complete and i s by no means • thoroughly understood. (Mitchell., 1929b) In the early stages of phys i o l o g i c a l i n v e s t i g a t i o n the amino acids absorbed by way of the i n t e s t i n e were thought to be almost immediately re-synthesized into proteins -possibly i n the i n t e s t i n a l wall i t s e l f . Improvement and perfection of methods for the detection of amino acids i n the blood, however, l e d to the general discard of t h i s theory. Rolin and Denis, (1912), were able to demonstrate f i r s t the normal presence of amino acids i n the blood, and second, a marked increase i n t h i s amino-acid content immediately a f t e r protein ingestion by the animal. Further investigations (Van Slyke, 1913) revealed the blood amino acid l e v e l to be r e l a t i v e l y low even i n cases of animals fed diets r i c h i n proteins or injected intravenously with amino acids, suggest-ing an almost immediate removal of these amino acids from the blood by the ti s s u e s . Here again the procedure was neither simple nor uniform: differences were observed i n the rates of absorption of various i n d i v i d u a l amino acids as well as differences i n retention by the various t i s s u e s . (Van Slyke, 1942) Functions of Nitrogenous Compounds i n the Tissues Several possible fates may await the amino acids taken up by the tissues. One immediately thinks of t h e i r re-synthe-s i s into body proteins to meet the requirements of growth i n - 9 -the young animal or of tissue repair and maintenance i n the adult but these are not t h e i r only uses. A certa i n amount of the amino acids, including those from protein consumed i n excess of requirements, are deaminized and used as a source of energy either immediately or at some l a t e r time. While provision of an energy source i s not usually considered as a major accomplishment of protein materials as contrasted with carbohydrates and f a t s , i t seems l i k e l y that i t may assume considerable proportions p a r t i c u l a r l y i n the case of animals which subsist on high protein d i e t s . The source of energy f o r muscular work,or conversely, the influence of muscular work on protein metabolism has long been a subject of controversy among phys i o l o g i s t s . At one time i t was believed because of the nitrogenous nature of the muscle tissues involved that protein i t s e l f supplied the t o t a l energy required f o r t h i s metabolic phenomenon. This view was discarded following completion of a nitrogen and energy balance experiment i n Switzerland which i s of at l e a s t s u f f i c i e n t h i s t o r i c a l i n t e r e s t to record. Two mountaineers, •weighing s i x t y - s i x and seventy-six k i l o s respectively, climbed the Faulhorn - a v e r t i c a l elevation of 1956 metres. Their t o t a l protein consumption during the period of the climb was found to be 22.09 gm. and 20.89 gm. which, even i f com-pl e t e l y u t i l i z e d could not supply the energy required f o r performance of the work involved (Cathcart, 1925). Although t h i s experiment could c e r t a i n l y not be judged s c i e n t i f i c a l l y precise by modern standards, nevertheless i t s main conclusion - 10 -i s well founded that "The substances by the burning of which force i s generated i n the muscles are not the albuminous constituents of those tissues but non-nitrogenous substances either f a t s or carbohydrates." More recent knowledge, as indicated l a t e r , does not make such sweeping claims but rather points to the use of protein under cert a i n conditions f o r the supply of at leas t a portion of the body's energy. I t may be of in t e r e s t to present at t h i s point a compari-son of the requirements of various animals i n order to i n -dicate the r e l a t i v e importance of protein as a dietary con-stit u e n t and as an energy source. Using the commonly accep-ted r a t i o of 2.00 mg. of endogenous nitrogen excreted per Calorie of basal heat produced, (Ashworth, .Brody, Smuts, Terroine and many others) the following protein requirements might be expected to apply: TABLE I: RELATIVE IMPORTANCE OF PROTEIN AS AN ENERGY SOURCE. Species Body Weight (grams) B.M.R. (Cal/24hr) Endogenous N Excretion mg. (calculated) Protein Equiv. gms. Calories Supplied : bv Protein No. Rat 400 33.2 I 66.4 0.415 1.70 5.12 Cat 3000 152.0 304.0 1.90 7.80 5.13 Dog 14000 485.0 970.0 6.06 24.84 5.13 Sheep 45000 1160.0 2320.0 14.56 59.50 5.13 Man 65000 1640.0 3280.0 20^50 84.05 5.13 Basal metabolism data are taken from Benedict, " V i t a l Energetics" (1938). Calories supplied are calculated on the basis of Rubner's 4.1 Cal/gm. - l i -l t i s i n t e r e s t i n g to note that i n a l l these animals the amount of protein that must be fed i n order to s a t i s f y the body's endogenous needs f o r nitrogen supply only approximately f i v e percent of the t o t a l c a l o r i c requirements. The f a c t that carnivora are included among these findings suggests the p o s s i b i l i t y that such animals do not necessarily require a large proportion of protein i n t h e i r d i e t and moreover that they may have become meat eaters through reasons of ecological advantage rather than phy s i o l o g i c a l necessity. Such an observation appears e s p e c i a l l y pertinent with regard to the formulation of p u r i f i e d rations f o r experimental use with mink. Further uses of the amino acids include the formation of various enzymes, hormones and detoxication products such as those formed by means of conjugation with glycine. I t i s doubtful i n the course of any of these functions whether the actual dietary amino acids are used as such. Rather, these amino acids are modified through decarboxylation, deamination and s i m i l a r processes and d i f f e r e n t fragments are re-conjugat-ed to give r i s e to the amino acids of the t i s s u e s . In the course of these transformations some of the non-nitrogenous residues may be converted to glucose, glycogen or f a t and stored i n the animal body. S p e c i f i c Dynamic Action Any discussion of nitrogenous foods i n the l i g h t of t h e i r energetic e f f i c i e n c y would be incomplete without men-tion of t h e i r c h a r a c t e r i s t i c S p e c i f i c A A c t i o n . E a r l y - 12 -n u t r i t i o n i s t s , i n attempting energy balance t r i a l s , found that when an amount of protein s u f f i c i e n t to meet an animal's basal energy requirements was fed, i t raised the heat output considerably over the previous l e v e l . Lusk (1931b) (15) i n his laboratory has measured heat production of mature dogs i n complete repose a f t e r consuming p u r i f i e d diets with the following r e s u l t s : 100 c a l s . ingested as protein of meat increase heat production 30 c a l s . 100 c a l s . ingested as f a t increase heat production 4.1 c a l s . 100 c a l s . ingested as glucose increase heat production 4.9 c a l s . These findings indicate that i n protein foods at l e a s t the energy l o s s of SDA i s of s u f f i c i e n t magnitude to warrant careful consideration i n c a l c u l a t i o n of n u t r i t i o n a l require-ments. The cause and nature of SDA has been the subject of much intense i n v e s t i g a t i o n . Mere mechanical i r r i t a t i o n of the i n t e s t i n a l t r a c t i s not the cause of l i b e r a t i o n of t h i s waste energy as evidenced by the experiments of Benedict and Emmes (1912) who fed humans cathartics and agar-agar with no subsequent increase i n heat production. Further, the sugges-tion that SDA might be caused by the work of digestion was discarded on the basis that amino acids injected into the animal body raised the l e v e l of metabolism equally with a s i m i l a r quantity of the same amino acids ingested by the animal (Weiss, 1924). Another theory that amino acids act as stimulants to c e l l u l a r metabolism has been rejected i n the l i g h t of recent work by Borsook (1936). The most commonly 13 -^accepted view today postulates that SDA arises as a r e s u l t of intermediary chemical reactions undergone by the amino acids and i s f a i r l y c l o s e l y correlated with the t o t a l energy involved i n the metabolism of those amino acids (Kriss, 1941). The SDA of proteins varies according to the consti.tu.egt amino acids and the "balance" of those amino acids i n the l a r g e r molecule. Maximum values for SDA are obtained at environ-mental temperatures of 25°C or over ; minimum values are shown at low temperatures of about 0 - 5°C, i n d i c a t i n g that the animal may make use of t h i s otherwise wasted energy to maintain i t s body temperature i n cases of environmental ex-tremes. Apart from i t s purely t h e o r e t i c a l connotations, SDA would appear to be of s i g n i f i c a n t importance i n the study of mink n u t r i t i o n by reason of the facts that the normal mink ra t i o n as now fed contains considerable protein and that the environmental temperatures under which these animals are kept are often below those minimums reported above. Protein Storage Actual storage of protein materials as such at f i r s t considered highly improbable has more recently become a sub-ject f o r intense inves t i g a t i o n . P o s s i b i l i t y of protein storage i n the animal body was postulated by Lusk (1931,c) i n an attempt to explain the continued nitrogen excretion of animals maintained 6n a nitrogen-free diet. The l a g i n attaining nitrogen equilibrium a f t e r an increase or decrease i n protein intake was observed by several investigators, i n -cluding Deuel (1928,a), Morrison (1942} and Ashworth and - 14 -Brody (1933) with the r e s u l t that protein retention has been generally c l a s s i f i e d under two broad headings: (Koster l i t z , 1946) 1. Nitrogen retained or l o s t i n conjunction with changes -i n protein intake. This nitrogen takes on the form of l a b i l e protein i n the cytoplasm of l i v e r and to some extent other t i s s u e s . 2. A more stable type of protein storage evidenced i n animals maintained on a protein-free d i e t or i n animals ex-posed to some abnormal nitrogen loss as i n cases of bleeding or thermal burning. More and more i n recent years the concept of the nature of stored or deposit "protein has changed from the early p i c -ture of an i n e r t "store" to that of a dynamic equilibrium. This l a t t e r view has been supported by the experiments of Borsook (1943) and more recently of Schoe)ieimer (1942) who made use of nitrogen isotopes as b i o l o g i c a l t r a c e r s . I t must be emphasized here and i t v a i l become more evident l a t e r that one of the more perplexing problems involved i n the study of the nitrogen metabolism of any animal i s the correct evalua-t i o n of the protein reserves of the body. While i t i s comparatively easy to theorize upon the general scheme of protein metabolism, i t i s correspondingly d i f f i c u l t to demonstrate the actual mechanics of the reactions involved i n the various phases of the^operation. For instance, one may point to protein synthesis i n the body as a mere con-jugation of amino acids, or indeed, as the reversible phase - 15 -of the reactions of p r o t e o l y s i s . In instances where the amino acid content of the dietary constituents and the end products of protein digestion are s i m i l a r , such may well be the case, but i f , as i s often true i n animal n u t r i t i o n , "complete" proteins are to be b u i l t from "incomplete", then obviously a more detailed procedure i s involved. The work of Wastneys and Borsook (19S5) has demonstrated the synthetic as well as hydrolytic effects of the g a s t r o - i n t e s t i n a l proteases: pep-s i n and t r y p s i n . I t appears, however, that these two diverse actions are regulated by r i g i d and s i m i l a r l y diverse condi-tions of temperature and pH, therefore, i t i s immediately obvious that the optima of hydrolytic and synthetic action cannot occur simultaneously. I t must be admitted that pro-t e i n synthesis i s an extremely complex procedure possibly including successive reductions, oxidations and polymeriza-t i o n s . Microbial Action A supplementary phase i n the metabolism of proteins i s carried out by the microorganisms l i v i n g within the alimentary canal of the host animal. M i c r o b i a l digestion occurs to a cer t a i n extent i n a l l animals i n the large i n t e s t i n e where amino acids and other protein residues that have escaped e a r l i e r absorption are exposed to b a c t e r i a l decarboxylation to form t h e i r corresponding amines. Many of the amines so formed are toxic, however, t h e i r creation i n a part of the digestive t r a c t from which, at l e a s t i n the l i g h t of present knowledge, absorption i s very s l i g h t , suggests that they have - 16 -but a doubtful b i o l o g i c a l significance ( C a h i l l , 1944,b). The puocess of deamination may also be brought about by microbial action; the method involved being apparently depen-dent upon conditions i n the i n t e s t i n e regarding oxygen supply, a c i d i t y and the l i k e . I t i s i n t e r e s t i n g to record the f i n d -ings of Hanke and Koessler (1920) regarding b a c t e r i a l action i n the intestine wherein they propose a buffering e f f e c t i n -herent i n such reactions. From t h i s work i t would appear that production of amines from amino acids by bacteria occurs only i n acid producing media while deamination r e s u l t s i n a buffered or alkaline medium. Another type of b a c t e r i a l a c t i v i t y that has recently assumed prominence i n n u t r i t i o n a l studies i s that of protein synthesis i n the digestive t r a c t s of some animals. In rumin-ants, f o r example, the rumen microflora are able to degrade protein from the ingesta, l i b e r a t i n g ammonia. Other bacteria, using t h i s ammonia as a s t a r t i n g point, are able to synthe-siz e proteins f o r t h e i r own use. The host organism, i n turn, from the bodies of such bacteria, i s able to obtain s i g n i -f i c a n t amounts of protein additional to that produced through the e f f o r t s of i t s own normal digestive a c t i v i t y . Possibly the most s i g n i f i c a n t feature of t h i s b a c t e r i a l action occurs i n cases where dietary protein i s low and hence where protein synthesis must exceed degradation. In such instances, the bacteria may u t i l i z e non-protein nitrogen sources as a basis f o r t h e i r synthetic a c t i v i t y thus increasing the supply of available nitrogen to the animal body (McNaught, 1947). - 17 -Apart from isuminants and those types of animals possessed of naturally enlarged caeca, however, animals do not harbour s u f f i c i e n t micro-organisms to produce s i g n i f i c a n t quantities of amino acids and proteins. The r e l a t i v e l y simple type of digestive t r a c t of carnivora greatly reduces the opportunity for synthesis and increases the dependence of the animal upon dietary sources of protein. Endogenous Nitrogen Metabolism The foregoing discussion has been concerned p r i m a r i l y with the metabolism of nitrogenous materials entering the body through the digestive t r a c t . Another aspect of t h i s metabolism e x i s t s , namely the transfers involved among the nitrogenous constituents of the tissues themselves. The existence of t h i s mode of metabolism was made known through the q u a l i t a t i v e differences i n the end products of protein digestion found i n the urine of animals maintained at wide-l y d i f f e r e n t l e v e l s of protein intake. Theories of Endogenous Nitrogen Metabolism Numerous investigators pondered the significance of these inconsistencies i n nitrogen excretion, however, i t was not u n t i l the c l a s s i s work of F o l i n , (1905), that a workable ex-planation was reached. F o l i n noted two main types of nitrogen excretion i n the urine - one constant, the other extremely variable. The former types represented i n the urine by such substances as creatinine and neutral sulphur, he termed en-dogenous nitrogen excretion. The l a t t e r , characterized by - 18 -formation of urea and inorganic sulphates, was c l a s s i f i e d as exogenous nitrogen excretion. While the exogenous quota appeared mainly concerned with the products of hydrolysis, the endogenous portion was taken as representative of oxidations occuring throughout the body tissues generally. F o l i n r s theory with the exception of minor modifications occasioned "by improvement i n techniques and subsequent advances i n the knowledge of separation and structure of the compounds involv-ed has received wide acceptance by biochemists, at l e a s t u n t i l very recently. The concept of endogenous nitrogen as postulated by F o l i n was broadened by l a t e r workers, including Lusk and Thomas, to embrace variable quantities of a l a b i l e or deposit protein. I t was noticed that animals fed nitrogen-free diets took a certain length of time often several days to reach a base (endogenous) l e v e l of nitrogen excretion i n t h e i r urine, (Smuts, 1939), and that t h i s time was roughly proportional to the nitrogen content of the pre-test d i e t . The thought n a t u r a l l y arose that a temporary store of l a b i l e protein existed i n the blood or c e l l u l a r f l u i d s of the body and was drawn upon during periods of negative nitrogen balance. S i g n i f i c a n t l y , the t r a n s i t i o n i n the products of nitrogen ex-cret i o n also lags behind the normal time of digestion when protein i s once more included i n the d i e t , thus suggesting the replenishment of t h i s nitrogen r e s e r v o i r . E s s e n t i a l l y , the F o l i n theory of endogenous nitrogen ex-cretion presumes the existence of an e a s i l y mobilized yet temporarily i n e r t store of nitrogenous material. More and - 19 -more i n recent years and e s p e c i a l l y following the investiga-tions of Borsook et a l , (1955) the concept of the so-called "deposit protein" reservoir has changed to one of a dynamic equilibrium. Borsook suggests the use of the term "continu-ing metabolism" with reference to the animal body's use of protein reserves; drawing a sharj d i f f e r e n t i a t i o n between t h i s quota and the "wear and tear" portion of F o l i n . Con-tinuing metabolism varies from one experimental animal to another, depending upon the previous dietary h i s t o r y and i n -volves continuous processes of amino acid degradation and synthesis. Measurement of Endogenous Metabolism This secondary nitrogen metabolism, i f i t may be termed such i n contrast to that involving the dietary constituents d i r e c t l y , i s obviously a reasonably constant measure of the basal l e v e l of nitrogen excretion by any animal. Assessment of basal nitrogen metabolism, (a convenient s t a r t i n g point i n nitrogen balance studies, j u s t as basal metabolism i s i n energetics) may be carried out by measurement of the c r e a t i -nine content of the urine of the experimental animals. Creatinine, a waste product, i s without doubt the most t y p i -c a l l y endogenous produot of nitrogen metabolism as the asso-ciated neutral sulphur excretion i s not altogether independent of dietary influences (Brody, 1954). Data have been produced to show that creatinine output of i n d i v i d u a l animals kept f i r s t on a high protein, l a t e r on an almost protein-free d i e t , i s p r a c t i c a l l y constant, (Hunter, 1928a), and although some - 20 -investigators such as Zwarenstein, (1926) claim to have noted marked variations i n creatinine output; i t i s nevertheless s i g n i f i c a n t that these variations cannot be correlated to t o t a l nitrogen output. The exact role played by creatinine i n metabolism i s yet to be described; however, Shaffer's concept that creatinine i s a product and an index of one phase of tissue catabolism (rather than of the e n t i r i t y ) and that t h i s phase takes place l a r g e l y within the muscles, o f f e r s at l e a s t a preliminary hypothesis (Shaffer, 1908). Lest the urinary excretion of creatinine be taken as an unimpeachable standard, several causes of v a r i a t i o n should be l i s t e d . Hunter (1928b) notes that creatinine output i s pro-foundly influenced by continued absorption of unusual d i e t s . For instance, i n humans fed a low protein, meat-free d i e t , the creatinine excretion declined gradually but s t e a d i l y . I t seems possible that such a lowering of creatinine output might be occasioned by a decline i n the t o t a l muscle mass. S i m i l a r -l y , animals maintained i n a f a s t i n g condition exhibit a slow but rather regular f a l l i n creatinine excretion. Addition of creatine, a precursor of creatinine to the d i e t may cause Increased creatinine formation as may also the "pre-mortal" r i s e a f t e r long periods of nitrogen starvation. In addition, a v a r i e t y of pathological conditions may cause departures from the normal l e v e l of creatinine output. Generally speak-ing, the constancy of endogenous catabolism should be evaluat-ed with respect to the exogenous catabolism rather than as an absolute i n v a r i a b i l i t y . - 21 -As studies on the minimum endogenous nitrogen catabolism are commonly conducted upon animals fed protein-free or pro-tein-low d i e t s , i t i s i n t e r e s t i n g to connect how these animals are able to maintain the i n t e g r i t y of t h e i r t i s s u e s . The mere fact that some nitrogenous materials are being excreted points to the continued d i s i n t e g r a t i o n of the body tissues yet obviously these tissues, i n certain s p e c i f i c cases at l e a s t , must be renewed from some source. In the early stages of protein i n a n i t i o n i t seems possible that the body's con-tinued losses of nitrogenous materials may be borne by the blood supply but i n time t h i s supply must be renewed. Indeed, at a l l times the blood can be considered to have a "wear and tear" requirement i n the most l i t e r a l sense of the term. I t must be admitted that i n the l i g h t of present knowledge the ultimate o r i g i n of the endogenous portion of the blood's n i t r o -gen remains a subject for speculation. In summary of t h i s extremely short survey of the l i t e r a -ture on. nitrogen and especially protein, metabolism, the fea-tures of exogenous and endogenous functions, of protein storage and of the constantly changing equilibrium e x i s t i n g among the nitrogenous constituents of the tissues must be emphasized. In an attempt to simplify to some extent an extremely com-plex picture and at the r i s k of appearing presumptuous, the wr i t e r has prepared the following schematic diagram of n i t r o -gen metabolism w i t h i n the animal body. INGESTION -PROTEINS, AMINO ACIDS, N.PN. i -GASTRIC HYDROLYSIS-ENZYME ACTION PEPSIN, RENNIN... I ^INTESTINAL I BACTERIAL PUTREFACTION DECARBOXYLATION, DEAMIN-ATION BROUGHTABfaUT BY INTESTINAL MICROFLORA^ MICRO-BIOLOGICAL ACTION RUMINANTS-PROTEIN SYNTH-ESIS FROM AMMONIA, N.P.N. DIGESTION^ ENZYME ACTION TRYPSIN.EREPSIN FURTHER CLEAV-v AGE BEGUN BY \ PEPSIN. FAECAL EXCRETION UNDIGESTED PROTEIN & PROTEIN RESIDUES. EXCESS EXCRETIONS OF THE DIGESTIVE TRACT, AMMONIA. INTESTINAL ABSORPTION LYMPH CIRCULATION-^ \ CATABOLISM DEAMIN ATION, DECARBOX-UREA, YLATJON ^-BLOOD CIRCULATION s~ CELLULAR ft PLASMA S PROTEINS-^ >  ANABOLISNK X- FORMATION OF-m ^ TISSUE PROTEINS. V . \ ^ENZYMES. HORMO^ESA CREATININES^: DETOXICAtlON PRODUCTS CONVERSION OF EXCESS NON-NITROGENOUS RES-IDUES, AND POSSIBLE USE FOR ENERGY SUPPLY. J NITROGEN LOSS NITROGEN STORAGE C Y T O P L A S M I C DEPOSITION OF PROTEIN IN LIVER, M U S C L E , & OTHER TISSUES FIGURE 2 : NITROGENMETABOLISMiN T H E ANIMAL BODY - 25 -The Significance of Nitrogen Balance , E a r l i e r i n t h i s paper reference was made to the form of experimentation known as "nitrogen balance", that i s , the comparison of the nitrogen content of the ingesta and excreta of various animals, A condition of nitrogen equilibrium i s said to exis t wherever the loss of nitrogen from an animal's body equals the nitrogen content of i t s food during a s i m i l a r measured i n t e r v a l of time. Where nitrogen excretion exceeds intake, the animal under consideration i s termed i n negative nitrogen balance, and conversely where nitrogen i s retained i n the body ( i . e . where intake exceeds excretion), the animal i s i n po s i t i v e nitrogen balance. Animals that are increasing t h e i r muscular tissues generally do not excrete as much n i t r o -gen as they take i n . Such animals include the young (growing) adults recovering from wasting diseases, animals undergoing muscle building exercise, and pregnant females. Sherman (1941), c i t e s experiments to show that the animal body tends to adjust i t s p rotein metabolism to i t s protein supply, and that once i t i s accustomed to any certa i n rate of protein metabolism, an appreciable length of time i s necessary to e f f e c t a material adjustment. Calculation of the State of Nitrogen Balance The state of nitrogen balance i s of course calculated by a measurement of the nitrogen content of ingesta and excreta of an animal over a defined period of time. Ingesta refers to the food intake as under normal conditions the nitrogen content of impurities i n the water used i s n e g l i g i b l e . - 24 -Excreta includes a l l those body wastes that might conceivably contain nitrogen: the faeces, urine, sweat, skin brushings and f a l l e n h a i r . Of these a l l but the f i r s t two names are commonly considered i n s i g n i f i c a n t , however, they may assume importance i n furred animals l i k e mink, e s p e c i a l l y during the shedding season. Further reference w i l l be made to t h i s p o s s i b i l i t y l a t e r . Many experiments and e s p e c i a l l y those dealing with n i t r o -gen minima are concerned s o l e l y with the urinary portion of the excreta and t h e i r r e s u l t s are tabulated as such. The general scheme of nitrogen balance t r i a l s involves f i r s t the attainment of a minimum l e v e l of nitrogen excretion and second the creation of nitrogen equilibrium through administration of nutrients of known nitrogen content. Two major requirements must be met i n the attainment of minimum nitrogen excretion: 1. Protein intake should be lowered, preferably to zero, -or at l e a s t to such a l e v e l as w i l l not influence the rate of nitrogen excretion by the kidneys. Such a l e v e l w i l l , of course, vary with the nature of the dietary protein. 2. Adequate energy intake should be provided through non-protein sources i n order to prevent the catabolism of nitrogenous constituents of the tissues as f a r as possible. In order to ensure an adequate c a l o r i c intake i t has been shown expedient to feed carbohydrates and fa t s i n amounts considerably greater than the actual basal metabolism would indicate necessary. The value of nitrogen balance experiments l i e s i n t h e i r - 25 -applicati o n i n the study of the protein requirements of animals. I t might be supposed that to meet an animal's basal needs f o r nitrogenous material a l l that would be_nejDjeaaary^" would be an amount equal to that l o s t by the animal under the conditions of minimal excretion previously described. Such i s not the case, however, due mainly to the diverse composition of the various native proteins that may be fed. Accordingly, modern nitrogen balance work has favoured the use of protein hydrolysates or amino acid mixtures as nitrogen sources on the assumption that the mixture supplying propor-tions of amino acids nearest the animal's requirements w i l l maintain nitrogen equilibrium at the lowest possible l e v e l (Kade, 1948), Such experiments serve a twofold purpose i n that they provide valuable data regarding amino acid composi-t i o n of various feed combinations i n c i d e n t a l to the informa-t i o n obtained on the state of nitrogen metabolism i n the animal. N u t r i t i v e Value of Proteins In order to properly appreciate the findings of nitrogen balance experiments, consideration must be made of the net value of d i f f e r e n t proteins to the animal under consideration. This concept of net worth of proteins has been commonly ap-proached under the heading of B i o l o g i c a l Value - an extremely important phase of p r a c t i c a l n u t r i t i o n . While i t i s r e a d i l y acknowledged that experiments concerned with i n d i v i d u a l amino acid relationships are indispensable from the point of view of fundamental knowledge, nevertheless such findings must be - 26 -supplemented by an accurate appraisal of the a v a i l a b i l i t y of such amino acids to the animal to be of much p r a c t i c a l use. Higher animals have depended i n the past upon native proteins as the main source of t h e i r dietary nitrogen, and i n a l l pro-b a b i l i t y w i l l continue to do so i n the future. Consequently, an experimental balance must be struck: chemical and b i o l o -g i c a l or a comparison of the precise requirements of an animal with the combinations that are l i k e l y to exi s t i n i t s natural dietary sources. The absolute e f f i c i e n c y of proteins i n feeds, that i s to say t h e i r b i o l o g i c a l values, may be expressed as the percen-tage of t o t a l intake of these nutrients a c t u a l l y u t i l i z e d by the body. A workable equation f o r the expression of such values i s that originated by Thomas i n 1909, l a t e r modified by M i t c h e l l (1924) as follows: N intake - (faecal N - metabolic N) - ( u r i n a r y N - endogenous N)xl00 N intake - (faecal N - metabolic N) This formula i n general use takes cognizance of the f a c t that the endogenous or metabolic portions of the t o t a l n i t r o -gen available have been u t i l i z e d by the body even though they are subsequently excreted. As examples of b i o l o g i c a l values, the following table i s quoted from Maynard (4£): TABLE l i t BIOLOGICAL VALUE OF THE PROTEINS OF HUMAN FOODS Food B i o l . Value <fo Food B i o l . Value i Whole egg 94 Whole wheat 67 85 Potato 67 Egg white 83 Rolled oats 65 Beef l i v e r 77 Whole corn 60 Beef heart 74 Wheat f l o u r 52 Beef round 69 N a v ^ lasted] 38 - 27 -Several features contribute to the ultimate b i o l o g i c a l value that w i l l be assigned d i f f e r e n t proteins. B r i g f l y , these include the relationship e x i s t i n g among the constituent amino acids, ( e s p e c i a l l y as regards the "essential"amino acids) the proportion of the protein moeity to the remainder of the d i e t and the s t r u c t u r a l composition of the entire food as related to ease of digestion. These factors seem of s u f f i -cient importance i n the determination of the n u t r i t i v e value of proteins to j u s t i f y the i n c l u s i o n of a b r i e f discussion. In the past, amino acids have been broadly c l a s s i f i e d under two main headings variously known as " e s s e n t i a l " and "non-essential" or "indispensable" and "dispensable". The essential type include those amino acids that cannot be syn-thesized i n the animal body i n s u f f i c i e n t quantity to meet the requirements for them and hence must form an indispensable" portion of the di e t . The non-essential amino acids are of course those which can be manufactured from other sources within the animal body. This l i n e of thought proposes that the b i o l o g i c a l value of any absorbed protein i s dependent on the proportions of esse n t i a l amino acids which i t contains. For example, i f one e s s e n t i a l amino acid i s completely lacking from a protein, i t w i l l prevent the f u l l u t i l i z a t i o n of the other amino acids and thus w i l l s e r i o u s l y lower the net value of that protein to the animal. I t i s in t e r e s t i n g to record that i n recent years the i n d i s p e n s a b i l i t y of at l e a s t some amino acids has been attributed to not the nitrogenous por-t i o n but rather the configuration of the elements carbon, hydrogen, and oxygen. Rose (1937) has demonstrated that - 28 -phenyl pyruvic acid may take the place of pSylalanine i n which case an animal could probably convert some of i t s pyruvic acid to the corresponding amino a c i d . E v e n so, one might be excus-ed i n naming pSylalanine e s s e n t i a l on the -grounds that the u t i l i z a b l e pyruvic acid i s not i t s e l f a natural component of foods. Among the naturally-occurring proteins, those composing the endosperm of cereal grains are considerably lower i n some of the esse n t i a l amino acids, notably l y s i n e , than most pro-teins of animal o r i g i n . Due to t h i s inconsistency, an erro-neous b e l i e f has been founded that plant proteins i n general are unbalanced and hence i n f e r i o r . T h i s concept i s untrue as many proteins from the l e a f y parts or the embryos of plants are b i o l o g i c a l l y equal and economically superior to animal proteins: a f a c t that i s important i n the formulation of rations both experimental and p r a c t i c a l . I n b r i e f , the com-binations that may e x i s t among the constituent amino acids i n any native protein are so diverse that generalization as to t h e i r n u t r i t i v e q u a l i t i e s i s unsafe. The e f f i c i e n c y of proteins i n meeting the requirements of i n d i v i d u a l animals i s also dependent i n no small measure upon the accompanying non-nitrogenous portions of the r a t i o n . B o t h carbohydrates and fats are able to diminish the cata-bolism of proteins, that i s , exert a "protein-sparing" action carbohydrates apparently being more e f f i c i e n t than f a t s i n th i s regard. The actual mechanics of t h i s action are not ea s i l y described. I t would appear that i n eases where animals do not receive s u f f i c i e n t carbohydrate to maintain the normal - 29 -glucose content of t h e i r blood the required glucose may be supplied by deaminized residues of amino acids (Landergren, 1907). A supply of carbohydrate i n such instances would of course avoid the use of proteins i n the formation of blood sugar and might be ef f e c t i v e at any l e v e l of intake due to the r e l a t i v e ease of oxidation of glucose. In a s i m i l a r man-ner f a t may spare protein by preventing i t s consumption f o r energy purposes. The lower e f f i c i e n c y of f a t may be explain-ed by the f a c t that f a t stores i n the body are less r e a d i l y depleted than are those of glycogen ( H i l l , 1924). The preceding discussion has stressed the importance of the chemical properties of the components of a r a t i o n i n the determination of i t s n u t r i t i v e value. Attention must also be paid to the physical properties of the dietary mixture, as unless the nutrients can be made available f o r absorption, they oannot be u t i l i z e d i n any way by the animal. A simple example of such a condition may be found i n the ooarse dry roughage feeds which made up a considerable proportion of the ration of herbivores. Ruminants and kindred types of animals are able, by reason of fermentation processes c a r r i e d out i n enlargements of t h e i r digestive t r a c t s , to s p l i t away the tough, c e l l u l o s e - r i c h sheath which protects the natural proteins of suoh forage. Those animals possessed of a simple digestive t r a c t , (and esp e c i a l l y carnivores) are unable to e f f e c t t h i s preliminary digestion, and are thus faced with the uncomfor-table s i t u a t i o n of having a supply of chemically-suitable proteins that they are unable to assimilate. Taking another extreme example, keratin, the protein of h a i r i s strongly - 30 -r e s i s t a n t to digestion and therefore must he considered of low b i o l o g i c a l value. I t can furnish but a n e g l i g i b l e amount of use f u l nitrogenous material to the body again f o r reasons of physical rather than chemioal structure. I t w i l l be evident from the b r i e f discussion of these two examples that two main factors play a part i n the physical aspect of the values of proteins: the actual gross structure of the food material and the species,differences which determine the digestive c a p a b i l i t i e s of the various animals. The foregoing pages should be considered i n the nature of a preois, almost an abstract, of the extremely extensive and involved l i t e r a t u r e dealing with the metabolism of n i t r o -genous compounds. Many other features might have been consi-dered, including the varying requirements f o r proteins during the successive stages of growth and development and the phy-s i o l o g i c a l c h a r a c t e r i s t i c s , both normal and abnormal. The i n c l u s i o n of such material, though i t i s c e r t a i n l y relevant, would not add m a t e r i a l l y to the underlying theme of t h i s thesis, namely the in v e s t i g a t i o n of the basal nitrogen meta-bolism i n adult mink. - 31 -EXPERIMENTAL A perfect experiment i n any f i e l d of science may be said to be one that has been planned and conducted i n such a way that the results obtained are susceptible of only one i n t e r p r e t a t i o n . - H. H. M i t c h e l l Some Considerations Involved i n Planning a N u t r i t i o n Experiment Several d i f f e r e n t methods have been attempted i n studies of nitrogen metabolism with varying degrees of sucoess i n operation. I t i s desirable before planning an experiment i n -volving t h i s subject to weigh the advantages and disadvantages of such methods as discussed i n the l i t e r a t u r e and evaluate them i n the l i g h t of c e r t a i n general considerations which must be met to ensure successful r e s u l t s . A preliminary e s s e n t i a l of any n u t r i t i o n a l experiment -indeed, any experiment - i s that of provision of adequate " oontrols." To obtain r e s u l t s that may be applied to normal animals, i t i s evident that one must work with normal animals either as the actual experimental subjects or as controls f o r comparison. This i d e a l of normality, so l o g i c a l i n theory , i s extremely d i f f i c u l t to a t t a i n i n practice and f a i l u r e of i t s attainment i s perhaps the outstanding contributory cause to experimental f a i l u r e . Many instances may be c i t e d of the dangerous breaches i n experimental data occasioned by i n s u f f i c i e n t attention to the aspect of normality. One of the most pertinent discus-sions of t h i s topic i s presented by Baldwin (1947,b) as follows: - 33 -In an i n t a c t , normal animal, to take a s p e c i f i c example, we cannot obtain much information about the metabolism of proteins by straightforward i n -vestigation of nitrogenous substances entering and leaving the organism. I f proteins are fed to a mammal, we f i n d that the ingoing protein nitrogen emerges again i n the form of urea or i n a b i r d i n that of u r i c a c i d . Very l i t t l e more can be d i s -covered. How the nitrogen i s detached from the protein and how i t i s b u i l t up into urea i n the one case into u r i c acid i n the other, we cannot discover without taking the animal more or le s s to pieces. I f , however, we take a mammal from which the l i v e r has been removed, i t w i l l survive f o r some days provided that proteins are witheld from the d i e t . I f a protein meal i s given, how-ever, the animal quickly d i e s . Closer examina-t i o n reveals that death i s due i n the main to poisoning by ammonia and that the blood and urine a l i k e contain ammonia but no urea. But whereas ammonia set free by deamination i s converted into urea i n the normal animal, urea production ceases with hepatectomy. This i s o l a t e d example i l l u s t r a t e s a ptly the need f o r some sort of derangement or abnormality on the one hand i n the i n -vestigation of fundamentals of metabolism,coupled with the necessity f o r careful i n t e r p r e t a t i o n i n the p r a c t i c a l applica-t i o n to normal animals on the other. In countless cases erro-neous conclusions have arisen as a r e s u l t of abnormalities introduced i n the experimental animals or as a r e s u l t of the experimental techniques adopted. Indeed, many of these ab-normalities were necessary i n the conduct of the investigations; the point of the matter i s that they ,were not recognized as such and so considered i n the f i n a l compilation of the data. A delicate balance i n experimental approach appears necessary between i n v i t r o and i n vivo techniques. Any of the questions posed by phenomena of intermediary metabolism can-not be adequately answered at the present time at l e a s t by experiments upon i n t a c t animals. By the same token, however, - 33 -i t must not be accepted that simply because a reaction takes plaee i n a P e t r i dish or t e s t tube, i t w i l l produce the same re s u l t s i n a l i v i n g organism. A combination of data i s i n -dicated, taking into consideration those gathered i n varying types of experiments, by d i f f e r e n t workers under diverse laboratory conditions; and above a l l i n c l u s i v e of s u f f i c i e n t numbers to be s t a t i s t i c a l l y s i g n i f i c a n t . Choice of F i e l d f o r Experimentation In planning an experiment on the n u t r i t i o n a l requirements of mink, one must always bear i n mind the almost complete lack of a background of s c i e n t i f i c research with these animals as contrasted to other species. From a p r a c t i c a l standpoint too the widespread v a r i a t i o n and disagreement exi s t i n g i n the composition of rations on economically successful mink ranches lends weight to the aura of uncertainty surrounding the physio-l o g i c a l needs of these animals. I t seems evident, therefore, that any preliminary inves t i g a t i o n should be directed toward attainment of the correct balance both economic and physio-l o g i c among those substances which are to form the major por-t i o n of the d i e t , namely the carbohydrates, fa t s and proteins. In t h i s s p e c i f i c instance of experimentation the inves-t i g a t i o n of protein requirements was undertaken because t h e i r Importance qua n t i t a t i v e l y as a r a t i o n constituent seems en-hanced by the p e c u l i a r p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the animal. Further, while the modern highly successful plan of investigating i n d i v i d u a l amino acid requirements might have been indulged i n , a note of caution seemed wise. Accordingly, - 54 -a plan of experimentation was drawn up dealing with the gross or o v e r a l l picture of protein usage as indicated by means of various nitrogen balance t r i a l s . Plan of Experiment The plan of experiment adopted may be divided into essen-t i a l l y two parts. F i r s t , a t r i a l was designed using two animals i n which the absolute minimum of urinary nitrogen ex-cretion was measured i n adult animals maintained i n a f a s t i n g condition. This preliminary i n v e s t i g a t i o n was believed nec-essary f o r the determination of the basal or minimal l e v e l of nitrogen excretion and as a guide to the length of time nec-essary to reduoe the body's main store of e a s i l y mobilized or l a b i l e protein. In addition, i t was f e l t that a compari-son of data obtained i n this way with the minimal figures f o r other species available i n the l i t e r a t u r e might be of great assistance i n an estimation of the basal metabolism of mink. The second and more extensive part of the experiment i n -volved tabulation of nitrogen balance data using as many animals as possible with the apparatus and time a v a i l a b l e . The aim of thi s d i v i s i o n of the experiment was to maintain a status of nitrogen equilibrium on a die t containing the minimum possible nitrogen content. I t was important to produce nitrogen equilibrium at t h i s basal l e v e l because as suggest-ed i n the e a r l i e r review, protein i s not stored to any appre-ciable extent i n the adult animal and hence i t i s altogether possible that equilibrium might be established at a higher than normal l e v e l . The information gathered i n t h i s way would - 35 -i t was hoped, serve as an accurate index of the protein re-quirements of the animal, at l e a s t insofar as the s p e c i f i c protein used i n the experimental r a t i o n was concerned. Further, i t was hoped that i n combination with basal metabolism data, the information r e s u l t i n g from t h i s experiment might serve as an i n d i c a t i o n of the t o t a l c a l o r i c needs of the animal. Crea-ti n i n e nitrogen determinations as well as t o t a l nitrogen de-terminations were made during t h i s l a t t e r period of experi-mentation as a form of check on the b i o l o g i c a l value of the protein used (Murlin, 1948), Method: The search f o r an absolute minimum i s l i k e the philosophers 1 search f o r the ab-solute truth. - E. P. Cathcart Actually, there i s not one minimum but several protein minima concerned with many factors that must be considered c a r e f u l l y when la y i n g down the method of experimentation (Melnick, 1936). These factors include: a. The nature of foodstuffs fed with the protein. b. The completeness of the d i e t - q u a n t i t a t i v e l y and q u a l i t a t i v e l y . c. The c a l o r i c value of the food. d. The stage of maturity of experimental animals. e. The a c t i v i t y of the experimental animals. f . The environmental temperature. g. The n u t r i t i v e condition of the animals p r i o r to the t e s t combined with adequate preliminary adjustment. These items give some hint of the precautions necessary i n setting up an experiment of t h i s type and at the same time serve as a warning against a too hurried i n t e r p r e t a t i o n of r e s u l t s - 36 -C e r t a i n f e a t u r e s o f t h e method a d o p t e d were common t o b o t h b r a n c h e s o f t h e e x p e r i m e n t and t h e s e w i l l be d i s c u s s e d f i r s t . I t must be emphas ized f r o m the o u t s e t t h a t f o r l a c k o f any p r e v i o u s d a t a on the s u b j e c t , c o n s i d e r a b l e e x p e r i m e n t a -t i o n was n e c e s s a r y i n t o the c o n s t r u c t i o n o f w o r k a b l e a p p a r a t u s . The e x p e r i m e n t a l a n i m a l s were m a i n t a i n e d i n a s e p a r a t e room f rom t h e m a i n c o l o n y o f t h e U n i v e r s i t y F u r A n i m a l U n i t and were t h e r e f o r e c o m p l e t e l y q u i e t and u n d i s t u r b e d e x c e p t f o r t he s h o r t p e r i o d d a i l y when u r i n e c o l l e c t i o n s were made and f e e d i n g , i f a n y , c a r r i e d o u t . E x t r e m e s o f t e m p e r a t u r e were g u a r d e d a g a i n s t b y adequate v e n t i l a t i o n and a v o i d a n c e o f any d i r e c t d r a u g h t s o f a i r a c r o s s t h e a n i m a l c a g e s . The cages t h e m s e l v e s were o f s u f f i c i e n t s i z e t o a l l o w f r e e movement t o t h e a n i m a l s y e t were c o n s i d e r a b l y s m a l l e r t h a n t h e n o r m a l r a n c h u n i t i n o r d e r t o r e s t r i c t t h e i r a c t i v i t y t o n e a r e r b a s a l c o n d i t i o n s . U r i n e c o l l e c t i o n s were made by means o f s t a i n l e s s s t e e l f u n n e l s o v e r w h i c h the cages were suspended e q u i p p e d w i t h w i r e mesh f a e c e s s c r e e n s and g l a s s w o o l f i l t e r s . G r a d u a t e d c y l i n d e r s (100 m l . ) were u s e d as c o n t a i n e r s f o r t h e u r i n e , a l l o w i n g f o r r a p i d and r e a s o n a b l y a c c u r a t e d e t e r m i n a t i o n o f t h e t o t a l volume o f e x c r e t i o n . U r i n e c o l l e c t i o n s were made d a i l y , u s i n g a t h i n o v e r l a y o f t o l u e n e i n the g r a d u a t e s as a p r e s e r v a t i v e . I n c a se s where samples were h e l d o v e r f o r a n a l y s i s , t h e y were m a i n t a i n e d u n d e r r e f r i g e r a t i o n , a g a i n u s i n g t h e l a y e r o f t o l -uene t o e x c l u d e a i r . T h r o u g h o u t a l l e x p e r i m e n t s an ample s u p p l y o f d r i n k i n g w a t e r was k e p t c o n s t a n t l y b e f o r e the a n i m a l s . D u r i n g t h e n i t r o g e n b a l a n c e t r i a l , c o n s i d e r a b l e d i f f i c u l t y - 37 -was encountered i n the preparation and administration of syn-t h e t i c rations. Again, the lack of previous information on the subject hampered investigations and several successive mixtures were attempted before a successful r a t i o n was found. In the preparation of the r a t i o n attention had to be paid not only to the chemical composition, p a r t i c u l a r l y as regards nitrogen content, but also to the physical nature as the con-sistency of the feed appeared of considerable importance i n assuring the desired l e v e l of intake. Administration too pre-sented i t s problems as a method had to be devised whereby the amount consumed by each animal could be accurately measured. Two workable schemes were devised i n t h i s connection. In the f i r s t , a semi-solid mixture was made of the ra t i o n , using d i s -t i l l e d water as a diluent and the r e s u l t i n g paste was extrud-ed to the animals through a hard glass tube. This method was found s a t i s f a c t o r y with rations wherein starch was the dominant carbohydrate as i t formed an adhesive g e l - l i k e mixture; how-ever, rations high i n sucrose were apt to go at le a s t i n part into solution and another method had to be devised i n order to avoid losses. The second type of feeder consisted of an attachable container with a projecting l i p to prevent inac-curacies i n estimation of feed intake caused by the itfink's natural habit of carrying o f f i t s feed before consuming i t . Details of the experimental r a t i o n composition and methods of administration are given at length i n Appendix I I . Analysis of urine samples f o r t o t a l nitrogen was made d a i l y i n the Animal N u t r i t i o n Laboratory using the Gunning Modification of the Kjeldahl method. I t was noticed early i n - 38 the course of experimentation that considerable quantities of h a i r and skin debris were shed by the animals. The p o s s i b i l i t y of an increase i n urinary N due to washing over t h i s matter suggested i t s e l f . An experiment was designed, therefore, to check nitrogen analyses of s i m i l a r samples before and a f t e r contact with such extraneous matter. Check tests were made from time to time f o r the detection of b i l e i n the urine as th i s would of course indicate a difference- i n the physiologic nature of the nitrogen content and f o r a s i m i l a r reason tests for albumin were carr i e d out p e r i o d i c a l l y . Creatinine determinations were made spectro-photometri-c a l l y , using a modified a l k a l i n e p i c r a t e procedure o r i g i n a l l y suggested by F o l i n and Jaff e (Peters, 1942). As previously mentioned, these tests were not run d a i l y but were made at def i n i t e i n t e r v a l s during the course of the experiment. As reference has been made i n the l i t e r a t u r e that glucose may i n -terfere with the Jaff e reaction, (Barclay, 1947), Benedict's tests were performed from time to time as a check on the v a l i -d i t y of the creatinine figures. The actual laboratory proce-dures adopted, including any modifications employed, are l i s t -ed i n Appendix I. - 39 -Observations and Discussions: Endogenous Nitrogen Excretion The data gathered i n the preliminary experiment (that i n -volving the f a s t i n g catabolism) are presented i n summary form i n Table I I I . A "nitrogen c o e f f i c i e n t " , arrived upon by d i v i d -ing the t o t a l d a i l y urinary nitrogen excretion ( i n grams) by the body weight ( i n kilograms) i s employed to give a more com-parative picture of the wastage of the protein reserves of the body. The body weight f o r purposes of these calculations was taken as the mean between s t a r t i n g and f i n i s h i n g weights. A graphioal representation of the urinary nitrogen excre-t i o n of the two animals involved i n t h i s experiment i s present-ed i n figure 3 (a). The f i r s t day's urinary nitrogen loss by animal number 1 i s indicated by a broken l i n e on the graph, as i t was believed to be abnormally high due to faecal washing. This possible source of error was immediately corrected by i n -s t a l l a t i o n of a succession of f i l t e r s as previously described. I t w i l l be noticed that a preliminary f a s t of 9 days was carried out i n order to overcome the effects of any previous feeding. Although t h i s period may seem lengthy i n comparison to those adopted f o r other species by Smuts (1935) i t was f e l t j u s t i f i e d due to the absence of any p r i o r information i n t h i s regard f o r the mink. After t h i s preliminary f a s t , a nitrogen-free d i e t was administered i n an attempt to s a t i s f y the animals' c a l o r i c requirements from some non-protein source. As a point of i n t e r e s t i t may be recorded that, while animal number 2 died a f t e r 12 days on experiment, animal 1 continued f o r 20 days, a f t e r which i t was removed and returned to the Fur - 40 -Animal Unit i n apparent good health. TABLE I I I : ENDOGENOUS NITROGEN EXCRETION DATA FOR MINK ON A NITROGEN-FREE DIET. Mink No. 1 Mink No. 2 Day Total T o t a l N Coeff. Total Total N Coeff. Urine N gms. Urine N gms. ml. gms. ml. gms. 1 105 B745 4.62 35 1.28 1.41 2 45 1.78 1.52 38 0.98 1.75 3 17 0.40 0.34 29 1.15 1.21 4 15 0.27 0.23 86 0.65 0.71 5 19 0.33 0.28 68 0.86 0.95 6 25 0.24 0.20 88 0.65 0.71 7 16 0.28 0.24 96 0.82 0.90 8 24 0.24 0.20 88 0.77 0.85 9 31 0.43 0.37 95 0.52 0.57 10 64 0.68 0.58 101 0.72 0.79 11 36 0.36 0.31 110 0.60 0.66 12 48 0.52 0.44 110 0.60 0.66 13 50 0.49 0.42 14 51 0.52 0.44 15 121 0.42 0.36 16 56 0.49 0.42 17 49 0.53 0.45 18 47 0.50 0.43 19 47 0.65 0.55 20 74 0.54 0.46 I t i s i n t e r e s t i n g to note that, i n common with other species, the urinary nitrogen excretion f o r mink dropped o f f sharply during the early stages of f a s t i n g and then tended to l e v e l o f f on a more-or-less basal l e v e l . A d e f i n i t e increase i n t o t a l nitrogen excretion was noticeable subsequent to the administration of a nitrogen-free d i e t due probably to the animals' increased water consumption and increased urinary ex-cretion occasioned by resumed ingestion of food. This increas-ed urine production by animals receiving feed a f t e r a f a s t was most noticeable both i n t h i s and the l a t t e r section of t h i s experiment and apparently outweighs any sparing e f f e c t that the carbohydrates and fats fed might have had upon the dimin-ishing protein reserves of the body. I t i s perhaps s i g n i f i c a n t - 4 1 -to record that of the many nitrogen excretion studies examined i n the l i t e r a t u r e , including those widely quoted works of Brody and Smuts, none l i s t e d f i g ures on t o t a l urine volume. One cannot help but wonder under the circumstances whether many of the variations noted i n urinary nitrogen excretion might not be due to fluctuations i n t h i s basic physical f a c t o r . Tor purposes of comparison, nitrogen excretion figures c i t e d by Lusk (52) from a starvation experiment and Deuel (53) from a nitrogen-free d i e t t e s t are presented i n fi g u r e 3 (b). Here the r e l a t i v e l y longer i n i t i a l period of sharply d e c l i n i n g n i t r o -gen excretion i s probably accounted f o r by the greater body size of the experimental subjects. During the mink experiment, a s l i g h t r i s e i n t o t a l urinary nitrogen excretion may be noticed throughout the basal period. This increase i s extremely gradual and gives no suggestion of the so-called "pre-mortal r i s e " even i n the case of animal number 2 which did die on experiment. An explanation of t h i s increased nitrogen l o s s i s d i f f i c u l t , however, i t seems possible that i t may be i n the nature of a compensatory reaction brought about to meet the acidosis caused by the catabolism (and i n -complete oxidation) of body f a t . As time permitted the use of two animals only on the pre-liminary nitrogen depletion phase of experiment, the r e s u l t s cannot be regarded as conclusive by any means; however, they do indicate c e r t a i n trends which may be of some s i g n i f i c a n c e . Considerable v a r i a t i o n existed i n the nitrogen c o e f f i c i e n t s exhibited by the two animals although the general excretion eurves were reasonably s i m i l a r , (see f i g . 3A). I t appears - 42 -that a reasonably stable l e v e l of nitrogen excretion i s reached a f t e r 4 days of f a s t i n g , and t h i s finding i s i n agreement with the calculations published f o r other animals from M i t c h e l l ' s laboratory. The establishment of an absolute basal l e v e l of nitrogen excretion i s more d i f f i c u l t to a t t a i n and while that plotted f o r animal number 1 (figure 3A) was reasonably constant, ce r t a i n discrepancies do e x i s t . The data appear to suggest, i n accordance with the theory of Borsook, that there i s no cle a r cut "endogenous" l e v e l of nitrogen excretion but rather a constantly changing nitrogen equilibrium which i s adjusted to the nitrogen metabolism of the animal. The maintenance of the l a r g e r animal i n a state of protein i n a n i t i o n was accomplished with X4D3 apparent hardship on the animal i t s e l f . The smaller animal, on the other hand, appear-ed to lose i t s v i t a l i t y rather quickly and as noted died during the course of the experiment. This difference seems l i k e l y due to the greater reserves of adipose tiss u e i n the former animal. While both mink refused a considerable proportion of t h e i r nitrogen-free energy source during the i n i t i a l stages of feed-ing, the la r g e r animal was able to meet at l e a s t part of i t s c a l o r i c requirements through the breakdown of body f a t . Nitrogen Balance Experiments To r e i t e r a t e b r i e f l y , the scheme of the nitrogen balance t r i a l s was to maintain nitrogen equilibrium at the lowest possible l e v e l , using d i f f e r e n t s p e c i f i c nitrogen sources. The animals were f i r s t fasted u n t i l c a l c u l a t i o n of t h e i r n i t r o -gen excretion indicated that they had reached a reasonably con-stant l e v e l ; they were then fed a nitrogen-free basal r a t i o n TABLE IV (A) URINARY NITROGEN EXCRETION ANIMAL NO: I s o g S3 3 i l l I ^ 5 j-I R 5 H I B IESI I E 9 raBlTOIBIIWllSElKS IB9SB I KB ESI i Egg • B 9 IE9I 844 815 190 56?, 390 T£2 4001 3991 740 7 5 5 77S 790 310 303 | 755 283 2951 715 1781 133 U75 183 SloU'52 80 95 565 190 USOI56D S<32 482 545 35B S19 510 339 438 485 317 418 440 319 4301455 327 4$t> 366 4oj 814 875 358 735 398 « o 710 401 507|68? £50 324 645 118 157 U o 8 200 %oZ OX Goo 115 148 595 2 6 4 5^0 585 226 283 580 540 £66 573 816 local 570 1140 1386 1KB IH 789 •  " 743 « 7511 « 7£o E2B3I cC o 760 547 756 552 735 530 lg»iu!S] 781 B 740 « 700 623 » too BLI^ lo^ 570 « 103 547 « Uo5 2^0 - 2,05 49o| » ho? S3 EH 1551 103 105 103 ICS BL £0S 191 W l 312, 440 415 618 560 540 >• 530 « 505 BLrt 103 490 ID'S 485 « 103 477 '« ICS 470 <» 10? 460 "BL 205 457 » i f t i 450 - 1<J1 442 .. y\\ 437 •« S?& 430 » SB 59 SSBS 1160 1148 11% 1113 Vt&S 1V68 1130 1130 loto 352 255 466 35C 4 % mo 487 276 228 222 269 29€ 24ft 546 Z<J8 276IZ40 317 26o 258 273 251 2AO 213 167 148 159 141 163 205 157 148 171 174 15o1152 am 20f 17-a" 176 2-SI 2-3?, 399 10O0 'i S83 383 5 3 2 584 584 855 3£f<93c 5"6o .VEX TO F^£^UHC>-T 1 I4*r| looo - 43 and again fasted to confirm the r e s u l t s obtained previously. Following t h i s i n i t i a l preparation, the mink were fed an amount of the. nitrogen-free r a t i o n s u f f i c i e n t to meet t h e i r c a l o r i c requirements as estimated from t h e i r body weights together with a measured amount of protein food i n an attempt to reaoh and maintain a state of nitrogen equilibrium. The oomplete picture of urinary nitrogen excretion obtained as a r e s u l t of these t r i a l s i s presented i n table IV" A. I t w i l l be noticed that i n many oases a time l a g existed i n the adjustment of nitrogen intake to the output l e v e l . This l ag was necessitat-ed by the time involved i n c o l l e c t i o n and analysis of the urine samples and the c a l c u l a t i o n of the nitrogen content therein. Towards the close of the experiment d i f f i c u l t y was encountered i n maintaining a s u f f i c i e n t l y high t o t a l feed intake and fresh l i v e r was substituted f o r the spray-dried l i v e r meal previously used, as a protein source. Addition of t h i s fresh product mark-edly increased p a l a t a b i l i t y of the r a t i o n , r e s u l t i n g i n weight increases i n the two larger animals and decreased losses In ; the others. The problems of formulation of rations that were at the same time chemically and physioally suitable were most d i f f i c u l t and sometimes involved departures from the planned techniques as i l l u s t r a t e d i n the example above. Conditions approaching nitrogen equilibrium were attained i n mink numbers 1, Sa, 3a and 4. The other two animals contin-u a l l y refused portions of t h e i r experimental rations with the r e s u l t that they evidenced steady declines i n body weight and corresponding increases i n urinary nitrogen excretion. I t w i l l be noticed (table IV A) that nitrogen was f i r s t added to - 44 -the basal r a t i o n at three l e v e l s corresponding to one, two and three grams of dried l i v e r meal d a i l y . (See appendix I I f o r d e t a i l s regarding analyses of r a t i o n constituents.) Nitrogen equilibrium between feed intake and urinary output was c l o s e l y approached by one animal at the 103 mg. l e v e l of nitrogen per animal per feeding and by three others at the 205 mg. l e v e l as i l l u s t r a t e d i n table IV B. TABLE IV B: URINARY NITROGEN BALANCE WITH DRIED LIVER MEAL. Animal No. 1 2a 4-Exp. Days 29-32 25 T27 30-32 18-26 liean N Intake (Feed) 205 mg. 103 mg. 198 mg. 205 mg. Sdean N Loss (Urine) 218 mg. 95 mg. 210 mg. 181 mg. N Balance -13 mg. +8 mg. -12 mg. f 24 mg. I t w i l l be noticed that mean figures f o r nitrogen intake and output over several days are quoted rather than the i n d i -v idual d a i l y values i n view of the considerable d a i l y f l u c t u a -t i o n . Only those days showing a reasonably close approximation to nitrogen equilibrium were considered. One s p e c i f i c instance of urinary nitrogen equilibrium was also noticed during the period i n which the animals received fresh l i v e r as t h e i r only nitrogen supplement. This case i s outlined i n a s i m i l a r manner to those above i n table IV C. TABLE IV C: URINARY NITROGEN BALANCE WITH FRESH LIVER. Animal No. 2a Exp. Days 31-33 Mean N Intake (Feed) 191 mg. Mean N Loss (Urine) 177 mg. N. Balance +14 mg. - 45 -I t would appear that a temporary urinary nitrogen e q u i l i -brium can be reached i n the adult mink by i n c l u s i o n of as l i t t l e as 103 mg. of nitrogen i n the form of dried l i v e r meal or 191 mg. of nitrogen i n the form of fresh l i v e r i n the d i e t . Further, a reasonably stable equilibrium can be maintained by the use of. ,205 mg. of nitrogen i n the form of dried l i v e r meal. These figures may be translated to represent protein supplements of 644 and 1289 mg. i n the case of the dried pro-duct and 1194 mg. i n the case of fresh l i v e r . Moreover, bas-ing calculations upon the Kjeldah! nitrogen determinations car r i e d out on these products i n the Animal N u t r i t i o n Labora-tory, (see Appendix II) one may state that urinary nitrogen equilibrium can be reached with these animals by the feeding of 1 gram d a i l y of dried l i v e r meal or 10 grams of fresh l i v e r and that t h i s condition may be maintained by the feeding of 2 grams of dried l i v e r meal with some suitable non-protein energy source. Some words of explanation are necessary at t h i s point regarding the use of the term "urinary nitrogen equilibrium." Normally, nitrogen balance experimentation implies a compari-son of nitrogen intake with nitrogen output v i a a l l routes i n -cluding faeces, h a i r and skin losses and the l i k e . In the present work t h i s concept was recognized at the outset but recognition was tempered by the a n t i c i p a t i o n of the d i f f i c u l -t i e s involved i n metabolic studies with a "new" animal. Con-sequently, the writer decided, somewhat on the p r i n c i p l e that, "half a l o a f i s better than no bread," to r e s t r i c t the analy-t i c a l phases of the i n v e s t i g a t i o n to the urinary portion of FIGURE 4 ( B ) N I T R O G E N B A L A N C E T R I A L D A T A : T O T A L U R I N A R Y N I T R O G E N E X C R E T I O N K E Y T O R A T I O N S ^ ( 2 A . B L = B A S A L + L I V E R 3 A . ' J 3 L M = B A S A L * D R I E D L I V E R M E A L . B A S B A S A L ( N F R E E ) 0 2 4 6 8 1 0 12 1 4 16 18 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 T I M E I N D A Y S 8 0 0 7 0 0 6 0 0 5 0 0 4 0 0 3 0 0 2 0 0 - 46 -the excreta. This method has c e r t a i n d e f i n i t e advantages es-p e c i a l l y i n a preliminary course of experimentation such as t h i s one because the urinary excretions allow f o r greater speed coupled with accuracy i n both c o l l e c t i o n and analysis than do the t o t a l excreta. The writer f e e l s j u s t i f i e d i n assuming a high degree of d i g e s t i b i l i t y i n the p u r i f i e d rations used, (see Appendix II) therefore i t i s l o g i c a l to assume that the major proportion of the animals nitrogen losses would occur through the kidneys and appear i n the urine. Ashworth (1933a) i n studies of nitrogen metabolism of other species stated that the f a e c a l nitrogen on a nitrogen-free d i e t was nearly constantly 20$ of the t o t a l nitrogen ex-cre t i o n . Morrison (1949) l i s t s the d i g e s t i b i l i t y of commer-c i a l dried l i v e r meal as 96.7$. I t seems probable,therefore, that even allowing f o r a d i g e s t i b i l i t y c o e f f i c i e n t of as low as 90$,which i s u n l i k e l y , the f a e c a l nitrogen should not con-t a i n more than 25$ of the t o t a l nitrogen excretion. I t i s now possible, by use of these assumptions, to estimate the t o t a l nitrogen l o s s that must be met i n order to e f f e c t a minimal equilibrium. Increase of 25$ to allow f o r faecal nitrogen losses would indicate probable establishment of n i t r o -gen equilibrium as l i s t e d i n table IV D. In t h i s table the nitrogen requirements have been translated into the more ea s i l y applicable protein complements, always bearing i n mind that the s p e c i f i c protein i n t h i s case i s supplied by dried l i v e r meal. - 47 -TABLE IV D: TOTAL NITROGEN BALANCE WITH DRIED LIVER MEAL. Animal No. 1 8A 3A 4 Experimental Days 85-30 85-30 85-30 85-30 Mean Urinary N Loss 178 mg. 183 mg, 801 mg 177 mg Mean Toal N Loss (calculated 837 " 164 » 868 " 2S6 n Theoretical Protein Loss 1,481 " 1,085 » 1,675 » 1,475 L i v e r Meal Equivalent 8,314 " 1,601 " 8,617 " 8,306 tt Actual L i v e r Meal Fed (mean) 8,400 " 1,166 " 1,166 " 8,900 S i m i l a r periods have been studied f o r the various animals i n order to give a comparative p i c t u r e . I t would appear on the basis of the mean of the i n d i v i d u a l animals studied that n i t r o -gen equilibrium should be established by the i n c l u s i o n of 1414 mg. of actual protein from l i v e r meal or 8809 mg. of l i v e r meal i n the d a i l y r a t i o n . Another i n t e r e s t i n g observation i s afforded by comparison of the t o t a l l o s s i n body weight of experimental animals with the losses recorded i n nitrogenous material. Using as samples animals 1 and 4 f o r which the longest continuous records are available, t h i s comparison i s presented i n table IV E. TABLE IV E: PROTEIN LOSSES CO] MPARED TO TOTAL WEIGHT LOSS Animal No. 1 4 Experimental Days Total Weight Loss 3-87 244 gm. 8-87 390 gm. [Jrinary N Loss Total N Loss (calculated) Food N (subtract) 5.15 gm. 6.86 gm. 3.88 gm. 5.78 gm. 7.63 gm. 2.67 gm. Corrected N Loss Protein Loss (dry wt. N x 6.85 Protein Loss (as tissue) % of Total Loss 3.58 gm. )88.37 gm. 31.16 gm. 18.7 <fo 4.96 gm. 31.00 gm. 41.33 gm. 10.6 io 60 jjj 4 0 J— .02 0 3 0 4 .05 .06 J07 0 8 0 9 C O N C E N T R A T I O N '• M G . C R E A T I N I N E I N S A M P L E RGURE 4 ( C ) S T A N D A R D C U R V E F O R C R E A T I N I N E ( A L K A L I N E P I C R A T E M E T H O D • F O L I N W J A F F J L R E A C T I O N ) - 48 -The dry weight of protein l o s t has been converted to re-present tissue protein on the basis of a 25$ dry matter content determined i n horse muscle f l e s h i n the Animal N u t r i t i o n Labora-tory. In these animals then an average of 11.6$ of the body weight losses sustained were i n the form of tissue protein. Variation between the two animals i s probably due to the higher condition of the heavier mink at the s t a r t of the experiment, meaning that a correspondingly greater proportion of i t s weight losses would be i n the form of f a t or water. Creatinine Excretion Estimation of creatinine excretion was attempted with two main purposes i n view. F i r s t , i t was thought that a comparison of the creatinine excretion of the mink with that tabled f o r other species i n the l i t e r a t u r e might y i e l d a valuable insight to the phy s i o l o g i c a l nature of th i s animal. Second, by compari-son of creatinine nitrogen and t o t a l nitrogen excretion data, an estimate of the b i o l o g i c a l value of the protein supplements used might well be reached. The general data regarding crea-t i n i n e excretion are presented i n table V. TABLE V: CREATININE EXCRETION IN MINK ON TEST Animal No, 1 2. 2A 3, 3A 4 Day.Ration Weight Gns. Urin.N , Mg. Creat Mg. Wt. Gms Jrin. »NMg, Creat. Mg. IflTt.T 3ns Trini Mg. Creat Mg. WV Gns: MB. ?ieat. "Mg. 2 1 F 8 B 10 F 15 BLM 27 BLM 33 BL 35 BL 1035 1005 920 808 761 810 823 '844 310 178 392 200 540 L140 16.32 16.51 14.58 13.74 12.17 12,77 13.09 790 755 675 560 154 89 189 121 12.1C 12.IE 10.36 8.4C 789 740 662 520 406 218 312 539 1144 11*32 9.66 a 32 L42E 138C 12 7E 1172 780 352 498 320 150 532 855 19.13 19.31 17.83 15.74 13. 52 13.70 13.43 60S 573 563 98 83 • 803 9.1? 8.7S 8.61 48* 442 430 187 156 780 6.98 6.42 6JB3 990 99€ 100C Average 880 515 14.17 580 328 8.86 452 374 6.75 1034 498 16. 09 Rations: F-Fast, B-Basal, BLM-Basal,Liver Meal, BL-Basal, L i v e r . - 49 -An examination of these figures (table V, see also figure 4A) leaves one with the impression that there i s l i t t l e ab-normality i n the nitrogen metabolism of mink, at l e a s t as f a r as may be demonstrated by t h e i r creatinine excretion. In general, the data obtained i n t h i s experiment f u l l y confirm the c l a s s i c a l theory of creatinine excretion. Variations are per-ceptible i n the d a i l y excretion of creatinine but i t i s s i g n i -f i c a n t to record that such variations do not correspond to changes i n protein intake. On the other hand, the l e v e l of creatinine i n the urine follows c l o s e l y any change i n body weight or perhaps,more aptly stated, i n muscle mass. (See figure 4A). Exceptions from t h i s general trend appear i n the cases of animals 2 and 3 where a rather sharp r i s e i n c r e a t i -nine excretion appears i n conjunction with a prolonged drop i n body weight. I t w i l l be r e c a l l e d that both these animals died as a r e s u l t of experimentation and there seems l i t t l e doubt that the f i n a l r i s e i n creatinine represented the "premortal r i s e " noted i n other species. Post mortem examination of these animals presented a picture of severe emaciation and wasting of muscle t i s s u e , (see appendix I I ) , thus bearing out t h i s theory. Following the indications that creatinine excretion may be most properly evaluated as a function of body weight, the figures i n table V show the average creatinine excretion for. the mink to be 15.58 mg. per kilogram of body weight. I t i s i n t e r e s t i n g to compare t h i s figure to the "Prediction Table" calculated by Brody, (1945) from his observations on numerous - 50 -species wherein he c i t e s a creatinine excretion of 13.2 mg. per kilogram f o r a 700 gram animal. The accuracy of Brody's predic-tions appears to be borne out once again by the close p r o x i -mity of h i s estimate to the actual a n a l y t i c a l data. A reason for the s l i g h t increase i n creatinine excretion i n mink above the expected i s a matter f o r conjecture, however, a strong p o s s i b i l i t y i s indicated i n the extreme a c t i v i t y , involving intense muscular action so prevalent i n these animals. One other possible cause fo r v a r i a t i o n e x i s t s , namely, the occur-rence i n the urine of mink of some stable colouring material which might a f f e c t the colorimeter readings i n creatinine de-terminations. The reasonable range of creatinine excretion arrived upon i n t h i s experiment would tend to discount such a p o s s i b i l i t y yet i t cannot be ignored u n t i l f u rther experi-mental evidence on the subject has been gained. Information i s also forthcoming regarding the e f f i c i e n c y of use by mink of l i v e r and l i v e r meal long regarded as s a t i s -factory protein supplements f o r these animals. In the l i g h t of the data presented i n table V, i t would appear that c r e a t i -nine contains an average of 5.9$ of the t o t a l urinary nitrogen on the l i v e r meal r a t i o n and 1.7% of the t o t a l urinary nitrogen on the fresh l i v e r supplement. The p r i n c i p l e involved here i s , i n b r i e f , that creatinine as a t y p i c a l endogenous urinary con-stit u e n t , w i l l vary inversely as a proportion of the t o t a l urinary nitrogen as the t o t a l changes i n conjunction with changes i n dietary protein. In other words, creatinine n i t r o -gen w i l l be a r e l a t i v e l y large proportion of the t o t a l urinary - 51 -nitrogen when a protein of high b i o l o g i c a l value i s fed. The figures c i t e d herein are merely r e l a t i v e and indicate a some-what greater net e f f i c i e n c y f o r dried l i v e r meal than from the fresh l i v e r as would be expected from the very physical nature of the materials. The p o s s i b i l i t y suggests i t s e l f , however, that continuous and systematic studies of creatinine and t o t a l urinary nitrogen excretion might lead to rapid and accurate assessment of the value of i n d i v i d u a l proteins to s p e c i f i c animals. This concept has been advanced i n the f i e l d of human n u t r i t i o n by Murlin (1948) and i s presently undergoing further inves t i g a t i o n i n h i s laboratory at Rochester. Summary At the time of i n i t i a t i o n of t h i s study, l i t t l e or no v a l i d information was available dealing with the nitrogen re-quirements of the mink. On the basis of p r a c t i c a l feeding ex-perience i t appeared that a d a i l y c a l o r i c intake of the order of 200 - 300 ca l o r i e s per kilogram of body weight would permit normal maintenance. The protein intake on a "normal" ranch r a t i o n varies tremendously and i t Is d i f f i c u l t to quote figures of any tangible meaning. On the other hand, i f one assumes that the mink f i t s the normal animal curve produced i n the studies of Ormsby, Benedict, Brody and others, one might predict a basal d a i l y requirement of 40 - 80 calories per kilogram and a maintenance requirement of approximately double these f i g u r e s . In a l i k e manner, one would expect an endogenous urinary nitrogen excretion of 2 mg. per c a l o r i e of basal heat which In numerical terms represents - 52 -80 - 160 mg. of endogenous urinary nitrogen per kilogram of body weight. With this experience for guidance, the present work has attempted to confirm or refute these generalities with respect to mink and to determine whether the apparently high level of feeding commonly practised is justified by the metabolic be-haviour of the animals. For purposes of clarity and brevity, the results of the present investigation can best be summariz-ed as follows: 1. The difficulties involved in design and operation of equipment for nutritional research with a "new" animal should not be minimized. Points whi3h received special consideration in the present work are listed hereunder: (a) Caging.- Special cages were constructed to allow for collection of urinary excretions with mink. These cages had of necessity to be escape-proof, and of a size to restrict yet not cramp the normal movements of the animals. Details of their con-struction are given in appendix II. (b) Feeding. Considerable experimentation was neces-sary in order to arrive upon a practical yet accurate method of feeding. Experimental rations and puri-fied diets used successfully with other animals proved unsatisfactory with mink. Certain modifi-cations in existing rations were made and workable mixtures as noted in appendix II were adopted. (c) Watering. The animals on experiment had to be - 53 -provided with s p e c i a l l y designed watering de-vices i n order to ensure adequate supply while at the same time avoiding s p i l l a g e and consequent change of urine volume. A closed water b o t t l e with drinking tube proved most s a t i s f a c t o r y pro-vided a short conditioning period was given the animals immediately p r i o r to the t e s t , (d) Urine C o l l e c t i o n . Stainless s t e e l funnels and strainers were adopted a f t e r a great deal of ex-perimentation. This material proved admirable f o r the purpose as i t allowed f o r accurate t o t a l urine c o l l e c t i o n , and rapid and complete cleaning. The writer f e e l s that the equipment and method f i n a l l y evolved i s suitable both from a p r a c t i c a l and a s c i e n t i f i c viewpoint f o r work with mink. A new animal i s thus available f o r laboratory experimentation and may lend i t s i n d i v i d u a l pe-c u l i a r i t i e s to the task of expanding the ever-increasing know-ledge i n animal n u t r i t i o n . 2. Endogenous urinary nitrogen excretion with mink has been shown to follow the general trends exhibited by other species. This observation i s important i n i t s e l f i n that i t tends to remove the mink from the sphere of an "unknown" and "abnormal" animal and place i t instead i n the ranks of those f o r which constant predictions may be made contingent on the accumulation of s c i e n t i f i c data. Actual endogenous urinary nitrogen excretion of the two animals tested averaged 565 mg. per kilogram of body weight d a i l y . Using the commonly accepted - 54 -r a t i o of 1 c a l o r i e of basal heat to each 2 mg. of endogenous nitrogen, t h i s would indicate a B.M.R. of 287 calories per k i l o or approximately 200 c a l o r i e s f o r a 700 gm. animal. I t would appear that a higher rate of heat production e x i s t s i n the mink than might be expected from the general "prediction" tables. (Brody, 1945) The causes of t h i s additional c a l o r i c increment are probably the extreme nervous temperament and high state of muscular a c t i v i t y exhibited by these animals and even under the c a r e f u l l y regulated experimental conditions a quiescent state was not attained. 3. Nitrogen Balance. Conditions of nitrogen equilibrium were attained by the i n c l u s i o n of 1414 mg, of actual protein i n the d a i l y r a t i o n of a 726 gm. mink, that i s 1947 mg. of protein per kilogram of body weight. In t h i s experiment as i n the one outlined i n part (2) above, the condition of the t e s t animals was c l o s e r to what i s known as the "maintenance" stan-dard than to a true basal l e v e l . On the basis of the experi-mental evidence gathered herein, i t would appear that an average sized female mink might be maintained i n nitrogen balance on a d a i l y dietary supplying s l i g h t l y l e s s than 2 grams of actual protein. I t must be emphasized once again that n i t r o -gen balance experiment figures are v a l i d only insofar as the s p e c i f i c protein employed i n feeding i s concerned and hence the figures cited above must be applied i n terms of l i v e r meal protein. 4. Creatinine Excretion. The results obtained following investigations into the rate of creatinine excretion of mink - 55 -were most i n t e r e s t i n g . As with other species, creatinine ex-cretion by minlr proved to be extremely constant and appeared to vary d i r e c t l y with body weight. No evidence was discovered to refute the theory that creatinine excretion i s l i t t l e af-fected by dietary nitrogen content. An average rate of crea-t i n i n e excretion of 15.58 mg. per kilogram of body weight was established f o r the animals under in v e s t i g a t i o n as compared to a predicted excretion from the l i t e r a t u r e of 13.2 mg. per k i l o -gram f o r an animal of s i m i l a r s i z e . The variance between ac^ tual and predicted values f o r creatinine excretion i s thought to be due to the intense muscular a c t i v i t y of the mink. 5. P r a c t i c a l Implications. While i t i s hoped that some small contribution to the s c i e n t i f i c knowledge of n u t r i t i o n has been made by t h i s present work, the writer f e e l s that the p r a c t i c a l applications from such knowledge, duly confirmed, could be most extensive. Without delving into d e t a i l , t h i s work would appear to indicate that the mink may shortly be subjected to r i g i d feeding standards i n common with other domestic animals. The suggestion i s put forward that very considerable overfeeding of protein has been indulged i n with these animals, probably on the premise that, as carnivora, they require diets r i c h i n muscle f l e s h . The experiments ci t e d herein indicate very d e f i n i t e l y that a nitrogen e q u i l i -brium may be maintained through the administration of extreme-l y small quantities of protein and a presumption may be made that t h i s protein need not a l l be of animal o r i g i n provided that the needs f o r e s s e n t i a l amino acids are met. One might - 56 -expand the theme ad infinitum yet l e t i t s u f f i c e to say that provided a sound basis i s b u i l t upon s c i e n t i f i c f a c t s , there i s no reason why the n u t r i t i o n a l requirements of mink may not be reduced to numerical terms and established as a matter of common knowledge. 6. Recommendations and Suggestions. C r i t i c i s m may be made of the present work on the grounds that the numbers of animals involved are i n s i g n i f i c a n t . The reason f o r t h i s pau-c i t y of numbers i s simple In that the time involved i n devis-ing equipment and experimental methods was so considerable that further experimentation became impossible. Moreover, the mere care and maintenance involved i n operation of the stock colony of animals was also extremely time consuming and yet t h i s labour v/as b a s i c a l l y necessary f o r the whole conduct of the experiments. The writer would urge that the preliminary i n -sight into the various requirements of mink, as contained i n th i s t h e sis, be exploited i n fur t h e r investigations so that the actual time available f o r research may be u t i l i z e d to the f u l l . Further i n v e s t i g a t i o n appears indicated i n the f i e l d s of nitrogen and energy balance, b i o l o g i c a l values (possibly as demonstrated through creatinine studies) and d i g e s t i b i l i t y of d i f f e r e n t i n d i v i d u a l nutrients, and feed mixtures. I t i s only through c o r r e l a t i o n of information gained by means of these devious methods that a sound basis for the n u t r i t i o n of mink may be f i n a l l y approached. APPENDICES The following relevant data are included i n Appendix form f o r reasons of spacing and arrangement. - i -APPENDIX I: PREPARATION OF REAGENTS AND LABORATORY TECHNIQUES. 1, Standard Acid Preparation: (Chemical Rubber Pub. Co., 1948) St a r t i n g with HC1 of density about 1.10, constant b o i l i n g HC1 i s prepared by d i s t i l l a t i o n and discard of the f i r s t f of the l i q u i d passing over. Correction must be made f o r v a r i a -tions i n atmospheric pressure. The following figures are sug-gested by Hollingsworth and Foulk: Barometric Pressure %HC1 by Weight Wt. HC1 f o r IN Solution 770 20.197 180.407 gm. 760 20.221 180.193 750 20.245 179.979 740 20.269 179.766 730 20.293 179.555 The amount of acid needed i s weighed out accurately, using a c a p i l l a r y or Pasteur type pipette to f i n i s h and i s dil u t e d to the required volume. For example: making 4 l i t r e s of N/14 acid from constant b o i l i n g HC1 at 770 l b s . pressure, use: 180.407 x 4 . 51.5448 gm. 14 2. Standard A l k a l i Preparation: (Hawk, 1947a) a. Preparation of carbonate-free NaOH: Shake up 110 gm. best quality NaOH with 100 gm. d i s t i l l -ed water i n a 300 ml. Erlenmeyer f l a s k to make a saturated .sol'n. Stopper, and allow to stand u n t i l the sodium carbonate s e t t l e s to the bottom leaving a layer of clear, saturated NaOH sol'n. p r a c t i c a l l y free from carbonate. b. Preparation of a Standard NaOH solution: For 4 l i t r e s of standard N/14 solution, measure out 17.96 ml. of the saturated NaOH sol'n. into a large f l a s k , (6 l i t r e Erlenmeyer) add 3000 ml. d i s t i l l e d water and mix thoroughly. Rinse a clean burette with the a l k a l i sol'n. pre-pared, f i l l , and t i t r a t e the sol'n. against the standard N/14 acid prepared as above, using 1% a l c o h o l i c phenolphthalien as indicator. Calculate the normality of the a l k a l i sol'n. from the t i t r a t i o n and d i l u t e u n t i l a N/14 sol'n. i s obtained. At a l l times shake the sol'n. thoroughly to ensure thorough mixing. Store the a l k a l i i n a stoppered, p a r a f f i n - l i n e d b o t t l e . A 4 l i t r e aspirator bottle i s convenient f o r use. 3. Mixed Indicator Preparation: (Zuazaga, 1942) Prepare a 6.1% sol'n. of Bromcresol Green. Prepare sep-arately a 0.1% sol'n. of Methyl Red i n 95% alcohol. Mix the two indicators i n the proportion of 5 parts Bromcresol Green to 1 part Methyl Red sol'n. - i i -4. Kjeldahl/Gunning Method for Nitrogen Determination, (Koch, 1934) with Modifications: Reagents: a. Standard HC1 and NaoH sol'ns. (N/14) b. Concentrated H2S04. c. C U S O 4 sol'n., 10%. d. K 2 S O 4 , reagent grade. e. Pumice, powdered, Kjeldahl grade. f . NaOH sol'n., 40%. g. Mixed Indicator as above. Procedure: Pipette accurately a 1 ml. sample of the urine into a Kjeldahl f l a s k , add 10 ml. H 2 S O 4 , 1 ml. C U S O 4 sol'n. and 5 gm. K 2 S O 4 . Also prepare a blank, using the same amounts of rea-gents but no urine. Place the f l a s k s on the digestion rack and heat gently, l a t e r intensely, u n t i l the reaction mixture i s a c l e a r , l i g h t green. When digestion i s complete, sw i r l f l a s k s to get contents on walls, d i l u t e with 100 ml. d i s t i l l e d water and set aside to cool. Measure c a r e f u l l y 50 or 100 ml. (depending on the amount of nitrogen presumed to be i n the sample) of standard N/14. HC1 into 250 ml. Erlenmeyer c o l l e c t i o n f l a s k s . Place these fla s k s under the t i p s of the adaptors on the d i s t i l l a t i o n shelf. Apply the heat on the heating elements to be used i n the d i s -t i l l a t i o n . To each Kjeldahl f l a s k containing the digestion mixture, add a generous spoonful of the powdered pumice to prevent bumping and pour evenly and slowly down the side of each f l a s k 40 ml. of the 40% NaOH sol'n. Connect the flasks with the traps on the d i s t i l l a t i o n apparatus and s t e a d i l y ro-tate them to ensure thorough mixing of the contents. Immed-i a t e l y place the c o l l e c t i o n f l a s k s so that the t i p s of the adaptors are beneath the surface of the contents, r a i s e the heat under the d i s t i l l i n g f l a s k s and d i s t i l about ^ the quan-t i t y over. Remove the c o l l e c t i o n f l a s k s and t i t r a t e the stan-dard acid remaining against the standard a l k a l i , using the mixed indicator previously described. Calculate the number of milligrams of nitrogen i n the sample. As the standard acid was N/14, each ml. of acid used up represents one mg. of nitrogen i n the form of ammonia. From t h i s f i g u r e , calculate the t o t a l number of mg. of n i t r o -gen excreted by the animal during the entire c o l l e c t i o n period. 5. F o l i n - J a f f e Method for Creatinine Determination with Modi-f i c a t i o n s (Peters, 1942): a. Preparation of P u r i f i e d P i c r i c Acid: (Hawk, 1947b) (Ordinary CP P i c r i c Acid forms too deep a colour f o r iaccurate photo-colorimetric procedures.) Transfer 500 gm. of moist p i c r i c acid to a Florence f l a s k of 1500 ml. capacity. Add 500 ml. acetone and shake with a - i i i -l i t t l e warming under hot tap water u n t i l a l l the c r y s t a l s have dissolved. Add 20 gm. Norit activated charcoal, shake, and f i l t e r into another f l a s k . Dissolve 250 gm. of anhydrous NagCOg and 100 gm. of NaCl i n 2500 ml. of warm water i n a large "beaker. S t i r slowly with an agate-ware spoon or glass rod and add the acetone sol'n. gradually to the a l k a l i n e s a l t sol'n. When the evolution of C0g has f i n i s h e d , l e t stand i n cold water f o r about % hour, and f i l t e r on a large (20 cm.) Buchner funnel. Wash with 2 l i t r e s of 7% NaCL sol'n., and suck as dry as possible. Return the ppt. to the beaker and add 2 l i t r e s of b o i l i n g water and 20 gm. of NagCOg. To t h i s hot sol'n. add gradually with s t i r r i n g s 150 gm. of NaCl. Cool, f i l t e r , wash as before with 7% NaCl, then with 2% NaCl and f i n a l l y with methyl alcohol to remove most of the remaining chloride and water. Dry at room temperature. P i c r i c acid i s prepared from the picr a t e prepared as above by treatment with d i l u t e HC1. Prepare 2 l i t r e s of d i l u t e HC1, (1 v o l . cone. HC1. to 4 v o l . water) and pour the acid over the p i c r a t e , s t i r r i n g with a glass rod to ensure complete ac-t i o n . F i l t e r again through the Buchner funnel, using hardened f i l t e r paper. Dry the p i c r i c acid c r y s t a l s i f they are to be used immediately. Temperatures up to 90°C may be s a f e l y used i n the drying of p i c r i c acid. b. Estimation of Creatinine i n Urine: (Peters, 1942) P r i n c i p l e : A tungstic acid f i l t r a t e of urine i s treated with a mixture of p i c r i c acid and sodium hydroxide. A red compound i s formed which, with the yellow of the excess p i c r i c acid, produces an amber coloured sol'n. Procedure: Transfer 5 ml. urine to a 100 ml. volumetric f l a s k , d i l u t e to volume and mix. Transfer 2 ml. of t h i s d i l u t -ed urine to a f l a s k , add 16 ml. of N/12 H 2 S O 4 . Mix. Add 2 ml. 10% Na2W04# Shake. F i l t e r through Whatman no. 40 paper. Transfer 5 ml. of the f i l t r a t e to an absorption c e l l , and 5 ml. of d i s t i l l e d water to a s i m i l a r c a l l f o r a blank. To each add 2.5 ml. of fresh a l k a l i n e p i c r a t e . (1 v o l . 10% NaOH to 5 v o l . 1.175% p i c r i c acid. This pic r a t e must be used within 5 minutes.) Mix thoroughly. Let the mixture stand f o r 20 minutes. Read i n a c o l o r i -meter or spectro-photometer, using a wavelength of 520 mu. In practice a standard curve was prepared using a standard creatine sol'n, d i l u t e d over the range expected to be encounter-ed. Measurement was made i n a Coleman spectro-photometer. 5. Detection of albumin i n Urine: (Hawk, 1947c) Place 5 ml. of Robert's reagent (1 v o l . cone H N O 3 and 5 - i v -v o l . saturated mg. S O 4 ) i n an i n c l i n e d t e s t tube and slowly pipette urine down the side of the tube. P r e c i p i t a t e d protein w i l l form a white l a y e r at the interface of the two sol'ns. 6. Detection of B i l e i n Urine: (Hawk, 1947d) Rosenbach's Modification of the Gmelin Reaction. F i l t e r 5 ml. urine through a small f i l t e r paper. Intro-duce a drop of cone. H N O 3 at the apex of the paper. Presence of b i l e pigments i s indicated by a succession of d i f f e r e n t colours spreading out from the centre. 7. Detection of Glucose i n Urine: (Hawk, 1947c) Benedict's Test. a. Preparation of Benedict's Sol'n: Reagents: Copper sulphate - 17.3 gm. Sodium c i t r a t e - 173.G gm. Sodium carbonate - 100,0 gm. D i s t i l l e d water to make 1 l i t r e . With heating, dissolve the sodium c i t r a t e and carbonate' i n about 800 ml. water. F i l t e r i n t o a graduate and make up to .850 ml. Dissolve the C U S O 4 i n 100 ml. water and add i t . s l o w l y to the citrate/carbonate sol'n. with constant s t i r r i n g . Make up to 1 l i t r e . b. Benedict's Test: To 5 ml. of Benedict's reagent prepared as above, add exactly 8 drops of urine. B o i l the mixture vigorously f o r 2 minutes, then allow to cool spontaneously. Presence of glucose iH indicated by a heavy curdy ppt. of varying colours; apple green, yellow, or red, depending upon the amount of sugar present. 8. General Laboratory Procedure: Analyses of urine f o r t o t a l nitrogen were made d a i l y , and i n duplicate. The mean value was taken as the actual nitrogen content. Creatinine determinations were carr i e d out on samples of urine collected on various predetermined dates throughout the experiment. Representative samples were taken during the pre-test, f a s t i n g , nitrogen-free, and complete synthetic d i e t periods. Repeat analyses were, of course, c a r r i e d out wherever close agreement was not attained i n analysis of the duplicates. In such cases where urine samples had to be kept overnight, they were placed i n a r e f r i g e r a t o r and under an overlay of toluene• 9. Investigation re Contamination of Urine Samples: I t was noticed that mink undergoing nitrogen balance de-terminations shed considerable h a i r , underfur and skin debris, es p e c i a l l y during the spring and early summer months. In an - V -, attempt to determine whether the N content of the urine would a l t e r i n passage over t h i s matter, the following experiment was i n i t i a t e d : a. The amount of h a i r shed hy one mink i n a day (including skin brushings and anything other than faeces and urine) was col l e c t e d and was found to weigh 0.2490 gm. b. A 40 ml. sample of urine (about an average day's excretion) was c o l l e c t e d , an ali q u o t analyzed f o r t o t a l N by the Kjeldahl method and the remainder poured over the above h a i r sample i n a 125 ml. Erlenmeyer f l a s k and allowed to stand f o r 24 hours. c. A representative sample was taken from t h i s "extracted" urine, f i l t e r e d , and analyzed f o r t o t a l N as previously. The res u l t s (mean of two determinations) are given below: Total Nitrogen (mg. per ml.) Sample 10.0 mg. 9.8 mg. Straight Urine Urine/Hair Extraction - v i -APPENDIX I I : ANIMAL TECHNIQUES 1. Housing and Care of Animals Cages were devised that would ensure a reasonable amount of space f o r the animals while at the same time r e s t r i c t t h e i r a c t i v i t y towards a basal l e v e l . These cages had to be designed to allow f o r complete c o l l e c t i o n of a l l excreta. The oages themselves were fabricated from squared wire fencing and meas-ured 18" i n height and s l i g h t l y l e s s than 20" i n diameter. Each cage was equipped with a s l i d i n g door to which feed trays could be attached and a 150 ml. water bot t l e with drinking tube. Animals were removed f o r examination or weighing by means of a box trap. Urine c o l l e c t i o n was c a r r i e d out by means of 20" diameter stainless s t e e l funnels placed immediately under the cages. These funnels were constructed with a short v e r t i c a l rim to ensure complete c o l l e c t i o n of excreta. Faeces were separated from the urine by means of two st a i n l e s s s t e e l wire mesh screens placed i n each funnel. Hair and skin debris was se-parated out by a l i g h t l y packed glass wool f i l t e r placed i n the narrow neck of each funnel. The actual urine containers were 100 ml. graduated cylinders hung d i r e c t l y under the fun-nels on pierced rubber stoppers. Toluene was used to exclude a i r from the urine samples during c o l l e c t i o n . With the ex-ception of the funnels and screens, a l l equipment was construc-ted and assembled by the writer at the Animal N u t r i t i o n Labora-tory. Immediately a f t e r measurement of the t o t a l d a i l y urinary excretion, samples were transferred to te s t tubes and kept i n a r e f r i g e r a t o r under toluene u n t i l analysis could be c a r r i e d out. A breakdown diagram of equipment used and a photograph of the u n i t i n operation are included as Figures 5 and 6. - W I R E M E S H C A G E ( 2 0 " D I A . , 718" H T , T ' S Q U A R E S ) - W A T E R B O T T L E " S P R I N G C L I P - C A G E D O O R ^ L I D I N G - S T E E L A N I M A L G U A R D • D R I N K I N G T U B E S T A I N L E S S S T E E L F U N N E L W I R E M E S H F A E C E S S C R E E N W I R E S C R E E N G L A S S W O O L F I L T E R G R A D U A T E D C Y L I N D E R F I G U R E 5 • D I A G R A M O F A N I M A L E Q U I P M E N T A D O P T E D F I G U R E 6 « P H O T O G R A P H O F U N I T I N O P E R A T I O N - v i i -2. Experimental Ration Preparation (a) Nitrogen-free energy source. Two nitrogen-free diets were compounded as l i s t e d below: i . Starch d i e t (used i n preliminary experiment) (Ashworth, 1933). Corn Starch - 74.0 gms. Lard - 8.0 " Cod L i v e r O i l - 2.0 » Sucrose - 10,0 " S a l t Mix, U.S.P. II - 4.0 " Cellulose - 2.0 " The cod l i v e r o i l used was a standardized product having a potency of 43,780 I.U. vitamin A per gm. Cellulose was provided by shredding the dry weight re-quired of Whatman no, 1. f i l t e r papers and pulping them i n a known weight of d i s t i l l e d water i n a Waring Blendor. This r a t i o n supplies approximately 4.2 c a l o r i e s per gram. i i . Sucrose d i e t (used i n the second experiments) (Frost, 1946) Sucrose 73 gms. Lard 20 « Corn O i l 3 i t Cod L i v e r O i l - 0.5 t i S a l t Mix (U.S.P. II) - 4.0 i t Choline Chloride 0.1 i t Agar 1.0 n Thiamin HC1 0.6 mg. R i b o f l a v i n 0.6 t t Nicotinamide 12.0 II Pyridoxine HC1 0.4 t i Ca. Pantothenate 1.2 t i I t w i l l be noticed that t h i s r a t i o n was strongly f o r t i f i e d with the B vitamins i n an e f f o r t to combat anorexia which normally occurred during long periods of nitrogen-free feeding. The cod l i v e r o i l used was a high-potency standardized product containing 43,780 I.U. vitamin A per gram. This d i e t supplies 5,1 c a l o r i e s per gram and was fed at a rate ensuring 200 calor-ies per kilogram body weight. Kjeldahl nitrogen analysis of the sucrose d i e t indicated a mean nitrogen content of 0,025%, (b) Nitrogen sources. The nitrogen source o r i g i n a l l y planned f o r t h i s experiment was a standardized spray dried l i v e r meal produced by the Valentine Meat-Juice Co., Richmond, Va., U.S.A. K j i l d a h l - v i i i -determinations on t h i s product showed a mean nitrogen content of 10.27%* The advantages of such a supplement were obvious -i t offered a uniform, powdered nitrogenous food which could be e a s i l y stored and accurately weighed i n small quantities. Certain disadvantages i n e i t h e r p a l a t a b i l i t y or physical tex-ture soon became apparent, however, and low t o t a l r a t i o n con-sumption l e d to the use of fresh hog l i v e r as a nitrogen source. The fresh l i v e r used was a.product of Canada Packers Ltd., branded as "hog l i v e r , inedible, f o r animal food only." The l i v e r was kept frozen u n t i l s h o r t l y before use and representa-t i v e samples were taken, avoiding the outer surfaces which might-have become dehydrated. For convenience i n weighing, the l i v e r samples were f i n e l y chopped i n the frozen condition e thus avoiding l o s s of moisture or blood. Mean of Kjeldahl de-terminations carried out on the fresh l i v e r showed a nitrogen content of 1.91% on a wet weight b a s i s . Dry matter content was established as 32.5%. 3. Administration of Rations A great number of t r i a l s were necessary before a s a t i s -factory method of feeding could be devised due to the habit inherent to mink of carrying food from any container before devouring i t . A most s a t i s f a c t o r y method consisted of mixing the r a t i o n with a known weight of d i s t i l l e d water to a pasty consistency and then expressing the-desired amount of the mixture to the animal through a hard glass tube. In t h i s manner the amount fed could be accurately regulated and there was p r a c t i c a l l y no l o s s . This method proved very s a t i s f a c t o r y with the f i r s t basal r a t i o n but was discarded with the second r a t i o n f o r fear of l o s i n g appreciable amounts of the sucrose i n s o l u t i o n . A box feeder was devised f o r use with the second r a t i o n and s p i l l a g e loss was subtracted from the amounts fed. Diagrams of the feeders used and a photograph of one i n operation are presented as figures 7 and 8. 4. Loss of Experimental Animals A l l losses of experimental animals Incurred i n both parts of the experiment showed the same general picture, as follows: i . Ante-mortem examination. The animals appeared normal up to a period of 3 or 4 days before t h e i r death a f t e r which they became l i s t l e s s and s t e a d i l y weaker. When offered feed of any nature, these animals refused i t completely. Samples of urine co l l e c t e d the l a s t two days ante-mortem showed evidence of haematuria and yielded increased nitrogen content figures on analysis. i i . Post-mortem examination. Autopsy of the animals showed - i x -them t o he e m a c i a t e d though n o t e n t i r e l y d e v o i d o f m e s e n t e r i c f a t . P e t e c h i a l haemhorrage s p o t s were e v i d e n t on t h e l i v e r and a c e r t a i n amount o f b l o o d and o t h e r f l u i d was found f r e e i n the a b d o m i n a l c a v i t y . S p l e e n s were somewhat e n l a r g e d and k i d n e y s appea red s l i g h t l y p a l e i n c o l o u r . The appea rance o f t h e l u n g s i n one o f t h e s e a n i m a l s was s u c h as t o i n d i c a t e t h a t pneumonia had been a c o n t r i b u t i n g cause o f d e a t h . F I G U R E 7 E X P E R I M E N T A L F E E D I N G M E T H O D S I. B O X T Y P E F E E D E R F I G U R E 8 P H O T O O F 2 ( A B O V E ) I N O P E R A T I O N APPENDIX II I ADDITIONAL DATA RE MINK NUTRITION 1. Natural Diet of the Mink. Any n u t r i t i o n a l study of a recently domesticated animal should include some mention of the p a r t i c u l a r animal's d i e t i n the wild state under more or less natural conditions. Such a d i e t should not be adopted as a- r i g i d standard since what we look on as "natural" conditions have undoubtedly been con-siderably r e s t r i c t e d by the inroads of our modern c i v i l i z a -t i o n ; yet the d i e t selected by the animal when i t has any de-gree of free choice offers a valuable guide to the p a l a t a b i l i -ty of various feeds to that animal. Also the i n s t i n c t f o r self-preservation i n the wild animal probably leads i t to the choice of a reasonably balanced diet,therefore,some informa-t i o n on the n u t r i t i v e requirements f o r the same animal under domestic conditions may possibly be gleaned from t h i s study. In the case of mink, most studies of the animals' d i e t i n the wild state have been conducted by the Wild L i f e services of countries to which the mink i s native. One of the foremost investigators i n t h i s f i e l d , ( B a i l e y , 1930), i n a study c a r r i e d out i n Yellowstone National Park wrote that the general di e t of wild mink consisted of f i s h , frogs, crustaceans and to some extent, mice, gophers, muskrats, ground s q u i r r e l s , chipmunks, birds and other small game. G r i n e l l (1937) i n his studies of C a l i f o r n i a Wild L i f e carried the examination s t i l l f a rther to include the gross percentage composition of the mink's stomach contents. He reports that laboratory examination of 149 mink stomachs from d i f f e r e n t parts of C a l i f o r n i a repealed the con-tents to be the following percentages by bulk: Canadian surveys,(Cowan,-1948), bear out the above f i n d -ings i n the main but indicate that the f a r northern type of Yukon mink d i f f e r i n pr e f e r r i n g rodents f o r food wren when f i s h i s r e a d i l y a v a i l a b l e . Observations of the habits of wild mink, (Bailey, 1936), have shown that the animal i s r a r e l y found f a r from water; therefore, i t may be assumed that i n many oases f i s h and other acquatic l i f e constituted a major portion of the d i e t . This same work names crustaceans as be-ing the f a v o r i t e food of wild mink and i n regions where they are abundant the p r i n c i p a l food the year around. According to traces observed i n the droppings of wild mink i n and near t h e i r dens, any game i s consumed p r a c t i c a l l y i n i t s entirety: bones, feathers, f u r , scales, s h e l l s and a l l . F i s h Birds Small Mammals Crayfish and Mussels Non-Food Material 39.6% 27.0% 21.5% 3.4% 8.5% - x i -2. Time of Passage Naturally the use of food materials by d i f f e r e n t species varies considerably according to the time available f o r action of the various digestive mechanisms on that food. A/search of the l i t e r a t u r e revealed no information i n t h i s regar&T^there-fore, a b r i e f experiment was set up to determine time of passage, using animals of the Unive r s i t y Mink Colony as sub-j e c t s . A reagent grade of Carmine (Merck) was used as an i n -dicator and was mixed with a normal stock d i e t i n the quantity of approximately 1 gm. to 150 gms. of the wet r a t i o n . T r i a l s were run i n duplicate on two d i f f e r e n t pairs of animals. The time of feeding was accurately recorded and appearance of the dye i n the faeces was taken as in d i c a t i v e of time of passage of food material. Aotual time of defacation was not recorded i n any case but the several t r i a l s agree on an approximate time within the range of 7 to 9 hours. 3. Weight-Growth Correlations. I t i s evident that once a maintenance standard has been set for mink n u t r i t i o n , supplementary requirements allowing for the processes of growth, production and reproduction must shortly be arrived upon. In order to assess the rate of growGi i n mink k i t s on a "standard" r a t i o n and hence l a y the basis f o r calculations of growth requirements, four l i t t e r s of mink from the Uni v e r s i t y Mink Colony were weighed weekly from b i r t h with the following r e s u l t s : *TFfoL€. N \ ^tvCMl ZlMM Cflft(leVM\o^ vA UTTERS of NovlHG, [AvM 0? itortlBUftL 9itfWtok\_S %, i ^ i 3 fife. \ WKS) i 4 5 c \A ME 1 L (A L s (A VI WW in si 5f 7? t\ 17 to IA I-41 XX 23 z 47 4r zz • £•57 ft ZS7 us 11 i«7 lOC) 1 0 1 2.1* 51 53 34 74 r7 12 f4 St - m III |0£ lo i $-n. ^7 lo? MS" let in J44 41 St) <?<? /// "7 /of 15T5" \3"l l£>7 \Ii 4.J7 |W IJo \?) I3t I4\ 154 4 if i n I 0 1 lo7 r /.?sr «7 /4? 11 ~ 5,9) l\\ IbO \S7, IS? IS4 I43 174 |7i III f - V | 141 131 I37J L 14? 1U So » t.D iLt> l \ \ 14? U \ X|f 24o 1 S 1 :rt> lof 141 fe.iT i n \f? 2£>o| 7 Zu SIS 32X in » 7.0 Si? SOD 256 7S7 S?7 243 3oT 2i4 171 ?.iq wr 17j- Iff f 43c JU 4 J u l . lq 34S 3io 177 Si4 W7 3tr 33f sir 173 34o 342 341 "3 ?77 406 4V 424 These data are untreated and are included f o r reference only. Mean values "M" .are given f o r each l e t t e r at each weighing. - x i i -4. Relation Between Organ Weights and Body Weight of Mature Mink. Structure and function are two aspects of the same thing. Each s t r u c t u r a l de-t a i l possesses i t s functional expression.... Carrel This project has been undertaken f o r the purpose of corre-l a t i n g organ weights to body weight i n mature mink thus para-l l e l i n g s i m i l a r studies already oompleted f o r other animals. In attempting research into the n u t r i t i o n a l requirements of mink, the writer has been hampered by the lack of a v a i l a b l e "normal" data on these animals which might serve as a basis f o r c a l c u l a t i o n s . D i f f i c u l t i e s have been encountered i n the formulation and administration of experimental rations to mink which suggest the p o s s i b i l i t y of physiologic and anatomic d i f -ferences between them and other experimental animals. The various body functions with which we are interested, such as energy and protein metabolism, tissue production (growth)," fur production, and reproduction a l l depend f o r t h e i r construc-t i v e materials upon the e f f i c i e n c y of the organs of the diges-t i v e t r a c t . A knowledge of the correlations of these various organs to body size may well be a convenient s t a r t i n g point i n a study of t h e i r function although i t must be remembered that changes i n h i s t o l o g i c a l structure (and corresponding physiolo-g i c a l action) may invalidate relationships based only on s i z e . I t i s hoped that t h i s present study may shed some l i g h t on the functional e f f i c i e n c y of mink and that i n c i d e n t a l l y i t may o f f e r a normal standard against which abnormal conditions may be contrasted and evaluated. Method. Weight, being a function of volume, was taken i n preference to length as a standard f o r comparison. Mature animals were chosen as subjects i n order to reduce v a r i a b i l i t y due to age. The animals chosen f o r the purpose of t h i s experiment were mature mink of both sexes and of the standard or dark type. They were k i l l e d by gassing i n an a i r t i g h t chamber immediately a f t e r which the complete body weight was recorded and the p e l t removed. The number of animals k i l l e d over a short period of time i n order to obtain the s a t i s f a c t o r y "primeness" of p e l t necessitated storage of the carcasses p r i o r to diss e c t i o n . This was effected through sharp-freezing and glazing of the carcasses with water i n order to prevent extreme evaporation. At the time of dissection, the v i s c e r a were c a r e f u l l y re-moved, separated where applicable and weighed immediately. Use of a team system whereby ce r t a i n operators were assigned to a balance or to the task of d i s s e c t i o n reduced the exposure time to a minimum. Weights were taken of the carcass with p e l t - x i i i -removed, l i v e r , heart, lungs, stomach, i n t e s t i n e , kidneys and spleen. Any excess blood was allowed to drain o f f on to ab-sorbent paper before the organs were weighed and as l i t t l e surrounding f a t as possible was removed with the digestive t r a c t . From the weights obtained percentages of t o t a l body weight were calculated and the re s u l t s tabulated. INTEST. WT Va'W 3.3  4Ut, 3.X  41 fI 3J 44. to J.i KIDNEYS WT % /O. /o o. IS 194 0.13 ib.i3 olo /OC? 07/ 19Z 0.7) 9J9 on fit c& 9-91 0.7S /Oil oi4 7.41 0% 113 6.9 f $:s o is 7.1 6. ft 4f AS1 HEART | LUNGS | STOMACH I S P L E E N L41 6. X i ± 73 Uf it 71 72 53 11 7j__U_ 70 ill 4.1  1) l4.v 41 17 n.o Cl lo fo.c f/  14 4ft 4* 71 4Lo <:./  13 VJt 41 _V 91 6J_ tl 4h 63 7/ H 4LL 72 &o I > 14.0 4,4 91 o U 0.93 WB1 o.t4 If. SI oil 11.34 0.IJ 6.13 U3 e.fi 931 Cit TMk OlX 6.74 L47 OS? I.K 0/4 3.00 0.21 B E 1 H H 2.30 0/1 o/6 J 31 on 9-/f 0 71 9-o oil 94 o% /24\ 6.71 on 14/ on WESmWUBSBEL I1l\ 0./f\ //(, O./f /.ill o.n\ nl 0J4 KHHaiEJIlHIHIiaEailH ti.F ns 94 OlD jt.x 114 1.1 0.S4 /ir llo ix CS9 ft2C 1.24 llK tjf /i.4 /.21 P.of Q.IS /J3f 1.21 90 0.(2, left >X4 II Oio l i t 1.41 11 biO J.I c./r 11 o>4 "2 1 t.ii J.I 0/1 IS on n c./4 13 O-li 6.1 c.tx wzm II. if Off EH na 16-SS 2.3 oil at 1.4 0/4 /I.O 01) *4J D.li 34.7 2.0 1.1 Oil So. 1 3.1-x 2-3 0-/C 1i 0.19 2.X on 19-3 2/3 2.0 D.2Z 1/4 Oil 13 M r n.9 0.11 4.0 6.24 114 /if 2.x o i4 ll4 m 3.4 t /9 ff.4 0 9? 2.1 til Oil / 3 O./i xiv 5 . Basal Metabolism Data for Mink During the course of experimentation, values for the basal metabolism of the mink were calculated from urinary nitrogen excretion. In order £ 0 check the accuracy of these figures, actual oxygen consumption of the last three test animals was measured over definite periods of time, using a Mc Donald College respirometer apparatus. A certain amount of d i f f i cu l ty was encountered in maintaining a quies-cent state i n the animals after a preliminary 24 hour fast, •however conditions reasonably near basal were attained. The data obtained are l i s t ed as follows: OXYGEN CONSUMPTION -IN MINK 23 JULY, 194-9 'Wol Weight 02/min. O2/24 hrs. BMR c a l . Cal/Kg Tests gm. cc. calculated calculated calculated 4 1000.0 16.8 23.19 1. 111.33 111.33 6 1 83O.O 19.2 27.65 132.74 159.93 6 2A 560.0 20.3 29.23 140.33 250.59 6 The thermal equivalent per l i t r e of oxygen was taken to be 4.801 calories, corresponding to an R.Q. of 0.8, i n accord-ance with figures cited by Brody, "Bioenergetics and Growth", p. 310. BIBLIOGRAPHY ASHWORTH, U . S . , and B r o d y , S . , ( 1933 , a ) U . M o . R e s . B u i . 1 8 9 , p p . 6 8 , 8 . ASHWORTH, U . S . , and B r o d y , S . , ( 1933 ,b ) U . M o . R e s . B u i . 1 9 0 , p p . 1 9 . B A I L E Y , V . , (1930) " A n i m a l L i f e o f Y e l l o w s t o n e N a t i o n a l P a r k " , U . S . G o v t . P r i n t i n g O f f i c e , W a s h i n g t o n , D . C . , p p . 1 4 4 - 1 4 6 . B A I L E Y , V . , (1936) "The Mammals and L i f e Zones o f O r e g o n " , i n t h e c o l l e c t i o n " N o r t h A m e r i c a n F a u n a " , 5 5 , p . 2 9 3 . BALDWIN, E . , (1947,a) "Dynamic A s p e c t s o f B i o c h e m i s t r y " , Cambrics U . P r e s s , 1 s t E d . , p p . 4 5 6 , 2 1 1 . BALDWIN, E . , ( 1947 ,b ) o p . c i t . 1 7 1 , 1 7 2 . BARCLAY, J . A . , Kennedy , R . J . , (1947) B i o c h e m . J " . , 4 1 , 4 , pp. 586-589. BENEDICT, F . G . , (1938) " V i t a l E n e r g e t i c s " , C a r n e g i e I n s t . Wash. , P u b . 503 , p p . 2 1 5 , 1 7 5 - 1 7 6 . BENEDICT, F . G . , Emmes, L . E . , (1912) Am. J . P h y s i o l . , 3 0 , p . 1 9 7 . BORSOOK; H . , (1936) B i o l . R e v . 1 1 , p . 1 4 7 . BORSOOK, H . , D u b n o f f , J . W . , (1943) A n n . R e v . B i o c h e m . 1 2 , p p . 1 8 3 - 2 0 4 . BORSOOK, H . , K e i g h l e y , G . L . , (1935) P r o c . R o y . S o c , 1 1 8 B , p p . 4 8 8 - 5 2 1 . BRODY, S . , P r o c t o r , R . C . , A s h w o r t h , U . S . , (1934) U . M o . R e s . B u i . 2 2 0 , p p . 4 0 . BRODY, S . , (1945) " B i o e n e r g e t i c s and G r o w t h " , R e i n h o l d P u b . C o r p . , 1 s t E d . , p p . 1 0 2 3 , 3 9 7 . . CAHZLL, W . M . , ( 1944 , a ) " O u t l i n e o f t h e Amino A c i d s and P r o t e i n s " , e d . M . S a h y u n , R e i n h o l d P u b . C o r p . , 1 s t E d . , p p . 2 5 1 , 1 7 9 . C A H I L L , W . M . , ( 1 9 4 4 , b ) o p . c i t . , 1 8 2 . CATHCART, E . P . , (1925) P h y s i o l . R e v . , 5 , p p . 2 2 5 - 2 4 3 . COWAN, I . M o T . , (1948) U n p u b l i s h e d D a t a , The D e p t . o f Z o o l o g y , The U n i v e r s i t y o f B r i t i s h C o l u m b i a . DEUEL, H . J . , S a n d i f o r d , I . , S a n d i f o r d , K . , B o o t h b y , , W . M . , ( 1 9 2 8 ) J . V B i o l . Chem. 7 6 , p p . 3 9 1 - 4 1 4 . BIBLIOGRAPHY - 3 F O L I N , 0 . , D e n i s , W . , (1912) J . B i o l . Chem. 1 1 , p . . 8 7 . F O L I N , 0 . , (1905) Am. J . P h y s i o l . 1 3 , p . 6 6 . FRANKEL, E . M . , (1916) J . B i o l . C h e m . , 2 6 , p p . 3 1 - 5 9 . FROST, D . V . , R i s s e r , W . C . , (1946) J . N u t . 3 2 , p p . 3 6 2 - 3 7 2 . G R I N E L L , J . , D i x o n , J . S . , L i n s d a l e , J . M . , " F u r B e a r i n g Mammals o f C a l i f o r n i a " , p a r t I , p p . 3 7 5 , 2 4 7 . "HANDBOOK OF CHEMISTRY AND P H Y S I C S " , (1948) Chem. Rubbe r P u b . C o . , 3 0 t h E d . , p p . 2 6 8 6 , 1 3 5 0 . HANKE, M . T . , K o e s s l e r , K . K . , (1920) J . B i o l . C h e m . , 4 3 , p p . 5 6 7 - 5 7 7 . HAWK, P . B . , O s e r , B . L . , Summerson, W . H . , (1947•a ) " P r a c t i c a l P h y s i o l o g i c a l C h e m i s t r y " , 1 2 t h E d . , B l a k i s t o n , p p . 1 3 2 3 , 8 0 7 . HAWK, P . B . , e t . a l . , ( 1947 ,b ) o p . c i t . , p . 1 2 3 1 . HAWK, P . B . , e t . a l . , ( 1 9 4 7 , c ) o p . c i t . , p . 7 6 3 . HAWK, P . B . . , e t . a l . , ( 1947 ,d ) o p . c i t . , p . 7 7 0 . HAWK, P . B . , e t . a l . , (1947 ,e ) o,p. c i t . , p . 7 5 8 . H I L L , A . V . , ( 1 9 2 4 ) , S c i e n c e , 6 0 , p p . 5 0 5 -HDNTER, A., ' ( 1 9 2 8 , a ) " C r e a t i n e and C r e a t i n i n e " , Longmans, G r e e n , p . 1 1 4 . HUNTER, A . , ( 1928 ,b ) o p . c i t . , p . 1 2 8 . JACKSON, R . W . , (1945) " C h e m i s t r y o f t h e Amino A c i d s and P r o -t e i n s " , e d . C . L . A . S c h m i d t , C o l l e g i a t e P r e s s , M e n a s h a , W i s . , 2nd E d . , p p . 1 2 9 0 , 9 7 5 . KADE, G . F . , P h i l l i p s , J . H . , P h i l l i p s , W . A . , (1948) J . N u t . 3 6 , 1 , p . 1 0 9 . KOCH, tf.C, (1934) " P r a c t i c a l Methods i n B i o c h e m i s t r y " , p p . 2 8 2 . 1 7 6 - 1 7 7 . ' KOSTERLITZ , H . W . , C a m p b e l l , R . M . , (1946* N u t . A b s . , and R e v . , p p . 1 -14 . K R I S S , M . , ,(1941) J . N u t . , 2 1 , p p . 2 5 7 - 2 7 4 . LANDERGREN, E . , (1916) i n C a t h c a r t , G . D . , B i o c h e m . J . , 1 0 , p p . 1 9 7 - 2 4 4 . BIBLIOGRAPHY - 5 LUSK, G., (1931,a) "The Elements of the Science of N u t r i t i o n " , W.B. Saunders Co., 4th Ed., pp. 843, 283. LUSK, G., (1931,b) op. c i t . , pp. 89,90. LUSK, G., (1931,c) op. c i t . , p. 92. McNAUGHT, M.L., Smith, J.A.B., (1947) Nut. Abs. and Rev. 17, 1, pp. 18-29. . MAXIMOW, A.A., Bloom, W., (1947) "A Textbook of Histology", W.B. Saunders Co., 4th Ed., pp. 695, 417. MAYNARD, L.A., (1947) "Animal N u t r i t i o n " , McGraw, H i l l Book Co„ 2nd Ed.,,pp. 494, 349. MELNICK, D., Cowgill, G.R., (1936) J . Nut., 13, pp. 491-423. MILLER, J.I., Morrison, F.B., (1942) J . Agric. Res., 65, p. 425. MITCHELL, H.H., and Hamilton, T.S., (1929,a) "The Biochemistry or the Amino Acids", The Chem. Catalog Co*, Inc., 1st Ed., pp. 619, 203. MITCHELL, H.H., Hamilton, T.S., (1929,b) op. c i t . , 247. MITCHELL, H.H., (1924) J . B i o l . Chem. 58,.pp. 873-903. MORRISON, F.B., (1949) "Feeds and Feeding", 21st Ed., Morrison Pub. Co., Ithaca, pp. 1207, 1122. MURLIN, J.R., Szymanski, T.A., Nasset, E.C., (1948) J . Nut.,36, 1, 171, 176. PETERS, J.P., (1942) J . B i o l . Chem. 146, p. 179. ROSE, W.C., (19.37) Science, 86, pp.298-300. RUBNER, M., (1931) c i t e d i n Lusk, G., "The Elements of the Science of N u t r i t i o n " , 4th Ed., W.B. Saunders Co., pp. 843,186. SANCTORIUS, "1614) "De Medicina S t a t i c a Aphorismi", Venice, c i t e d i n Lusk, op. c i t . , 17, SCHOENHEIMER, R., (1942) "The Dynamic State of Body Constitu-ents", Harvard U. Press. . SHAFFER, P.A., (1908) Am. Jour. Physiol., 23, pp. 1-22. SHERMAN, H.C., (1941) "Chemistry of Food and N u t r i t i o n " , She Macmillan Co., 6th Ed., pp. 611, 195. SMUTS, D.B., Marais, J.S.C., (1939) Onderstepoort J . Yet. S c i . , 13, 1, pp. 219-225. BIBLIOGRAPHY -, 4 SMUTS, D . B . , (1935) J . N u t . , 9 , p p . 4 0 3 - 4 3 3 . VAN S L Y K E , D . D . , M e y e r , G . M . , (1913) J . B i o l . Chem. 1 6 , u p . 1 8 7 -1 9 7 . VAN S L Y K E , D . D . , (1948) S c i e n c e , 9 5 , p p . 2 5 9 - 8 6 3 . WEISS , R . , R a p p o r t , D . , (1924) J . B i o l . C h e m . , 6 0 , p p . 5 1 3 - 5 4 4 . WILSON, S . J . , W a l z e r , M . , (1935) Am. J . D i s . C h i l d . , 5 0 , p . 4 9 . ZUAZAGA, G . , (1942) J . I n d . Chem. A n a l . , 1 4 , p . 2 8 0 . ZWARENSTEIN, (1926) B i o c h e m . J . , 2 0 , p p . 7 4 2 - 7 5 0 . 

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