INTERACTIONS BETWEEN VITAMIN BI!’ CORTISONE, INSULIN AND ALLOXAN DIABETES ON PROTEIN, CARBOHYDRATE AND VITAMIN B}2 METABOLISM IN RATS Thais for 1h. Doom of Ph. D. MICHIGAN STATE COLLEGE Yu-Sheng Louise Fang 1954 III II III II IIIIII 903 66661 d This is to certify that the thesis entitled _ Ip- :1 .5 «A 5 '“ Interactions Between VitaminB J Cortisone, Insuiih ana’Alloxan””~””” Diabetes on Protein, garbohydrate_n__ and Vitamin 312 Metabolism in Rats .~— “~-- ‘. 3:: r .._aw.¢.~-'HM‘ Ipresented In] Yu-sneng Louise Fang has been accepted towards fulfillment of the requirements for _M degree in _Eh§csiology I / Major professor Dme October 53 1954 0-169 PLACE ll RETURN BOX to remove thle checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Ie An Afflnnettve ActionlEquel Opportunlty lnetttution W INTERACTIONS BET’IIETIN VITAMIN B CORTISONE, INSULIN 12’ .AJII).ALLOXAN DIABETES ON PROTEIN, CARBOHYDRATE AND VITAMIN B12 METABOLISM IN RATS BY Yu-Sheng Louise Fang AN ABSTRACT shitnnitted.to the School of Graduate Studies of Michigan fitate College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology Year lQSu Approved by Yu-Sheng Louise Feng 1. When young rats were fed a vitamin Blz-deficient diet, supplementation with this vitamin increased appetite and body weight gains, slightly increased blood glucose, greatly increased glucose tolerance, but slightly decreased urinary nitrogen excretion. When one to four mg. of corti- sone acetate daily were injected into vitamin BIZ-deficient rats, there was a progressive increase in urinary nitrogen, increased hyperglycemia and glucosuria, decreased glucose tolerance, reduced body weight gains and decreased appetite. When 200 mcg. of vitamin B12 per kilogram of diet was fed to cortisone-injected rats, and they were permitted to eat EB libitum, increases in urinary nitrogen losses were largely prevented, the hyperglycemia and glucosuria were reduced, glucose tolerance was increased and body growth was increased. Vitamin B12 was ineffective in these respects when food intake was restricted to that of animals receiving cortisone without vitamin B12. It is concluded that large doses of vitamin B12 can partially counteract the protein catabolic actions of cortisone by increasing appetite, increasing the availability and utilization of carbohydrate by the organism and reducing gluconeogenesis from protein. 2. Large doses of cortisone partially interfered with the favorable action of vitamin 812 in increasing the effi- ciency of food utilization for body growth. This was accompanied by hyperglycemia and glucosuria, and was related Yu-Sheng Louise Feng to increased insulin resistance. Less carbohydrate was therefore left available for transformation into body weight gains (probably fat). 3. Alloxan—diabetes reduced body growth and the feed/gain ratio on the vitamin B -deficient but not on the 12 vitamin Blz-adequate diet. In the latter rats there was much higher blood glucose, more urinary glucose, increased glucose tolerance but about the same urinary nitrogen losses as in the former animals. It is concluded that vitamin 812 can act independently of insulin insofar as its effects on glucose utilization and body growth are concerned. u. Single injections of insulin (2 units in most cases) were much more effective in reducing blood glucose in normal, alloxan-diabetic and cortisone-treated rats on a vitamin B12- adequate than on a vitamin BlZ-deficient diet. This indi- cates that an ample supply of vitamin B12 is essential for maximum.insulin action . By far the greatest resistance to insulin was found in the cortisone-treated rats on the vitamin BlZ-deficient diet, confirming the findings that cortisone increases insulin resistance. 5. (a) Injections of large doses of cortisone (2 to u mg. daily) increased the urinary excretion of radioactive vitamin B12, particularly in rats fed a vitamin BIZ-deficient diet. On a diet meeting only normal requirements for vitamin B12 (20 mcg./kilogram), cortisone did not increase urinary Yu-Sheng Louise Feng vitamin B12 until u mg. were injected daily. In general, the amounts of radioactive vitamin B12 lose in the urine were shown to be directly related to the dose of cortisone administered. (b) Intraperitoneal injections of 750 mg. of glucose did not change urinary losses of vitamin B12 in alloxanized rats fed either a vitamin 812-adequate or -deficient diet. Apparently blood glucose was already being used to the maximum.extent possible in these rats. (0) In normal and cortisone-treated rats on a vitamin BIZ-adequate but not on a vitamin BlZ-deficient diet, intraperitoneal injections of glucose decreased the loss of urinary vitamin B12. This is believed to reflect greater glucose utilization in the former animals. (d) Insulin injections (3 injections of 0.5 unit each in 2h hours) greatly reduced urinary radioactive vitamin B12 losses in normal, alloxanized and cortisone- treated rats whether on a vitamin Bla-adequate or ~deficient diet. This is believed to reflect greater glucose utili- zation in these animals. The decreases in urinary vitamin B12 were less on the vitamin-deficient diet, particularly in the cortisone-treated animals, and is believed to reflect the reduced effectiveness of insulin on glucose utilization in these rats. INTERACTIONS BETWEEN VITAMIN B12, CORTISONE, INSULIN AND ALLOXAN DIABETES ON PROTEIN, CARBOHYDRATE AND VITAMIN 312 METABOLISM IN RATS By Yu-Sheng Louise Feng A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology 195M ”(I-{€8.13 TABLE OF CONTENTS INTRODUCTION. 0 0 O O O O O O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . Adrenal Cortical Hormones . Diabetes and Insulin . . Vitalnin B12 e e e e e e e EXPER IMEN TAL O O O O O O O O O O O O O O O O O O O O 0 Experiment I. Prevention by Vitamin.B of Protein Catabolic Action of Cor isone . . . , Experiment II. Prevention by Vitamin B of Protein Catabolic Action of Cor isone . . . Experiment III. Effects of Cortisone, Vitamin 812, Insulin and Alloxan Diabetes on Blood Glucose and Urinary Glucose andNitrogen............ Experiments IV and V. Glucose Utilization in Normal, Alloxan-Diabetic and Cortisone- treated Rats as Influenced by VitfllflinBl eeeee ee e Experiment VI. Effects of ortisone on Distribution of Vitamin 312 in Blood, liver and Urineeeee eee e e e Experiment VII. Excretion of Radioactive Vitamin B12 in the Urine following Injection of Cortisone at Different Levels . . Experiment VIII. Effects of Alloxan-diabetes, Cortisone and Vitamin B12 on Exeretion of Radioactive Vitamin B Experiment IX. Effects of Glucose Administration on Excretion of Vitamin B Alloxan, Cortisone and ViIamin B12- treated Rats . . . . . . . Experiment X. Effects of Insulin Injections on Vitamin Bl Excretion in Normal, Alloxan an Cortisone-treated Rats . 12 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . APPENDIX . . . . . . . . . . . . . . . . . . . . . . . Page mop: r H 23 3O 35 AB 55 59 6A 67 70 7M 80 & m6 TABLE I. II. III. IV. V. VI. VII. VIII. IX. X. XII. LIST OF TABLES Effects of vitamin B12 and cortisone on urinary nitrogen and food intake . . . . . Effects of vitamin B1 and cortisone on urinary nitrogen ang food intake. . . . . . Effects of vitamin B1 on body weight and , alloxan and cortisone ood intake . . . . . . Effects of alloxan, cortisone, and vitamin B12 on blood and urinary glucose and urinary nitrogen O O O O O O O O O O O O O O O O 0 Effects of insulin on blood glucose after pretreatment with alloxan, cortisone and VitaminBlzeeeeeeeeeeeeeeee Effects of vitamin Bl , alloxan and cortisone on glucose tolerance test . . . . . . . . . Effects of vitamin B , alloxan and cortisone on glucose tolerance test . . . . . . . . . Effects of cortisone and vitamin B 2 on distribution of CORD-vitamin 1312 in blood, liver and urine . . . . . . . . . Effects of different levels of cortisone on excretion of radioactive vitamin 812 Effects of alloxan, cortisone and vitamin B12 on excretion of radioactive vitamin B12 in urineeeeeeeeeeeeeeeeeeee Effects of glucose injections on vitamin B12 excretion in alloxan, cortisone, and vitamin Blz-treated rate . . . . . . . . . Effects of insulin injections on excretion of radioactive vitamin B 2 Cortisone-treated. rat% 0 e e e e e e e e e in urine in normal, alloxan and Page 28 31+ 38 MO A9 S2 S7 61 65 68 72 ACKNOWLEDGEMENT The author wishes to express her sincere gratitude to Dr. J. Meites, Professor of the Department of Physiology and Pharmacology, for his patient guidance and advice throughout the course of this work and during the prepar— ation of the manuscript. She also wishes to express her appreciation to Dr. B. V. Alfredson, head of the Department of Physiology and Pharmacology, for providing facilities and laboratory space to carry on these experiments, and to Dr. L. F. Wolterink, Professor of the Department of Physi- ology and Pharmacology, for his valuable criticism. Many thanks are due Miss Pauline Ho for her laboratory assistance, and Mr. John Monroe for care of the animals used in these experiments. Grateful acknowledgement is made to Drs. L. Michaud and C. Rosenbloom of Merck and Co., Rahway, New Jersey, for supplying cortisone acetate, crystalline vitamin B12 and radioactive vitamin B12, and to Dr. K. K. Chen of Eli Lilly and Co., Indianapolis, Indiana, for supplying zinc insulin (Iletin). Finally, the writer is indebted to the Michigan Agri- cultural Experiment Station and the United States Public Health Service for providing financial support to the project under which this work was carried out. INTRODUCTION Large doses of cortisone have been shown to produce catabolic effects characterized by reductions in body, hair and thymus growth and by increased gluconeogenesis from protein. There is also considerable evidence that large doses of cortisone stimulate pancreatic islets function (Baker, 1952; Franckson 35 3;. 1953). but at the same time interfere with the action of insulin on carbohydrate utili- sation.(Inglelgtlg;. 19h5; Franckson'gt‘gl. 1953). Vitamin 312 has recently been demonstrated to favor transformation of carbohydrate into fat (Chow‘gt‘gi. 1951, 1952), a function which is also characteristic of insulin. The foregoing suggests that the action of cortisone, insulin and vitamin B12 any be interdependent insofar as their effects on carbo- hydrate and protein metabolism are concerned. In a series of reports Meites (1951, 1952a, 1952b) observed that large doses of vitaminB12 partially counter- acted the inhibitory effects of large doses of cortisone on body, hair and thymus growth in young rats. These beneficial effects were invariably accompanied'by increased food intake and by greater efficiency in converting food into body weight gains, but at the same time cortisone prevented vitamin.B12 from exerting its effects in full. It was therefore of interest to attempt to discover how these interactions :‘ between cortisone and vitamin.312 were produced, and to determine what the role of insulin.might be in this process. Specifically, this thesis will deal principally with the following questions. 1. 2. 3. h. 5. 6. By what means do large doses of vitamin.BlZ partially counteract some of the catabolic actions of cortisone? Does vitamin.812 prevent increased gluconeogenesis from protein? If so, through what mechanism? How do large doses of cortisone partially interfere with the ability of vitamin.Blz to transform food into body weight gains (1.6.. transform carbohydrate into fat)? Does vitamin.Blz require insulin to increase food intake, body growth and glucose utilization or can it function independently of insulin? Does insulin require vitamin 812 for its action on carbohydrate metabolism or is its action independent of vitamin 812? What are the reactions to glucose tolerance tests of nonlal, alloxan-diabetic and cortisone-treated rats on a vitamin BIZ-deficient or -adequate diet? This should indicate to what extent these rats can utilize glucose. 'Hhat are the effects of cortisone, alloxan-diabetes, insulin or glucose injections on vitamin.Blz require- ments, as measured by urinary excretion of injected radioactive vitamin B12? Of course complete answers to the above questions were not forthcoming in the research reported here, but it is believed that some of the interrelationships between corti- sone, insulin and vitaminB12 have been clarified. Several if not many additional questions have arisen as a result of the findings recorded in this thesis, and only further investigation can be expected to resolve them. Of course complete answers to the above questions were not forthcoming in the research reported here, but it is believed that some of the interrelationships between corti— sone, insulin and vitaminB12 have been clarified. Several if not many additional questions have arisen as a result of the findings recorded in this thesis, and only further investigation can be expected to resolve them. LITERATURE REVIEW Introduction Inasmuch as this thesis deals with interactions between vitmmin.B12, cortisone, insulin and diabetes as related to protein, carbohydrate and vitamin B12 metabolism, it is pertinent to briefly review some of the salient actions of the former on the latter. The writer found it necessary to select from a vast literature, and for the most part the articles reviewed here were chosen because of their direct bearing on the thesis problem. Adrenal Cortical Hormones Effects of cortisone and ACTH on b°qia hair and tgymus growth The adrenal cortical hormones play an important part in the maintenance of normal growth processes. This function appears to depend primarily upon the effects exerted by the adrenal cortical hormones on protein and carbohydrate metabo- lism. Protein synthesis is retarded in adrenalectomized animals, but is not altered further during stress. Ingle (l9h9) found that during severe stress induced by bone fracture or burns in the adrenalectomized rats, the break- down of protein was not accelerated as it was in normal animals. Injection of high doses of ACTH or cortisone 1‘ causes impairment of growth because of inhibition of new protein formation and protein catabolism. The growth- inhibiting potency of ACTH or cortisone parallels the magnitude of the negative nitrogen balance (Ingle, 19h6). .Administration of ACTH (Baker‘gt‘gl, l9h8) and cortisone (winter'gt‘gl. 1950) to rats suppressed growth of hair. Ingle (19h9) stated that the prolonged local application of adrenal cortical extract or of cortisone to the skin of rats reduced the cellularity of the dermis. Dougherty and White (19h5) found that treatment with ACTH or cortisone induced involution of the thymus, lymph-nodes and spleen. They stated that lymphocytes underwent lysis within a few hours after treatment. Baker gt 5;. (1951) also found that new formation of lymphocytes was suppressed as indicated by a reduction in mitosis and immature cells, and destruction of reticular tissue cells from which lymphocytes originated. Effects of cortisone and ACTH on carbggydrate and protein Wish - ~ The possible relationship between adrenal cortical function and carbohydrate metabolism was first appreciated by Britten (1932). In adrenal cortical insufficiency the carbohydrate content of the blood and tissues was decreased below normal. In contrast, administration of cortisone or ACTH has been shown to increase liver glycogen, and produce hyperglycemia and glucosuria. Long, Katzin and Fry (19h0) found that administration of adrenal cortical extract or crystalline carbohydrate-active adrenalsteroids to fasted adrenalectomised or to normal or hypOphysectomized animals resulted in a ten-to-forty-fold increase in liver glycogen, and increases in blood glucose and urinary nitrogen excretion. Ingle (19h0) noted a diabetogenic effect of cortisone in partially depancreatised and normal rats as indicated by hyperglycemia, glucosuria and increased excretion of non- protein.nitrogen. Large doses of either ACTH or of cortisone have been observed to produce a negative nitrogen balance in laboratory animals and in man (Long :3 :1. 191m; Ingle, 191m). Engel (1951) stated that it was probable that the adrenal cortical hormones acted predominately at the level of whole protein rather than at some intermediary stage of nitrogen.metabo- 11mm. Support for this statement comes fromlthe work of Ingle‘gt‘gl. (1950) who found that cortisone accelerated the rise of amino acids in the blood of liverless rats. The classical work of Long, Katzin and Fry (l9h0) formed the basis for the present knowledge of the effects of adrenal cortical hormones and ACTH upon carbohydrate and protein.metabolism. The observation that the rise in glucose in the blood and tissue was paralleled by a rise in the excretion of non-protein nitrogen led tjam to believe that. the hormones possibly stimulated gluconeogenesis from tissue proteins. {Adrenalectmmised animals showed an inadequate blood glucose level when exposed to circumstances demanding an increased rate of protein.metabolism. Ingle (19h5) induced severe glucosuria in normal rats by administering cortisone, but elieved that the extent of the glucosuria was too great to be completely accounted for by gluconeo- genesis from protein. Albright (l9h3) stated that cortisone inhibited the synthesis of protein rather than accelerated protein catabolism. The balance between the rate of break- down and resynthesis of tissues could be shifted in the direction of tissue depletion by either stimulation of catabo- lism or inhibition of anabolism» Hoberman (1950) and Clark (1953) reported evidence for both an acceleration in protein catabolism and an inhibition of anabolism in experiments involving the use of HIS-labeled glycine in rats given either cortisone or ACTH. Engel (1949) reviewed the evidence that carbohydrate and other foodstuffs could decrease the catabolic effect of the adrenal cortical hormones and suggested that the hyper- phagia which frequently occurred during treatment with ACTH or cortisone represented a homeostatic response by the body which tended to sustain nitrogen balance. Long,.Katsin and Fry (19h0) and.Engel (19h9) noted that administration of carbo- hydrate to cortisone treated rats prevented the protein-catabolic effect of the latter. This suggests that the adrenal steroids may also act directly on carbohydrate. As will be seen later, these observations have a direct bearing on the results reported here with vitaminB12 in cortisone-treated rats. \ Relation of cortisone and ACTH to function of the_pgncreas Since the level of blood sugar appears to control the production of insulin by the beta cells of the islets of Langerhans, Jensen (19kb) stated that any agent which induced hyperglycemia could be expected to stimulate this gland indirectly. Baker gg‘gl. (1952) found that ACTH cuased hypertrOphy, degranulation and an increase in number of beta cells in the islets of Langerhans in rats. Kobernick and More (1950) and Franckson at 21. (1953) found hydropic degeneration of the islets of rabbit and rat after long-term treatment with cortisone. Ingle‘gg‘gl. (l9h5) reported that normal rats made diabetic by either cortisone or hydrocortisone were highly resistant to insulin. Adrenal steroid diabetes with insulin resistance was described by Sprague (1950) in a patient with Cushing'e syndrome who excreted large amounts of hydro- cortisone in the urine. Franckson at 21. (1953) found that cortisone administration was followed by a transitory diabetes in rats, characterized by hyperglycemia, lessened glucose tolerance and.marked resistance to insulin. They<3oncluded that steroid diabetes was due to reduced glucose utilization because of inhibition of insulin activity. In confirmation of these results Boutwell and Chiang (195h) reported that large doses of cortisone depressed oxidation of glucose in the mouse. Diabetes and.Insulin Effects of insulin on carbqudrate,_protein and fat metabolism The primary manifestation of insulin action 12.X$12 is a lowering of blood sugar level. This may be elicited by decreasing the production of blood sugar by the liver or by increasing the utilization of glucose in the organs and tissues. The energy provided by the reactions of inter- mediary metabolism is used by the cell for the performance of work, including growth and reproduction, as well as mechan- ical work such as muscular contraction. The overall process involves the oxidation of foodstuffs to carbon dioxide and water. The energy so generated is not released all at once as it is when sugar is burned in air. Instead, the original assimilated foodstuff molecules undergo a series of inter- mediate reactions. In each step, energy is absorbed or liberated by the synthesis or cleavage of the chemical bonds present in the intermediate compounds. Insulin is the major hormone in the body that is able to accelerate the removal of glucose from the blood, as well as its trans- formation and ultimate utilization by the tissues. Since skeletal muscles are the largest organs concerned with glucose utilization, it is the effect of insulin on this tissue that has been most studied although its effects on glucose metabo- lism are concerned with other organs, particularly the liver. Long (l95h) stated that insulin either directly or /u 10 indirectly accelerates the rate of glucose utilization by three major metabolic pathways: (1) polymerization of glucose to glycogen both in.muscles and liver; (2) conversion to fatty acids both in liver and in adipose tissues; and (3) an ultimate increase in the preportion of glucose or glycogen that is oxidized to carbon dioxide and water. Stédie, Haugaard and Marsh (1952) studied the isolated rat diaphragm and found that the effect of insulin increased with increasing glucose concentration. Bouckaert and de ‘Duve (l9u7) measured quantitatively the amount of glucose which disappeared in the liver and in the peripheral tissues under the action of insulin. By comparing normal and hepa- tectomized animals with respect to the amount of glucose needed to maintain a constant level of blood sugar, they found that liver accounted for a large fraction of total glucose utilization. They concluded that insulin promoted the net uptake of glucose by the liver, since hepatectomy greatly diminished the amount ef glucose necessary to maintain the blood sugar level after a large dose of insulin. Bouckaert 35 51. (1911.7) and Wick _e_§_ 51;. (1951) worked on intact and eviscerated animals and concluded that the primary physio- logical effect of insulin in lowering blood sugar was its increase in utilization of glucose in the organs and tissues of the body,_and in decreasing the net production of glucose by the liver. In the absence of insulin the diabetic organism.excretes 11 abnormally large amounts of nitrogen in the urine (Luck 33 gl_. 1933; Duncan _e_t 31. 1914.2; Macleod, 1926). This indicates that insulin must act to inhibit protein catabolism at some point. Bach and Holmes (1937). using liver slices 33 _v_i_t_r_o_, showed that insulin inhibited the deaminationof amino acids. This was accompanied by a decreased rate of appearance of carbohydrate, leading to the conclusion that insulin inhibited gluconeogenesis from amino acids and therefore from protein. This nitrogenous sparing effect of insulin was further demon- strated by Gaeblcr and co-workers (1914.5) who found that whereas extracts of the anterior pituitary administered to normal animals resulted in nitrogen retention, the same treatment in diabetic animals caused an increased nitrogen excretion. Lotspeich (1915.9) suggested that insulin promoted protein synthesis 3.3 219. This view was based on results which indicated that insulin accelerated the disappearance of amino acids from the blood stream in about the same pro- portion as these amino acids occurred in muscle protein. More recently Best (1952) showed that it was possible to induce the hypOphysectomized rat to grow by treatment with insulin when food intake was not controlled. He concluded that insulin enhanced the appetite and accelerated protein anabolism. There are many indications that insulin influences the metabolism of fat. Stetton and Boxer (19141;) demonstrated that one of the main defects in diabetes was an inability to 12 synthesize fat. Liver slices from diabetic rats were found to have a greatly diminished ability to synthesize fat from acetate (Brady'ggflgl. 1951) or glucose (Chernick 23 2;. 1950). On the other hand, Brady'gtigl. (1951) found that insulin accelerated the incorporation of Clublabeled acetate into long-chain fatty acids. Control of insulin secretion Since insulin secretion is stimulated by an increase in.blood glucose, any factor which serves to increase blood sugar will increase the function of the pancreas. Anderson, Lindner and Sutton (19h?) studied.the insulin content of perfusate coming from the isolated pancreas, using the hypo- physectomized, adrenal-demedullated, alloxan-diabetic rat. They reported an increase in the output of insulin by the isolated pancreas when the glucose concentration of the para fusing fluid was elevated. Soskin and Allweiss (l93h) found that it required a constant injection of insulin to maintain a normal blood sugar level in the depancreatized dog. Two hormones which are concerned indirectly with the secretion of insulin are growth hormone from the anterior hypOphysis and glucagon from the pancreas. The major effect of growth hormone appears to be a suppression of carbohydrate utilization by the peripheral tissues, particularly by skeletal muscles (Long, 195h). Anderson and.Long (19h?) also demonstrated that growth hormone inhibited insulin secretion 13 by the isolated perfused pancreas. Krahl and Park (l9h8) reported that growth hormone injections depressed the glucose uptake of the rat diaphragm. It was concluded that growth hormone inhibited the utilization of glucose and diminished. the response to insulin. In the year following the isolation of an hypoglycemic factor from the pancreas by Banting, Best and Collip (1922), Kimball and.Murlin (1923) reported a transient hyperglycemia following the administration of crude extract of pancreas containing insulin, and named the hyperglycemic factor or factors which they detected "glucagon". Sinn and Behrens (1953) reported that glucagon is a protein and can be crystallized from pancreas. Under continuous intravenous infusién of glucagon accompanied by insulin in amounts which alone would produce hypoglycemia, glucagon.was found to be capable of preventing hypoglycemia and even of maintaining an hyperglycemia for at least six hours (de Duve, Hers, Bouckaert, l9u6; Weisberg, Caren, Huddlestun and Levine, l9h9; Tyberghein, 1952, 1953; Myers 23 2l° 1953). Bernstein, Reid and Goring (1951) administered growth hormone to rats and cats and injected portal blood from these treated animals into adrenalectomized~hyp0physectomized, alloxan-diabetic ‘rats. This procedure elicited hyperglycemia in the test animals, but not when blood from a peripheral vein was injected. Support of their observations was provided by Foa (‘1 11L and cedworkers (1953) who reported that following injection of purified growth hormone in donor dogs, an hyperglycemic factor appeared in blood from the pancreas but not from peripheral blood. It was concluded that growth hormone administration caused the liberation from the pancreas of a hyperglycemic factor, presumably "glucagon", which was rapidly destroyed in normal blood. Relation of insulin and diabetes to B-vitamins The problem of maintaining preper nutrition in controlled diabetes appears to bees quantitative relation between the amount_of insulin and the amount of carbohydrate in the diet which can be utilized. 'With decreased utilization of carbo- hydrate in insulin-deficient individuals, there apparently is a decreased need for the accessory factors associated with carbohydrate metabolism. Thiamin, niacin, and perhaps pantethenic acid act as co-enzymes in carbohydrate oxidation systems. According to Samuels (l9h8), the need for these vitamins is reduced in the diabetic just as it is in the normal animal on a high fat, low carbohydrate diet. 0n administration of insulin the intake of B-vitamins becomes high, since the tissue concentrations have to be restored as well as provide for the increased utilization of carbo- hydrate. Not only is the need for B-vitamins dependent upon the available insulin, but the effectiveness of insulin appears to be influenced by any deficiency of these factors. Martin 15 (1937) found that depancreatized dogs on a vitamin B-deficient diet became resistant to insulin. Elsom, Lukens, Montgomery and Jones (l9h0) reported a progressive decrease in the response to insulin as a deficiency of the B-complex was produced experimentally in a woman. 0n feeding riboflavin and thiamin, this subject became abnormally sensitive to insulin. Biskind (19u5) reported that vitamin therapy was effective in decreasing the hormonerequirement in insulin- rcsistant diabetes. Best 23.2l' (1939) found that in thiamin deficiency reduction of insulin production occurred. Apparently this was due to the inanition rather than to thiamin lack £22 53, since animals limited in food intake but receiving ample thiamin showed a similar drOp in insulin content. Since there is good evidence that vitamin 312 is necessary for normal carbohydrate metabolism (Chow :3 51. 1952, 1953, 195h). there is a possibility that insulin may increase the need for this vitamin. Evidence to support this view will be presented in the experimental data. Vitamin B12 Effects on protein.metaboligg Cary and Hartman (19113-1914.?) showed that an "animal protein factor" was present in natural materials such as whole:milk, cheese and liver extract but absent in yeast and vegetable foods. They also noted that extraction with hot alcohol removed this factor from commercial casein and 16 that the deficiency could be made more acute in rats by adding hot alcohol-extracted casein to a soybean.mea1 diet. They claimed that an "animal protein factor" was needed for the mmtabolism.of protein at some stage, since a deficiency of this facterwwas accentuated by raising protein levels in the diet. They found it possible to concentrate this factor from liver extracts. 0tt, Rickes and Wood (l9h8) reported that crystalline vitamin B12 had "animal protein factor" activity in chicks on all-vegetable diets, and subsequently it was believed that these two factors were the same. How- ever, there is some doubt that all ”animal protein factors“ contain only vitamin.Biz. Hsu and Combs (1952) claimed that a vitamin.B12 deficiency in chicks increased the blood levels of non-protein nitrogen, amino nitrogen, urea nitrogen, creatinine, and glucose as compared to chicks receiving crystalline vitamin_812. The level of uric acid was not consistently affected. It was suggested that vitaminB12 is involved in nitrogen.metabolism in the chick. Cheng and Thomas (1952) attempted to deter- ‘mine whether vitamin.BlZ played a part in utilization of protein.for the growth of animal tissues. Under rather rigid conditions of vitamin.Blz depletion, it was demonstrated that vitamin.812 injections increased the utilization of protein as judged by its capacity to increase nitrogen retention in rats. The evidence indicated that vitamin.Blz aided in the conversion of the amino acid homocystine to methionine. In i, 17 the case of soybean protein, which is low in methionine, vitamin.B12 helped the animals more completely to utilize this food. Charkey 35 El. (1953) investigated the possibility that vitamin 812 may enhance utilization of amino acids in chicks. They found that both vitamin B12 and ”animal protein factor” promoted growth and increasedblood levels of argi-l nine and.methionine, but had no effect on blood tryptOphane, lysine and histidine. Effects of carbohydrate and fat metabolism Animal responses to several of the B-vitamins can be altered by changing the dietary compositions with regard to the three major foodstuffs: fat, carbohydrate and protein. Information gained in this type of study has been of prime importance in elucidating the mechanism of action of these vitamins. It was therefore considered worthwhile by Boss- hardt (1950) to apply this method of nutritional investigation to vitamin.B12. Variations in dietary composition with regard to fat, carbohydrate and protein were shown to have an influence on vitamin B12 needs in the growing mouse. A decrease in the fat level of the diet with a corresponding increase in carbohydrate intensifiedthe growth retardation due to a deficiency of vitamin.Bla. This growth retardation was partially corrected by the feeding of fat or administra- tion of vitamin 812, and was completely overcome by adminis- tration of both fat and vitamin 312. 18 Chow at El‘ (1951, 1952) reported that when rats re- ceiving supplementary vitamin 312 by injection were pair-fed to controls, no growth stimulation followed administration of vitamin.BlZ; but if the controls were force-fed to match the intakes of the supplemented animals eating 39 libitum, the former animals gained.much more weight during the experimental period. However, under neither condition was any differb ence in nitrogen balance detectable. They concluded that vitamin 312 was without effect on the retention of nitrogen over a broad range of nitrogen and caloric intakes. By analyzing carcass composition they showed that vitamin 812- deficient weanling rats had a high water and low fat content. Under the influence of vitamin.312, these values returned to normal levels quite characteristic of those recorded for healthy animals on good stock diets. But no effect was seen on the preportion of protein in the carcass. They claimed that vitamin.812 was, in some as yet unknown.manner, involved in transformation of carbohydrate to fat, and seemed to play no direct part in protein metabolism. They also concluded that the role of vitamin.312 appeared to be regulatory, since animals receiving abundant quantities did not become obese but tended to revert in carcass composition to generally accepted normal values. In confirmation of the above, Arnrich ‘gg‘gl. (1952) reported that when vitamin.Blz or aureomycin or both were added to a purified diet and fed to dogs, nitrogen metabolism was unchanged but the dogs gained more weight. I". 19 These dogs had greater carcass fat and more fat-rich adipose tissue. They concluded that the increased weight gains were due to increased far deposition. Ling and Chow (19Sh) studied the effects of vitamin.B12 on carbohydrate and far metabolism by glucose tolerance tests and by estimation of the phospholipid content of blood and tissues. It was found that a vitamin B12 deficiency prevented normal carbohydrate utilization, as indicated by an abnormally high blood glucose level following intravenous injections of glucose. The blood glucose levels returned to normal at a much slower rate than in vitamin-adequate animals. They concluded that vitamin.B -defieient rats 12 lost part of their ability to transform carbohydrate to fat. Their data showed further that abnormally small amounts of phospholipids were found in tissues of vitamin BIZ-deficient rats and in blood of patients with pernicious anemia in relapse. Administration of vitamin.812 to the above resulted in.marked increases in the phospholipid content of the blood and tissues. Vitamin.Blz-deficient rate also showed a marked dimi- nution in levels of soluble sulfhydryl compounds in blood, which rose with the administration of this vitamin.(Ling and Chow, l95h). It was suggested that this was due to the change in concentration of glutathione. This was corrobo- rated by Register (l9Sh) who also found a marked decrease in the levels of liver and blood sulfhydryl groups when rats 20 were fed a soybean ration deficient in vitamin B12. It was agreed that this was due primarily to a glutathione deficiency. Administration of glutathione or vitamin.B12 lowered the blood glucose levels of rats with hyperglycemia induced by a high carbohydrate, low fat diet or by glucose injections. Interactions between vitamin B12 and adrenal cortical hormones Heites (1951, 1952a, 1952b) reported that cortisone depressed body, hair and thymus growth in growing rats fed a soybean meal or a semi-synthetic diet. These effects were completely or partially prevented by incorporating 200 mcg of vitamin 812 per kilogram of diet or 0.005 percent aureo- mycin. It was also found that vitamin.Blz was more effective than aureomycin, and that the combination of the two sub- stances was more effective than either alone. The favorable action of the vitamin and antibiotic were accompanied by an increase in food consumption and greater efficiency in con- verting food into body weight gains, although the latter effect was reduced from that found in non-cortisone treated animals. It was concluded that large doses of cortisone increased requirements for vitamin B12 in the young rat. Rupp and Paschkis (1951) reported that when vitamin 312 was administered to force-fed hyperthyroid rats on a constant food intake, the weight loss was identical with that of hyperthyroid animals not receiving vitamin.312. However, vitaminB12 decreased the loss of nitrogen resulting from 21 the catabolic action of thyroid. They also reported (1953) that vitamin.Blz failed to influence cortisone-induced protein catabolism in rats when food intake was limited. Ershoff (1951, 1953) reported that liver residue contained a factor other than vitamin B12 which counteracted the growth-retarding effect of thyroid powder or cortisone fed to young rats. ‘Wahlstrom and Johnson (1951) reported that large doses of cortisone injected into baby pigs on a vitamin 312' deficient diet increased the urinary excretion of vitamin B12, as determined by microbiological assay. Chow‘gt‘gl. (1953) performed the same type of experiment in rats. They reported that vitamin.Blz activity in Zh-hour urine specimens of the cortisone-treated animals was twice that of control animals, as measured either by microbiological assay or by concenp tration of radioactive vitamin.BlZ. The tissue analysis demonstrated that in each instance the organs of the corti- sone-treated animals retained less radioactive vitamin.B 12 than the controls. Interactions between vitamin.Bla and diabetes Harte, Chow and Barrows (1953) observed a marked reten- tion of radioactivity in the pancreas following injection of radioactive vitamin.Blz into rats, and suggested a possible role for vitamin.Big in pancreatic diseases which might be manifested by an abnormal excretion of this vitamin. Further 22 studies by Chow at 3;. (1953) indicated a possible corre- lation between the urinary excretion of administered vitamin 312 and diabetic retinopathy (sometimes seen in advanced stages of diabetes). The diabetics with retinOpathy excreted.much more radioactive vitamin.B12 in the urine than diabetics without retinOpathy. Since the dietary history of these patients was not given, it is difficult for the writer to determine to what extent the two forms of diabetes influenced vitamin B12 retention. EXPERIMENTAL Experiment I.- Prevention by Vitamin B12 of Protein Catabolic Action of Cortisone 08. Since it had been demonstrated that large doses of vitamin.312 could partially counteract certain catabolic effects of cortisone in young rats (Meites, 1951, 1952, 1953), it was of interest to determine whether this was :mediated to any extent by preventing excessive protein breakdown. The effect of cortisone on urinary nitrogen losses was determined on rats fed diet deficient or ex- cessive in vitamin B12. He thods . Forty Garworth male weanling rats were fed a stock soybean.meal diet deficient in vitamin.312 for a preliminary period of 30 days. The composition of the diet is described_ in the appendix. ‘Water and food were available at all times. The rats were housed in metal cages, 10 to a cage, at a mean room temperature of 76° :_1° F. Artificial light was avail- able daily from 7:30 AM to 9:30 PM. Twenty-four hour urine speeimens were collected every ten days and total urinary nitrogen was determined by a standard.micro-Kjeldahl pro- cedure (Hawk'gg‘gl. 1951), described in the appendix. After the 30-day depletion period, the rats were divided into four uniform groups and were treated as follows for an additional 30 days: Group 1. No vitamin.BlZ 2. 200 mcg. vitamin.Bla/kilogram of diet 3. Cortisone u. Cortisone + vitamin.312 Groups 3 and h received 1 mg. of cortisone acetate (from here on referred to as cortisone) daily by subcutan- eous injection for the first 10 days, 2 mg. daily for the second 10 days and h.mg. daily for the third 10 days. A total of 200 mcg. of crystalline vitamin.Blz was mixed with one kilogram of the‘stock diet. This amount of the vitamin represents approximately ten times the normal requirement (Stokstad‘gg‘gl. l9h9; Zucker gt‘gl. 1950). Body weight and food consumption were measured every two days. Urinary nitrogen was determined every five days. For urine collection, five rats were placed in a single metabolism cage in which water was available at all times and food was present in non-scatter’metal feeders. The animals were kept in the cage for 2h.hours and the urine was collected in flasks containing one gram.of citric acid as a preservative. All the specimens were placed in a refrigerator at the end of 2h.hours. In.this and all subsequent experiments the standard 25 error of the mean was determined by the following formula: 2 3m: 9 n(n-l) Results It can be seen in Fig. l thatthe to rats averaged 1&5 grams each at the end of the 30-day depletion period. The rats which received vitamin.BlZ (Group 2) reached an average body weight of 288.2 grams each as compared to the vitamin-deficient rats in Group 1 which averaged only 205.8 grams each. The rats in Group 3 which were treated with 1 mg. of cortisone daily for 10 days without vitamin.Blz, gained only 3 grams in body weight; when 2 mg. of cortisone were injected daily, growth was completely suppressed: and h.mg. of cortisone daily resulted in loss of body weight. ‘When vitamin B12 was added to the ration (Group h), cortisone did not prevent growth although the body weight did not reach the level of the rats given vitamin.B12 alone (Group 2). The rats fed vitamin.BlZ (Groups 2 and h) had the highest food intake and efficiency of utilization for body growth. The total food intake in Group 2 was 730 grams while in the cortisone-treated vitamin Bla-deficient rats (Group 3), the total food intake was only h28.8 grams. Food intake was increased in Group h when vitamin B12 was added to the diet, but the efficiency of food utilization was well below the two groups which were not treated with cortisone (Groups 1 and 2)e WWOQTI/ MA 729 I BwflFIC/ENT . 11 BL- 200 mm fl TISGNE .- U {(2 f CORT-(SW [50 (WW/WY ”Imam (”WWW/2411,.)Ioo Y z. 5 DAILY F000 2" M’ TAKE (yrs/mt ) IO 230 ...... ' 24o BODY WEIGHT (5/1215) 200 ’60 I. e .. .Wo ..e e... e. IMO COR TISONE 2M9 CORTISONf 4 ’77? CORT/50M DAILY 90A!” DAILY ’20 6 l2 /3 at 30 25 DAYS Fig.1 Effects of vitamin B|2 and Cortisone on urinany nitrogen and food intake. 27 The average daily excretion of urinary nitrogen per 100 grams of body weight (Table I) appeared to be greater in the vitamin.Bla-deficient animals (Group 1) than in the vitamin.BlZ-adequate animals (Group 2). However, there was no increase in urinary nitrogen after vitamin B12 administra- tion to Group 2. Cortisone progressively increased the urinary nitrogen excretion in the vitamin.BlZ-deficient animals (Group 3). After the third 10-day period of treat- ment with cortisone, the nitrogen excretion per 100 grams body weight per day was almost three times as high as in the pro-treatment period. Vitamin.B12 largely prevented this increase of urinary nitrogen excretion induced by cortisone (Group h). Conclusion; n VitaminB12 largely prevented these protein catabolic actions of cortisone under 32 libitum feeding. Therefore it may be concluded that vitamin.B12 can prevent increased urinary nitrogen, probably by increasing appetite and there- by enhancing the availability of carbohydrate to the organism. This is in agreement with the work of Longlgt‘gl. (l9h0) and Engel (19h9) who showed that administration of large amounts of carbohydrate together with adrenal cortical extract to rats prevented the protein catabolic effects of the latter. In addition, large doses of cortisone markedly inhibited the ability of vitamin B12 to transform food into body weight III I. |.|.rl.ll.llll 28 uad>hopda hsouoa as some» moonwam n no ewsaobflt Tmmu foam m. m+ do“ m4“ 04m.“ 0.3“ «am 55»; mm.mma ao.:oa m. m wo.oo am.eo ma.ama a:m.o:a om.aa m.mmm + .comassoo : 2w m+ m. mm... m3? to“ 5. mm“ Goa” mm. mm mm. o. Nmm mo. emm mm.oom om. wed * m.oma a- m.m~: econuusoo n .qansd It .wa m In! .ms H onomapaoo m.~H ISM 9m“ mam? m.o+ 9.3“ 4.3“ ms Réo 2.5 amino +6.2. 92: 5A.: $8.2: A3m Q62. m 35»; m o.mm.+. tow.“ 04mm he.“ RAH down 9mm.“ NH mmJS méwa 3.03 3.43 mm.mom 2.53 $3.12 +86 0.3: m 553» on H '1 (1| .& [ o.” m oa m 0H m escapees» .amfimmon .5 when ad moaned poosneeaa eaomom .am hem Hence noesnseaa macaw .93 smegma... Hoop. .3 8S aas £5 33.: 38 .22 chIHll H MHdaflH Q00m_Qz¢ zmcomBHz Hm ho macabhm H wands I! D l I {'0 0' ’ | ' - II I “I III .I I I I e! | 0.. I ' i I 8 .l I - ' .f i . I I '( lll'- - "J .l. V l l l O .l O r '. 0 ‘0 .1. u ' ' .l ’(I I IV .' I..." I'll!» .- (III-0" Ilr‘Il'll gains. A partial explanation for this phenomenon will be provided in subsequent experiments. 29 30 Experiment II. Prevention by Vitamin.812 of Protein Catabolic Action of Cortisone ose In this experiment an attempt was made to confirm the results of Experiment I and in addition to determine whether vitamin.Blg could prevent the increase in urinary nitrogen losses produced by injecting large doses of cortisone under limited food intake, as it did in the rats which were fed 3.9 libitum. Methods Fifty rats were used in this experiment. The proced- ure*was essentially the same as in Experiment I except for ,_one additional group of rats. After being on the vitamin BIZ-deficient diet for 20 days, the animals were divided into five uniform groups and were treated as follows for 30 days: ’ ' Group 1. No vitamin.BlZ 2. Vitamin B12 -- 200 mcg,/kilogram of diet 3. Cortisone h. Cortisone + Vitamin B12 \ 5. Cortisone + Vitamin 312, but pair-fed to Group 3 Groups 3, h and 5 were treated with 1 mg. of cortisone daily for the first 10 days; 2 mg. daily for the second 10 days and h.mg. daily for the third 10 days. Urinary nitrogen was measured every week during the depletion period and every 31 five days during the treatment period. Body weight and food consumption were measured every two days. Results The results are Shown in Fig. 2. The first four groups followed essentially the same pattern as in the first experi- ment. The rats averaged 58.3 grams at the beginning of the depletion period and 9h.9 grams at the end of the depletion period. Group 1, which continued to receive no vitamin.B12 showed a final avergge body weight gain of h5 grams. Group 2 whose ration was supplemented with 200 mcg. of vitamin.B12 per kilogram of diet, showed an average weight gain of 97.5 grams per rat. Cortisone (Group 3) caused a marked suppression of body growth. ‘When vitamin.Blz was added to the ration of the cortisone-injected rats (Group h) it partially pre- vented the growth inhibition. These rats gained an average of 50 grams less than.the vitanin.Blz-supplemented rats in Group 2. 'When food intake was limited in Group 5 to that consumed by Group 3, vitamin.Blz did not at all prevent the growth inhibition induced by cortisone. Food intake and efficiency of utilization for body growth were greatest in the rats treated only with vitamin 312 (Group 2) and least in rats given cortisone without vitamin.Blg (Group 3). Food intake was increased when vitamin.Blz was given to the cortisone-treated rats (Group h) but efficiency of food utilization was well below that of either Group 1 or 2. r3 I 312 [IFICIENT [Bn'axMQAy 25,. 1 009713075 1 COIPTISOVE * BIZ IRIMRY I CURTISMP 8.2 (mm FED) P'fieweeegm.~.ml 42/19 cmnmw r .,.—-’I ’0’” O D’. /—.———I ----- I ----‘---.‘l "'-. Museum... ee .21 5 ID A; vitamin Br: and food Fig-Z Effects of nitrogen and 20 25 30 cartisanc on udnar, 33 Table II shows that the average daily excretion of urinary nitrogen per 100 grams of body weight during the vitamin.Blz-deficient period was not altered after the vitamin was added to the diet in Group 2. Nitrogen values in Group 1 were slightly higher than those of Group 2, although these differences were not as marked as in the first experi- ment. All levels of cortisone significantly increased urinary nitrogen losses in the vitamin.Blz-deficient rats and the largest dose of hormone doubled the initial nitrogen values (Group 3). The addition of vitamin 312 to the ration prevented any increase in urinary nitrogen excretion by cortisone under ad libitum feeding (Group h) but was com- pletely ineffective when food intake was limited (Group 5). (gonclusiong . Vitamin.B12 prevented the protein catabolic actions of cortisone under‘gg libitum feeding, but was ineffective when food intake was reduced to that of rats given cortisone without vitamin.Blz. As explained in the first experiment, this action of vitamin B12 can be attributed to the greater amount of carbohydrate made available as a result of the increased food intake. 34 nasphepca hunts. as noxsp someway m we emshe>4* l I I I I I I no.“ name ~:.o+ m.oa+ oo.e+ ms.HH+ oa.o+ mH.o+ oo.m+ m m assess» o>.o- ss.mmm om.aom sm.moa o:.m:a m.m:a *:H.QHH u- o.amm + oceanssoo m mean mod“ mad“ amen. 1:. u... 5.5.." RAJ“ mam 55s; mo.~ma Hm.::a :m.::a 00.3mm :s. a 20.5mm sso.oma me.o o.~mm + ososassoo : so.mu m 9:“ oa.mmu mm.~nu as.onu m:.m¢u_ em.:u m~.e- m .Hmm mm.moa oo.m~a m:.oea mo.mma szm.an u- o.amm .sousssoo m .mfi: .wE N .mE H enomwuaoo om.¢u .sm.mu c:.nu ms.nu .so.nu om.mu m:.mu mm.oHH ma.~m~ ee.oma mm.mma mm.HmH H~.oma .Ho.oma Ha.m o.emm mam.nasu»«> N omen 2.5“ 8.9... Twas Séfl $3” ~m.m u m oH.emH so.mma Ho.ssfl sw.o:H me.H:H Hm.cma aom.~ma ~:.~ e.emm Hm assess» an a v1) .sm 3 m 3 m 3 m s a .9: mean. .sw wwwo ea poahea.pacausoha udwnwmmma .Ewamwm Hench anefivecae ozone .uss sm\sswfios_~wwn .sw.ooa\z .ws...p< osapsn soon ..>4 mm so assumes HH memes . 35 Experiment III. Effects of Cortisone, Vitamin.Blz, Insulin and Alloxan-diabetes on Blood Glucose and Urinary Glucose and Nitrogen ose This experiment was designed to provide information on the following: 1. Through what means does cortisone partially prevent vitamin.Blz from exerting its full effects on efficiency _ of food utilization and body growth? 2. Does insulin require vitamin.B12 for its action? 3. Does vitamin B12 require the presence of insulin for its action on food intake and body growth? Methods Sixty weanling male rats were fed the vitamin.Blz- deficient stock diet for 60 days, when their body weights reached an average of approximately 160.0 grams each. They were then divided into six groups of 10 each and were treated as follows for 30 days: Group 1. No vitamin.Blzp/’ 2. Vitamin.B12 -- 200 mcg./kilogram of diet V“ 3. Alloxan -- 17.5 mg./100 grams h. Alloxan + Vitamin.Blz 5. Cortisone -- h.mg./day/rat 6. Cortisone + Vitamin.B12 Food intake and body weight were measured every two days. In.accordance with standard procedure, all rats which were to 36 receive alloxan were starved for 72 hours. In order to maintain the same body weight in all rats, the rats which were not treatedvvith alloxan were also starved for 72 hours. At the end of this period, Groups 3 and h were injected with alloxan monohydrate. At the end of five days, hyperglycemia was established. Blood glucose was determined on the 5th, 10th, 20th and 30th days by the micro-method of Folin and.Ma1mros (Hawk 'gt'gl. 1951). This procedure was used because less blood was required and lower blood levels could be determined than with the Hartman, Shaeffer and Somogyi micro-method. After each initial collection of blood, 0.5 units of insulin were injected into the rats of Groups 1 and 2 and 2.0 units were injected into the rats of the other four groups. Blood samples were collected four hours later.” The insulin dosage was less in Groups 1 and 2, because in preliminary experiments, it was found that 2.0 units of insulin injected into vitamin.BlZ-deficient and -adequate control rats produced a severe and often fatal hypoglycemia. ‘Urinary glucose was determined by the method of Hawkins and Van Slyke (Hawk et a1. 1951) and urinary nitrogen by the micro-Kjeldahl procedure previously described. The rats were starved 12 hours prior to insulin injections and for four hours subsequently. *Sugar determinations were made four hours after insulin injection because it was found that blood glucose fell to the lowest point at this time. See appendix for experiment in which this was determined. 37 Results 1. Effects on body weight and food intake (Table III and Fig. 3). The rats in Group 1 which were fed the vitamin B12- deficient diet gained an average of h3 grams each as compared to the vitamin-fed rats in Group 2 which gained an average of 88 grams each. Alloxanslowed body growth in the vitamin- deficient rats (Group 3). and the average total gain was only about 20 grams. In the vitamin.BlZ-adequate animals given alloxan (Group h), the average total gain was approxi- mately 77 grams. Cortisone .decreased body growth more in the vitamin.Blz-deficient animals (Group 5) than in the vitamin.Blz-adequate rats (Group 6). The latter is in agreement with the results of the previous experiments. 'Food intake was reduced in all groups which did not receive vitamin.Blz, and was increased when the vitamin was added to the diet. It will be noted that in the alloxanp diabetic rats fed vitamin.Blz (Group h), food intake, body weight and efficiency of food utilization were about the same as in the normal rats fed vitamin.B12 (Group 2). 'As in the two previous experiments, it can be seen that cortisone reduced the ability of vitamin B 2 to convert food into body 1 weight gains (Group 6). 2. Effects on blood and urinaryglucose and urinary nitrogen (Table IV and.Figs. 3 and h). The average blood glucose in the vitamin-deficient rats 38 TABLE III EFFECTS OF VITAMIN B12, ALLOXAN, AND CORTIS NE 0N BODY WEIGHT AND FOOD INTAKE Group Treatment Body weight Food intake smo sm- Initial Final Total Efficiency 1 No vitamin 312 176.7 219.8 302.6 7.02 2 Vitamin B12 169e8 25701 “.0208 (1.60 200 mcg./kilo- gram 3 Alloxan 170.7 191.0 285.2 lh.0h h Alloxan + 169.6 2h6.1 398.2 5.20 Vitamin B 12 5 Cortisone 170.8 lh8.3 257.2 -- u mso/day 6 Cortisone + 169.9 215.3 3&5.3 7.60 Vitamin B 12 --VIT. Bu DEFICIENT —._ ALLOXM +VIT. Bu ----VIT. Bu M/ K6 —- CORTISONE “WI/DAY —ALLOXAN l7.5f‘6/l006fl m»- CORTISOK +VIT. Bu URINARY NITROGEN 4- fl6/l00 6H BODY VII/Z4 HRS. Z” w .eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeOO 00“ ““m"°“°. " a=: :" .e e0. 2’ ’00 .. ~::::;:‘0-0 ---------------------------- nom-.-._._._.-.-._ Q d 15 AVERAGEFOODINTAKE men ‘--"""'::.:_.. ,0 ‘~‘-—“ 4—. 8 . I . .\‘:—./\——\ MM sow WEIGHT I, IN 6" .I 0" I 0' 0 w b .v" j 5" 0’ 0“ . ,’ "' .’.’ §e§.-.-.—.-.’ 0' 0’ "’.’ ' e’ .-.-e-e-e-'-°-.-O-e’°.’ I ’ "' ’ e O O O" ” e0...” e". ." «.00'“. ’I. e’.’ 0 . 200 x. a.» » "o’." ..e° V g .0... * e0 ’ 14"" " ' 0" e 06’ ... ... .I .e’ l ;_— [0 DAYS 20 30 Fig. 3 Effects of alloxan , cortisone and vitamin B |2 on body weight, food intake and urinary nitrogen. 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The former averaged approximately 8h.5;: 5.0 mg. percent and the latter about 115;: h.0 mg. percent. Alloxan did not induce as much hyperglycemia and glucosuria in the vitamin.BlZ-deficient rats (Group 3) as in the vitamin.Blz-adequate rats (Group h). The blood glucose in the former increased from 82.8 to 169.h mg. percent on the 5th day, to 183.5 mg. percent on the 10th day, to 188.9 mg. percent on the 20th day and to 188.3 mg. percent on the 30th day. In the latter rats, the blood glucose reached 257.5 mg. percent on the 5th day, 273.9 mg. percent on the 10th day, 277.2 mg. percent on the 20th day and 287.5 mg. percent on the 30th day. Cortisone increased the blood glucose level to a high of 380.2 mg. percent in the vitamin.Blz-deficient rats (Group 5), while in the vitamin.BlZ-adequate rats cortisone increased blood glucose to only 213.9 mg. percent at the end of 30 days of treatment. The urinary glucose values followed those in the blood. Group 5 had higher blood glucose level and higher urinary glucose level while Group 6 had lower'blood glucose level and less glucose in the urine. In general, the nitrogen excretion pattern in the urine followed the results obtained in the previous eXperi- ments. It should be noted that in the vitamin BIZ-deficient rats (Group 5) cortisone increased blood glucose and urinary glucose and nitrogen to a greater extent than in the vitamin- #3 adequate rats (Group 6). Alloxan increased urinary nitrogen excretion about the same under both dietary treatments. 3. Effects of insulin (Table V and Fig. h). Comparisonsof Groups 1 and 2 after insulin injection show that the vitamin BIZ-deficient animals were less reactive to this hormone than the vitamin-adequate rats. Blood glucose fell only about 33 to 37 percent in the former as compared to S3 to Sh percent in the latter. In the alloxan diabetic rats insulin reduced blood glucose only one-half as much in the vitamin.Blz-deficient (Group 3) as in the vitamin-adequate rats (Group h); however, the percentage reduction was about the same in both groups. The vitamin.BlZ-deficient rats treated with cortisone (Group 5) showed the greatest resist- ance to insulin of any group, with only an average reduction in the three trials of about 2h percent in blood glucose, while the vitamin.Blz-adequate animals treated with cortisone (Group 6) showed an average reduction of about 66 percent. Conclusions 1. VitaminB12 increased body growth by increasing appetite and increasing efficiency of food utilization. Alloxan slowed body growth in the vitamin.B 2-dericient 1 rate, but not in the vitamin-adequate animals. This was accompanied by increased food intake and food utilization which was practically equal to that of the normal vitamin Biz-treated rats. This demonstrates that insulin is not "'Il. mm 0.30 N.:o o.mw cadence fleece . Ihumma IH.mma Ic.>:a eedmrwou .wm H.MHN.m~ o.mHN.m~ mom+mozm I. I. caaauna hound m 3+0 mam m.o+m.oam H.0mflh.mom o.m+m.~ma :.:+:.mw GHHSuaa enouom Nam casspa> + enouapaoo o N..Nm m.HN :.mm eueeaoou pneeaem . INHom Im.m> Ih.o> emeonoec .m: m.oaH0.:om m.maHm.m~N o.cHo.m0H I. I. cuasmca hophd e ea+m own m.ma+m.amm w.w+:.oem o.m+m.mmH o.~+a.mm assumed .eoeom edoaauhoo m m.do 1.30 c.mo eeeoheoc uneasem . Imnima Im.mwa Im.m~a eueeaoeo .wz w.HHHc.m0H m.HHH>.w® M.NHH:.HOH I. I. cHHSmca hound o Ha+m paw H.m+m.-m o.w+¢.m~m o.:+m.~mm :.:+e.mm quasmnfi .eoeom mam eaeuua> +_n¢soaa< : >.H~ m.m~ 3.00 cadence aeoao . I.udma m.wma IM.NNH euewaweu .wm ~.mu .mm m.:me.om m.:na.em I. I. assumed souo< m n+0 was m.e+o.mwa o.~+m.mma m.oa+:.oea H.m+w.mm ensues. osoeom deuoaad n N.mm N.mm m.mm oueeheoc uneasem Ic.mo IN.No Ic.oc eneehooc .mz m.mnm.mm e.NH~q:m a.mnb.mm I. I. cadence nouu< m.e+a.maa o.m+o.oHH o.:+e.maa H.m+e.mo m.m+o.om usages“ onoeom , mam_easaes> m :.:m o.mm m.om oncehoou uneeaem Ib.om IH.mN NN.HM oneehoou .wz :uenm.om :.mne.»m :.mum.mm I. I. assume“ nopc< a m+m.om m.:+a.mm e.m+m.sm o.:+~.:m p.:+s.:o em and .nocom m assess» an a .aae on name om .aue o” aaue m Heapsem anospaosa cease R .wa enoosdm vooam mam szsaH> nae mzomuamoo .zaxonus maH: azmza Human. #5 essential for the growth promoting action of vitamin B12. Cortisone retarded body growth and food intake in the vitamin BIZ-deficient rats to a.much greater extent than in the vitamin-adequate rate. It will be noted again that cortisone partially interfered with the action of vitamin 312 on food efficiency and body growth. 2. Vitamin.B12 maintained blood glucose levels somewhat higher than in the vitamin.Bla-deficient animals. Alloxan diabetes raised blood glucose to a much higher level in the vitamin BIZ-adequate than in the vitamin-deficient rate. This can be attributed to the ability of vitamin Biz to increase food intake. The urinary glucose was also higher in the vitamin-treated animals. It is of interest, however, that these alloxan-diabetic animals fedvitamin.B12 weighed more and used their food almost as efficiently for making gains in.weight as the normal vitamin.Blz-fed rate. This indicates that the ability to convert food into body weight is not seriously interfered with in the absence of insulin if a sufficient amount of vitamin.B is available. It will 12 be noted that in the absence of insulin, vitamin B did not 12 alter urinary nitrogen excretion. The greatest hyperglycemia and glucosuria was produced by cortisone in the vitamin B12- deficient rats, apparently by interfering with insulin action (Ingle at 21. 19h6) and by increasing gluconeogenesis. Vitamin.312 partially counteracted the catabolic action of cortisone by reducing gluconeOgenesis from protein. Thus h6 both urinary nitrogen and blood and urinary glucose were reduced by treatment with vitamin B12. This is believed to further explain how large doses of vitamin.BlZ can partially counteract the protein-catabolic effects of cortisone. 3. Insulin was generally less effective in reducing blood glucose in the vitamin.Blz-deficient groups than in the vitamin-adequate groups with possible exception in the alloxan-diabetic rats. This indicates that the vitamin is essential for the full and maximum effect of insulin to be manifested, and in this respect it is similar to some other B-vitamins (Samuels, l9h8). It is probable that the ability of insulin to favor the transformation of glucose into fat depends in part on the presence of vitamin 312‘ By far the greatest resistance to insulin was encountered in the corti- sone-treated rats on the vitamin BIZ-deficient diet (Group 5). This apparently was due to: 2) an interference by cortisone with insulin action, and is in agreement with similar obser- vations by Ingle.2t g}. (19h5) and b) to the decreased effectiveness of insulin in the absence of vitamin B12. u. Cortisone partially interfered with the favorable action of vitamin B12 on efficiency of food utilization and body growth, apparently by inducing hyperglycemia and glucos- uria and thereby leaving less carbohydrate available for conversion into body weight gains. The hyperglycemia and glucosuria are undoubtedly related in part to cortisone- induced insulin resistance. In normal rats fed vitamin 312, 1+7 there was no loss of sugar in the urine. It would have been interesting to see whether injections of insulin into cortisone-treated, vitamin.BlZ-fed rats would not have further prevented decreases in body growth. hB Experiments IV and V. Glucose Utilization in Normal, Alloxan-diabetic and Cortisone-treated Rats as Influenced by Vitamin B12 ose Ch°".25.5£0 (195h) demonstrated that glucose tolerance was lower in vitamin.BIZ-deficient than in vitamin.Blz- adequate rats. In these experiments, it was intended to see whether these results could be confirmed and also to deter- mine the effects of alloxan-diabetes and cortisone on glucose utilization in vitamin BIZ-deficient and «adequate rats. Methods . _ _ - > The 60 rats from Experiment III were used in this study. At tne end of 33 days of treatment they were starved for 12 hours, blood.samples were taken from each rat, and initial glucose values were determined. A total of 750 mg. of glucose in 5 ml. of physiological saline was injected intra- peritoneally into each rat. Blood samples were taken one and two hours later for glucose determinations. Twenty-four hours later a similar dose of glucose was injected into each rat and urine was collected for six hours for glucose assays. The preceding was repeated after an interval of six days. Results of Experiment IV The results are shown in Table VI and Fig. 5. Appar- ently the vitamin.BIZ-deficient rats (Group 1) had little ability to utilize the injected glucose, since their blood H) .‘nN 1fin ~ o.amH eucehona uueoaem I I I mam 5.939 mm; mm.em+-..$: mm.m~+mo.m.£. o.3+ma.£~ + .3on: m.NmH H.Hom oneoaocfi uceoacm l l I .BE OOH\.wE m.NH 3.0 o.mm+:o.o§ om.ma+mo.m£. 3.3363 5534 0.0H d.ow smashed“ uceeaem I. I I .mwmewea oom 30.0 mm.m+oa.ema maéxcoémm eo.m+o~.mma rm ease»; mono: ¢.mmo eneohocu accenem 36 msemumeem: 8.83933 34.393 Nam case»? .2 .93 {.5 ooa\.am Econ m 58m; 73E _ enoosam hhccdhb R .wa enoofiaw cooam paeapeeas Emma mozgdos MmOUDam zo mzomHBmoo n52 ZSSQQ .NHm 2528“; he meumhhm Hb Hands 61me raERMCE (1551 I ) I VII Ba DEFICIENT lIVlT.B.z sz/KG' I ALLOXAN I'LSMG/IOOGM NALLOXAN + VIT. 8.2 Y CORTISONE 4MG/ DAY YICORTISONE +VIT. BIZ I 80 I z ”OZ/RS Fig. 5 Effects of vitamin Ba , alloxan and corEiSOne on glucose tolerance test. 51 glucose reached levels almost three times as high as in the vitamin-adequate animals (Group 2). Also the former excreted about four times as much glucose in the urine as the latter. Both the vitamin BIZ-deficient and -adequate alloxan-diabetic rats (Groups 3 and h) showed the same elevated blood glucose levels, but the percentage increase was much greater in the vitamin-deficient rats. There was somewhat more glucose excreted in the urine of the latter rats. Cortisone ele- vated glucose to a much higher level in the vitamin B12- deficient rats (Group 5) than in the vitamin—adequate rats (Group 6), despite the fact that blood glucose was initially much higher in the former. There was also about twice as much glucose excreted by the vitamin Biz-deficient as by the vitamin-adequate rats treated with cortisone. Results of Egperiment V It can be seen in Table VII and Fig. 6 that the results are essentially the same as in Experiment IV. Conclusions These data confirm the report of Chow 22 El. (l95h) that a vitamin BIZ-deficiency interferes with the utilization of glucose by rats. This was also shown to be true in alloxan-diabetic and cortisone-treated rats. In general, these results are believed to suggest that vitamin B12 may increase glucose utilization independently of insulin. Large amounts of cortisone can partially interfere with 52 Comm :.:> ouaohoaa acoonom I I I mam 55»; 00.0 ma.ma+ms.omm Hm.0a+mo.amm mo.m+00.:0a + ocomappoo comm dooia ondohoaa pGoOQom i; mm.ma+m0.m00 3.3003300 0:.3H00.$m 2830.30 b.0o m.moa omwonona unconom I I I Nam 55»; 0m; fi.mm+mm.:0: 5.01.262. 3.3+0mé0m + 55H? momma o.oam oncogena unoohom 00.0 00.0mufi.03 3.3..”855 mmfiumméma fined... ~.o meow onuonoaa pcoohom 08.0 0m.mumo.0ma 0H.mnmm.mmm 34.3033 mam ease»; moomm >.omo oudohona pcoohom mma figures: gauge? sesammfim 2m can? on .93 {.5 00H\.am W33 w» .33 H H333 onooaam hhdauns R .wE ouoosam vooam vcosndoha Emma Moz mo maommmm HH> Hands -v" “- Gil/(50815 TOLERANCE (TEST 1) ALLOXAN + V! T. 8.2 — VIT.B.2 DEFICIENT --- ms. zooms mmCORTISONE 4fl6/DAY ALLOXAN mus/moan common: HI”. 3.. M Ian 2 HOURS vitamin 8.2, test, alloxan anal cortisone Fig. 6 Effects Of glucose tolerance on URINARY Gil/€0.51: Gfl/IMG/‘I BODY DWI/6‘ [IRS rIVlT. 3.13m mam HI". 3.. F— IVII 3.. zoom Icomsoue 4W [.5 , [Mm Him ICMTISONE +V|I8n r F 1.0 » _ _ r r 0.5 ' F— r 1 [i] I l! I n I [I] l I I l 61 UCOSE TflER/VICE TES TS mm. oer. [mow + ms... I INITIAL IIII.a..zoo./II6 II CURTISOIIE Ina/w DIST m um mus/m ICORHSONE+ VIT. 5.. mom I I - F ”I F ._ - 7" " I“ IEfi- I I I I I I I I Effects of vitamin Bsz , alloxan and glucose tolerance test. co rtisone on Sh this action of the vitamin. The actions of the pancreas and cortisone on glucose metabolism do not appear entirely independent of vitamin B12. Thus a vitamin B12 deficiency enhances the ability of cortisone to increase gluconeogenesis from protein and hampers insulin in increasing glucose utilization. On the other hand, an excess of vitamin B12 decreases the action of cortisone on gluconeogenesis and enhances insulin function. Since adrenal cortical action on carbohydrate formation is unopposed in diabetic animals, it is suggested that large doses of vitamin 812 may par- tially substitute for insulin in maintaining carbohydrate utilization and body growth. The latter is purely conjec- tural and may have to be demonstrated. SS Experiment VI. Effects of Cortisone on Distribution of Vitamin B12 in Blood, Liver and Urine Purpose Chow _3 El. (1951) determined the distribution of radio- active vitamin B12 in normal and vitamin Blz-deficient rats, and found that the former retained less of this vitamin than the latter. Wahlstrom and Johnson (1951) stated that large doses of cortisone injected into baby pigs increased urinary excretion of vitamin B12. It was desired in this experiment to study the effects of cortisone on the distribution of radioactive vitamin B12 in blood, liver and urine of vitamin Blz-deficient and -adequate rats. Methods At the end of Experiment II, five rats from each group were injected intraperitoneally with S moo. of radioactive vitamin B12 (labeled with 0060) and were placed in metabolism cages for collection of Zu-hour urine specimens. At the end of this period, they were killed by decapitation, and 1 ml. of blood was collected from each rat. Whole liver weights were recorded and approximately 250 mg. samples from each rat were removed for counting radioactivity. Both blood and liver samples were ashed in a muffle furnace for two hours. Urine samples were prepared by drying 2 ml. of urine in a crucible cover. All samples were counted under a Geiger-Muller end window counter for ten minutes. Corrections 56 were made for background. The results were calculated as counts per second per ml. of blood, counts per second per 100 mg. of liver and counts per second per Zh-hour urine specimen per 100 grams of body weight. Results In Table VIII it can be seen that vitamin BIZ-deficient rats (Group 1) retained considerably more radioactive vitamin B12 in their tissues and excreted less than half as much in the urine as the vitamin Bl2-adequate rats (Group 2). The cortisone-treated, vitamin BIZ-deficient rats (Group 3) had approximately the same amount of radioactive vitamin 812 in the blood, but less was concentrated in the liver and about three times as much was lost in the urine as in Group 1. When cortisone was given to vitamin B -adequate rats (Group h), 12 the distribution of the radioactive vitamin was about the same as in Group 2, indicating that the hormone did not alter the retention of vitamin 812 in these rats. When food intake was limited (Group 5), cortisone slightly increased the ex- cretion of vitamin 812 in the urine. Conclusions These results confirm the reports of Chow gt 3;. (1951) and Wahlstrom and Johnson (1951) that more vitamin is retained in animals which are deficient in this vitamin, and that cortisone increases the excretion of vitamin B12 in vitamin Blz-deficient animals. Although there may be some questions 57 TABLE VIII EFFECTS OF CORTISONE AND VITAMIN B12 0N DISTRIBUTION OF Co6o-VITAMIN 312 IN BLOOD, LIVER AND URINE Distribution of Cozo-Vitamin B12 Group Treatment Blood Liver Urine cps7fiI. cps7mg. cps/100 gm. body weight/2h hrs. 1 No vitamin.BlZ 0.219 0.100 10.87 2 Vitamin 312 0.109 0.078 2h.69 200 ug./kilogram 3 Cortisone 0.21h 0.083 35.66 6h Cortisone + 0.179 0.096 26.88 Vitamin.B 12 S Cortisone + 0.187 0.093 30.65 (pair-fed 58 as to whether the activity measured was actually vitamin B12, this can be assumed since Chow 33 El~ (1951, 1953) showed that biological and radioactive vitamin 812 values in tissues and urine of rats were in close agreement. It should be recalled that the amount of vitamin B12 fed the rats in Groups 2, u and S was about ten times above normal require- ments, and hence it is not surprising that cortisone failed to alter markedly the distribution or excretion of vitamin B12 in these animals. 59 Experiment VII. Excretion of Radioactive Vitamin B12 in the Urine following Injection of Corti- sone at Different Levels Purpose This experiment was intended to provide further infor- mation on the effects of cortisone on urinary vitamin B12 losses. Specifically, it was desired to determine the effects of different levels of cortisone in vitamin B12- deficient rats and in rats fed only normal vitamin 812 requirements. Methods Thirty young male rats were initially fed the vitamin Blz-deficient stock diet for 60 days and were then divided into six uniform groups of five each. Three groups were continued on the stock soybean meal diet and the other three groups were fed the same diet supplemented with vitamin B12 in amounts of 20 mcg. per kilogram of ration for 10 days. Twenty mcg. of vitamin B12 per kilogram of diet is believed to represent the normal requirement for growing rats (Stokstad gt 3;. 19h9; Zucker gt 2l° 1950). Beginning on the llth day, the rats were treated for 20 days as follows: Vitamin Blz-deficient groups: Group 1. Controls 2. 2 mg. Cortisone daily 3. u mg. Cortisone daily. 60 Vitamin BIZ-fed groups: Group u. Controls 5. 2 mg. Cortisone daily 6. h mg. Cortisone daily Food intake and body weight were measured every two days. Urinary nitrogen was determined every five days. After 20 days of the above treatment, the rats were injected with 0.1 mcc. of radioactive vitamin B intraperitoneally. 12 Twenty-four-hour urine specimens were collected and vitamin‘ B12 activity was determined as in the previous experiment. Results Body weight, food intake, food utilization per gram of body weight and urinary nitrogen excretion are shown in Table IX and Fig. 7. 0n the whole, these results are quite similar to those reported in Experiments I and II. There- fore only the urinary vitamin B12 excretion values will be considered here. In the vitamin BlZ-deficient rats cortisone greatly increased the excretion of the radioactive vitamin. Two mg. of the hormone daily doubled the loss of the vitamin and h mg. daily tripled its loss in the urine. 0n the vitamin- adequate diet, only the u mg. level of cortisone increased the loss of vitamin B12 in the urine. .. H.mHm c.0ma o.aoa a.:ma sawae onouwpnoo .ms.:. 0 .: o.Hmm o.oom o.aoa :.mma sagas oeouwpnoo .ms m m oe.: m.mmm o.e:m o.moH 0.:mH Hotneoo : _eou-mflm casuua> a: 3.0:H 76.:ma o.ooa o.mma haaee oeomwppoo .mg.: m a: «.mma o.ooa o.m~a o.:ma hands .eoudpaoo .mg m m mm.» m.oma 0.:oa o.o~H m.:ma Hoapeoo H peoaoam.eamam awaapw> hoaoaoauuo Hung onospaoha ”confides” doom Haven. mudgamom team uaospaohn. @995 unumm .xuuaw econ .sw pnwwos seam abs. 2H Nam awn—“Eda“; EH8040H3 mo zogoxfl zo NaomHamoo ho mg.“— EmMEHQ mo maog NH wands o 1"..." ..I 'II".I’ I qu '..||b~n.hl' . 61 I .. a L,JHI-.| 'l' ". p L Djih‘q‘k‘a Ei‘jaIUp HmmN.H :.~ma N.oma o.~:a haawo onouapnoo .mE 3 o o:mm.o :.»ma m.HmH s.mma sagas oeoaaaeoo .wa m m momm.o H.oo m.mm m.mo Honpeoo : newsmam ewsspa> mmmm.a H.mmm 5.:ma m.mma hawae .eomwpnoo .ma : m oopa.a H.H:H :.o:H o.o:a sagas oeomwuaoo .mg m m pasm.o 0.00 o.~o H.Hma Hoppeoe H peoaoweoeumam casspw> .m.H .voa H.o chap Na when 0H uhsv m .unn :N\.Ew 00fi\mao aflofip¢0ha dsoaw ends: ca Nam danuabl o0 .nae :m\»nmaoa been om .aw oom\z .ws .o>« 605“.“ nfloo NH "ma—EB mm rooo I. CONTROL fix mm VIT. BaDEF.{I. zm'c. ma. 1.2m. IN G" 1C 4HGto C m. \ as coansowE mum mm .,,_.-.—-- 1c m/nooen BODY mm HRS. lit—~— .’.’ v Maggooooo.ooooooooooooooooooooooogog:g'g AVERPGE BODY WEIGHT IN G" / .. 0000000 0'... ...oo........ooooooul. §. §. ’0 .§. /I£ §°=o=0=°=o =. fl“. §,§,=.=0‘='§°§oak . Q s a. ..-I. 0.. . ---..-----.--...--.-----.....a §. §. § pun-II... Q o ‘0‘. .~.~.-°-.-.-.-.-.-.-'-I¢ .5.- ia Is" ' 75 DAYS Fig. 7 Effects of different levels of cortisme on 5.4, weight, food intake and urinary nitrogen. 63 Conclusions 0n the whole, these results confirm those in the previous experiment. In addition however, they show that urinary lossesof vitamin B12 also depend on the level of cortisone in the body. In the vitamin-deficient rats, 2 mg. of cortisone doubled and h mg. tripled urinary vitamin B12 losses. In the rats which received only a normal vitamin B12 intake, there was no increase in urinary vitamin B12 until u mg. of cortisone were injected daily. These results are believed to reflect an interference by cortisone of carbohydrate utilization, hence reducing the retention of vitamin B12 in the body. ‘3 "7.’ in‘ “AH 'I-P‘fifih‘g , _ u ‘ n H‘ l i I 6h Experiment VIII. Effects of Alloxan-diabetes, Cortisone and Vitamin B12 on Excretion of Radio- active Vitamin B12. Purpose In this experiment it was particularly desired to study the excretion of vitamin B12 in alloxan-diabetic rats fed a vitamin B12-deficient or~-adequate ration. In addition, further data were obtained on the effects of cortisone on the urinary excretion of vitamin 812. Methods Some of the rats employed in Experiment III were used in this study after having been treated as previously described for 32 days. Five rats from each group were injected intraperitoneally with a dose of 0.1 mcc. of radio- active Co60 —-abeled vitamin 812 before they were placed in the metabolism cages. Twenty-four-hour urine specimens were collected and the radioactivity of each urine sample was determined by the same procedure employed previously. Results It can be seen in Table X that the vitamin B -deficient 12 animals (Group 1) retained more of this vitamin than the vitamin BlZ-adequate rats (Group 2), as was found in the previous experiments. The alloxan—diabetic, vitamin 812' deficient rats (Group 3) retained more while the alloxan- diabetic, vitamin Biz-adequate rats (Group A) retained less o "". {all It} liltl .Itli .qyzll. I: .[lllII‘II ill . ; 1J4!) JI.I$,:I. ...l 1‘ hi .5 65 TABLE I EFFECTS OF ALLOXAN, CORTISONE AND VITAMIN B12 ON EXCRETION 0F RADIOACTIVE VITAMIN B12 IN URINE Group Treatment COSO-Vitamin B12 in urina cps/100 gm./2u hr. 0.1 no. IOPO 1 No vitamin.BlZ 2.667h 2 Vitamin 1312 11.3936 200 mcg./kilogram 3 Alloxan 2.7735 17.5 mg./100 gm. h Alloxan + vitamin.B12 n.6286 S Cortisone 6.3852 Ll- mg./day 6 Cortisone + vitamin B12 #05790 1.1441115... 1-. .. .I "—- 66 of this vitamin. The amounts of vitamin B12 found in the urine were similar to those of the corresponding first two groups. Cortisone increased the excretion of vitamin B12 in the urine of the vitamin BIZ-deficient rats (Group 5), but not in the vitamin Blz-adequate animals (Group 6). This is in agreement with the previous experiments. Conclusions Alloxan did not alter the urinary excretion of Vitamin B12 in either the vitamin Biz-adequate or -deficient rats. This indicates that alloxan—diabetic rats can utilize vitamin B12 as well as normal rats, and is in agreement with the other data in Experiment III showing that in alloxan- treated rats, vitamin B12 can increase food.intake, efficiency of food utilization and body weight gains. It is concluded therefore, that vitamin B12 can act independently of insulin insofar as the foregoing effects are concerned. “:0 ’ ' . 13-3 *5 u‘w.)\-..-bu " . P! ’.m’ f . g..,7: ' 67 Experiment IX. Effects of Glucose Administration on Excretion of Vitamin 812 in Alloxan, Cortisone and Vitamin BlZ-Treated Rats Purpose In this experiment, it was desired to ascertain the pattern of vitamin B12 excretion in the urine after glucose FR administration to normal, alloxan-diabetic and cortisone- r: treated rats. It was hoped that this would provide further information on vitamin 812 metabolism as influenced by the foregoing treatments.l g; I Methods At the end of 35 days of treatment, five rats from each group in Experiment III were starved for 12 hours and were injected intraperitoneally with a dose of 750 mg. of dextrose in 5 ml. of physiological saline. This was immediately followed by another intraperitoneal injection of 0.1 mcc. of radioactive CoéO-labeled vitamin B12. The rats were placed in the metabolism cages for 2h hours for urine collection, and the radioactivity in each urine sample was counted. Results The results are shown in Table XI. Approximately the same amounts of vitamin B12 was excreted in the urine of the vitamin BlZ-deficient rats (Group 1), as in previous experi- ments. However, much less vitamin B12 was excreted in the 68 TABLE XI EFFECTS OF GLUCOSE INJECTIONS ON VITAMIN B12 EXCRETION IN ALLOXAN, CORTISONE AND VITAMIN BlzaTREATED RATS Co6o-vitamin 812 in urine Ops/100 5:11.121; hrs. Results from table Present (Without glucose)x results , Group Treatment No vitamin.Blz 2.667h 2.3276 Vitamin 1312 M3936 2.91165 20Cmog./kilogrem Alloxan 2.7235 2.6827 17.5 mg./100 gm. Alloxan + vitamin.B12 h.6286 n.5u66 Cortisone 6.3852 6.7698 h nee/day n.579o 2.35m Cortisone + vitamin.B12 §-! . :Qf‘x xnxx :I'A-‘ n—u—g 1 v I l v’. ‘7': II_ 69 urine of the vitamin BlZ-adequate animals (Group 2) than in previous experiments. In Groups 3 and h, about the same amount of vitamin B12 appeared in the urine as in Experi- ment VIII. The administration of glucose did not alter Vitamin B12 excretion in the cortisone rats fed the vitamin Blz-deficient diet (Group 5), but decreased the loss of I“ vitamin B12 in the vitamin-fed animals (Group 6). E”‘ i s : 1 Conclusions § 0 The decreased excretion of radioactive vitamin B12 in éd . 7:, 5* Group 2 is probably a reflection of increased glucose utili- zation in these rats. Glucose administration did not alter urinary vitamin B12 losses in either of the alloxan-treated groups. Perhaps this can be attributed to the fact that blood sugar was already high and was being used to the maximum but limited ability of these animals. Hence the additional injection of glucose did not alter vitamin B12 metabolism. The glucose injections also did not alter the loss of the vitamin in the cortisone-treated, vitamin B12- deficient rats, probably because glucose was also being used to the limited maximum in these rats. The presence of large doses of cortisone together with a deficiency of vitamin 812 inhibited glucose metabolism. In Group 6 how- ever, extra glucose could be utilized because these rats were receiving vitamin 812 in their diet. Hence less vitamin B12 was excreted into the urine. 70 Experiment X. Effects of Insulin Injections on Vitamin B12 Excretion in Normal, Alloxan and Cortisone- treated Rats Ppgpose Since there were strong indications that vitamin B12 was required for full insulin action in the previous experi- ments, it was desired to determine the effects of insulin on the excretion of radioactive vitamin B of normal, 12 alloxan-diabetic and cortisone-treated rats. Methods Thirty weanling rats were fed the vitamin Bla-deficient stock diet for 60 days. At the end of this period, the rats were divided into six uniform groups of five each and were treated as follows for 30 days: Group 1. No vitamin B12 2. Vitamin B12 -- 200 mcg./kilogram of diet 3. Alloxan -- 17.5 mg./100 grams h. Alloxan + Vitamin B12 ‘ 5. Cortisone -- h mg./day/rat 6. Cortisone + Vitamin 812 At the end of 20 days, all rats were starved for 12 hours and initial blood samples were collected for glucose determinations. Three doses of 0.5 unit of insulin were injected into each rat at eight-hour intervals during a period of 2h hours. A dose of 0.1 mcc. of radioactive 0060- 1abeled vitamin 812 was injected following the first insulin 71 injection into each rat, and all injections were by the intraperitoneal route. Urine samples were collected for 2h hours and radioactivity was determined as before. Food was withheld during this period. Results The results are shown in Table XII. During the period of insulin administration the rats in Groups 1 and 2 were in a semi-conscious condition because of hypoglycemia, and consequently the data from these rats may not be entirely valid. {The rats of the other four groups were not adversely affected by the insulin injections. Insulin apparently produced retention of the injected radioactive Vitamin B12 in all groups, since less of this vitamin appeared in the urine than.was found under similar treatment but without insulin injections in Experiment VIII. It will be noted that the decrease in vitamin B excretion in Group 5 was 12 much less than in the other groups. Conclusions When the results of Table XII are compared with Table X it can be seen that insulin decreased the urinary losses of vitamin B12 in all groups irrespective of previous treatment. This seems logical since the foregoing experiments have indicated that insulin increases vitamin 812 requirements by the body. The particularly small percentage decrease in 72 TABLE XII EFFECTS OF INSULIN INJECTIONS ON EXCRETION OF RADTOACTIVE VITAMIN 312 IN NORMAL, ALLOXAN AND CORTISONE-TREATED RATS (0.5 u. of insulin/rat at 8-hour interval) Coéo-vitamin.Blz in urine cps/100 gm./2h hrs. Results from table}? Present Percentage (Without insulin) results decrease Group Treatment 1 No vitamin 1312 2.66711 1.25111 53.09 2 Vitamin 312 n.3936 1.3698 68.82 200 meg/1:11 ogram 3 Alloxan 207235 1.fl572 #5039 17.5 mg./100 gm. h Alloxan + n.6286 1.6270 6h.8h vitamin.Blz 5 Cortisone 6.3852 h.83h5 2h.28 h.mg./day 6 Cortisone + h.5790 l.h689 67.92 vitamin.Blz . 73 vitamin B12 excretion seen in the cortisone-treated rats on the vitamin-deficient diet (Group 5) is believed to reflect a greater insulin resistance in these animals. In short, insulin was less effective in the presence of corti- sone, less glucose was utilized and more vitamin B12 was excreted into the urine. A further comparison of these two tables shows that in every case, the decrease in vitamin 812 excretion was more marked in the vitamin-adequate (Groups 2, h and 6) than in the vitamin-deficient rats (Groups 1, 3 and 5). This again appears logical, since insulin was more effective in the vitamin-adequate rats, more glucose was metabolized and hence more vitamin B12 was retained in the body. It should be recalled that the rats in Groups 2, h and 6 were receiving 200 mcg. of vitamin B12 per kilogram of diet, or ten times more than their normal requirements. It is remarkable therefore, that the insulin injections should have altered the excretion pattern of vitamin B12 in these rate, since this suggests that insulin increased vitamin B12 needs by perhaps ten fold or more. It would be interesting to repeat this experiment but feed only normal vitamin B12 requirements. DISCUSSION In the reports from this laboratory dealing with inter- actions between cortisone and vitamin B12 (Meites 33 El. 1951, 1952a, 1952b, 1953). it was suggested that large doses of cortisone increased requirements for vitamin 312. This view was based on the findings that (a) large doses of cortisone aggravated the condition of rats on a vitamin Bla-defiCient diet, as indicated by inhibition of body and hair growth, decreased appetite and increased nitrogen losses in the urine; and (b) when normal intake of vitamin B12 was permitted, it had relatively little or no ability to overcome the catabolic actions of cortisone, and an intake at least ten times greater than normal was necessary to produce any marked counteraction of cortisone. It was also observed that while vitamin B increased appetite and 12 the efficiency with which food could be converted into body weight gains, it was prevented from.doing so to its fullest capacity by cortisone. It became of primary interest there- fore, to attempt to discover why large doses of cortisone increased vitamin B12 needs. The results presented in this study and the related observations of other workers are believed to provide some answers to the above questions. First it has been shown that large doses of cortisone increase the secretion of insulin .l.l I. I1. Jill)! I .. .w \ 75 by the pancreas of the rat and guinea pig (Franckson pp 3;. 1953, Hausberger 32 pl. 1953). Second, insulin increases the need for vitamin B 2, as demonstrated in the present 1 experiments. Thus, (a) insulin was more effective in reducing blood glucose in the presence of adequate vitamin B12 than on a deficient diet, (b) insulin reduced the excretion of radioactive vitamin B in the urine, and (c) injected 12 glucose was more easily metabolized in vitamin BIZ-adequate than in vitamin Biz-deficient rats. Third, cortisone pro- duces insulin resistance, as was observed here and by others (Francksonlpp‘gl. 1953). This is believed to further increase the need for vitamin 812, since even in the presence of,_ cortisone vitamin B12 is able to augment the effectiveness of insulin. Fourth, it was confirmed that large doses of cortisone increase the urinary losses of vitamin B12, par- ticularly in rats whose diet is deficient or just meets normal needs for vitamin B12. Whether this represents a direct effect of cortisone on vitamin B12 metabolism, an effect on the kidneys or circulation, or a change in the metabolism of carbohydrate can not be adequately answered at present. Most of the evidence in this thesis favors the latter possibility. Large amounts (ten times normal) of vitamin B12 were able to partially overcome the catabolic actions of excessive doses of cortisone under 3g libitum feeding but not on limited food intake. Apparently, these effects of the 76 vitamin were produced by inhibiting gluconeogenesis from protein by cortisone and by increasing glucose utilization. This depended on the ability of the vitamin to increase food intake. Long 33 El. (l9h0) and Engel (l9h9) similarly observed that administration of large amounts of carbo- hydrate to rats injected with adrenal cortical hormones counteracted the protein catabolic action of the latter. The observation that vitamin B12 was ineffective against cortisone under limited feeding conditions is in agreement with similar findings by Rupp and Paschkis (1953). Indeed it has been noted that vitamin 812 does not elicit any growth effect in rats when food intake is limited (Rupp pp 51. 1951; Baker, 1953). Since vitamin B12 is concerned principally with carbohydrate metabolism.(Bosshardt pp 51. 1950; Chow 33 gl. 1952; Black pp_§1. 1952), it appears likely that the decreased ability of cortisone to induce gluco- neogenesis from protein in the presence of vitamin B12 is by way of a direct action on carbohydrate metabolism. This remains to be elucidated. Large doses of cortisone partially interfered with the ability of vitamin B12 to increase the efficiency of food utilization for body growth. This is believed to be due in part to the loss of sugar in the urine and in part to increased insulin resistance, both of which were demonstrated in the present study. Although insulin does not appear to be essential for vitamin B12 function, as seen in the 77 alloxan-diabetic rats, it may act synergistically with the vitamin in favoring lipogenesis from glucose. The latter has been shown to be an independent function of both sub— stances. Thus cortisone, by decreasing the effectiveness of insulin, would depress its synergistic action with vitamin 812. Studies of fat formation from labeled glucose will help to determine whether this is actually true. The fact that in alloxan-diabetic rats vitamin B12 was albe to increase food intake, efficiency of food utilization for body growth and glucose metabolism deserves further comment. It will be recalled that vitamin B12 was practially as effective in these respects in the alloxan-treated as in the normal rats. Sturtvant pp 3;. (195h) have similarly reported that when food intake was increased in alloxan- diabetic rats, there was increased hyperglucosuria accom- panied by greater body growth. These observations are of considerable interest since it has been claimed that diabetic animals have practically no capacity to convert glucose into fat (for review of lipogenesis in diabetes see Gurin, l95h). Unfortunately no direct measures of lipo- genuds were made in the present work, and a carcass analysis of the alloxan-diabetic rats would have been particularly informative. If it is presumed, however, that vitamin B12 did favor lipogenesis in these rats, the possibility arises that the vitamin, particularly in large doses, can substitute TV» I I,‘ ' P L's-'L'v 'u 78 for one of essential functions of insulin. This deserves further study. There is evidence that vitamin B 2 may not only be able 1 to function independently of insulin, but also of the adrenal cortical hormones. Meites (1953) and Ralli‘gpigl. (1952) found that excessive doses of vitamin B12 were able to maintain life and partial body growth in adrenalectomized rats. This was accompanied by increased food intake and efficiency of food utilization. Cortisone and insulin may also be able to function in the absence, or at least in the presence of only a limited intake of vitamin B It is 12' clear that the functions of these two hormones are easily modified by the concentration of vitamin B12 in the diet, and it is regretable that no intermediate levels of vitamin B12 were used in the present study. However, it was shown that on a vitamin Biz-deficient diet, insulin was less effective in metabolizing glucose and cortisone was more effective in producing gluconeogenesis from protein. The reverse was true on a vitamin BIZ-adequate diet. This is believed to demonstrate the importance of vitamin B12 in maintaining normal carbohydrate metabolism under the delicate but opposing actions of cortisone and insulin on blood sugar levels. Vitamin B12 is perhaps more important in cortisone- insulin interactions than other B-vitamins, although little information is yet available of the relation of other vitamins :71 I 1'... “P “I!“ _ 79 to these two hormones. It has been noted, however, that a "vitamin B-complex deficiency" produces insulin resistance in animals and human beings (Martin, 1937; Elsonn pp 21. l9h0; Biskind, l9h5; Samuels, l9h8) and that large doses of cortisone may aggravate the condition of young rats on a thiamin-deficient diet (Wilwerth and Meites, 1953). The need for further studies with other B-vitamins is clearly indicated, since there is ample evidence that each does not have the same role in carbohydrate, fat or protein metabolism. In conclusion, it has been demonstrated that cortisone, the pancreas and vitamin B12 all interact on carbohydrate, protein and vitamin 812 metabolism. A change in the body level of any one of these factors modifies the function of the other two. It is believed that further studies of these and other vitamin-hormonal inter-relationships will increase our understanding of the intricate machinery of the body, and may even lead to more rational and effective hormone and vitamin therapy in man and animals. SUMMARY 1. When young rats were fed a vitamin BIZ—deficient diet, supplementation with this vitamin increased appetite and body weight gains, slightly increased blood glucose, greatly increased glucose tolerance, but slightly decreased urinary nitrogen excretion. When one to four mg. of corti- sone acetate daily were injected into vitamin BlZ-deficient rats, there was a progressive increase in urinary nitrogen, increased hyperglycemia and glucosuria, decreased glucose tolerance, reduced body weight gains and decreased appetite. When 200 mcg. of vitamin B12 per kilogram of diet was fed to cortisone-injected rats, and they were permitted to eat pg libitum, increases in urinary nitrogen losses were largely prevented, the hyperglycemia and glucosuria were reduced, glucose tolerance was increased and body growth was increased. Vitamin 812 was ineffective in these respects when food intake was restricted to that of animals receiving cortisone without vitamin B12. It is concluded that large doses of vitamin B12 can partially counteract the protein catabolic actions of cortisone by increasing appetite, increasing the availability and utilization of carbohydrate by the organism and reducing gluconeogenesis from protein. 2. Large doses of cortisone partially interfered with the favorable action of vitamin B12 in increasing the 81 efficiency of food utilization for body growth. This was accompanied by hyperglycemia and glucosuria, and was related to increased insulin resistance. Less carbohydrate was therefore left available for transformation into body weight gains (probably fat). 3. Alloxan-diabetes reduced body growth and the feed/gain ratio on the vitamin B12-deficient but not on the vitamin BIZ-adequate diet. In the latter rate there was much higher blood glucose, more urinary glucose, increased glucose tolerance but about the same urinary nitrogen losses 1‘ ‘l. as in the former animals. It is concluded that vitamin B12 can act independently of insul insofar as its effects on glucose utilization and body growth are concerned. A. Single injections of insulin (2 units in most cases) were much more effective in reducing blood glucose in normal, alloxan-diabetic and cortisone-treated rats on a vitamin B - l2 adequate than on a vitamin Bla-deficient diet. This indi- cates that an ample supply of vitamin B is essential for 12 maximum insulin action. By far the greatest resistance to insulin was found in the cortisone-treated rats on the vitamin Blz-deficient diet, confirming the findings that cortisone increases insulin resistance. 5. (a) Injections of large doses of cortisone (2 to h mg. daily) increased the urinary excretion of radioactive vitamin B12, particularly in rats fed a vitamin BlZ-deficient diet. On a diet meeting only normal requirements for 82 vitamin B12 (20 mcg./kilogram), cortisone did not increase urinary vitamin B12 until h mg. were injected daily. In general, the amounts of radioactive vitamin B12 lost in the urine were shown to be directly related to the dose of cortisone administered. (b) Intraperitoneal injections of 750 mg. of glucose did not change urinary losses of vitamin B12 in alloxanized rats fed either a vitamin Bl2-adequate or -deficient diet. Apparently blood glucose was already being used to the maximum extent possible in these rats. 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Blood Glucose Levels after a Single Injection of Insulin in Alloxan-diabetic Rats Purpose Preliminary to the experiments in which insulin was used, it was important to determine at what period of time blood glucose would fall to the lowest level after a single injection of insulin. Methods Eight adult male rats were used in this experiment. They were starved for 72 hours, only water being permitted during this time. At the end of this period, a dose of 17.5 mg. of alloxan monohydrate per 100 grams of body weight was injected subcutaneously (Bailey, 19M9). Diabetes was definitely established five days after the injection as indicated by a marked hyperglycemia. It was found that blood glucose values were between 320 mg. to M00 mg. percent. Blood glucose was determined by the Hartman, Shaeffer and Somogyi micromethod (Hawk 3373;. 1951). A volume of 0.2 ml. of blood was collected with a Folin-Wu micropipette from the tail of the rat, after first cleaning it with 80 percent alcohol, and then cutting off the tip. Two units of insulin were injected intraperitoneally into each rat. Food was removed 12 hours prior to the injection. After this, blood was collected every two hours for glucose determinations. Food was withheld during the collection period. 108 Results Figure 8 shows that the average initial blood glucose level was 380 mg. percent. After insulin administration, blood glucose fell gradually reaching the lowest level of about 125 mg. percent in four hours. Blood glucose re- turned to 250 mg. percent 10 hours after the insulin in- jection. Conclusions These data showed that with the dose employed, the greatest insulin effect could be expected four hours after injection. Consequently this time interval was used in all ~experiments in which insulin was employed. 400 O Q "3 f;f>S*Z/9(7 Ila an: 200 - 71937ng I00 110 2. Blood Glucose Determinations a. Somogyi-Shaffer-Hartman Method (Hawk 23 5;. 1951) A volume of 0.2 m1. of blood was drawn from the tail of each rat by a Folin micropipette and was mixed into 5.8 m1. of water in a 25-ml. Erlenmeyer flask; the pipette was then rinsed several times with the lacking water. A volume of one ml. of 1.8 percent of zinc sulfate and one m1. of 0.1 N of sodium hydroxide were added and mixed. The flask was stoppered, shaken and the contents were filtered through No. 1 dry filter paper. Five ml. of the Shaffer-Hartman copper reagent were measured in a 25 x 250 mm. test tube, and 5 m1. of the blood filtrate was mixed into it, shaken, and covered with a glass bulb. The test tube was placed in a boiling water bath for 15 minutes. It was then cooled, 1 m1. of 5 N sulfuric acid was added, and it was titrated with 0.005 N sodium thio- sulfate. Starch was used as an indicator. A blank was run on 5 ml. of the copper reagent after boiling with an equal amount of water. In the calculations, the blank titration was subtracted from the titration of the unknown. This gave the m1. of thiosulfate required for the unknown. For the glucose equivalent, the Table (page 525) in Hawk pg E1. (1951) was consulted. Since this table applies to the usual 1:10 dilution of blood, and in the present case, a 1:MO dilution was used, the mg. of glucose in 100 m1. of blood given in the table were multiplied by four. 4!: .8: 15% .k, 112 b. Folin and Malmros Method (Hawk 3; 31. 1951) With a Folin micropipette 0.1 ml. of blood was drawn from the tail of a rat and was transferred to a centrifuge tube containing 10 m1. of dilute tungstic acid. This was mixed and centrifuged. Four m1. of the water-clear super- natant fluid were transferred to a test tube graduated at 25 m1. To this, 2 m1. of O.M percent potassium ferricyanide- carbonate solution were added. The contents were heated in boiling water for 15 minutes and cooled in running tap water for 2 minutes. Then 5 ml. of ferric iron solution were added and mixed. Two minutes afterwards, the contents were diluted with water almost to the 25-ml. mark, two drops of alcohol were added to prevent foaming, and water was added exactly to the 25-ml. mark and mixed. It was read in a Fisher electrophotometer 20 minutes later. A green plate filter of 525 mue.wavelength was used. The photometer was initially set to zero density with water. For preparation of the standard solution of sugar, a stock solution of 1 percent glucose was made up in saturated benzoic acid. The stock solution was diluted to 0.01 mg. to 0.1 mg. per 0.1 m1. of water. The optical density for each amount was read on the photometer, and a linear line was drawn from these values. The calculation of blood glucose was as follows: mg. percent glucose = density of unknown x O 0h x 10 x 100 density of standard ° M. 0.1 O Mr: . . . U r i \ ‘ . .— . - 7 . s v v y a I ‘ ) a ,- ‘ ‘ .- 7. \ ‘ f‘ L ‘ . . i ‘ . l 7 v: ‘ .1 x l 1 i f ‘ l ‘ ‘ 0‘ I f‘ . ‘ . I . . , - . —. ‘ - x x V ' Q * o ' ‘ . , . , y 7 f! 7 ‘ : ' \ Ir ‘ 1 I I v . 4 . H! :2 fl -. ‘ ‘ ' i D ' . .' .C I I . ‘n ' ‘ . y ‘ . s . I U . , , I“: 7‘ ‘ l. '. r -. v ‘- , 1‘ . \' '. ' " . u J . ‘ ,_ L . l r ‘ l . - I ‘ . b ,, . 3 0 . ‘I . , ' l . ‘. ‘ ‘- . \ . 7 I‘ . . ,. 1 f ,- o ' _ ’ . , I _ . z . . ~ I . 4. ; , ‘ - '- *r: a - - 7 I f , . 1 't I . . ‘ - ,. I e ,7 - J 4 I” f r ' I f.- . a v ‘ . ‘ - — a 7 V' ‘ ’ "I . . . ' ‘ . , t t ' ' . . u I v ‘ n ' . D ‘ I I \ —. r- . v. ‘ ’c 1 fl - F " (~- I . I I ‘ . 7 K . ‘_ y' . I I O I I C t I - 7 1', — . ” ‘I X ‘ ‘8‘ ' , . ~ 1.. r- _. I _ O 8 7 . O «.99 (NV .3379 36.3.56 9m 0% as 90 Pm 7 3‘7 am. 9w 3 d c A O\ 4 ON a . do 7» 1 o. . 9 P 7 1 am, 1 U 9 .u no 7. . 0‘ 2, X 4 9W 1 Q.“ [ 115 3. Determination of Total Urinary Nitrogen Koch and McMeekin Method (Hawk 3; _a_l_, 1951) One ml. from.a 2h0hour urine specimen was diluted to 50 ml. and mixed in a volumetric flask. One ml. of the dilute solution was pipetted into a micro_Kjeldahl flask, and one ml. of 50 percent sulfuric acid was added and mixed. The flask was heated over a gas flame under a hood for 10 minutes, after which 3 drOps of 30 percent hydrogen peroxied were added. The flask was heated 6 more minutes until all the sulfuric acid fumes disappeared. It was then cooled for 30 minutes and diluted to 75 ml. with water. A total of 15 ml. of Nessler's reagent was added and the whole was diluted to 100 ml. This was left to stand for 10 minutes and was then read on a Fisher electrophotometer in which a green plate filter of 525 mu. wavelength was used. For a standard nitrogen preparation, 0.0714 gram of ammonium sulfate was dissolved in one liter of water together with a few drops of concentrated sulfuric acid as a preser- vative. This contained 1 mg. of nitrogen per 10 ml. It was used in amounts of 0.1 ml. per 1 ml. of the stock solution, and was diluted with 15 ml. of Nessler's reagent and water to 100 ml. The values were read on the photometer and a linear line was drawn. The calculations were as follows: reading‘of standard . N i t d d 1 reading of unknown x mg n 8 an ar x ur ne volume body weight Total nitrogen was eXpressed as mg./lOO gm. body weight/Zh-hour urine specimen. ’\ ~- -Aa-p—b‘. ._ .--— . .. a8 n l L I L c Q Q Q g \9 V: 8 \ [jgyvaa 793?20b 117 h. Determination of Urinary Glucose Hawkins and Van Slyke Method (Hawk §£_§l. 1951) One ml. of the Zh-hour urine specimen was diluted to 50 ml. in a volumetric flask, and 2 ml. of this was pipetted into a pyrex test tube (1h x 125 mm.). Two ml. of ferri- cyanide solution were added and mixed. The flask was immersed in a beaker of boiling water containing a similar test tube with water alone for comparison. A white back- ground was made on the sides of beakers into which the solutions were poured by pasting on white paper. The time in seconds required for the last trace of yellow to dis- appear was determined with a stopwatch. From the chart on page 865 of Hawk 33 El. (1951), estimations of glucose in gm./lOO gm. body weight/ZM-hour urine were calculated, i.e.: gm. of glucose x 50 x urine volume body weight ilx I" h Kilogram Stock Soybean Meal Diet Yellow corn meal (Thoman) Ground whole wheat (Thoman) Alfalfa leaf meal (Thoman) Brewer's yeast (Strain G) (A. Busch) Iodized salt Soybean.meal (low fiber, solvent extracted, containing 50% protein, Ar cher-Danie .l s -~Midland) lhOO gm. lOOO gm. 2&0 gm. 120 gm. uO gm. 1200 gm. 118 _,, .rz ROOM USE Ci‘éLY‘ a ’56 ‘3‘ 3 J “'1 7‘ I. ‘ ’fif“ . shim-"f" "75172 173 HICHIGQN STRTE UNIV. LIBRRRIES llHIWHINHII!HIIHWIHIWNWHIWIINIH‘IHIIIHI 31293010666661