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W” has been accepted towards fulfillment ol the requirements for t degree in jor professor [WW 0169 m _ thesis entitled K . . Z2? W 454% 5/2 :. Z 4: / , / presented bg f-J‘I- -‘l’l‘ ‘ . r _, 'l "' f . - . o '~l,vl y "7“: . I k ' ‘4"? f‘ I ‘9. "'1‘ w I‘ ‘ ‘ 5 k ' ‘ ' \ >- v “1"‘1‘5 :1 '1 ‘ . . f ‘ ‘, s ,' " I " . *. c D‘, 1 , . ‘ 's ‘ t. >.- ' 6", '4 - ‘ 6. ‘ . a g ' h. “£131", 3 3 «in? - "I .' in} .l v "- x j ‘ gt?“ l . f . . at. ~~. A STUDY OF THE INTERACTIONS OF VITAMIN B13 AND DESOXYCORTICOSTERONE ACETATE IN UNILATERALLY NEPHRECTOMIZED RATS BY William P. Baker 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 MASTER OF SCIENCE Department of Physiology and PharmacolOgy 1953 ACKNOWLEDGMENTS The author is grateful to Dr. Joseph Meites for his assistance in carrying out this work and in preparing this manuscript. The demonstration of histologic technique by Mrs. Rosa Ogle is also greatly appreciated. The author wishes further to acknowledge the helpfulness of other members of the Department of Physiology and Pharmacology for their valu- able suggestions, and to thank Dr. B. V. Alfredson for making available the facilities of the department. TABLE OF CONTENTS PAGE INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF DESOXYCORTICOSTERONE . . . . . . . . . . . Replacement for the Adrenal Cortex . . . . . 4 Effects on Electrolyte and Water Metabolism . . . 5 Desoxycorticosterone and Hypertension . . . . . 8 Effects on Tissue and Organ Pathology . 9 REVIEW OF VITAMIN 312. . . . . . . . . . . . . . . . . 16 Effects on Hemopoiesis . . . . . . 16 Effects on Growth and Protein Metabolism . . . . 18 Interactions Between Vitamin 312 and Hormones . . 20 EXPERIMENTAL Experiment I: Relation Between Desoxycorticosterone Acetate and Vitamin B13 in Unilaterally Nephrectomized Rats Procedure . . . . . . . . . . . . . . . . . 25 Results . . . . . . . . . . . . . . . . . . 2? Experiment II: Relation Between Desoxycorticosterone Acetate and Vitamin B13 in Unilaterally Nephrecto- mized Rats Procedure . . . . . . . . . . . . . . . . . 31 Results . . . . . . . . . . . . . . . . . . 33 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 36 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 45 REFERENCES . . . . . . . . . . . . . . . . . . . . . . 49 LIST CF Ttilis TABLE PAD: I. Tieatments of Kate in Experirent I.......... 2? II. EffGCt Of Vitamin Dln an-fi/cr Dec 3x37- cortices tero he .cetate on Grouth, Feed and Saline Consumption of Unilaterally Neph- TECtOTCiZE.‘ #9.: 08.0.0...00.00.00.0000000000000 29 III. Effect Of Vitam in 313 an.1/Qr Decoxy- corticostercne Acct *Ce on the Organ h'eigbts of Unilaterally Neprrectomized R1ts......... 30 IV. Treatments of Rats in Experinent II......... 3? V. Effect Of "itamin 512 arc/’or D550yy- cortico ste rone acetate on GroxMt , Food and Saline Consumption of Unil te ally Neph- l‘aCtO'ALzed EatSCOOOOI.OOOOOCOOOOOOOOOOOOOOOO 55 VI. Effect of itamin B13 anj/op Decgyv- corticosterone AC9tLt€ on the Orgin Teights of UniL .ter ally Neohrectomized Rats.......,. 37 VII. Effect of Vitamin 310 and/or De goxy_ corticosterone acetate on the Water Content of Tissues of Unilaterally Nephrectsmized FatSOOIOIOQ0000.00.00.0000000000000.00.00... :8 INTRODUCTION In a recent review of the relationship of nutrition and stress Goodhart and Jolliffe (1952) stated, 'it can be said without fear of contradiction that optimal nutrition with vitamins and other necessary nutrients helps to main- tain the organism's resistance to stress at its maximum". Ascorbic acid and the B vitamins, especially pantothenic acid and vitamin 313, were mentioned by these authors as being of great importance in protecting the organism against stress. Many of the metabolic diseases increase the dietary requirement of the afflicted organism. Stresses such as fever, drugs, toxins, pregnancy, lactation, hyperthyroidism, hyperfunction of the adrenal cortex and related conditions may increase the requirement for certain dietary elements. In hyperthyroidism, diabetus insipidus, fever, cardio-renal disease and similar conditions, increases in basal metabolic rate accompany the increased requirement for many substances. This indicates that these changes in requirement are brought about through some alteration in the rate of certain metabolic processes. Strenuous physical activity, aside from increas- ing the caloric needs of the individual, also elevate the requirement for vitamins of the B complex. When the role of the B vitamins in carbohydrate metabolism is considered, it seems within the realm of possibility that such a change should occur. 2 Various hormone imbalances often result in an altered vitamin requirement. Hyperthyroidism in both man and experi- mental animals is accompanied by a need for greater quantities of vitamins, including vitamin 312. The administration of large doses of estrogens (Meites and Shay 1951) and cortisone (Meites 1952) produce elevated needs for vitamin 312' Although much interest has been shown in the vitamin requirements as they are altered by stresses such as endo- crine hyperfunction, many studies have been conducted in which the effects of the vitamins on endocrine function have also been studied. Morgan (1951) has reviewed this relation- ship as it pertains to the adrenal cortex. The changes in form and function of the arenal cortex in pantothenic acid deficiency indicate a depletion of the hormone-forming capacity of this gland in the absence of this particular vitamin. There is no evidence at present to indicate whether the impaired metabolism seen in this condition is a result of a primary effect on the adrenal or is secondary to an effect produced in the altered metabolic processes of the tissues. The interrelationship of hormones and vitamins is an interesting one. The recent use of desoxycorticosterone acetate (DCA) in producing patholOgical lesions and the questions regarding the etioIOgy of this pathology led to the undertaking of this problem. Since increased levels of vitamin B13 will protect the animal from thyrotoxicosis 3 and will prevent the growth depressions seen when large quantities of cortisone or estrOgen are given, it seemed lOgical to determine whether or not vitamin 313 would exert a protective influence against DCA pathOgenesis. REVIEW OF DESOXYCORTICOSTERONE Desoxycorticosterone was first synthesized by Steiger and Reiohstein (1937) and was later found in very minute quantities in adrenal cortical extract by Reichstein and von Euw (1938). There is still some question however, whether desoxycorticosterone is a true adrenal cortical hor- mone (Hartman and Brownell, 1949). Because of its ease of synthesis and relative abundance, desoxycorticosterone has been studied in great detail. The results of these studies have shown this 11-desoxy steroid to have such important biological qualities as partial replacement for the adrenal cortex, as a powerful regulator of salt and water metabolism and as a factor which can produce hypertension and many types of pathology. Replacement for the Adrenal Cortex. Remington, Perkins, Swingle and Drill (1941) were able to maintain adrenalectomized dogs on DCA (desoxycorticosterone acetate) for long periods of time. Hartman and Brownell (1949) main- tained an adrenalectomized deg on DCA for the greater part of four years. However, there were times during this period 'when it was necessary to administer whole adrenal cortical extract because of a loss of appetite and the appearance of other cortical insufficiency symptoms. Thorn and Firor (1940) have used DCA successfully in the clinical treatment of 5 Addisonian patients. Although DCA will suffice to maintain adrenalectomized animals, and has been used successfully in the treatment of Addisonians, the indications are that it is not a complete replacement for the adrenal cortex. Britton and Cline (1941) have compared the effects of whole adrenal cortical extract and DCA in the treatment of adrenalectomized cats and rats, and have found the whole extract to be superior therapy. The animals in these experi- ments which were maintained on whole cortical extract main- tained higher blood sugar levels and liver glycogen contents than the animals receiving DCA. These findings led the authors to postulate that DCA probably has no direct effect on carbohydrate metabolism. The work of Ingle and Thorn (1941) in which they compared the diabetogenic properties of cortisone and DCA, also indicate that DOA is practically inert with respect to carbohydrate metabolism. Ingle (1941) has also shown that DOA is relatively inactive in its effect on the work performance of adrenalectomized rats. Electrolyte and Water Metabolism. Although little or no carbohydrate activity can be attributed to DOA, this steriod has been shown to be a very effective factor in salt and water metabolism. Electrolyte and.water metabolism are so intimately related and interdependent that it is extremely difficult to discuss them separately or devise experiments which will eliminate one of these factors as a variable. For 6 this reason this discussion will consider them simultane- ously. Hagan, Ferrebee, Phyfe, Atchley and Loeb (l940) con- ducted experiments in which they studied the effect of DCA in a normal female dOg. Prior to the administration of DCA this dog drank 400 ml. of water and excreted an equal vohume of urine per day of specific gravity of 1.045. After six days of DCA administration the dOg was drinking 1000 m1. of water per day and excreting 1000 ml. of urine of specific gravity 1.025. This polydypsia was further exaggerated by the administration of sodium chloride in the drinking water. There was no evidence of excessive fluid retention in these animals since the water consumption paralleled the fluid lass by excretion. Also there was no significant gain in weight in any of the animals. The treatment of these animals with DCA was accompanied by an elevation of serum sodium and a decline in the level of serum potassium. It is also of interest to note that the serum protein concentration and blood gIUCOSe levels were not effected by this experimental regime. Kuhlman, Ragan, Ferrebee, Atchley and Loeb (1939) reported similar findings in other dOgs. Thorn, Howard and Emerson (1939) noted in Addisonians that overdosages of DOA would produce edema if sodium chloride was included in the therapy. Ferrebee, Hagan, Atchley and Loeb (1939) reported similar findings. Although it is not 7 well understood why DOA should produce an edema in some cases and a diabetes insipidus—like syndrome in others, it is gen- erally believed that the nature of the diet, the quantities of DOA given and.the duration of the DCA administration are all important factors. Gaunt and Birnie (1951) have recently reviewed a por- tion of the literature dealingwith the hormones and salt and water metabolism. These authors discuss some of the physio- logical factors which may effect the manifestation of DOA overdosage. The conditions resulting from the administration of large quantities of DOA is best summarized by a diagram taken from Gaunt's review: DOA Overdosage \ Enhanced renal tu lar Inhibited renal tubular Reabsbrption of salt reabsorption of water \ thirst polydypsia (L Increased extracellular v volume (edema) polyuria 8 Desoxycorticosterone in_Hypertension. Hartman and Brownell (1949) reported that Loeb, Atchley, Ferrebee and Ragan (1939) were the first investigators to note the effect of a DOA overdosage on the blood pressure of a human. Rodbard and Freed (1948) administered DOA to normotensive, spontane— ously hypertensive and Goldblatt hypertensive dogs and pro- duced elevated blood pressures in each of these cases. Grollman, Harrison, Williams (1940) and Friedman, Friedman, and Polley (1948) have produced hypertension in rate also by administering DOA. Swingle, Parkins and Remington (1941) studied the cir- culatory dynamics of normal and adrenalectomized dOgs follow- ing DOA treatment. The administration of DOA was followed by a plasma volume increase and an elevation in blood pressure in both cases. However, the increased blood pressure could not be attributed to the increased plasma volume since no blood pressure elevation was seen when normal animals were transfused with normal serum. Ziegler, Anderson and McQuarrie (1944) have studied the electrolyte and water content of tissues from hypertensive animals and have found an increased level of water and sodium in these tissues. It is beyond the sc0pe of this literature review to survey the attempts which have been made to determine the etiolOgy of DOA-induced hypertension. However, the sodium retaining property of DOA and the popular use of low sodium 9 diets by Perera and Blood (1947) and Kempner (1944) in treat- ing hypertension indicates that this quality of DOA may be responsible for its role as a hypertension-producing substance. Effects 9g Tissue and Organ Pathclcgy. DOA has been shown to produce many characteristic changes in the various tissues and organs. Selye (1941) reported that in short term experiments in which rats were given DOA, an involution of the thymus, spleen and adrenal cortex resulted, accom- panied by renal hypertrophy. The results of long term experiments revealed similar findings except that enlarge- ment of the thymus and spleen occurred. Selye and Pentz (1943) in another experiment noted that cardiac and hepatic enlarge- ment were also produced by DCA and that these effects and the renal hypertrophy were augmented by unilateral nephrectomy. Hall, Finerty, Hall and Hess (1951) likewise reported an adrenal cortical atrophy following treatment with DOA. How- ever, since this atrophy was not accompanied by a reduction in cortical ascorbic acid, these authors postulated that the action of DOA could not be considered as a stress. Selye, Mintzberg and Rowley (1945) studied the effects of various electrolyted upon DOA-induced patholcgy, and found sodium chloride to add greatly to DOA-induced pathogenesis. Selye, Stone, Timiras and Schaffenburg (1949) have shown that when DOA is administered to unilaterally nephrectomized 10 animals maintained on a normal diet and 1 percent saline, patholOgical lesions similar to those seen in man during hypertension often result. When unilaterally nephrectomized rats were kept on a regime of 6 mg. of DOA daily and l per- cent saline for 27 days, he found nephrosclerosis, periarter- itis nodosa in the mesentric vessels, and degenerative changes and Aschoff bodies in the myocardium. Selye, Sylvester, Hall and Leblond (1944) also stated that they produced a hyperemia and swelling in the tarsal joint region of the hind feet of rats maintained under similar experimental conditions. At autOpsy, signs of nephrosclerosis, periarteritis nodosa or rheumatic nodules in the hearts of most of these animals were apparent. It is of interest to note that all of these path- ological develOpments were exaggerated by adrenalectcmy or thyroidectomy. Timiras, Fairbault and Selye (1949) employ- ing similar experimental procedures but using larger doses of DOA, produced severe brain lesions in rats. These lesions were characterized by an edema of the brain and a consequent volume increase in both cerebral hemispheres, a narrowing and sometimes obliteration of the sulci and a flattening of the gyri. They also noted histological evidence of peri- arteritis nodosa of the cerebral arterioles accompanied by a pronounced hyalinization of the vessel walls, venous stasis, congestion and multiple small thromboses. Degenerative changes in the nervous tissues were also reported. ll Selye and Stone (1943) were able to elicit a neph- rosclerosis, generalized tissue edema and cardiac dilatation similar to the cardiovascular changes seen in human hyperten- sives by simply placing chickens on a 2% saline drink. When DCA was administered, much smaller quantities of NaCl were required to produce these changes. The pathology of the kid- neys of these animals were characterized by hypertrOphy of the convoluted tubules, cloudy swelling and the development of hyaline casts. The glomeruli were enlarged and some of the glomerular capillaries showed hyalinization and proliferation in the region of the epithelial cells. It is of interest that progesterone produced similar results. Other workers in trying to reproduce lesions similar to those reported by Selye, Sylvester, Hall and Leblond (1944) have met with varying degrees of success. Rosenberg, Woodbury and Sayers (1952) reported that from'the large num- ber of rats which they kept on experimental regimes similar to that utilized by Selye, not one animal developed any articular pathology. Harrison (1946) was likewise unable to produce any signs of arthritis in unilateral nephrectomized rats receiving DOA and saline. This author suggested that a direct or indirect infection may have been the cause of the experimentally produced arthritis reported by Selye, since it was reported that a large number of Selye's animals suc— cumbed because of pneumonia. It was also suggested.that low 12 air temperatues might have been a factor in the etiology of the nephrosclerosis and rheumatic cardiac lesions. Darrow and Miller (1942), however, were able to demon- strate cardiac lesions in rats rollowing repeated injections of DOA. It was postulated that this effect was due primarily to the low content of potassium in cardiac muscle as a result of DOA administration. Guillemin and Fortier (1951) produced myocardial nodules, periarteritis nodosa and macroscropic signs of nephrosclerosis in unilaterally nephrectomized rats receiv- ing saline and DOA. The kidneys of these animals showed glo- merular hyalinosia intense tubular enlargement, and a cellular vacuolization which was predominant in the proximal segment of the tubule. This pathology was greatly reduced and in some cases prevented by Phenergan, a compound known to alter mem- brane permeability. Since the sodium content of the diet is known to be critical in DOA-induced pathogenesis, and since sodium chloride alone will produce typical lesions in some Species, these authors interpreted their results to indicate that the sodium chloride was responsible for the path010gy, and that DOA only facilitated the accumulation of large quanti- ties of sodium chloride in the tissue. Rosenberg, Woodbury and Sayers (1952) have recently pro- duced.such pathology as focal glomerulitis, interstitial myo- carditis and periarteritis nodosa in unilaterally nephrecto- mized rats receiving DOA and saline drink. These workers were 13 able to reduce or prevent this occurrence of lesions by administering cortisone of ACTH. These authors believe that the probable action of the adrenal cortical steriods in pre- venting collagen diseases is due to general inhibition of all inflammatory processes regardless of the causative agent. It is also stated that there is no reason to conclude that the collagen diseases of man and DOA-treated rats have common etiology simply because both respond to cortisone and ACTH therapy. Woodbury, Rosenberg and Sayers (1950) have likewise shown that simultaneous administration of ACTH and DOA results in an inhibition of the pathoIOgical changes normally induced by DOA. These authors postulate that the exogenous DOA in- hibits the production of ll-oxygenated steriods by the adrenal and consequently lowers the resistance of the animals to stress. ,Selye (1946) has still another explanation of DOA- induced pathogenesis which is perhaps broader and more in— clusive than any of the hypotheses so far mentioned. This theory has been named the "general adaptation syndrome". Selye (1946) defined the "general adaptation syndrome" as the sum of all non-specific systemic reactions of the body which follow a long continued exposure to stress. It is postulated that the etiology of such diseases as hypertension, gastrointestinal ulcers, nephrosclerosis, nephritis, rheuma- tic fever and periarteritis nodosa may be a result of this l4 phenomenon. The general adaptation syndrome has three phases: (1) the alarm reaction, (2) a period of resistance and (3) a stage of exhaustion. The alarm reaction represents the re- sponse of an organism to any stimuli to which it is not adapted. Factors which may elicit such a response are in- fections, burns, hemorrhage, exposure to extreme temperatures, trauma, drugs, toxic substances, dietary deficiency, hormonal agents, etc. The period of stress is followed by a phase of resistance during which the animal has acquired adaptation to the alarming stimuli. This period is characterized by hyper- acmivity of the adrenal cortex. While it is postulated that many of the adrenal steriods serve to protect the tissues during this stage, it is also believed that the increase of some of the endogenous hormones may be the etiological factor in producing certain cardiovascular, renal and joint diseases. A stage of exhaustion ensues when the organism is no longer capable of maintaining resistance. Functional changes are considered the principal manifestation during the course of the general adaptation syndrome. These include such fac- tors as blood pressure changes, blood clotting time changes, absorption of various substances and metabolic changes in- volving enzymatic activity. The pathological changes seen include atrophy of the thyroid, gonads, lymph nodes, thymus and spleen, while the pancreas and adrenal cortex hypertrOphy. 15 Characteristic morphological changes are also seen in the liver, kidney, lungs, heart and lens of the eye. 16 REVIEW OF VITAMIN B13 Effects gg_Hem0poiesis. Rickes, Brink, Konisuzy, Wood and Folkers (1948a) of the Merck Research Laboratories first announced that a compound had been crystallized from liver extracts which was active in the treatment of Addisonian anemia. E. L. Smith (1948) conducted experiments during this same time and isolated from four tons of ox liver one gram of a red.amorphous material which was likewise effective in treatment of pernicious anemia. Shorb (1947) studied the microbiological requirement for these liver preparations and found that Lactobacillus lactis Dorner required a factor present in the liver extracts. It was also reported that the therapeutic value of the dif— ferent factors paralleled the potency of the preparations as assayed by this particular strain of bacteria. The sugges- tion was made that these factors were identical. In a later publication Shorb (1948) stated that crystalline vitamin 813 was extremely active when measured by the microbiological assay, and his data also indicated that vitamin 812 was par- tially if not entirely responsible for the activity of the liver preparations in Lactobacillus lactis Dorner. When vitamin 813 became available, many laboratory and clinical studies were conducted to determine its exact therapeutic and physiologic significance. Some of the very early clinical investigations were conducted by West (1948) 17 in which he elicited favorable responses in all of three pernicious anemia patients treated with vitamin 813. In one patient who received 150 micrograms of vitamin 812 in a single intramuscular injection, the red blood cell count rose from 1.5 million to 3.4 million in twentybthree days. The other two patients received much smaller doses and showed a positive but less dramatic response. The hematological effectiveness of vitamin B13 was , soon established but a question remained regarding the dif- ference between this substance and other known substances such as folic acid which were also known to stimulate erythro- poiesis. Berk, Denny-Brown, Finland and Castle (1948) re- ported a case in which a victim of pernicious anemia had been treated with pteroylglutamic acid and had responded favorably hematologically but had developed the neur010gic complications normally associated with this disease. The subsequent treat- ment of this patient with vitamin 813 resulted in an allevia- tion of this neurolOgic syndrome and led these authors to postulate that pteroylglutamic acid was not responsible for vitamin B12 activity. Chemical evidence by Rickes, Brink, Konisuzy, Wood and Folkers (1948b) indicated that vitamin 813 was a complex containing cobalt and phosphorus and therefore must be a distinct vitamin entirely different from any other known vitamin. 18 Effects 2Q_Growth anlerotein Metabolism. Animal studies prior and subsequent to the discovery of vitamin 812 have contributed greatly to the understanding of the nature and biological significance of this substance. Hammond (1944) in an attempt to find a substance which would promote growth in chicks maintained on a vegetable diet, found cow manure, ruuen contents and sardine fish-meal to be effective. Bird, Rubin and Groschke (1948) were successful in concentrat- ing the growth factor present in cow manure. Zucker and Zucker (1948) found a growth factor present in animal protein but absent in plant materials which they called 'Zcopherin' and which later became known as the “animal protein factor”. Nestler, Byerley, Ellis and Titus (1936) showed that the chick growth factor found in cow manure was evidently the same as the factor necessary to maintain the hatchability of eggs when hens were kept on vegetable diets. Ott, Rickes and WOOd (1948) demonstrated that vitamin 812 had growth activity in chicks maintained on the soybean protein diets used by Bird, Rubin, Whitson and Haynes (1946) in studying the “animal protein factor”. Lillie, Denton and Bird (1948) compared vitamin B12, purified cow manure growth factor and liver extract, using the chick growth as a cri- terion. Their data indicated that these factors are very similar if not identical. l9 McGinnis, Hsu and Graham (1948) studied the blood non-protein nitrOgen and body growth in chicks maintained on a vegetable diet. When this diet was supplemented with vitamin-free casein or alpha protein, the animals receiving this diet exhibited an elevated blood non-protein nitrOgen and a decreased growth rate. An alcoholic extract of liver corrected both of these factors. When the chicks were fed the basal ration supplemented with fish meal, blood non- protein nitrOgen was lower and the growth rate higher than when the vegetable ration alone was fed. These authors con- cluded that an unidentified factor present in liver is nec- essary for normal protein metabolism. Charkey, Wilgus, Patton and Gassner (1950) found that vitamin B13 depressed the blood level of non-protein nitrogen and at least seven amino acids when administered to vitamin BIZ-deficient chicks. The increased growth rate and feed utilization efficiency accompanying this depression of circulating levels of amino acids, led these authors to postulate that vitamin B13 affects growth by enhancing the utilization of the circulating amino acids. Rose and Schweigert (1952) determined the desoxyribo- nucleic acid and ribonucleic acid content of tissues from vitamin Blg-sufficient or deficient animals. Their data re— vealed that the livers of animals deficient in vitamin 812 contained less desoxyribonucleic acid and ribonucleic acid 20 on the basis of percentage of fresh weight, dry weight or “total nitrogen than did the livers of the animals receiving vitamin B12' The content of ribonucleic acid and desoxyribo- nucleic acid per cell was the same for the two experimental groups. These results indicate that the decreased rate of nucleic acid synthesis is accompanied by a decreased rate of cell division in the livers of the vitamin BIZ-deficient rats. It is apparent from the literature cited that most authors place great importance on vitamin B12 insofar as it effects protein metabolism. It is of interest to note, how- ever, that Chow (1952) has shown that vitamin BIZ-deficient animals have a low fat, high water but normal protein content and that these abnormalities can be corrected by the admin- istration of vitamin 313. This author and also Bosshardt, Paul and Borne (1950), McCollum and Chow (1950) and Rupp (1951) support the hypothesis that vitamin 812 is involved primarily in the utilization of carbohydrate and synthesis of fat from carbohydrate rather than in protein metabolism. Interactions between Vitamin 212 §3g_Hormones. Ershoff (1947) reported that liver counteracted the retardation of body growth and inhibition of ovarian deve10pment in immature rats fed toxic amounts of thyroid. This author interpreted these findings as indicating that normal body growth and gon- adal development require a substance present in liver. The requirement for this substance is increased by feeding thyroid. 21 Betheil and Lardy (1949) also found vitamin B12 to be active in counteracting retardation of growth in thyroid-treated rats. Ershoff (1949) conducted experiments in which he studied the relative effectiveness of vitamin B12 and liver in inhibiting thyrotoxicity in rats fed a casein-sucrose diet. The results revealed that liver was very active in promoting growth under these conditions while vitamin B12 had no effect at all. Robblee, Nichol, Cravens, Elvehjem, and Halpin (1948) demonstrated that induced hyperthyroidism increased the effective range of the chick assay for an unidentified growth factor present in fish solubles, whole liver powder, and other liver fractions. Meites (1950a) found in rats that thyroid weight and rate of radioactive iodine uptake by the thyroid was not in- fluenced by vitamin 813. These same experiments revealed that vitamin 313 was capable of eliciting an increased body weight gain in hyperthyroid animals. Nichol, Dietrich, Cravens and Elvehjem (1949) showed vitamin B13 to be very similar if not identical to factors assayed by Robblee, Nichol, Cravens, Elvehjem and Halpin (1948). It was shown that the inclusion of iodinated casein in the assay diet was beneficial. These authors state that the thyrotoxicity produced by feeding iodinated casein could be completely counteracted by orally or parenterally administered vitamin B12 . 22 Bolene, Ross and MacVicar (1950) found that greater quantities of vitamin B13 were needed to counteract the thyrotoxic effects of iodinated casein in female than in male rats. In both sexes greater responses were obtainable from liver than vitamin B13. Frost, Fricke and Spruth (1949) reported that the sex of the animal had no effect on the response of the hyperthyroid rat to vitamin B13. Watts, Ross and Whitehair and MacVicar (1951) studied the growth response of normal and castrate hyperthyroid rats to vitamin B13 and liver. Both substances were found to be more effective than liver. These authors postulated that the male sex hormone may act synergistically with a factor present in the liver which is not vitamin B13. It is also possible that the female sex hormones act antagonisti- cally with such a factor. Meites and Shay (1951) have shown that vitamin 312 will partially inhibit the growth retarding effects of di- ethylstilbestrol and thyroprotein. While vitamin 812 in- creased the food intake of these animals, it did not reduce the effectiveness of these two hormones in depressing testes weight or the tendency of diethylstilbestrol to increase the weight of the seminal vesicle. It was suggested that one reason for the effectiveness of vitamin 812 in these experiments was its ability to increase food consumption. 23 Monroe and Turner (1949) presented data indicating that vitamin B13 may increase the catabolism of thyroxine, which could also explain the observations of Meites and Shay (1951). This seems unlikely, however, since the latter work- ers found that vitamin 812 has no effect on normal thyroid function. Meites (1951b) and Libby and Meites (1952) have shown that vitamin 813 counteracts the growthéinhibitory effects of thiouracil in rats and chicks. Vitamin 313 was found not to alter thyroid function in these species. . Wells and Kendall (1940) demonstrated that growth in immature rats is inhibited by large doses of cortisone. Ingle, Higgins and Kendall (1938) showed that a negative nitrogen balance can be induced by cortin treatment. Meites (1952) found that hair growth is greatly retarded when ani- mals are given large doses of cortisone. Since all of these disturbances are largely protein in nature, and may well be due to an increase in protein catabolism it seemed 10gi- cal that the effects of vitamin B13 upon cortisone over- dosage should be studied. Meites (1952) conducted such a study and found that vitamin 812 or aureomycin partially or completely prevented the inhibition of body, hair and thymus growth which results from cortisone treatment. While either substance was effective in cnunteracting these effects of cortisone, a combination of the two was even more effective. Both vitamin B13 and aureomycin elicited an increase in food 24 consumption and increased the efficiency of converting food into body weight gains. Although it is not well understood how vitamin 812 or aureomycin function in producing these changes, the enhancement of food intake and possibly a specific effect on protein metabolism may account for their beneficial effects in hormone-treated animals. The two experiments which are described in this thesis were carried out to determine the effect of vitamin B13 on the cardio—renal manifestations of a DOA overdosage. These experiments were also designed to determine to what extent the saline consumption might influence the actions of DCA and vitamin 312 on kidney and heart, and to ascertain if the action of vitamin B13 was secondary to an increase in food consumption. 25 Experiment I. Relation Between Desoxycorticosterone Acetate and Vitamin_§12‘;n Unilaterally Nephrectomized Rats. Procedure: Forty-seven male rats of the Carworth strain, weighing 51 g 12 grams each, were used in this experiment. These rats were maintained on the following vitamin Biz-deficient diet‘ for thirty-two days. Yellow corn meal 35% Ground wheat 25% Linseed oil meal 10% Soybean oil meal 20% Alfalfa leaf meal 6% Brewers yeast 3% Sodium chloride 1% Immediately following this thirty—two day period the rats were unilaterally nephrectomized and divided into eight groups. Each of these groups was given 1 percent saline to drink and the above mentioned diet supplemented with various quantities of crystalline vitamin B12‘*. Groups number 2, 5, S, 7 and 8 received subcutaneous injections of an aqueous suspension of descxycorticosterone acetate**‘ twice daily, ’ This diet is a modification of a diet used success- fully by Dr. C. A. Hoppert of the Chemistry Dept. at Michigan State College, East Lansing, Michigan. ‘* Crystalline vitamin B13 was kindly furnished by Merck and Company, Inc., Rahway, New Jersey. "* Desoxycorticosterone acetate was kindly supplied by Dr. W. J. Haines of the Upjohn Company, Kalamazoo, Michigan. 26 totaling 3 mg. per rat per day. The experimental treatments for each group are summarized in Table I. Table I Treatments of Rats in Experiment I. W Group No. of Rats DCA (mg. per Vit. B12 (ug. No. per Group Rat per Day) per kg. of diet) 1 10 0 O 3 9 3 O 3 5 O 50 4 5 0 400 5 4 3 50 6 5 3 100 7 5 3 300 8 4 3 400 Each group of rats was kept in a separate cage and the largest groups in two cages, intmder that their daily food and saline consumption could be measured. The rats from each group were weighed every four days to determine their growth rate. At the end of twenty days, all of the animals were sacrificed by severing the spinal cord. These rats were weighed and autopsied. The heart, kidney, adrenals and thymus were removed and weighed on a Roller- Smith balance. The heart, kidney, thymus and duodenum 27 fromgtwo animals of each group were fixed, sectioned, and stained for microsCOpic exannnation. Each animal was also examined at autopsy for any gross path010gy which might exist. The data obtained from this experiment and the subse- quent experiment were analysed statistically using the following formula: 3. E. = .zdz HST-'17 RESULTS: The results of this experiment are summarized in Tables II and III. Table II shows the average body weight gain, saline intake, food consumption and food utilization efficiency of the respective groups. The average kidney, heart, thymus, and adrenal weights are shown in Table III. It is apparent from the data of Table II that the animals receiving vitamin 312 (groups 3-8) grew faster, con- sumed more food and utilized their food more efficiently than did these rats (groups 1 and 2) receiving a diet which was deficient in this vitamin. These results also indicate that DCA greatly increased the saline consumption, especially in those rats (groups 5-8) which were receiving a diet sup- plemented with vitamin B13. The percentage increase in saline consumption was considerably greater than the increase in food intake. The DOA-induced polydypsia (group 2) was marks edly inhibited by a vitamin B13 deficiency. 28 It can be seen from Table III that DCA (groups 2, 5-8) produced a renal and cardiac hypertrophy. This effect was less pronounced in the absence of vitamin 312 (group 2). BOA produced little or no adrenal atrophy (group 2) and had no significant effect on the weight of the thymus gland. The results of the histological study of the kidney, heart, thymus and duodenum revealed that vitamin BIB.had no effect on either the thymus or duodenum, but the renal and myocardial hypertrophy seen in the DOA—treated animals was partially inhibited by a vitamin 313 deficiency. DCA had no demonstrable effect on the thymus or duodenum, except that some evidence of arteriosclerosis in the arterioles of the latter was noted. DCA produced a myocardial hypertrophy and an arteriosclerosis in the small coronary blood vessels. Nephrosclerosis and hyaline casts in the renal tubules were also seen following DCA treatment. Cloudy swelling was characteristic of the kidneys of all the animals receiving DCA. All of these pathological findings have previously been reported by other authors using similar procedures (Selye and Stone, 1943; Rosenberg, Woodbury and Sayers, 1952). Ow 0Q .‘ 1.89: mo .Hoaam .dhmwwamum all. .Qsoam amp mpms mo genes: esp mumOHWCa mammnpcmmma 2H mesam> * peas .ma\mam .eaa .aa woe m.aa m.nH mm as m.eaa ea m.mea ase\nom .ean flea m peas .MM\0Hm .eaa .0: men e.ne o.eH om Ha m.eaa e w_ema ass as .aan “ea e peas .ma\mam .eaa .ne ooH o.na e.HH me e w mas o m_aaa asexaaa ..at flea e peas .ma\mam .pH> .m: on e.em n.ma ooa ma w.oaa ea.w nus ate too .ms flea n peas .ms\mam e.Ha H.eH he as m_eaH ea w.enH .eaa .ae ooe any a pmaw .um\mam n.ea o.eH as n w.ema m m mHH .eae .ae on Ana n n.eH e.a on e w.ona n w.nma asexnoa .msn “my m n.mH H.0H mm m m.mea e.m HNH naeeeae AoHa H .Em UmESmcoo .Em .HE .sw .aw peas no .am mm©\pma\mxmpca hmw\pmn\mmmpcfi assay awapaca .o: ocakuamm .pa .m>w coop .m>¢ m:HHMm .m>fi .p3 Neon .mam pcompmmae macaw oat DOOR mHmm Qt ... 19:29» U 20 ME4.Bm1.HOGIBrOLHBDOUHLw ammHzoemmmmmmz haunmmsrstb as a6He.:nmaoo meutW mmo m0\o 0 EH... 1... .-BH> rmO 80.3th .csma Mo moans oasocopmess .pzmHma moon .m OCH sea .m& CH mpcmHma cmmao pcmmmamma mosam> smacqss .mpanms compo Hoopom pcmmmsmma mosamb momma: eeHe .ma\mHm mH ooH me W one. on W oeo.H a? .ms ooe n m.oe no m.oou Hm emo H Hem ooo.m aae\noo .mao m pmHo .wM\mHm 3 non mH W oHo on W oHH.H .3» .ma oom H w.om on w.non om moo eHm nnH.m ass\noo .oao a . peas .ma\mHm eH oeH a one oe ooH H .eHe .ma oeH - .- . w 1 ) a ( e.w em on m.ccs on moo so eee H aao\noo .mam o pmHo .ma\mam oH emH as w men. on w neoHH .3» .ma on m m.ne mm m.ooa on one H eom ooo m aee\noo .mso o S. 4 EH on w aoe ee W own peas .mifim m m.ma me a new on eon on waH.H .eea .ne ooe e I oH mmH en W was HH W see peas .ma\mHm a x no Hm m.,un om oHe an oom.H .ea» .6: on m 3 SH 2 W woe Ho u oHa H m_Ha oH m.oam m ooo oe v_noo.H aee\too .mso m - oH , omH ea m.ome mm m.eHe as a m_na ma m_omm on m.Hoe aaaoe_w omo.H* nHeaeaoo H .ma .ma .ma .m. . .0: .pa Hmcmaom .pa msamne .pa pammm .ps mmcoHM pmmapmmae macaw meta. Qmm 3...??er winmmaedba: a0 mamawma Ewmo mus zo gramme ma. amamoomaaoowxomma do}? mHm “Sums: a6 meta 31 Experiment II. Relation Between Desoxycorticosterone Acetate and‘Vitamin B13 in Unilaterally Nephrectomized Egpg. Procedure: A second experiment was conducted to determine the reproducibility of the results of experiment I and to fur- ther examine some factors not demonstrable in the data of the first experiment. Forty-five male rats of the Carworth strain, weighing 50 : 10 grams each, were used in this experiment. These rats were maintained on a vitamin BIZ-deficient diet identical with that used in the first experiment, for twenty-eight days. At this time the rats were unilaterally nephrectomized and divided into six experimental groups. Each group received the above mentioned diet with or without vitamin 813. Each group was fed ad libitgm except group 4 which was pair—fed to group 1. All groups were given 1 percent saline to drink except group 3 which received tap water. Each animal in groups number 2, 3, 4, 5 and 6 received subcutaneous injec- tions of an aqueous suspension of descxycorticosterone acetate twice daily, totaling 3 mg. per rat per day. The various treatments of the rats are summarized in Table IV. 32 TABLE IV Treatments of Rats in Experiment II Group No. of rats DCA (mg. per Vit. B13 (ug. No. per group rat per day) Drink per kg. diet) 1 8 O saline 0 2 7 3 saline 0 3 8 3 tap water 0 4 8 3 saline 50 5 7 3 saline 50 6 7 3 saline 20 Each group of rats was kept in a separate cage and their daily consumption of food and drink was noted. These animals were also weighed every four days to determine their growth rate. The animals were maintained under these experi- mental conditions for twenty days. During the last twenty- four hours of this twenty-day experimental period, four rats from each group were placed in separate metabolism cages and the volume of urine excreted was measured. At the end of the twentieth day all of the animals were weighed and then sacrificed by severing their spinal cords. The kidney, heart, adrenals and thymus were removed from each rat and weighed on a Roller-Smith balance. The kidney, heart and a sample of muscle and skin from each aninal was dried 33 at 100° C. to constant weight, and the water content was then determined. RESULTS: Tables V, VI and VII summarize the results of this experiment. Table V shows the average body weight gain, food and water consumption and food utilization efficiency of each of the six groups. The average kidney, heart, thymus and adrenal weights are summarized in Table VI. Table VII shows the average water content of the kidney, heart, muscle and skin of the rats of each group. It is apparent from the data of Table V that vitamin 313 (groups 4-6) increased growth rate, food consumption and feed.utilization efficiency above that of the controls (group 2). These increases, however, were not as marked as in the first experiment. It can be seen by comparing group 2 to group 1 that DCA slightly reduced the growth rate and food.utilisation efficiency, but increased saline consumption. The substitution of tap water (group 3) for saline drink slightly increased food utilization efficiency but greatly decreased water intake. Pair-feeding group 4 with group 1 decreased food.utilization-efficiency and growth rate in comparison to groups 5 and 6 which were allowed to eat ad libitum. It can be seen that the groups receiving vitamin B13 and DCA (groups 4—6) also consumed much greater quantities of saline than did group 2, which received DOA but no vitamin 54 B13. Groups 2 and 4 ate similar quantities of food but group 4 drank much greater volumes of saline, indicating that vitamin B12 increased saline consumption independentof its effects on food consumption. Comparisons of groups 2 and 4 also indicate that vitamin B12 enhances the effects of DCA in increasing saline consumption. The data in Table VI indicate that DCA caused a renal and cardiac hypertrophy, a reduction in adrenal size and no change in thymus weight. The rats (group 3) given tap water instead of saline (group22) exhibited smaller hearts, kidneys and adrenals than did the animals of the latter group. The rats in group 4 whose food intake was restricted, developed less severe cardiac and renal hypertrophies than were seen in the animals of group 5 which were treated similarly bd allowed to eat gg_libitpm. The animals (groups 5 and 6) which received both vitamin B12 and DCA exhibited a more pronounced cardiac hypertrOphy than did the rats which received DCA but no vitamin 813. Kidney weight, however, was not noticeably altered by including vitamin 313 in the diet of DCA treated rats. The data in Table VII show that the water content of the kidney, heart, muscle and skin did not change as a result of DCA administration, vitamin B12 adequacy or the inclusion of salt in the drinking water. The only possible exception to this is the slightly increased water content of the kidney as a result of DCA administration. This is not conclusively 35 demonstrated since group 6, which also received DCA, showed a low renal water content. 5 NC .cmms ho soasm osmocmpm es .osoam pom mama mo popes: map mpsOHocH mHmmnpcoaoa CH mmzam> t peas .ma\mHm .eHa .ma om e.mH o.mH on m m_eeH aim eoH asextoo .men “so o peas .ma\mHm .eHa .me on w.oH m.oH mm a w_eeH n.w mHH aee\eoo .mao Asa o H .oc mooam , , QpHB omm mama peas .mM\ch .pae .eeion m.eH o.eH no o m_HoH o_w oHH asextoo .ann Amv e aeHae _ ampma map m.aH e.oH em e_w noH o w_o0H aeexeoo .mao “mo n m.oH e.eH so o w.ooH o w.oHH aeexeoo .meo “no N m.eH o.eH me o m.moH see w_ooH eHeneeeo *Am H .sm1 omszwmoo .sw .Ha .am .sw peas so .am see\pea\eaeeeH aee\eea\easpea Hesse HeHeHeH .ee ooa\2Hom .pa am>< ooom .wbd popes .mam .ps Hoop .mwm pcmapmoae msoao ‘ _ mama QHNHEOBommmmmz qudmmaquZD a0 ZQHBmmszoo»MZHAwm 924 000m .mekomw zo mememot mzommemooHamooMxome m0\ 02w dam zHasBH> mo Bommmm b 113‘ '— o7 .pgmHma Hoop .am OOH poo .ma :H mmeHoa cameo mpanoB compo Hoopom .cmma mo soaao opmocspm pommmaaoa moSHse aoaoq £11.. ** pcmmmaaoa mmsHmb amass * .. - mHn pmrw . HM\C HH Hem HH w em on. w oHa .2» .ma om a. w oH en w em...” on one eHH Home; 2348 .mao o eeHe .na\mHm mH noH Hm W one. on N ooH.H .pHa .ma 8 e m H... me we new no .8 H mam m omo.H aegnoo .me o H .o: asoaw 0p omm AHHQ eeHe .ae\cm NH an HH u mom on w eoo .eHa .ma om o w om. on non as v oHo mo Hon; seekoo .msn e 2 HmH HH W on- oe W omm safe amass and. H m pH 3 w eon a... Hee Ho oeaH agnoo .65.... e. 3 ooo He W one on u So; ..... m oa 3 m. men 8 Has me We owe; one}? can m 3 EH HH w moe mm w own. as m w. ma mm m mam. oe ooo tion HOH H* 3858 H .wa .ma owe .a .oc .ps Hmcoaom .93 magmas .pz pammm .pz mocon pcmspmmas macaw. mean omnH oeomn sat. adsamaflHao 2 no mane 2 who who ro me So - uaoeaamooanooiommo mo\ 9am eHm ”:59? so somaam H> mqmte eeHe .mm\eem .eHa .ee-om o.oeo o.mse o.ooo m.nee aao\too .eao o peHe .ne\mHH .pH> .Us 0m H.oeo e.nee o.ooe n.oee Han eon .mao o H .oc macaw eeHs see aHea eeHe .na eHm .eHe .ma on )0 \. (.0 \u. {.1 01) 54.. c .94 a nun o one n cos n nee \eoo nae e HCHao pmumz .mu m.eeo n.ese o.mom o.ome aee\eoo .n.o o m.oeo m.nen m.Hom e.omn e\eoo .maw m m.HHo o.oee e.eme o.ooe mHoaeeeo H :me oHomza panes HosJHm .onll nonmme mo .cmleEu pneumoo amps: pcmaummae onaw mane omNH oaomnaama wHuem HH.o no we mHa ao Hamauao.o muses was no maeemo e maor meooOHanoo>xommo mo\ozs meHm ZHmteHs Homaam HH> mHmeH 39 DISCUSSION These experiments were designed primarily to study the interactions between vitamin B13 and descxycorticosterone in unilaterally nephrectomized rate. It is important in interpreting the data obtained from these experiments that the reader keep in mind that all of the animals, with the exception of those in group 3 of experiment II, were given 1 percent saline to drink. This increased sodium consumption undoubtedly altered the animals' salt and water metabolism. Sodium has also been shown to render unilaterally nephrec- tomized rats hypersensitive to DCA toxicity (Selye, Mintz- berg and Henley 1945 ). The results of both experiments indicate that vitamin B13 increased the growth rate of all the groups receiving this substance. This was much more noticeable in the first than in the second experiment. It is possible that the rats in the second experiment had greater initial body reserves of vitamin BIZ and hence were stimulated less when this vitamin was added to the diet. The increase in growth rate is due in part at least to the increased food consumption which resulted when vitamin 812 was included in the diet. When a group of animals (group 4 of experiment II) maintained on a vitamin Blg-sufficient diet was pair fed to a group receiving no vitamin B13, the two groups showed identical 40 growth rates. This would indicate that vitamin B13 was ineffective in stimulating growth in the absence of increased food intake, an observation in agreement with the generally reported actions of vitamin 813 in pigs and fowl. The data from these two pair-fed groups is not absolutely conclusive, however, since the group receiving vitamin B13 also received descxycorticosterone. A second factor contributing to the increase in growth rate of the animals receiving vitamin 312 is the increased food-utilization efficiency. All of the groups receiving vitamin B13 exhibited greater body weight gains per one hundred grams of diet consumed than did any of the groups not receiving this essential dietary factory. This seems to be a very likely possibility when the role of vitamin B13 to protein synthesis is considered (Rose and Schweigert, 1952; Charkey, Wilgus, Patton and Gassner, 1950). Chow (1952) and Rupp (1951), however, have reported findings which indicate that vitamin B13 may be even more effective in influencing fat and carbohydrate metabolism than in stimulat- ing protein synthesis. The increased food-utilization efficiency accompanying vitamin B13 administration can per- haps be partially attributed to the facilitation of certain transmethylation reactions in which this vitamin is known to act as a co-enzyme (Schaffer, Knowles and Salmon, 1951). Therefore, vitamin Blg-sufficient animals are capable of 41 utilizing substances for the synthesis of metabolites which animals deficient in this vitamin cannot use. It thus seeps reasonable that an increased feeding efficiency should result from vitamin B13 administration. Animals receiving quantities of vitamin 813 greater than 50 micrOgrams per kilOgram of diet did not consume more food or grow at a faster rate. This indicates that 50 micrograms of vitamin B13 was sufficient for optimal growth under these experimental conditions. When a group of animals (group 3 of experiment II) was given tap water to drink, they did not deveIOp the severe polydypsia and polyuria seen in the rats given saline to drink. The food consumption of the group receiving the tap water was slightly less than that of the group receiving the saline, but the food utilization efficiency of the ani- mals getting tap water was somewhat greater than that of the rate given saline. The same is true for the contrsls (group 1 of experiment II) which were given saline but no DOA. This group developed a less severe polydypsia than the animals receiving both DCA and saline. The over-all effect of DCA alone or DCA and saline on the organ weights was to produce a renal and cardiac hypertrophy and a slight adrenal atrophy with no discernable effect on the thymus. These results are in agreement with those of Friedman, Friedman and Nakashima (1950) and also 42 Guillemin and Fortier (1951). The increased kidney weight may have been due to DCA-induced hypertension or to a direct renal injury (Grollman, Harrison and Williams, 1940). The greatly increased load of the kidney in these experiments may also account for the renal hypertrOphy seen here. In these experiments the kidney weight did parallel the volume of urine being excreted. The fact that the kidney is capable of responding to an increased load in this manner is evi— denced by the compensatory renal hypertrophy which normally follows unilateral nephrectomy. The cardiac hypertrophy is probably a response to DCA- induced hypertension. Since the procedure employed in this experiment is very similar to that used in producing experimental hypertenakwuit is quite possible that an elevated blood pressure was present in these rats. A cardiac hypertrophy is a normal and sensitive response to hyperten- sion and is preferred by some authors as an index of blood pressure. Although Ingle (1940) has reported a slight thymus atrophy following the administration of DOA, the results of this experiment are inadequate to confirm or contradict this finding. The error inherent in these experiments because of the small number of animals employed, made impossible the demonstration of such small differences. Vitamin B13 seemed to act synergistically with DCA in producing renal and cardiac hypertrOphy. If the cardiac and 43 renal hypertrophy is a growth process involving the metabolic pathways employed in normal body growth, it is possible that vitamin 312 may have facilitated this hypertrOphy while a ' vitamin 813 deficiency retarded the growth of these organs. However, another possibility must be considered. The animals receiving vitamin 812 also consumed and utilized more food. Handler and Bernheim (1950), using hypertensive rats, have shown that the blood pressures of such animals can be greatly reduced by decreasing the caloric intake, and can be even more dramatically reduced by decreasing the dietary protein of these animals. The error in protein metabolism or the decreased food consumption brought about by a vitamin B13 deficiency may be capable of reducing the blood pressure and consequently the renal and cardiac hypertrophy normally seen in hypertension. Since group 4 of experiment II ate the same amount of food as did group 2 of this same experiment, the increased saline consumption of group 4 can be attributed to an effect of vitamin B13 which is independent of food con- sumption. This hypothesis is further substantiated by the fact that group 5 of this eXperiment ate more food than group 4 but consumed the same quantity of saline. The pathological lesions seen in the animals of this experiment were similar to but less frequent and severe than those reported by Selye and Stone (1943). This is not surprising since these workers used much larger doses of DOA 44 and ran experiments of longer duration than reported here. It is conceivable that the rats of the Carworth strain are less sensitive to DCA pathogenesis than the strains of rate used by Selye and Stone (1943), but Rosenberg, Woodbury and Sayers (1952) also reported only little pathogenesis in uni- laterally nephrectomized rats treated with DCA. The errors inherent in quantitating path010gical data with the small number of animals used in these experiments make difficult any exact interpretations of the effects of vitamin BIZ upon DCA-induced histological changes. However, the data from this study point to the probability that vitamin B13 enhanced rather than prevented the deleterious effects of descxycorti- costerone and saline drink on kidney and heart size. These data indicate, therefore, that a deficiency of vitamin B13 may be beneficial for preventing the effects of DCA and saline in enlarging heart and kidney size. 45 SUMMARY Two experiments were conducted to study the interactions between vitamin B13 and descxycorticosterone acetate in unilaterally nephrectomized rats. The animals used in the first experiment were maintained on a vitamin B13 deficient diet for thirtybtwo days and in the second experiment for twenty-eight days prior to unilateral nephrectomy. Following the removal of the left kidney the animals of each experiment were grouped and given rations containing various levels of vitamin 313' Some of the groups of each experiment received subcutaneous injections of 1.5 mg. of DCA twice daily. All of the animals in these experiments, with the exception of one group in experiment II, received 1 percent saline instead of tap water to drink during the period of the experiment following unilateral nephrectomy. The food intake, saline consumption, and growth rates of each group was followed for twenty days. At the end of this twenty-day period the animals were sacrificed and the kidney, heart, thymus and adrenal weights determined. In the first experiment microscopic sections of heart, kidney, thymus and duodenum from two rats of each group were prepared for histologic study. The heart, kidney and a sample of skin and muscle were removed from each rat of the second experiment and dried at 100° C. to determine the water content of these tissues. 46 In the first experiment vitamin B13 increased food con- sumption, foodrutilization efficiency and growth rate. DCA produced a polydypsia and polyuria which was made more severe by vitamin B13 administration. DCA also induced a renal and cardiac hypertrophy which was also more pronounced when vitamin B13 was included in the diet. Although DCA produced a nephrosclerosis and a myocardial hypertrophy, no gross articular pathoIOgy such as swelling of the tarsal joints was seen following DCA treatment. There was no evidence of rheumatic heart lesions following DOA administration. With the exception of renal and cardiac hypertrOphy the effects of DCA on the tissues were not effected by the vitamin B13 adequacy of the diet. In the second experiment, vitamin B13 again increased food consumption, food utilization efficiency and growth rate. These effects were less marked than in the first experiment. This can be at least partially attributed to the animals of the first experiment being maintained on the vitamin BIZ-deficient diet longer prior to begin— ning the experiment than were the rate used in the second experiment. As in the first experiment, DOA induced a polydypsia and polyuria which was made more severe by supplementing the diet with vitamin B13. The increased fluid consumption accompanying DCA treatment was greatly 47 reduced but not completely prevented by giving the animals tap water instead of saline to drink. DCA pro- duced a renal and cardiac hypertrophy. This effect was also less marked in the animals receiving tap water instead of saline to drink. The restriction of food intake had a similar but less marked effect in reducing this cardio—renal manifestation of DCA treatment. As in the first experiment DOA produced a greater increase in heart weight in the animals receiving vitamin B12 than in the animals which were deficient in this vitamin. The DCA- induced renal hypertrophy was not greatly effected by the vitamin B12 level of the diet. This may have been due to more adequate body stores of vitamin B12 in the animals of the second experiment. The water content of the kidney, heart, skin and muscle of the animals of this experiment was not altered appreciably by DCA treatment, by 1% saline or by the vitamin B12 adequacy of the animals. In these experiments DCA was more effective in producing a polydypsia and cardio-renal hypertrophy in animals receiving vitamin B13 than in the animals which were defi- cient in this vitamin.' One reason for this may have been the increased food consumption resulting from vitamin B13 administration. The increase in saline consumption result- ing from including vitamin B12 in the diet of DOA-treated animals, was much greater than the increase in food con- sumption. In the second experiment two DOA-treated groups 48 were given equal quantities of food, while only one of the groups was given vitamin 812° The group receiving vitamin 813 drank more saline and exhibited larger kid— neys and hearts than did the group receiving the vitamin BIZ-deficient ration. These findings indicate that vitamin B12 augments the effect of DCA by increasing saline consumption and this may occur independently of an increase in food consumption. 49 REFERENCES Berk, L., Denny-Brown, D., Finland, M. and Castle, W. 1948. Effectiveness of vitamin B12 in combine system disease. New England Journal gf_Medicine 239:328. Betheil, J. J. and Lardy, H. A. 1949. Comparative effec- tiveness of vitamin B12, whole liver substance and extracts of high APA activity as growth promoting materials for hyperthyroid animals. J, Nutrition 37:495. Bird, H. R., Rubin, M. and Groschke, A. C. 1948. A chick growth factor in cow manure VII. Its stability and solubility. ‘g. Biol. Chem. 174:611. Bird, H. R., Rubin, M., Whitson, D. and Haynes, S. K. 1946. Effectiveness in dietary supplements in increasing hatchability of eggs and viability of prOgeny of hens fed a diet containing a high level of soybean oil meal. Poultry Science 25:289. Bolene, 0., Ross, 0. B. and MacVicar, R. 1950. 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