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UBC Theses and Dissertations

The amelioration of experimental chronic hypertension by vitamin E Rixon, Raymond Harwood 1950

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THE AMELIORATION OF EXPERIMENTAL CHRONIC HYPERTENSION BY YITAMIN E. BY RAYMOND -HARWOOD RECON II So Submitted as Partial Fulfilment In a Major In Biology and Botany for the Degree of Master of Arts In the University of British Columbia. The University of Brit i s h Columbia *T0 A p r i l 1950. ABSTRACT Chronic hypertension was induced i n female albino rats. Amelioration by means of daily feeding of histidine and ascorbic acid, histidine and vitamin E, histidine and urease, vitamin E, urease and ammonium chloride was attempted. Blood pressures were determined under sodium pentathol anesthetic by the indirect method using the foot. Chronic hypertension was produced by two methods, (1) Injections of DCA. This method was found to give inconsistent results. Several modifications of this procedure were tried unsuccessfully. C2) The use of a choline deficient diet. This method proved to give consistent results i n producing chronic hypertension. Vitamin E was the only substance found useful in ameliorating the experimental chronic hypertension. This amelioration resulted only in the presence of excess vitamin IT. This fact was thought t;o give further evidence for the vasodilator properties of vitamin E. An hypothesis was advanced that DCA may inhibit the sympathetic nervous system, to sensitize the vascular responsiveness to pressor and depressor substances. ACKN01LEDGEMENTS \T wish to give acknowledgment to Dr. A. H. Hutchinson, Head of the Department of Biology and Botany, under patronage this thesis was written. To Dr. J . Allardyce, under whose capable guidance this project was undertaken and directed. To Mr. J. Salter, co-worker in the problem, for his unstinting help and collaboration* To Miss L. Gowie, Mr. E. Fung, Mr. ¥. Rivers, Mr. G. Morrison, Mr. P. Jones, fellow research workers, who were a l l very helpful In Innumerable ways during the actual compiling of the data. TABLE OF CONTENTS I. INTRODUCTION 1. A. THE PATHOGENESIS OF HYPERTENSION 2. 1. Role of the Kidney In Hypertension 2. (a) Renin - Angiotonin Pressor System 3.. Weaknesses, of the Renin Theory 4. (b) Effects of Nephrectomy 6. (i) Unilateral Nephrectomy 6. Cil) Bilateral Nephrectomy 8. (c) Hepato-Renal Vasotroplc Factors 9. 2. Role of the Adrenals 10. 5. Neurogenic Significance In Hypertension 13.• B. STATEMENT OF THE PROBLEM OF THIS THESIS 15. 1* Methods used i n the Production of Experimental Chronic Hypertension 15. 2, Methods of Ameliorating Experimental Chronic Hypertension 17. 3. General Delineation of this Thesis 19. IX APPARATUS 20. A. DESCRIPTION OF THE APPARATUS 20. 1. Lighting 20. 2. Microscope 20. 3. Cuff 21. 4. Cuff Pressure Source 21. I l l METHODS 22.. A. THE PROCEDURE OF MEASURING BLOOD PRESSURE 22. 1. Anesthesia 22. 2. Blood Pressure Determination 24. B. METHODS- IN EXPERIMENTAL CHRONIC HYPERTENSION. 25. IT RESULTS 29. ¥ DISCUSSION OF RESULTS 32. A. METHODS _IN PRODUCING EXPERIMENTAL HYPERTENSION 32. 1. Desoxycortlcosterone 32. 2. Choline Deficiency 36. B. AMELIORATION OF HYPERTENSION VXTAMIN E 38. C. CORRELATION INADEQUACIES BETWEEN THE DATA OF ACUTE AND CHRONIC HYPERTENSION 40. D. NEGATIVE RESULTS OBTAINED WITH UREASE AND AMMONIUM CHLORIDE 41. E. SUGGESTIONS FOR FURTHER RESEARCH 42 . VI CONCLUSIONS 44. Yi r SUMMARY 45. LITERATURE CITED 47. ABSTRACT 55. THE AMELIORATION Off EXPERIMENT AX CHRONIC  HYPERTENTION BY ¥ITAMIN E I INTRODUCTION One of the most controversial issues of present day medical research i s hypertension. The enigma of this problem has kept some of our keenest medical minds i n constant research since the year 1934 when Goldblatt showed It was experimentally possible to Induce a hypertensive state. This i n i t s e l f was a major step forward because i t made feasible the f i r s t controllable experiments on hypertension under carefully supervised laboratory condit ions. Previous to this time the amount of work done on this problem, qualitatively and quantitatively, was from a co-ordinated viewpoint, negligible. There were various men l i k e Bright, who precipitated the problem of hyper tension which, even now, i s only p a r t i a l l y resolved. But on the whole investigation Into the problem was the exception rather than the rule. Hypertension was at one time considered a hemodynamic response to aging o f the blood vessels - a f i n a l effort on the part of the body to maintain perfusion of blood through narrowed vessels. But this view has long since been superceded and to-day we firmly believe that elevation of blood pressure i s only a symptom of some preceding disorder and, as such, has numerous causes. This change i n thought coupled with the a b i l i t y to a r t i f i c i a l l y create hypertension has been undoubtedly responsible for the recent acceleration of research. The various ramifications of the problem of hyper tension are now approached from a number of bases. The fi e l d s of Investigation are usually classified as (1) the renal origin of hypertension (£.) the humoral pressor mechanisms C3) the neurogenic origins and C4) endocrine origins of hypertension. These classifications are for convenience only and must not be mistaken for separate and non-overlapping research. But even with this category simplification and corresponding research human hyperten sive disease remains a baffling and mystifying disorder. The more time, thought, work and effort that i s devoted to the problem itfc <R*W£ we discover the complexity of the and- problem^of i t s ultimate etiology. A. THE PATHOGENESIS OF HYPERTENSION 1. ROLE OF THE KIDNEY IN HYPERTENSION There i s no reasonable doubt that some cases of human hypertension are of renal origin. The unsolved problem i s whether many or most of those s t i l l referred to as n essential" are also of renal origin CEO). 3. Essential hypertension has usually been defined as the persistent elevation of systolic and diastolic pressures without accompanying renal disease, and has therefore excluded the renal origin of hypertension by definition. However, numerous mechanisms have arisen which centre about the kidney as the possible Initiator of hypertension. (a) RENIN - ANGIQT.ONIN PRESSOR SYSTEM. The publications of Richard Bright (6) (1827-1836) were the f i r s t to link the role of the kidney and hyper tension, but i t was Tigerstedt and Bergmann In 1898(83), who f i r s t demonstrated a blood pressure raising principle from a saline extract of renal tissue which they called "'renin1*. However, i t was not u n t i l such investigators as Houssay, Plentl, TaguinI, Fasciolo, Braun - Merendez, Page, Munoz, Leloir, Kohlstaedt and Helmer (1957 - 1941) (9, 37, 38, 39, 47, 48, 61,. 62) that the pressor response was found due to a substance they called "angiotonln" or "hypertensln". Angiotonln proved to be the result of a chemical reaction between a pseudaglobulin of the plasma and the substance "renin" found by Tigerstedt and Bergmann. Investigations by the above mentioned authors have revealed a fundamental scheme for the renal pressor mechanism. Renin i s produced by the kidney cozttex, of which the exact site i s unknown, but i t has been associat ed with the juxtaglomerular apparatus and the macula 4. densa of the kidney. Renin has been found to have the property of a proteolylic enzyme (9, 58} and a protein. Renin i s physiologically inert, but possesses the a b i l i t y to break down a substrate compound known preferably as "renin - substrate" (12) or as hypertensinogen, into an active vasopressor material "angiotonin" (or hypertensin). Renin - substrate i s a protein produced by the l i v e r and i s found in the alpha - 2 - globulins of the blood. Renin- substrate has a relatively large molecule, which under the influence of renin i s decomposed to a smaller polypeptide, "angiotonin" Angiotonin i s decomposed by the action of another enzyme "anglotonase" (or hypertensinase). Anglotonase is found in'various tissues namely, kidney, plasma, erythrocytes, and intestinal wall. It i s probable that angiotonin i s destroyed by a number of other enzymes, such as, proteinases of renal and intestinal origin, by amino polypeptidases found In kidney extracts and in blood plasma, by tyrosinase, by carboxylpolypeptidase as well as by oxidizing systems (15). WEAKNESSES OF THE RESIN THEORY. Using the most sensitive methods of detection, some renin has been found during the acute phase of hypertension in experimental animals (30) (Haynes and Dexter 1947), 5. and also in some patients with acute hypertension due to glomerulonephritis and toxemia of pregnancy. In dogs with chronic hypertension of three months to four years duration, no renin has been detected, nor in the systemic blood of patients with chronic hypertension (12) (Corcoran 1948). Renin i s no more commonly present in the renal venous blood of patients with essential hypertension than i t i s in others with no hypertension (Haynes et a l 1947) (31). Failure to demonstrate the presence of renin in hyperten sion might be due to the lack of sensitivity of the methods (12). If this were the case, Leloir (41) raised the question as to how such a small concentration of renin in the blood could lead to such marked hypertension, as i t has been shown that the methods are adequate to show i t s presence when in concentrations sufficient to increase ar t e r i a l pressure in normal animals (12). But Page (51) has found a greater sensitivity to renin injections in hypertensive animals. Verny and Yogt (84), and Brown and Maegraith (8) have also found a greater sensitivity of hypertensive animals to pressor substances such as adrenalin and tyramine. (.55, 59). The phenomena of renin tachyphylaxis i s another weakness in the renin - angiotonln theory. It is, well recognized that i t i s d i f f i c u l t or impossible to maintain increased pressure by infusion of renin for periods of more than a few hours (33, 52, 82). This tachyphylaxis 6 to renin was thought to be due to a disappearance of blood hypertensinogen or renin - substrate. Page (51) has found however, that injection of renin - substrate does not restore the response to renin and has shown that exhaustion of the substrate i s only part of the phenomena of tachyphylaxis (57, 53, 54). (See "Neurogenic Significance"). (b) THE EFFECTS OF NEPHRECTOMY The view that chronic hypertension as observed in the experimental animal, results from the elaboration of a circulating pressor substance has been rather generally accepted. However the experimental data of Grollman and co-workers cited i n the following sections, not only are contrary to this view, but present definite evidence against i t . XI) UNILATERAL NEPHRECTOMY It has been generally assumed that unilateral nephrectomy has no effect on normal animals (23). Although this i s usually true, a certain percentage of animals develope a moderate but definite increase in blood pressure which is evident some weeks following removal of one kidney (28, 25). Similar results have been shown to occur with unilateral operations on the kidney by application of a cloth capsule,figure-of-eight 7 etc. This rise may, however, not be evident for weeks or even months following the operation (Grollman et a l (28,35) ) However, Halpert and Grollman (29), have recently shown that the induction of hypertension as a result of unilateral nephrectomy and unilateral operations i s apparently dependent upon the existance of some lesion in the contralateral kidney. A study of kidneys of rats with chronic hypertension revealed the presence of local lesions not only In the kidneys to which a figure-of-eight had been applied but also similar lesions in the contralateral unoperated kidney. This finding i s cited as evidence that unilateral injury induces hypertension only in the presence of some anatomical lesion In the contralateral kidney. Otherwise, application of the figure-of-eight and removal of the remaining kidney i s necessary to induce chronic hypertension. Grollman (26) has cited these experiments as incompatable with the view that hypertension i s due to the elaboration of a pressor substance by the injured kidney. They are better ex plained by assuming that constriction of the kidney Interferes with a normal function of this organ essential for the maintenance of normal blood pressure. Grollman (25,28), Patton and associates (60) have found that i f the blood pressure of experimental animals i s elevated to hypertensive levels by the application of a figure-of-eight, cellophane wrapping, etc., to one 8 » kidney, the subsequent removal of this kidney f a i l s to restore the blood pressure to normal levels. Also, following the removal of the single remaining kidney in a hypertensive, unilaterally nephrectomized animal, the blood pressure remains elevated for several days, u n t i l a few hours before death of the animal. They state that were the injured kidney responsible for the liberation of a pressor substance, i t s removal should obviously result, in. a return of blood pressure to normal levels. (11) BILATERAL NEPHRECTOMY. The effects of bilateral nephrectomy on the blood pressure i s d i f f i c u l t to determine, since animals deprived of a l l renal tissue succumb from uremia before any effect of the deficiency of renal tissue on the blood pressure can manifest i t s e l f (26). Following the removal of the remaining kidney from a hypertensive animal Grollman (28,25) has found that the blood pressure remains at i t s elevated level, declining only as uremia sets i n during the last days before death. However, the Insufficient time offered by complete nephrectomy has somewhat been overcome by Grollman and Rule (27) by ut i l i z a t i o n of the parabiotic preparation. When one of the parabions was bilater a l l y nephrectomized, this animal became hypertensive, while the blood pressure of the parabiont with intact kidneys remained at a normal level. More recently (Grollman (23) 9 has shown that bilat e r a l l y nephrectomized dogs, maintained by an a r t i f i c i a l kidney, show an elevation of blood pressure to hypertensive levels. These experiments have been stated as evidence for the absence of renal tissue rather than the presence of an abnormal kidney that i s responsible for the developement of hypertension. According to Grollman (24) a l l the experiments cited above are consistent In showing the possibility of induc ing hypertension by methods which reduce the total amount of renal tissue and the possibility of the existence of hypertension In the absence of any renal tissue in the organism. Grollman's alternative hypothesis to explain the role of the kidney In the pathogenesis of hypertension i s to assume that this organ elaborates a humoral agent essential for the well-being of the organism, In the absence of which hypertension results. (c) HEPATO-RENAL VAGOTROPIC FACTORS A pressor-antipressor system has been described by Shorr & Zweifach (79) and other workers (11,86,81,80). Their experiments have shown the regular participation in the hypertensive syndrome i n man and animals of a pair of vagotropic principles of hepatic and renal origin, whose opposite action on the vascular bed are of'"'such a nature that they are said to constitute a circulatory homeostatic system. This system consists of a vasoexcitor substance (VEM) released from the kidney under stress, and which i s 10. apparently neither renin or angiotonln, and a vasodepressor substance (VDM) formed i n the l i v e r . The vasoexcitor apparently acts by increasing the vasomotion of the metaarterloles, and enhancing these vessels to epinephrine. The vasodepressor exerts an opposite effect. VEM has been shown present in the blood of animals during acute experi mental renal hypertension, and both VEM and VDM are present In high, neutralizing concentrations In the chronic stage of experimental renal hypertension. Derangements i n the balance of these factors have been postulated as a possible mechanism in the pathogenesis of hypertension. 2. ROLE OF THE ADRENALS IN HYPERTENSION The only endocrine organ which may possibly play a significant part In hypertension, even i f only a secondary one i s the adrenal system. According to GoldbLatt (20), Best and Taylor (4), there i s no evidence that the adrenal medulla plays a part In hypertension, but recently ffiylon and Heller (46) 1948, have found that "angiotonin" appar ently needs minute quantities of epinephrine, tyrosine and other tissue products to make i t effective. However, there are more definite indications that the adrenal cortex plays a role i n high blood pressure, as i t s secretions have been found essential to the experimental establishment and maintenance of hypertension (56j34). 11 Goldblatt (20) has found that the excision of both adrenals in dogs interferes with the developement of hyper tension due to the constriction of the main renal artery; and unless supportative therapy i s given high blood pressure i s not established. Ogden and associates (49) and Anderson et a l (3) 1944, have shown that excision of the anterior pituitary i n hypertensive rats results i n a f a l l of blood pressure, but becomes normal only i f hypertension i s of less than one month duration. Other workers have found no definite effect (20) . Also Houssayband Bexter (34) and Munozetal (47) have shown that bilateral adrenalectomy, although It has no immediate effect, Is followed by a pro gressive decrease i n the response to intravenous injections of renin. These inhibiting effects of adrenal insufficiency are possibly due to a decreased concentration of renin- substrate, since Lewis and Goldblatt, (42) have shown that the level of renin-substrate i s reduced i n adrenalectomy. The adrenal secretions are postulated as necessary for the production of the substrate. Recently Zweifach and associates (85) 1947, have found that the kidneys of adrenaleetomized rats,.eats and dogs progressively lost the capacity to produce the vaso excitor principal (VEM) which appeared i n the blood of hypertensive animals (See "Role of Kidney"). The adminis tration of desoxycortlcosterone acetate (DCA) restored the capacity of the kidney to produce VEM. They state that the 12. question remains as to whether the withdrawal of the adrenal hormones fromnthe circulation produces a specific lesion in the kidney VEM mechanism, or, whether the general ized cellular dysfunction, characteristic of adrenal insufficiency, i s the factor responsible for the decline in the blood pressure of both normal and hypertensive individ uals. Selye (69,70) has postulated on the basis of his work on the "adaptation syndrome", the hypersecretion of mineralo-corticold hormones of the adrenals as a possible cause of hypertension. Experimentally i t has been demon strated that chronic exposure to non-specific damaging agents produces adrenal cortical enlargement with an over production of cortical hormones and simultaneous nephros clerosis with hypertension in the rat. At present though, there i s no direct evidence indicating that hypersecretion of the animal rs own cortex leads to the developement of high blood pressure (70). However, hypertension has been produced experimentally by Selye and co-workers (68,71,72, 75„?4,75,?6) and others (40,7) by the administration of large amounts of desoxycortieosterone acetate (DCA) particularly when combined with a high salt diet and unilateral nephrectomy, with pathological changes simulating renal hypertension. Recently Selye (701 has found that doses as small as 1 mgm. of DCA per day are sufficient to produce the same results. But l i t t l e evidence exists that 13. injections of cortical extracts or the administration of adrenocorticotropic hormone (ACTH), which mediates Its effects through the adrenal cortex, w i l l produce hyperten sion (16). However, both adrenal cortical extracts and ACTH have been found by Dougherty (16) to exert similar effects on the alteration of the juxtaglomerular apparatus with the similar effects ascribed to DCA, although DCA was found more effective. 3. NEUROGENIC SIGNIFICANCE IN HYPERTENSION. There have always been suspicions that nervous factors were important In the genesis of hypertension, but crucial experimental evidence was lacking, and for a time i t seemed decisive that hypertension was due to a humoral mechanism. However, as w i l l be seen below, a number of investigators'have thrown doubt upon the purely chemical nature of experimental hypertension. Dock. (17) has shown that pithing a hypertensive rabbit results In a drop In.blood pressure. The blood pressure of the pithed rabbit, however, s t i l l Increased by the administration of renin. It was concluded that the mechanism responsible for the maintenance of high blood pressure i s not dependant upon circulating renin and angiotonin, and suggested the participation of the nervous system i n the mechanism of renal hypertension. 14. Similar experiments have been carried out by Page (57) i n which he found sectioning of the brain at various levels, widespread direct injury to the central nervous system, and severe shock;, abolished the pressor response of angiotonln. Even more recently Page and Taylor (57) have shown a further connection between the central nervous and the renal pressor systems. They found that previous administration of tetraethyl ammonium chloride (TEA), an agent which blocks the transmission of impulses through autonomic ganglia, overcame the phenomena of renin tachyphylaxis and augmented the response to angiotonln. TEA augmented the effects of both pressor and depressor substances. Corcoran (IS) has taken this action of TEA to mean some central influence which limits vascular responsiveness. He has postulated two states of tachyphylaxis, the f i r s t resulting from the exhaustion of renin-substrate as seen i n hepatectomized animals, and the other caused by some autonomic influence which Inhibits the action of renin. Evidence has been obtained by Ogden and associates (50), showing that a neurogenic mechanism may be involved in chronic hypertension. From studies on the sensitivity of experimentally hypertensive animals to sympathoparalytic agents such as phenobarbital, yohimbine* and F 933. They considered i t l i k e l y that renal hypertension proceeds i n two phases. (1) The f i r s t phase i s of humoral origin depend ant on excess liberation of renin. This phase i s resistant to depressors. (2) The second phase i s assumed to be one 15. i n which neurogenic influences predominate. In this phase the blood pressure i s depressed more by agents which affect the central nervous or sympathetic ac t i v i t y . These effects have been confirmed by. other workers (63, 65). More recently Corcoran and associates (13,14)1948, have demonstrated the dependance of c l i n i c a l and experi mental renal hypertension on central nervous act i v i t y . They found functional denervation (high spinal and caudal anaesthesia) of the renal vascular system w i l l lower the blood pressure of most hypertensives to normal levels. This response was thought to arise from renal vasodilation. B. STATFJVIENT OF THE PROBLEM OF THIS THESIS. The problem of this thesis consists basically of three parts (l) to learn the efficient use of the apparatus uti l i z e d by the previous workers of this laboratory for the determination of rat blood pressures, namely, the indirect method using the foot. (2) To find a suitable method for the establishment of chronic hypertension in female rats and (3) to find an agent for amelioration of this high blood pressure. 1. METHODS USED IN THE PRODUCTION OF EXPERIMENTAL CHRONIC HYPERTENSION. Success by Semple (77) and Fitch (19) of this 16 laboratory i n producing an acute, transitory hypertension by a 1 mgm injection of desorycortlcosterone acetate (DCA) led to the method of producing chronic high blood pressure i n rats by repeated administration of DCA. However, other methods, including modifications in the use of DCA, were decided upon. These were (1) a starvation diet and sub sequent Injectionswith DCA, (2) Injections of epinephrine and DCA given simultaneously, followed by daily injections of epinephrine. (3) The use of a choline deficient diet, with the possible acceleration of i t s effects by a DCA injection. The f i r s t modification, namely, the use of a star vation diet was i n i t i a t e d by Samuels (66) work on estrogens. He observed that obese women frequently showed irregular menstrual cycles and found evidence that estrogens when i n high concentrations i n the circulating fluids, were absorbed by the fatty tissue, and diffused out when i n low concentra tion i n the circulating f l u i d s . A similar effect was postu lated for desoxycorticosterone, because of i t s si m i l a r i t i e s i n chemical properties. This action would minimize i t s effects to produce hypertension. The second modification was the administration of epinephrine with DCA. This method of raising the blood pressure was thought possible because of the recently dis covered importance of epinephrine in the vasotropic factors of Zweifaeh and Shorr, and In the renal pressor system (See "Role of the Kidney" and "Adrenals" p.9,10 ) 17 The last procedure decided upon for the production of chronic hypertension was the use of a choline deficient diet. Best and Hartroft (5) discovered that hypertension developed i n weanling rats approximately 4 to 7 months after the Initiation of a 5 to 6 day choline deficient diet. Histological sectioning of livers and kidneys of the above rats showed irreversible damage. The kidneys had widespread tubular lesions, which were thought respon sible for the elevation of blood pressure. Because of the limited time at our disposal, DCA was administered along with the diet with hope that i t would accelerate the inchoate hypertension. 2. METHODS OF AMELIORATING . EXPERIMENTAL  CHRONIC. HYPERTENSION. Success by the previous workers of this laboratory (1) In ameliorating acute hypertension with the administra tion of histidine and ascorbic acid, led the way to further investigation i n the use of this amino acid. Since the effects of histidine were studied only on an acute,temporary stage of hypertension, i t was decided to investigate i t s depressor effects on chronic high blood pressure. The possible ameliorating effects of ammonium chloride were also investigated. Histidine was found to have l i t t l e depressor effect without the simultaneous use of excess ascorbic acid, a 18. strong reducing agent. Histidine, therefore, was thought to be converted to histamine i n the body, probably by the action of several strains of B. Coll in the intestines; and the depressor action of histamine was thought to be protected from the oxidative enzyme, "histamlnase", by the reducing powers of ascorbic acid. With this principle i n mind, the replacement of ascorbic acid by some other agent was deemed possible. Two other substances were decided upon, namely: Vitamin E and urease. Vitamin E (tocopherol) has been found essential for numerous phases of metabolism (32) (45). The action of vitamin E in many of these biochemical systems seems to be greatly dependant upon Its powerful antioxidant action. Tocopherol, therefore, was thought to be a suitable re placement for ascorbic acid. The apparent c l i n i c a l success of Shute and associates (78) In vitamin E therapy of many cardiovascular diseases was sufficient to assume that vitamin E may play an Independent role In the amelioration of hypertension. , It has been reported that urease was helpful in the hypertensive stage during pregnancy (2). Because of the possible reducing effect of urease by the-production of ammonia from urea, urease was postulated as a possible agent i n replacing ascorbic acid. It i s well established that an overdosage of desoxycortlcosterone acetate produces nephrosclerosis and hypertension. Biochemical studies of changes precipitated 19. by DCA overdosage i n animals revealed striking disturbances in the electrolytic metabolism and particularly a marked rise in the serum Na/ €L ratio (70,73). The Na/d ratio could be p a r t i a l l y restored to i t ' s normal by the adminis tration of ammonium chloride which at the same time prevents the production of nephrosclerosis and hypertension. It was decided to confirm further this action of ammonium chloride. 3.. THE GENERAL DELINEATION OF THIS THESIS. In light of the foregoing considerations, a formal summary of our problem can be stated as follows: (1) To become proficient i n determining blood pressures of albino rats by the indirect method of using, the foot. (£) To try to induce chronic hypertension by repeated injections of DCA, and to try numerous modifica tions of this procedure, namely; the administration of DCA with a starvation diet, daily Injections of epinephrine, and a choline deficient diet. (3) To discover the ameliorating action of histidine and ascorbic acid, histidine and vitamin E, histidine and urease, Vitamin E, urease, and ammonium chloride on chronic high blood pressure. II APPARATUS The apparatus used for determining the blood pressure of rats i n this investigation u t i l i z e d the indirect foot method. The basis for this, method i s described by G r i f f i t h and Farris and the numerous modifications used have been reported by Allardyce, Semple and Fitch (I), previously of this laboratory. A. DESCRIPTION OF THE APPARATUS. The apparatus has been f u l l y reported by Fitch (1.9), therefore the following sections on the apparatus w i l l be dealt with briefly, and w i l l Include a few modifications decided upon to improve the accuracy of the pressure determinations. A photograph of the apparatus is shown in figure I. 1; LIGHTING The source of light used was from a 100 watt diaphram spotlight. The light was passed through the microscope condenser to the web of the foot under consider ation. The f i e l d was found to be well and regularly illuminated, particularly i f the condenser was regulated to give the most diffuse l i g h t . a. MICROSCOPE The microscope was f i t t e d with a mechanical stage 21. onto which a wooden platform was attached for supporting the rat. There i s an aperature i n the platform coinciding with the passage of ligh t . This aper&ture i s surrounded by plasticine onto which the web of the foot i s spread and fixed to visualize the capillary movement. Because of the supporting arrangement for the web, i t i s only possible to use a low power objective. However, for more accurate observation and less strain on the eyes, the use of a 15 X eye-piece (magnification 150X) was found helpful. 3. CUFF. The making of the cuff i s adequately described by Fitch (19). In this investigation the cuff was made longer than previously reported to ensure complete encircling of the rat thigh. This procedure was found to give more definition i n determining the systolic pressure, and thus more accuracy. 4. CUFF PRESSURE SOURCE. Briefly, the pressure i n the cuff i s obtained through the use of a mercury column. The mercury tube i s attached to the cuff, a pressure guage (aneroid manometer) and a syringe, for aiding the i n i t i a l pressure to the cuff. The mercury column i s so assembled as to be used for fine adjustment• 22 III METHODS The previous work done In this laboratory Investi gated and established two very important problems, namely, finding a safe and effective means of anesthesia and the mastery of the microscopic technique Involved In deter mining accurate blood pressures. The establishment of these results aided greatly i n the development of our own technique. The solution to these two problems i s adequately described by both Semple (77) and Fitch Cl9). •A. THE PROCEDURE FOR MEASURING BLOOD PRESSURE. 1* ANESTHESIA. The anesthesia found most safe and effective by the previous investigators was sodium pentathol (monosodium salt of 5 - ethyl - 5 - (1 - methylbutyl) thlobarbiturle acid), and was the anesthetic used throughout our entire experimental research. Sodium Pentathol i s obtained In ampoules containing .5 gms. Since i t i s necessary to have a fresh solution of pentathol daily for blood pressure determinations, with a 2.5$ solution as the most reliable, i t was found convenient to subdivide the .5gms into five groups of 100 mg., and store i t In a refrigerator i n dry, sterile bottles especially f i t t e d with hypodermic rubber stoppers. When required, 4 ec. of water were added to make up the 2.5% 23 solution, an amount sufficient to do 8 - 10 rats. From the previous work of Semple (77) and Fitch (19) and our own observations, the therapeutic dose for most WisfcfcfrSk was established. Contrary to Fitch and Semple, who did not find the administered dose influenced by such factors as body weight, we did find the dose of sodium pentathol was most dependant upon the factor of body weight. Other factors such as recent meals also effects the dose. The value of the therapeutic dose was well defined by Semple; n that volume of 2±5% sodium pentathol that i s required to completely anesthetize the rat for a period lasting over 30 minutes but not longer than one hour." However, with the development of a more rapid technique, i t was possible to lower the period of anesthesia from 20 - 40 minutes, thus lowering the possible effect of an overdose. Table I shows the therapeutic dose found for most aiHwiasts.. If the rat f a i l s to succumb to the regular dose, i t i s permissable to give only .1 cc more of the anesthetic at that time. I f more than .1 cc of sodium pentathol i s given, the rat usually succumbs to respiratory f a i l u r e . Sodium pentathol i s injected interperitoneally. Care must be taken that the needle of the hypodermic i s in the peritoneal cavity and not i n the abdominal wall or some visceral tissue. The administration of the anesthetic was previously <|tf«*;«^iii&&Wpan assistant to hold the rat, 24. and an operator for tho Injootiono, but with practice an adequate method can be developed fo:ie>k\ch 'ri& \n<§lff ««3f£cjoi|recJ indivi(?rr-"., 2. BLOOD PRESSURE DETERMINATIONS. A rat i s anesthetized and l e f t for about 5 - 1Q minutes. After the anaesthetic has taken effect, the animal i s l a i d face down, and the thigh of the l e f t leg (found most convenient) Is wrapped somewhat firmly with the cuff. Holding the cuff In position, the rat i s l i f t e d onto the adjustable platform and placed In position, namely, with the web to be examined (preferably between digits 4 & 5) spread over the light aperature on the stage and fixed In place with plasticine. The web Is searched under low power for a suitable f i e l d of capillaries with a rapid flow of corpuscles. When the most satisfactory f i e l d has been located, the pressure in the cuff i s raised by means; of the syringe to approximately 110 - 115 mm. of Hg. The pressure i s then raised by the use of the syringe and by allo?jing mercury to run into the U-tube, until the blood flow and movements have been arrested. The pressure i s released, and a second pressure reading i s recorded. The average of these two values is taken as the systolic pressure. It i s necessary that several groups of vessels be observed simultaneously to make sure that the blood flow has ceased throughout the web. 25. B. METHODS IN EXPMIMENTAL CHRONIC HYPERTENSION. RUN I: For the f i r s t run, 11 female rats were chosen, and their normal blood pressures established for 5 consecu tive days. Of the 11 rats, two were set aside as normal controls, the other 9 were then injected intramuscularly with 1 mgm. of desoxycorticosterone (DCA). The day of DCA injections were termed day V . Subsequent readings, starting from day no'* were taken up to day 25. Since the desired chronic hypertension was obtained by one injection of DCA, the rats were divided as follows: GROUP (a) Two rats set aside at the start of the run for normal controls. They received no treatment. GROUP (b) Two rats which received 1 mgm. of DCA, and received no further treatment. GROUP (o) One rat, which received 20 mgm. of histidine and 70 mgm. of ascorbic acid dally in powdered fox chow (Purina Mills) This was; prepared as follows: A quantity of special diet in the form of a mixture consisting of 140 of powdered fox chow, 140 mgm of histidine, and 490 mgm of ascorbic acid was prepared for a seven day period and was divided into 7 equal portions. GROUP (d) One rat, which received 20 mgm of histidine i n powdered fox chow, and 10 mgm of vitamin E in o i l (alpha-tocopherol, British Drug Houses) 26 were added daily. The histidine and powdered fox chow were mixed as described above. GROUP (e) One rat, which was given 10 mgm. of vitamin E daily on powdered fox chow; GROUP (f) One rat, which received 20 mgm of histidine and 25 mgm of urease daily in powdered fox chow. A mixture of powdered chow, 140 mgm of histidine and 175 mgm of urease was pre pared as for group (c) GROUP (g) One rat, given 25 mgm of urease daily in powdered fox chow as described in group (c). GROUP (h) One rat, which received 2$ ammonium chloride (NH4 CI ) daily i n powdered fox chow. Ammonium chloride was prepared with powdered fox chow as described for group (c). A l l special diets were started on day 27. Pressure readings of these groups were taken up to day 48, when the special diets of groups (c) to (h) were stopped. A reading of a l l - r a t s was again taken on day 62, and the diets of only groups (d) and (e) were resumed. Subsequent readings on these two groups were taken on days 71 and 78. The results of this run are shown i n table 2, and figures 3 - 6 . RUN II; Eor this run 12 female rats were selected and their normal blood pressures established as in the previous experiment. Two rats were kept as normal controls and received no treatment. The remaining 10 rats were injected 27* with 1 mgm of DCA on day "o". Failure to produce high blood pressure resulted in further injections of DCA (Imgm.) on days 11 and 16. The pressure readings were continued until day 38. The results are shown in table 3 and figure 7. RUN. III; 10 female rats were chosen and their normal blood pressure determined as before. On day "o" a l l rats were injected with 1 mgm of DCA. Failure to obtain hypertension resulted in a second injection of 1 mgm of DCA on day 12. Pressure readings were taken up to day 32. The results are shown in table 4 and figure 7. RUN IV: The procedure for this run was the same as for runs II and III. DCA (Imgm) injections were given to 12 female rats on days "o", "9", "29f and "52". Blood pressure readings were established up to day 66. The results are shown in table 5 and figure 7. RUN V: Failure of runs II, I II and IV i n establishing hypertension by repeated injections of DCA, resulted in a modification of i t s use (See introduction p.15^. In this run 8 female rats were chosen and their normal blood pressures established for 2 days. Two of these rats were kept for normal controls and received no treatment. The 6 remaining rats were placed on a "starvation diet" consisting of 12 gms of the regular diet (fox chow), an amount found adequate in reducing the weight. This diet was continued for 17 days with intermittent blood pressure 28. readings. "The rats were found to lose an average of 35 gms In this 17 day period. On day 17, the six rats were Injected with limgm of DCA. Subsequent readings were taken up to day 33. The results are shown i n table 6 and figure 8. RUN VI: This run consists of a further modification in the use of DCA i n producing high blood pressure. For this run 5 female rats were obtained and their normal pressures recorded. Two rats were set aside as normal controls and were given no treatment. The remaining 3 rats were i n  jected with '1 mgm. of DCA and .002 mgm. of epinephrine on day M o n . The adrenalin injections were given daily up to day 24. Nine pressure readings were established up to day 24. The results are shown in table 7 and figure 9. RUN VTI: Unsuccessful attempts to obtain hypertension since run I, culminated i n using a choline deficient diet. Five female rats were used. After their normal pressures were established, they were placed on a prepared choline deficient diet (see figure 2) for 9 days starting on day "o", On day 9, 4 of the 5 rats were injected with 1 mgm of DCA. Readings were taken of a l l rats up to day 29. Results of this run are shown in table 8 and figure 10. RUN VIII: This run was composed of 12 female rats. The normal blood pressure of each rat was established for two days. On day n o n a l l rats were placed on a 7 day choline deficient diet (figure 2). Chronic hypertension was established in a l l rats by day 20, and were then divided • Sucrose 67.5% *Salta 5.0% Beef fat 12.0% Gelatin 7.0% Celluflour 2.0% Cod liver o i l 1.0% Oaesin 5.0% "Vitamin mix 1.0% . Fibrin 1.0% Cystine 0.5% * Salts (N.S.O. salt mix No.2) NaCl 4.55% Mgso^ 15.70% NaH2P04 8.72% KzPO^ 25.98% Ga(H2P84) 15.58% Ferric Citrate 2.97% Calcium Lactate52.70% * Vitamin powder/100 gm. Thiamin 500 mg Riboflavin 250 mg Pyridoxine 200 rag Ca Pantothenate 1 gm. Nicotinic acid 1 gm. -added to 997.05 gm of powdered.sugar. |]g$|bj£V Showing the constituents of the choline deficient diet used for the production of chronic high blood pressure. 29. into the following groups. GROUP (a) Two rats used as hypertensive controls and received no further treatment. GROUP (b)- Four rats, which were given 20 mgm of histidine and 70 mgm. of ascorbic acid dally /rat. i n powdered fox chow. The mixture was given as described i n Run I, group (e). GROUP Cc) Six rats, which received 40 mgm of vitamin E in o i l added daily /rat. to powdered fox chow. Both supplemented diets were continued for 13 days. On day 33 the special diets were stopped. The test continued for 39 days. The results are shown in table 9 and figure 11. 17 RESULTS The results of this investigation are li s t e d in tabular and graphic form. Figures 3 - 6 (table 3, Run I ) show a rise of blood pressure of ten female rats injected with 1 mgm of DCA on day no". The blood pressure rose in four days, 170 mm of Hg above normal, to systolic pressures of over 300 mm of Hg. Pressures over 300 mm of Hg could not be recorded by the aneroid manometer. Figure (3) shows the amelior ative effect of histidine and vitamin E and vitamin E 30 supplement. The pressures dropped to an average of 232 mm of Hg with both diets. These diets were stopped in 21 days, after which the hypertensive levels returned to their previous state. The diets were reinstituted on day 62 for 18 days. The pressures again lowered to an average of 215 mm of Hg. Figures 4, 5 and 6 show the negative results of histidine and ascorbic, histidine and urease, urease, and ammonium chloride respectively. Figure 7 (tables -3 - 5) shows the negative response of Runs II, III and IT to 1 mgm injections of DCA. The rats showed no response to DCA injections with the ex ception of Run IV, i n which a temporary r i s e of 25 mm of Hg was observed. Figure 8 shows the negative response of 6 rats to a 17 day starvation diet followed by 1 mgm injections of DCA to raise the blood pressure. Figure 9 shows the negative effects on the blood pressure of 3: female rats after 24 daily injections of adrenalin with an i n i t i a l 1 mgm injection of DCA on day Mo". Figure 10 shows the rise in systolic pressure of 5 rats placed on a choline deficient diet for nine days starting on day "o M. 4 rats were injected on day 9 and their systolic blood pressure rose to an average of 210 mm of Hg on the 14th. day. These 4 rats remained at this level u n t i l day 20. On day 20 the uninfected DCA rat had a pressure of 200mm of Hg. From day 20 to 26 the systolic 31 blood pressure of a l l rats rose to 300 mm of Eg. Figure 11 shows the effects of histidine and ascorbic acid and vitamin E on chronic hypertension produced by a 7 day choline deficient diet. Most of the rats had systolic pressures of over 300 mm of Hg. 2:0 days after the start of the choline deficient diets. ¥itamin E i s shown effective in lowering the blood pressure to an average of 205 mm of Hg. Time in Days •S 3 9 13 16 20 25 27 30 35 38 41 44 48 71 78 a 126 a 129 126 124 13l| 132* 132 * 134 134 132 155 134 132 152 - 1361 154 154 133 15 150 153 15^ 154 156 152 133 135 138 140 - 136 155 134 152 HI 126 122 i4o 230 300 300 300 300 300 500 300 300 500 - 300 300 500 300 151 130 8 145 300 5001300 I 300 300 500 300 c 108 109 108 116 114 116 117 e 125 128 128 f 116 118 120 g 120 122 124 116 118 119 173 212 300 300 300 300 300 500 300 500 300 500 500 500 144 210 300 300 300 300 300 500 300 300 300 £ 260 242 — — — 156 275 500 500 500 500 300 500 300 300 500 S. 280 265 254 148 227 255 300 # £ 246 250 \o 500 o-" 280 215 |T3 ST? 255 235 \ t 300 ST? 284 220 I56 215 288 500 300 300 300 300 300 300 300 154 297 300 500 300 300 300 1 500 500 300 300 148 190 260 300 300 300 300 300 300 500 300 500 300 300 500 500 500 300 500 300 300 |500 1500(500 1500 .BOO Table 2. Showing the results of the daily feeding of hietidine and ascorbic acid(groupo),histidine and vitamin S (group d), vitamin S (group e), histidine and urease (group f ) , urease (group g.), and ammonium chloride (group h) on the chronic hypertension of female rats (Run I) produced by a 1 mgm. injection of desoxycorticosterone acetate (DCA) on day ' 0 ' . (see figures 5-6) * not properly anesthetized. i ; - no reading. ' # died under anesthetic. 5 2 Time in Days 11 I 11 | 1A | 16 ! 145 144 145 £ 146 150] 140 146 • 140 138 16 20 22 26 _5p_J^___28_ 158 155 15* 156 158 15* J 2 145 140 3 144 145 4 145 14^ 5 140 144 6 158 142 7 145 145 8. 150 152 9 145 • 10 155 1>6 11 142 140 12 1>4 I32 I38 «3 o • 1 1*5 8 (0 140 » 155 4 o 140 £ 3 140 1* 155 135 J k j o 125 » 150 13A 132 13* 156 1361 150 140 160 130 o 125 126 *3 135 140 158 142 140 143 o • ' 1 . . . 130 150 130 125 155 155 155152_ 125 140 150 152 0 144 135 5 _ 1 o 150 132 t 136 146 145 142 144 146 130 132 " 138 128 130 156 140 140 a y . o 150 135 • 125 JL35 3 125 # 130 134 I 12ojl25 I 122 132 156 138 142 138 ~ ^ ~ ^ > " 120 115 * 152 L50 13^ 128 150 132 £ * 140 155 142. % 1*° 1 4 6 § £ 150 126 136 155 * & »' 144 140 142 146 140 140 r+ , , • ' 8 150 125 13A 150 131 15* 140 150 138 126 130130 H 5 i 5 0 _ 130 l g 13* 132 136 138 128 126 130 122 128 13A 13* 152 136 155 Table 3 . Showing the reeulte of 1 mgm. (DCA) on the blood preesure of female rata * not properly"anesthetized. # died under anestetic. irtjectione of desoxycorticoaterone acetate (Run II ). (see figure 7)» nam Time in days 8 12 12 § 3 2 1 0 » : ^ ^ = = = ______ , i •„.  1 1 26 130 130 130 125 152 1 34 CD <? 2 126 128 150 3 132 126 130 134 S o «<! o o 5 13* * 132 £ 1 2 8 1 2 5 135 132 3. O H * 4 128 154 126 £ 130 126 I52 150 g a> ft *t a 5 130 132 134 § 134 132 I36 130 § <B 6 132 134 138 S 136 134 152 136 g C*- CD B> C- 7 134 132 128 • 150 126 154 128 £         7 134 152 128 8 155 156 13* 9 138 140 136 10 125 152 128  *i 15* § 134 i o c+  O 3  5     CD ^ 130 126 134 128 £ II 34 * 136 130 «| 0 40 138 142 156; £ 128 126 I30 132 16 19 24 27 52 134 130 152 130 126 130 132 126 127 128 131 134 152 150 126 158 132 128 128 132 129 132 13* B 132 130 136 130 134 130 133 136 152 154 152 134 155 I30 13* 138 13* 138 138 140 155 125 128 122 130 128 Showing the results of 1 mgm. injections of desoxycortico- sterone ( DCA ) on the blood pressure of female rats (Run III) (see figure 7 ) . not properly anesthetized. Time in days 3 1 2 D 0 9 20 25 29 36 40 45 52 56 60 66 138 §" 134 s? CO 01 o o 155 *& 134 % o o 110 h- # 2 o o 118 £ 132 J 1 *1 o o 134 § 158 £ 130 124 128 122 132 128 128 126 124 125 1 S  145 160 <? I65 150 145 <? 158 140 134 125 170 120 140 125 160 o <+ O o  I o § 120 • 120 § 126 g 130 g c+ 160 § 140 130 126 120 130 152 170 Showing the results of 1 mgm, acetate (DCA) on the blood pressure 140 135 138 150 145 135 130 155 140 150 125 128 140 I55 132 160 152 135 135 132 154 155 128 130 150 124 128 injections of of female rats *S 145 142 158 o 1 £ 156 130 128 •1 o S 136 I38 140 ® 120 122 125 p a 140 130 128 3 g" 135 150 132 «* 3 130 128 128 122 125 124 152 125 120 desoxycortico- (Run IV) (see f i g . 7) Time in Days D 0 11 17 17 23 128 B E 150 128 B 127 129 • 129 128 129 B 132 124 CD 130 130 155 158 150 tarve 126 129 132 > 155 152 it ion 134 128 130 3 a 134 125 a 124 128 130 ctior 125 158 155 155 156 *•* 142 15* 152 151 126 156 n 126 128 128 128 155 158 152 > 154 150 a 155 155 ctior 152 126 140 158 159 140 Table 6. Showing the results of a starvation diet (group b ) with subsequent 1 mgm. injections of DCA on the blood pressure of female rats (Run V). (seefigure 8) no reading. 2 1 o 2 5 6 10 13 15 20 24 Time in days 145 145 - 142 140 143 143 142 144 l4o 142 135 158 159 156 135 136 135 134 146 142 140 158 145 142 145 156 145 144 141 144 138 140 159 136 159 142 140 157 138 138 142 135 Table 7. Showing the results of a 1 mgm. injection of desoxycorticosterone acetate (DCA) on day ' 0 ' , with with daily injections of JKBflBfl of epinephrine(adrenalin) (group b) on female rats(Run VI). (see figure 9)   152 130 145 142 142 142 136 158 9 I 9 I 14 16 20 24 27 I 29 a 1J4 132 I 150 135 - 140 1^ 0 205 210 5OO 300 g 142 150 g 185 185 180 190 300 5OO «. * - , - - I J 142 144 js 180 200 195 225 300 500 ; 0 9 1 ;  b I36 13€ » 155 136 §• 205 210 205 215 296 298 o o : b 140 14c 138 142 250 235 245 260 300 300 Table 8. Showing the results of a 9-day choline deficient diet (group a) with a lrngm, injection of DOA on the blood pressure of female rate (Run VII) . (see figure 10). • no reading. a 125 124 - a 1?6 140 - 142 140 o DJ 145 142 £ e 152 150 g> H j 156 138 £ a c 135 «< c 123 126 0 144 142 c 140 144 125 125 c 13S 140 Time in Days 8 16 20 I 20 I 27 29 I 33 3^ *0 126 138 144 I?? 200 300 146 300 J 300 300 - 500 293 - 296 154 l r : 2 215 142 220 125 14C 130 280 II 156 205 300 145 134 21S300 138 153 i?o 295 141 144 210 300 300 300 300 500 300 300 300 CB 500 f t- 1 1 294 P. £ 300 293 290 296 300 300 300 240 220 210 " 230 220 215 200 285 240 225 215 295 245 255 220 270 215 200 190 236 220 210 200 290 Table 9. Shoving the effects of histamine and ascorbic acid (group b), and vitamin E (group c) on the chronic blood pressure of female rats produced by a choline deficient diet(Run VIII). (see figure 11) 30 40 5o fco 7o ?o f o f floi (-meeting of- i)i>^<Jinc o t i J V»/Qtn«T> (L ^ a n d tfi/amin £ trrt « . t i 9 i o < r&te inje<Je<J ob>iH» J>€5o«^cor^cosfcrcme ace/ate. ^]m^tn| on </a»j *o' cwej-aije o f -2r\»is , TiormaJ cortfcvl 0 <jroup(j) l r a l ' / huhJi-nt |jo T*l<)^ ) a r » J V i f e w r x * F (tO-m^r^ daily ¥. DISCUSSION OF RESULTS« A, METHODS IN PRODUCING EXPERIMENTAL  HYPERTENSION. 1. DESOXYCORTICOSTERONE. The use of desoxycortieosterone acetate has shown extremely varied results In the production of chronic hypertension. In the introduction, i t was postulated on the basis of previous work, that repeated Injections of deoxycorticosterone acetate (DCA) would be necessary for the production of chronic high blood pressure. But, as shown by the results of Run I (figures 3 — 6), a very marked hypertension occurred in a l l rats with a single, injection (lmgm) of DCA. Values of 300 mm of Hg and over were recorded. Aside from the ameliorating of blood pressures in two rats with vitamin E therapy, a l l rats remained chronically hypertensive for the remainder of the test which lasted 68 days. Two rats from this run that were histologically sectioned by Logan (43) were s t i l l chronically hypertensive eight months after administration of DCA. These results d i f f e r from Fitch (19) and Semple (77), who obtained only acute, transitory hypertension by the same injection of DCA. Runs II, III, and HT (figure 7) showed even more 33. divergence than the preceding run. No significant rise i n blood pressure occurred with single or repeated injections (1 mgm) of DCA. These latter results were the ones obtained by Salter (64) of this laboratory, who worked primarily with male rats. The negative results of these experiments, however, were more in keeping with the literature, whichii^ fi«k»Jthat very large repeated injections, or implants of DCA are required, with a diet supplement of sodium chloride (see introduction p. 12 ). Although some workers are finding that lower amounts of DCA w i l l produce the same effects, these amounts are s t i l l much higher than the dosage used In this experiment. Histological Inspection of renal tissue taken from animals subjected to overdosage of DCA has revealed that kidney damage i n the form of nephrosclerosis i s necessary for the production of hypertension. This explanation may account for the negative results i n Runs II, III, IV" (figure 7) i n which 34 rats failed to reach hypertensive levels. However, i t i s inadequate to explain the results of Run I, since sectioning of kidney tissue from two rats i n this Run by Logan (43), failed to show any kidney damage, even after hypertension of 8 months duration. It is clearly shown from this evidence that the hypertension produced in Run I was possibly due to some extrarenal mechanism. If i t were possible to repeat the production of hypertension after the method of Run I by DCA injections, certain experiments could be carried out to determine any 34. neurogenic influence, namely, the use of sympatholytic drugs. These have been successfully employed by Ogden and associates ( see introducion p.14 ) in showing the influence of the nervous system in chronic hypertension. The i n a b i l i t y to produce even a marked rise i n blood pressure by the Intramuscular injection of DCA made i t necessary to modify the technique. The modifications were ( l ) , a starvation diet with subsequent injections (1 mgm) of DCA, and (2) the injection of epinephrine and DCA simultaneously, with subsequent daily Injections of epinephrine (see introduction p. 16). As can be seen from figures 8 and 9, 6t>3o& modifications of these experiments proved unsuccessful. However certain factors were established. Mr. Salter (64), a co-worker in these investigations, observed that both the starved rats which received DCA injections and also those which obtained no treatment with DCA, produoed hypertension 3 months after the i n i t i a l starvation, with average pressures of 195 mm of Hg. This evidence points to a possible dietary deficiency as an i n i t i a t o r of high blood pressure. Such a possibility has been suggested by various workers. Calder (10) and Durlacher and Darrow (18) have shown the production of hypertension by a diet deficient i n the heat-stable fraction of the vitamin B complex, which can be reversed by restoring this fraction. However, the involvement of a choline deficiency could be evident here, as w i l l be seen in •35. the following sections. The experiments with epinephrine and DCA may show that desoxycorticosterone plays a role i n counteracting the nervous system. Work: done in conjunction with Mr; Salter (64) (reported i n Mr. Salter's thesis) has shown that DCA injections sensitize rats to the pressor response of epinephrine. The results show that rats increase their sensitivity to the pressor response of epinephrine from 9 to 16 mm of Hg, over a 5 day observational period. But rats that were given daily Injections of 1 mgm of DCA previous to a small amount of epinephrine showed a higher pressor response Increasing from 16 to 32 mm of Hg. over the same observational period. These results might possibly indicate the participation of the nervous system i n this pressor response. Page and Taylor (57) have recently found that injections of tetraethyl ammonium chloride (TM), an agent which blocks sympathetic ganglia, increases the response of both pressor and depressor substances. Among these substances they found a manifold increase In the pressor response to epinephrine and angiotonin and In the depressor response of histamine. Corcoran (12) has taken this action of TEA to mean some central nervous influence which limits vascular responsive ness to pressor substances. DCA may have a similar effect i n the inhibition of this neurogenic mechanism, thus increasing the response to epinephrine. However, more experimental evidence i s needed to j u s t i f y this hypothesis. 36 This w i l l he referred to la t e r . 2. CHOLINE DEFICIENCY. As can be seen from figures 10 and 11 this method, as found by Best and Hartroft (see introduction p. 17 ), was the only definite means of inducing; experimental hypertension. Best and Hartroft found that i n a group of weanling rats which had been subjected to a 5 and 6 day choline deficient diet, hypertension occurred 4 to 7 months later, along with irreversible l i v e r and kidney damage. However figures 15 and II show that In the case of this investigation only a few weeks was necessary to induce hypertension by this method. Specifically in the case of Run VII, 17 days, and Run VIII, 13. days. The mean pressure of the most severe group obtained by Best and Hartroft was 195 mm of Hg, from which we can estimate the severest systolic pressure as being 260 mm of Hg, whereas systolic pressures i n these experiments were consistently above 290 with the majority over 300mm of Hg. However, several factors could possibly account for these differences i n results: (l) The period of deficiency was longer in both t r i a l s ; Run VII having a 9 day choline deficient diet and Run VIII a 7 day deficiency. (2) The use of weanling rats by Best and Hartroft rather than adult rats ( 6 months) as used In this Investigation. Young rats may be more resistant to such changes. (3) The last factor i s that the choline 37 deficient diet, mixed for the purpose was not identical to that used by Best and Hartroft. A comparison w i l l show a lack of "arachin" and "corn o i l " . However i t does not seem l i k e l y that the unsaturated and saturated fatty acid metabolism would enter into the problem for such a short deficiency period. Figure 1G represents the f i r s t attempt to obtain hypertension by a choline deficiency supplemented by DCA. As the graph shows the injection of DCA at the end of the choline deficient diet, raised the blood pressure infive days to a hypertensive level. However, this hypertensive level, an average of 205 mm of Hg, remained constant for 6 days un t i l the blood pressure of the control, which received no DCA, rose to meet this level, at which time the blood pressures of a l l rats rose concomitantly beyond readable levels (300 mm of Hg). The manner in which these hypertensive levels were reached raises a very Interesting point. Is the mechanism which raises the blood pressure in choline deficient rats the same as in choline diet deficient DCA injected rats ? We have two possible Interpretations. If we assume that renal lesions produced by the choline deficienty ini t i a t e s the renal pressor system as suggested by Best and Hartroft, then two possible mechanisms are involved in using DCA. (1) DCA may act on the renal tissue causing an 38. earlier secretion of renin thereby allowing the renin- angiotonin mechanism to come into play more quickly. (2) DCA may function i n inhibiting a neurogenic -tr mechanism as discussed in a previous section, in which i t was noted that the inhibition of this mechanism sensitized the pressor response to angiotonin B. AMELIORATION OF HYPERTENSION WITH  VITAMIN E. Of the various ameliorating agents used in this investigation, only vitamin E or alpha-tocopherol seemed to be of value in chronic hypertension. In Run I (figure 3) rats which had been made hypertensive by a 1 mgm DCA injection, were given vitamin E and vitamin E in combination with histidine (see Introduction p.17-18). In both cases the effects were similar In pa r t i a l l y reducing the blood pressure. The ameliorating effects of the histidine and vitamin E combination were i n the i n i t i a l stages faster, although somewhat more irregular than vitamin E alone, possibly indicating the participation of histamine*. However the f i n a l ameliorating results of the above pro cedure was similar to that obtained by vitamin E, and was considered as being primarily caused by vitamin E therapy. In Run VIII (figure 11), results almost identical with the f i r s t Run were obtained with alpha-tocopherol treat ment on rats made hypertensive by a choline deficiency. 39 Although these latter finds seem more effective, i t i s explanable on the basis of a higher dosage of alpha- tocopherol. The cessation of the vitamin E supplement re established the hypertension to i t s former level. This fact shows very clearly that the presence of Vitamin E i n excess amounts is needed for amelioration. To further substantiate this fact the special diets of the rats in Run I (figure 3) were reinstltuted with previously noted results. This singular effect of vitamin E indicates i t s independent action, particularly If the hypertension induced by a choline deficiency and DCA are assumed to be different. Recently Shute and associates (45) 1949, have shown a similar ameliorating effect by vitamin E therapy on hypertensive patients.. They state that a l l their evidence indicates vitamin E i s a capillary dilator. However, they have found that alpha-tocopherol does not always show ameliorative action, and have come to the conclusion that some hypertensives have constriction higher i n the vascular tree, perhaps at arterialar levels. They have found some evidence that estrogens may act as arteriolar dilators. In this respect, combinations of alpha-toco- pherol and estrogens have proved useful. From the results of our experiments this dilatory action of vitamin E seems quite probable, Also, the 40; presence of arteriolar constriction could very possibly account for the partial amelioration effects of vitamin E on these chronically hypertensive rats. It would therefore be of interest to find the effects of combined vitamin E and estrogens. Mr. Salter (64) of this laboratory, however has found estrogen injections capable of inducing high blood pressure. Nevertheless McGrath and Hermann (44) and Kaiser (36) have found prolonged and considerable dilation of capillaries resulting from intra muscular injections of estrogen. C. CORRELATION INADEQUACIES BETWEEN THE DATA  OF ACUTE AND CHRONIC HYPERTENSION. Attempts to ameliorate chronic hypertensions on the whole, have been unsuccessful. Fitch (19) and Semple (77), who worked exclusively on acute hypertension had definite ameliorative success. These workers found that histidine and ascorbic acid had a definite effect on lowering the systolic pressure of acute hypertension; In comparison, these substances were found to be ineffective i n chronically hypertensive rats (Run I and VIII; figures 3 and 11) of a DCA and choline deficiency origin. The explanation probably l i e s i n the basic dissimilarities between these two forms of hypertension. 41. D. NEGATIVE RESULTS OBTAINED WITH UREASE AND  AMMONIUM CHLORIDE. The Individual effects of urease and i t s effective replacement of ascorbic acid in combination with histidine (see introduction p. 18) has been found unsuccessful in ameliorating DCA - chronically hypertensive rats (Run I, figure 5). The use of histidine and ascorbic acid was found ineffective in the chronic stage but successful in ameliorating the acute stage of hypertension; therefore the i n a b i l i t y of urease in replacing the reducing action of ascorbic acid and i t s singular effect in ameliorating acute hypertension i s yet to be determined. Ammonium chloride was also unsuccessful in ameliorating chronic hypertension produced by a DCA injection (Run I, figure 6). The literature finds that acidifying salts, such as ammonium chloride did lower the blood pressure of hyper tension produced by larger doses of DCA. This hyperten sion, however, was always accompanied by nephrosclerosis. The effects of ammonium chloride are attributed to i t s balancing effects on the sodium metabolism (see introduction p.18). Since the DCA - chronic hyperten sion of Run I (figure 6) no such Kidney damage was observed (Logan 43), therefore i t would seem that sodium metabolism was not involved in this type of experimental hypertension. E. SUGGESTIONS FOR FURTHER RESEARCH. Examination of the results of this investigation and their subsequent discussion, fsfeM&es- the following suggestions for further research: 1, The use of vitamin E In ameliorating hyper tension needs to be investigated further, fa) Experiments could be conducted to discover the effects of vitamin E i n preventing the production of experimental hypertension, and i t s ameliorative effects In acute high blood pressure. (b) To discover the maximum ameliorating action of vitamin E by prolonged therapy on hypertension. (c) To discover the effectivness of combined estrogens and vitamin E therapy on chronic hypertension. Cd) To discover further the vasodilator action of vitamin E, by testing i t s a b i l i t y to lower the blood pressure of normal rats. Such i n  formation would add further confirmation to this action. 2. The effect of histidine and ascorbic acid has been found ineffective in chronic hypertension and a comparable 43 situation arises in the replacement of ascorbic acid by urease. However, histidine and ascorbic acid were successful i n ameliorating acute hypertensive. An experiment could then be carried out -to establish the effectiveness of histidine and urease in acute high blood pressure. 3. To investigate the pos s i b i l i t y of a dual mechanism between acute and chronic hypertension produced by (l) a choline deficienty and (2) DCA injections. Such experi ments could be carried out by the use of sympatholytic drugs such as phenobarbitol and yohimbine. This procedure would possibly determine the influence of the nervous system on the problem, particularly in chronic hypertension. 4. Evidence in the effect of DCA i n Inhibiting the nervous system, as previously discussed, could be obtained i n paralleling the response of pressor and depressor substances In rats singularly injected with either DCA and TEA (tetraethyl ammonium). The pressure response of angiotonln and adrenalin, and the depressor action of histamine might be determined. VI CONCLUSIONS. 1. The production of experimental hypertension by 1 mgm injections of desoxycorticosterone acetate has been found to be unreliable in female albino rats. 2. A choline deficient diet was found dependable in producing severe hypertension in adult female albino rats. 5. The daily feeding of vitamin E to female rats was shown to be p a r t i a l l y effective i n ameliorating experi mental hypertension caused by both desoxycorticosterone acetate injections and a choline deficient diet. 4. The part i a l amelioration of hypertension resulted only in the presence of excess vitamin E. This fact was thought to give further evidence to the vasodilator properties of vitamin E. 5. The feeding of histidine in combination with ascorbic acid and with urease were ineffective i n ameliorating chronic hypertension produced by a choline deficiency and DCA injections. 6» The feeding of urease and ammonium chloride individually have been shown i n effective In DCA - chronic hypertensive rats. The hypothesis was advanced that DCA may Inhibit the sympathetic nervous system to explain the Increased pressor response of epinephrine In rats Injected with desoxycorticosterone acetate. 45. SUMMARY This Investigation deals with the possibility: of ameliorating chronically hypertensive female rats by means of the daily feeding of histidine and ascorbic acid, histidine and vitamin E, histidine and urease, vitamin E, urease, and ammonium chloride. The apparatus used in determing the blood pressures was the indirect method using the foot, of G r i f f i t h and Farris, as modified by Allardyce, Fitch and Semple. Sodium pentathol was used as anesthetic. Two methods were used to Induce chronic hypertension Cl) desoxycorticosterone acetate (1 mgm) injections. This method was found to give very diverse results, and was disregarded as unsatisfactory. Modifications of this method were used involving a starvation diet and epinephrine injections. Both of these modifications failed to produce hypertension. fS) The feeding of a choline deficient diet. This method proved consistant i n producing a severe hypertensive state. A DGA injection was tried with the purpose of Initiating an earlier hypertensive state. Vitamin E was the only substance found useful in ameliorating the experimental chronic hypertension. This amelioration resulted In the presence of excess vitamin E. This fact was thought to give further evidence for the vasodilator properties of vitamin E. An hypothesis was advanced that DCA may inhibit the sympathetic nervous system, to sensitize the vascular responsiveness to pressor and depressor substances. 47. LIT.ERATPRE CUED. 1. Allardyce J., F. Fitch, R. Semple, 1948 Trans. Royal Soc. Can. 42: 25 - 35. 2. Allardyce T.» personal communication. 3. Anderson E., E.W.Page, d H. L i , and E. Ogden, 1944 Am. J. Physiol. 141, 393 - 96. 4; Best C.H., and N.B. Taylor, 1950 Physiological Basis of Practical Medicine P /Li 5. Best C.H., and W. S. Hartroft, 1949 Federation Proc. 8: 610-617. 6. Bright, Richard - cited from conference on Experimental Hypertension,N.Y.Acad Scl, 3. 1^47 7. Briskin H. L., F.R.Stokes, C. I. Reed and Mrasek, R.G. - Am. J. Physiol. 1943 138: 385 - 90. 8. Brown G.M., and B.G. Macgraith, 1941, J. Physiol 99: 304. 9. Braun - Merendez, E., J.C. Fasciolo, L.F. Leloir, J.M.Munoz, Am. J. Physiol. 1940: 98: 2:83. 10. Calder R.M., J . 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