EFFECT OF DIET AND CONCENTRATION ‘ 0F NaCl IN DRINKING SOLUTIONS 0N BLOOD PRESSURE, PULSE RATE , SODIUM AND POTASSIUM CONTENT OF SELECTED TISSUES IN RATS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY CHRISTINE GUSTARA 'BEEBE 1975 ABSTRACT EFFECT OF DIET AND CONCENTRATION OF NhCI IN DRINKING SOLUTIONS ON BLOOD PRESSURE, PULSE RATE SODIUM.AND POTASSIUM CONTENT OF SELECTED TISSUES IN RATS By Christine Gustara Beebe There is evidence that the type of dietary carbohydrate ingested by rats may alter blood pressure (Hall g;_;;, 1966, Yudkin 1972). There is also evidence in a number of studies of a positive blood pressure elevating effect of sodium chloride in rats fed chowhtype diets (Dahl I961, 1967, 1972, Sapirstein g§_§;. I950, Lenel 2£_§;. I948). The purpose of the present study was to examine the effect of various diets on blood pressure and pulse rate and to note any effect of a salt-diet interaction on these parameters. In addition ex- amination of growth rate, specific tissue size and tissue electrolyte concentration was determined in an effort to explain any blood pressure changes observed. Sixty-four male Osborne Mendel rats were divided into # groups of 16 animals each. Each group was fed 1 of u diets consisting of primarily grain, fat (51.7%), sucrose (67%) or cornstarch (67%). The diets were allowed ad libi- tum beginning at 21 days of age for a period of 10 weeks. (13>;“‘” Christine Gustara Beebe \\ Each group of rats was further divided into 2 groups of 8 rats each and offered a drinking solution of either 2% NaCl and water or distilled deionized water. Mean systolic blood pressure and pulse rates were recorded weekly for the 10 week feeding period. At the end of the feeding period the animals were sacrificed for body composition analysis and determination of tissue electrolyte concentration. In a second experiment, 24 rats were divided into h groups of 6 rats each and fed 1 of the k diets used in the previous experiment. The rats were offered a 1% NaCl and water drinking solution for 9 weeks post-weaning followed by a 1.5% NaCl solution for 9 additional weeks. Mean systolic blood pressure and pulse rates were recorded weekly until the rats were sacrificed after 18 weeks of feeding. Total diet consumption expressed as kcal. was nearly equivalent in rats given distilled deionized water regard- less of the type of diet fed. Total kcal. consumption was less in rats allowed the 2% NaCl solution irrespective of the type of diet fed. This decrease in food consumption ranged from 20% in grain-fed rats to 58% in rats fed the high sucrose diet. Total fluid consumption was similar in rats given dis- tilled deionized water regardless of diet, in contrast how- ever, consumption increased 3-fold in rats given the 2% NaCl solution. Rats given the 1-1.5% NaCl solution con- sumed approximately the same quantity of fluid regardless of the type of diet fed. When total sodium intake was Christine Gustara Beebe calculated relative to body weight, sodium intake of rats given the distilled deionized water was similar and ranged from 1.7 to 2.1 gm/1OO gm body weight regardless of the type of diet fed. In contrast, rats given the 2% NaCl drinking solution exhibited greater variability in sodium intake ranging from 26.5 gm/IOO gm body weight in rats fed the high fat diet to 53.# gm/IOO gm body weight in rats fed the high sucrose diet. Rats fed either grain or the high cornstarch diet consumed 32.9 and 29.9 gm/IOO gm body weight, respectively. In general, growth rates for all rats reflected the total amount of energy consumed. Rats given the 2% NaCl solution had very low body weights when compared to rats given distilled deionized water. In contrast, rats allowed the 1-1.5% NaCl drinking solution did not appear signifi- cantly stunted in rate of growth. Body composition revealed that rats given the 2% NaCl solution had very little body fat (6-9%) regardless of the diet fed. Rats given the distilled deionized water con- tained larger and more variable amounts of fat; i.e., 9%, 2#%, 20% and 13% for rats fed grain, high fat, high sucrose and high cornstarch diets, respectively. Total body water remained similar in rats given either distilled deionized water or the 2% NaCl drinking solution irrespective of diet. Final mean blood pressures were elevated to hyper- tensive levels in rats given the 2% NaCl solution and fed the high fat, high sucrose or high cornstarch diets (15h, Christine Gustara Beebe 178 and 150 mmHg respectively). Blood pressure was elevated in rats fed the grain diet (12? mmHg), however, not to the hypertensive level. Rats given the distilled deionized water remained normotensive. However, rats fed the high sucrose diet and givendistilled deionized water had sig- nificantly higher blood pressures than rats fed the grain diet (P<.01). A similar trend was observed in rats offered the l-l.5% NaCl drinking solution, i.e., blood pressures .remained within the normal range for rats fed grain, high fat and high cornstarch diets but were elevated to the range for hypertension in rats fed the high sucrose diet. Pulse rates were variable in rats given the 2%.Na01 solution. Regardless of the type of drinking solution con- sumed, the pattern of pulse rate over time was similar in all groups of rats, i.e., rats fed the high sucrose diet maintained the highest pulse rate. Relative to body weight, the heart, right kidney and right adrenal gland were hypertrophied in those rats offered the 2% NaCl drinking solution. The greatest amount of hyper- trOphy of the heart and kidney was observed in rats fed the high sucrose diet whereas adrenal hypertrophy was greatest in rats fed the high fat diet. Sodium and potassium concentrations in the serum and heart remained constant in all animals regardless of the type of diet or drinking solution. Concentration of sodium in the kidney increased with increase in sodium intake and therefore was largest in rats offered the 2% NaCl drinking Christine Gustara Beebe solution. Concentration of potassium remained unchanged in the kidney. The bone of the rat appears to be a labile source of sodium since sodium concentration in the femur increased markedly above normal normal values of 139-156 meq per gram of dry fat-free bone (57-236%) as sodium intake reltive to body weight increased. For the most part, con- centration of potassium in the femur decreased as sodium concentration increased. EFFECT OF DIET AND CONCENTRATION OF NaCl IN DRINKING SOLUTIONS ON BLOOD PRESSURE, PULSE RATE SODIUM AND POTASSIUM CONTENT OF SELECTED TISSUES IN RATS By Christine Gustara Beebe A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1 975 To my husband Dick and my son Nathan 11 ACKNOWLEDGEMENTS My appreciation and gratitude is extended to the fol- lowing individuals who contributed to this study: My academic advisor Dr. Rachel Schemmel for guidance throughout the course of the study. My committee composed of Dr. Schemmel, Dr. Olaf Mick- elsen, Dr. Wanda Chenoweth and Dr. Rudy Bernard for their time and advice. Dr. Duane Ullrey for use of his laboratory facilities. Dr. Pao Ku, laboratory technician for Dr. Ullrey for his time and patient assistance in instructing me as to the laboratory technique for wet ashing tissue. Janet Grommet for assisting with dissection of the rats. Mr. Larry Bowdre, Assistant Museum Curator for use of the Dermested Beetle colony. Dick and Nathan, my neglected family who proded me along when I needed it. National Institute of Health Traineeship #GMO 1818 and Michigan Agricultural Experiment Station for financial support. 111 TABLE OF CONTENTS Page DEDICATION . . . . . . . . . . . . . . . ii ACKNOWLEDGMENTS . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . ix REVIEW OF LITERATURE . . . . . . . . . . . . 1 Hypertension--Implication8 to Héalth e e e e e 1 Cardiovascular Risk Factor . . . . . . . . 1 Defining Hypertension . . . . . . . . . 2 Classifying Hypertension . . . . . . . . 3 Age and Hypertensive Disease . . . . . . . 5 Ob881ty and Hypertension e e e e e e e e 6 Diet and Essential Hypertension . . . . . . . 7 Alteration in Fat, Protein and Carbohydrate . . 7 Salt and Essential Hypertension e e e e e e e 9 Salt APPGtite e e e e e e e e e e e e 9 Human Hypertension . . . . . . 11 Salt in Experimental Hypertension . . . . . 12 Sodium. Potassium Ratio in Experimental Hypertension e e e e e e e 14 Salt and Renal Mass in Experimental Hypertension . a e e e e e 15 Possible Mechanisms for Salt-Induced Hypertension e e e e e e e e e e e e 17 Blood Volume Theory . . . . . . . 17 Imbalance of Electrolytes Theory . . . . . . 18 Electrolytes in Tissues of Hypertensives . . . . 19 Total Body Sodium e e e e e e e e e e e 19 Tissue SOdium e e e e e e e e 20 Na: K Ratio and Electrolyte Concentration» . . . 21 INTRODUCTION . . . . . . e e e e . e e e 23 MEI-‘HOD O O O O O O O O O O O O O O O O 2 6 Experimental Design . . . . . . . . . . . 26 Experiment I e e e e e e e e e e e e 26 Experiment II e e e e e e e e e e e 28 Experimental Conditions . . . . . . . . . 28 Blood Pressure and Pulse Rate Measurement Procedure for Sacrificing Rats and Removing Organs and Fat Depots Body Composition Analyses . . . . . MOiBture Content 0 a o a o o e e BOdy Fat and Lean e e e e e Preparation of Samples for Flame Emmission SpectroPhotometry e e e e e e e Serum . e e e e e e e e Heart and Kidney . . . . . . . . Bone e e e e e e e e e e 0 Diet Rations e e e e Flame Emmission Spectrophotometry . . Analyses Of Data e e e e e e e 0 RESULTS AND DISCUSSION 0 . e e e e e 0 Experiment I . . . . . . Diet and Kcal. Consumption . . . . . Fluid and Sodium Consumption . . . . Growth Rates 0 e e e e e e e e e BlOOd Pressure e e e e e e e e e PUlSe Rate a e e e e e e e e a Body Composition 9 e e e e e e e BOdy Fat e e e e e e e e e Lean BOdy Mass e e e e e e e e BOdy Water 0 e e e e e e e e 0 Organ Weights e e e e e e e e a Heart e e e e e e e e e e e Kidney e e e e e e e e e e e Adrenal Gland . . . . . . . . . Sodium and Potassium Concentration . . serum e e e e e e e e e e 0 Heart a e e e e e e e e e e Kidney e e e e e e e e e e 0 Bone e e e e e e e e e m 0 Experiment II e e e e 0 Diet, Kcal. and Sodium Consumption . . Growth Rate . . . . . . . . . PU159 Rate e e e e e e e e 0 Blood Pressure . . . . . . . . CONCLUSIONS . . . . e e e . e . SUGGESTIONS FOR FURTHER STUDY . . . . 0 Sodium Chloride Absorption . . Concentration of Sodium in Kidney and Bone Fructose and Blood Pressure . . . . . Appetite Depression . . . . . . . 100 100 101 101 Page LITERATURE CITED . . . . . . . . . . . . . 103 APPENDICES O O O O O O O O O O O O O O O 1 10 vi Table I. 2. 3. 1+. 5. 6. 7. 8. LIST OF TABLES Composition of rations . . . . . . . . . Composition of grain ration . . . . . ». . Cumulative diet and fluid intakes of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water or 2% NaCl for 10 weeks . . . . . . . . Cumulative sodium intake of male Osborne Mendel rats fed either a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water or 2% NaCl for 10 weeks e e e e e e e 0 Final blood pressure and pulse rate of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in con- junction with a drinking solution of distilled deionized water or 2% NaCl for 10 weeks . . . Body weight and body composition of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water or 2% NaCl for 10 weeks . . . . . . . . Heart, right kidney and right adrenal gland weights for male Osborne Mendel rats fed a grain1 high fat{ high sucrose or high cornstarch diet n conjunc ion with a drinking solution of distilled deionized water or 2% NaCl for 10 '8 elm O O O O O O O O O O O O O 0 Sodium content of serum, heart, right kidney and right hind femur of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water or 2% NaCl for 10 weeks . . . . . . . . . . vii 75 76 77 78 79 80 81 82 Table Page 9. Potassium content of serum, heart, right kidney and right hind femur of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water or 2%1NaCl for 10 weeks . . . . . . 83 10. Cumulative dietary and fluid intake of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a 1-1.5% NaCl drinking solution for 18 weeks e e e e e e e e e e e e e 9 8O 11. Cumulative sodium intake of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a 1-1.5% NaCl drinking solution for 18 weeks . . . . 85 12. Multivariate Analyses of Variance for Repeated Measures--effect of time, salt and diet on FUlSC rate 0 e e e e e e e e e e e 86 13. Multivariate Analyses of Variance for Repeated Measures--effect of time, salt and diet on b100d pressure e e e e e e e e e e e 87 viii LIST OF FIGURES Figure Page I. 2. 3. A. 5. 6. 7. 8. Blood pressure and pulse rate apparatus . . . 88 Mean body weight of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water for 10 weeks post- weaning e e e e e e e e e e e e e 89 Mean body weight of male Osborne Mendel rats fed a grain, high fat, high sucrose or high corn- starch diet in conjunction with a 2% NaCl drinking solution for 10 weeks post-weaning . 90 Mean systolic blood pressure of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water for 10 weeks post-weaning . . . . . . . 91 Mean systolic blood pressure of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a 2% NaCl drinking solution for 10 weeks post- weaning............. 92 Mean pulse rates of male Osborne Mendel rats fed a grain, high fat, high sucrose or high corn- starch diet in conjunction with a drinking solution of distilled deionized water for 10 weeks post-weaning . e e e e e e e e e 93 Mean pulse rates of male Osborne Mendel rats fed a grain, high fat, high sucrose or high corn- starch diet in conjunction with a 2% NaCl drinking solution for 10 weeks post-weaning . 9A Mean body weight of male Osborne Mendel rats fed a grain, high fat, high sucrose or high corn- starch diet in conjunction with a 1-1. %.NaCl drinking solution for 18 weeks post-weaning . 95 ix Figure Page 9. Mean pulse rates of male Osborne Mendel rats fed a grain, high fat, high sucrose or high corn- starch diet in conjunction with a 1-1.3% NaCl drinking solution for 18 weeks post-weaning . 96 10. Mean systolic blood pressure of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a 1-1.5% NaCl drinking solution for 18 weeks p08t'weaning e e e e e e e e e e ~e e 97 REVIEW OF LITERATURE Hypertensign-Implications to Health Cardiovascular Risk Factor Elevated blood pressure is one of the major risk factors contributing to cardiovascular disease. Hyper- tension, as it is commonly called, is a result of various known or unknown factors which increase cardiac output or more often increase periferal resistance in the circulation (Ganong 1971). Regardless of how hypertension is initiated the heart must work harder for each volume of blood pumped through the system. Therefore, any disease or complication in the heart has more severe consequences in hypertensive individuals. Hypertensives are also predisposed to thrombosis of cerebral vessels and cerebral hemorrhage (Ganong 1971, McMahon 1973). In addition renovascular disease (Leding- ham 1971) and premature onset of atherosclerosis (Brest and Meyer 1967) are greatly increased in hypertension. Statistics have dramatically highlighted the earlier mortality and increased morbidity of individuals with elevated blood pressure as compared to individuals with normal blood pressure levels. Actuarial survival rates published by life insurance companies (Actuarial Society of America 19AO and 1941) indicate that at any age in the adult I 2 the risk to life from the complications of high blood pres- sure increases steadily as pressure exceeds the mean of the papulation. Defining Hypertension The term hypertension has been difficult to define since a temporary rise in blood pressure can occur under a variety of situations such as physical and emotional stress. Customarily the term applies to the situation of an increase in mean arterial blood pressure in a patient at rest in both mind and body (Ledingham 1971). Herein lies a problem since it is difficult to determine when an individual is at rest in mind. Hypertension has also been defined as a level of blood pressure in an individual which lies 2, 3 or more standard deviations above the mean blood pressure in the population from which the individual is drawn, allowing for age and sex (Ledingham 1971). This definition appears illogical since the pOpulation as a whole, due to heredity or environment, may have a higher blood pressure than other populations. Various limits of normal blood pressure have been established by various individuals in numerous investiga- tions. Dahl (1972) measured blood pressure of employees at Brookhaven National Laboratories and established 140 mmHg systolic and 90 mmHg diastolic pressure as the upper limits for normal blood pressure in the subjects. A study of hypertensives in the inner city of New Orleans (McMahon 1973) defined hypertension as a pressure greater than 150/90 mmHg. 3 A blood pressure of 160/95 mmHg has been used in some in- stances to indicate hypertension (Chiang g§_§;, 1969). Studies using rats in developing experimental hyper- tension have defined hypertension as a systolic pressure of 150 mmHg (Molteni and Brownie 1972), 140 mmHg (Dahl 1961, Goldman.§t_g;. 1972), or 156 mmHg (Sapirstien.gt_gl. 1950). Obviously, the frequency of hypertension in a given population is dependent upon the level of blood pressure one chooses to define as the line between "normal" and "ab- normal". This appears to vary between investigators. The fact remains however, that an increase in blood pressure above normal is recognized as detrimental to the health of the individual (McMahon et a1. 1973, Ledingham 1971). Classifying Hypertension Hypertension can be classified into either primary (essential) or secondary hypertension. Secondary hyper- tension involves individuals with elevated blood pressure due to a variety of diseases such as renal failure, glom- erulonephritis and adrenocortical diseases (Ledingham 1971, Ganong 1971). Quite often this type of hypertension can be alleviated if the affecting disease is cured. Hypertension due to any of a variety of diseases can enter an accelerated phase in which necrotic arteriolar lesions develop and eventually lead to a malignant form of hypertension. Essential hypertension is the most common form of hypertensive disease and involves 90% of persons with the disease (Goldman.g§_gl. 1972). As the name implies, this I... elevation in blood pressure is characterized by diffuse art- eriolar constriction of unknown cause (Ganong 1971). In an effort to define the etiology of the disease several con- tributing factors have been implicated, of which heredity and environment are the two most often pursued. The importance of heredity to the development of hypertension has been demonstrated in both human and animal studies. The incidence of hypertension in the American Negro for example, has been shown to be more frequent than in the American White for both sexes (McMahon.§t_g;. 1975). It was once thought that the American Negro acquired this pr0pensity for hypertension upon entering western civili- zation. A study of healthy Bantu males in South Africa indicated the contrary however (Dahl 1972), when a high incidence of hypertension was found in this population group. Several strains of laboratory rats have been select- ively inbred to produce offspring with genetic spontaneous hypertension of greater than 150 mmHg systolic pressure (Smirk and Hall 1958, Okamoto and Aoki 1965, Dahl g£_g;. 1967). Dahl et al. (1967), through selective inbreeding developed two strains of rats with opposite genetic prop- ensities for hypertension when fed a high salt diet. The sensitive or (S) strain developed hypertension quite readily whereas the resistant or (R) strain did not. Data from parabiotic union of R and S rats allowed Dahl to postulate that the difference between the two strains was the result 5 of an undefined humoral mechanism. Further evidence to verify this postulation has not been produced. Stress, physical work and diet have been implicated as the major environmental factors contributing to the dev- elopment of essential hypertension (Ledingham 1971). Diet has been approached with the greatest degree of vigor and has involved such parameters as coffee drinking, obesity, trace elements, fat, protein and carbohydrate intake and salt ingestion. Age and Hypertensive Disease McMahon.gt_gl, (1975) studied 11,509 human subjects of differing age, race and sex and found that hypertension in- creased with age (arterial pressure greater than 150/90 mmHg). Of the subjects in the 50-59 age category, 57% of them were hypertensive. Of those in the 60 and above age category, 70% were hypertensive. Russek.gt_§;. (19h6) studied blood pressures of 5,551 men between the ages of 40 and 95 years. He observed that the frequency of systolic hypertension rose sharply with advancing years. Diastolic blood pressure however, showed little variation after the sixth decade. Blood pressures of Wistar Albino and Gray rats were studied over time by Durant (1927). He found systolic blood pressure increased with age in the Albino rat but not in the gray strain. Accurate data for blood pressure changes in the rat over time has been reported by Heyman and Salehar (19A9). They noted that systolic blood pressure increased 6 with age in the growing rat. Within the first 2 months up to a body weight of 150 grams the blood pressure increased rapidly. A slower but more progressive rise was noted until A months of age or 550 grams of body weight after which time blood pressure stabilized. Obesity and gypgrtension Clinical studies have indicated a definate correlation between obesity and increased systolic blood pressure (Wood and Cash 1939). Kannel and his coworkers (1967) reported information obtained from the Framingham study concerning the development of hypertension. They noted that over a 12 year period, normotensive obese individuals developed hypertension more often than normotensive individuals near ideal body weight. The risk of hypertension was 8 times greater in those individuals 20% overweight as opposed to those 10% underweight. A study by Hartman and Grist (1929) demonstrated a steplike rise in systolic blood pressure in subjects ranging from 25% underweight to 25%.overweight. The greatest dif- ference in blood pressure occurred between normal weight and 25%.overweight individuals. Obese subjects at least 2% times their ideal body weight were examined by Alexander (1965) and found to have a 50% incidence of hypertension to a slight or moderate degree. Direct intra-arterial blood pressure measurements were used. Ten percent of the individuals were severely hypertensive (blood pressure of 200/120 mmHg or greater). 7 One problem with correlations between blood pressure and body weight has been determining whether body weight itself is involved or the percentage of body fat. Kannel gt_§;. (1967) observed that blood pressure was more strongly associated with body weight than body fat in human subjects. Meyer g§_§l. (1969) also supported this concept when he pointed out that skinfold thickness and blood pressure are not as strongly correlated as are body weight and blood pressure. The extent to which overweight measures body fat- ness varies with body build. The frequency with which blood pressure and overweight coexist suggests that there is a causal relationship be- tween them. Chiang g§_g;. (1969) concludes that weight gain constitutes one kind of environmental stress that brings a genetic predisposition toward hypertension into the open. Diet and Essential gypgrtension Alteration in Eat, Protein and Carbohydrate Kempner (1944 and 1948) pioneered interest in the re- lationship between diet and blood pressure when he fed a rice-fruit-sugar diet to 500 hypertensive patients. Blood pressure decreased in 6A%>of the individuals. Hatch.g§_§;. (1954) conducted one of the few experimental studies in which nutritional components of a diet were varied and tested for the effect on blood pressure. The study involved modification of Kempner's rice-fruit diet. Modification included the addition of protein, fat and vegetables to the 8 basic diet. No loss of beneficial effects was noted when 12 to 50 grams per day of low-sodium protein, 20 to 40 grams per day of fat and 200 grams per day of vegetables were added singly or in combination to the diet. The study was conducted with hospitalized hypertensive individuals. Interestingly enough, the addition of 5 grams per day of salt to the diets prevented any blood pressure lowering effect of the diet. . A 5% sucrose solution when given in conjunction with a 1%.saline solution to rats by Hall and Hall (1966) produced a more dramatic rise in blood pressure than did a 5% glucose and I% saline solution or a 1% saline solution alone. Hypertension appeared in the saline group by the 218t day of the experiment and eventually affected 60% of the rats in that group. In contrast, there was a 50%.incidence of hypertension in the sucrose-saline group by the 1hth day which increased to 90%»at 21 days. None of the glucose- saline group were hypertensive by the-Ihth day however, some were in the prehypertensive range for blood pressure (140- 1h9 mmHg), Eventually 60% of the latter group were hyper- tensive by the 21st day. Cardiac weights in each saline group of rats were greater than water controls but not dif- ferent from each other. Kidney weight was increased in rats in the saline and sucrose-saline groups when compared to controls. Kidney weight was not increased in rats given the glucose-saline drinking solution. Ahrens (1974) supports the view that sucrose stimulates 9 sodium retention and in turn hypertension, and that the effect of sucrose is mediated through the fructose molecule. Salt and Essential gypgrtension Salt Appetite The custom of adding salt to food is ancient. Not only has salt been used by humans for palatability and preserva- tion of food but also for ritual and monetary exchange. It must be remembered however, that salt appetite, as it is commonly expressed, is acquired. An exception of course would include sodium deficiency (Blair-West et al. 1970) when an inherent requirement for salt would increase salt appetite or in the case of herbivorous animals desiring salt licks (Dahl 1972). Actual metabolic requirements for sodium are small; 6- 8 meq per day in the human infant and adult (Committee on Nutrition, American Academy of Pediatrics 197A). Average per capita sodium consumption in the adult in the United States is 150 to 200 meq per day (Committee on Nutrition, American Academy of Pediatrics 197k). In contrast, many people of the world living in the arctic, jungle and desert (primarily vegetarians) average 2 to 10 meq per day (Dahl 1972). The ability of humans to adapt to a wide range of sodium intakes is due to the renal-endocrine system respon- sible for regulating body sodium. This is done by varying urinary sodium excretion according to sodium intake and IO nonrenal sodium losses (Pitts 1970). The kidney and renin- angiotensin hormonal system are key factors in this physio- logical regulation and the regulation of blood pressure as well. The human is not the only species which indulges in the use of large quantities of salt. When offered water and saline concurrently, rats show preference for saline in concentrations between 12 and 250 meq/L (0.0007 and 1.A6%) (Blair-West 2.13.2;- 1970). McConnell and Henkin (1973) found a preference for 0.50 M EaCl solutions in both spontaneously hypertensive female rats and normotensive controls using a 2-bott1e preference test. The spontaneously hypertensive rats showed an increased preference for the salt solution as blood pressure increased to the hypertensive range through- out a 16 week period. The normotensive rats did not show an increase in preference with time. Results similar to the preceding were secured by Hall et al. (1972). They found that Sprague-Dawley rats consumed much more of’a 1%Isaline solution than Long-Evans rats. Hypertension developed in the former but not in the latter. Saline consumption increased as blood pressure increased. Hypertensive humans have not been shown to have a higher mean taste threshold for salt than normotensives (Joossens 1973). Joossens believes that chronic and pro- longed use of salt may effect the taste buds and thus impair the function of taste. II Human gypgrtension Evidence relating salt intake to human hypertension is indirect and derived from three sources; (1) therapeutic effects of salt restriction, (2) effects of salt elimination by diuretics, and (5) epidemiological correlations between salt intake and hypertension. The blood pressure lowering effect of Kempner's rice- fruit diet has been shown by Hatch.gp_pl, (195a) to be due to the low sodium content of the diet. Dahl (1972) con- ducted a study using rats which further documented the effects of sodium restriction on blood pressure. Among rats, the level of salt consumed after hypertension had been established was critical, whether the disease had been pro- duced by dietary salt or some other process. When rats were restricted in salt consumption the disease became more be- nign and life span was lengthened. Human weight reduction studies conducted by Dahl g£_§;. (1958) document that the decrease in blood pressure accomp~ anying weight reduction was not the result of weight loss but the restriction of salt that usually accompanied caloric restriction. Several diuretics used in the treatment of hypertension derive their therapeutic action from the elimination of salt in the urine (Joossens 1975). Hypertensive patients who have responded to diuretic drugs have responded equally as well to a salt restricted diet (Dustan.pp_pl. 1974). Group correlations in different geographical areas of 12 the world have suggested a dose response between salt intake and prevalence of hypertension. A study of various primi- tive peoples in different areas of the world (Brazil, New Guinea and the Cook Islands) has indicated that blood pres- sure does not increase with age and hypertension does not deveIOp as frequently as in some populations (Joossens 1973). In contrast, several population groups (United States, Japan and Sweden) consume consistently more salt than metabolically required and in turn exhibit elevated blood pressures (Dahl 1961, Joossens 1975). The northern Japanese for example, consume nearly twice as much salt as the southern Japanese (26 gm/day vs. 14 gm/day) resulting in an increased incidence of hypertension (40% in the north vs. 21% in the south) (Dahl 1961). Salt in Egpgrimental Hypertension Indirect evidence is the only basis for a correlation between salt intake and hypertension in man since only a small number of studies have been conducted with human sub- jects. One such study was conducted by Gros and coworkers (1971) in which 6 grams of salt was supplemented to the normal salt intake of 7 untreated mildly hypertensive patients and 5 normal subjects. They found no edema or sig- nificant change in body weight or blood pressure in either group. In the rat and other experimental animals a cause and effect relationship between salt intake and hypertension has been fairly well established. Lenel and associates (1948) I} maintained 6~week old chicks on a 0.9% NaCl drinking solu- tion and observed a higher blood pressure than in control chicks given tap water. When the drinking solution was changed to 1.2% NaCl, a greater increase in blood pressure was noted and associated with dehydration, diarrhea and subsequent loss of body weight. Blood pressure decreased when tap water was substituted. Sapirstien pp_§l. (1950) were successful in producing arterial hypertension in male rats by the substitution of a 2%.NaCl solution for drinking water for a 6 week period. Autopsy revealed an hypertrophy of the heart and kidneys relative to body weight. Self-sustaining hypertension has been induced in female rats by chronic feeding of salt at 8% by weight of the diet (Dahl g§_g;. 1968). Rats were maintained on the high salt diet for one year during which time 80% became hypertensive. A four month follow-up period of salt restriction failed to lower blood pressure in two-thirds of the rats. The effect of excess salt ingestion on blood pressure was self-sustain- ing even after the original stimulus was removed. The effect of salt on the development of hypertension in the spontaneously hypertensive rat has been examined frequently since these rats are considered to be the best model for investigating human essential hypertension (Aoki gp_g;. 1972, Koletsky 1958). The relevance of heredity and environment to the development of hypertensive disease is easily demonstrated in these rats. 14 Aoki pp_g;, (1972) found that spontaneously hyperten- sive rats deve10ped hypertension (170 mmHg) whether fed a high or low salt diet. No difference in magnitude of blood pressure was noted between the two groups. However, when a 1%Isalt solution was substituted for drinking water and given to the rats already on a high salt diet the effect on blood pressure was marked. Hypertension developed earlier and reached a higher level (204 mmHg) in these rats. The same study indicated that growth rate and life span were significantly decreased in rats given the high salt diet and 1% saline. Louis and coworkers (1969) employed a different strain of spontaneously hypertensive rat and observed hypertension with or without added salt in the diet. Gross increases in sodium intake (4% of the diet) increased the severity of the hypertension and depressed the growth rate as measured by body weight. A greater sensitivity of young animals to excess sodium chloride has been documented by Dahl gp_p;. (1967). The earlier and longer that rats received a diet containing 8% NaCl, the more severe was the hypertension that developed and the more difficult it was to reduce blood pressure with sodium restriction. Moderate to severe lesions in the kid- neys of these rats were observed (Jaffe et al. 1970). SodiumzPotassium Ratio in Egpgrimental Hypertension The ratio of sodium to potassium in the diet has been implicated as affecting the development of hypertensive 15 disease (Gros gp_g;. 1971, Meneely p£_5;. 1961). Meneely gp_p;. (1961) conducted an interesting experiment in which rats were fed either a diet high in both sodium and potas- sium or high in sodium and low in potassium. After 8 weeks on the diets, blood pressure was higher in rats given the high sodium-high potassium diet than in rats fed the high sodium-low potassium diet. At 12 weeks blood pressures were equal in both groups. By 19 and 27 weeks the blood pressure of the high sodium-low potassium group was climbing and became significantly higher than pressure in the high sodium high potassium group. Meneely concluded that when the pot- assium level of the diet was sufficiently high to restore the Na:K ratio to one, significantly lower blood pressure could be maintained. Gros gp_§;. (1971) failed to observe the same results in humans given a diet with a NazK ratio of one. Salt and Renal Mass in Egpgrimental Hypgrtension Supplemental salt intake in conjunction with a reduced kidney mass has been shown to accelerate the onset of hyper- tension in both rats and dogs (Molteni and Brownie 1972, Coleman and Guyton 1969, Langston.gp_p;. 1965, Douglas §£_g;. 1964, Koletsky 1959). Langston.gp_g;. (1965) removed 70% of the renal mass from dogs and observed a 50-40% increase in blood pressure within 48 hours after ingestion of a 0.9% NaCl solution. Blood pressure decreased to normal levels when tap water was resumed. Normal dogs with both kidneys intact were given 16 the 0.9% saline solution to drink; blood pressure increased by 10%. In addition, urinary sodium excretion doubled in the normal dogs indicating the intact kidney was necessary to handle the salt load. Douglas gp_g;, (1964) also removed 70% of the renal tissue of dogs maintained on a normal diet and tap water. Arterial blood pressure rose to hypertensive levels within one week after 1.2% saline was substituted for tap water. Blood volume increased initially but decreased to normal within 2 weeks. After a 55-day period of drinking 1.2% saline, tap water was again resumed and blood pressure re- turned to normal. Identical results were obtained by Cole- man and Guyton (1969) in a similar experiment with dogs. Mononephrectomy is another popular technique for re- ducing renal mass. When rats of several strains had one kidney surgically removed and were allowed free access to a 1% NaCl drinking solution; each strain.was found to react differently (Molteni and Brownie 1972). Some of the strains became hypertensive and others did not. When hypertension was evident it was often accompanied by high plasma levels of sodium and renal and cardiovascular lesions. Other studies have noted these same lesions in the face of hyper- tension (Jaffe gp_p;. 1970, Koletsky 1959). Koletsky (1959) postulated that the greater the degree of renal ablation the more responsive the animal is to the hypertensive action of salt. Salt itself was more often effective in producing elevated blood pressure in Koletsky's studies than was a decrease in renal mass up to 50%. 17 Possible Mechanisms for Salt-Induced Hypprtension The usual sequence of events in salt-induced hyper- tensive vascular disease have been observed as; (1) in- creased periferal vascular tone, (2) hypertension, (5) gen- eralized vascular disease, and (4) renal disease (nephro- sclerosis) (Koletsky 1961). The exact cause of the vascular lesions is not clear but is felt to be a consequence of the high blood pressure itself producing wear and tear on the vascular wall resulting in structural damage (Brest and Meyer 1967). Another possibility is that the salt acts directly on the vascular wall (Koletsky 1961). Renal dis- ease develops from severe vasoconstriction in the kidney (an autoregulatory response). Irreversible structural changes are associated with the narrowing of these renal vessels (Ledingham 1971). Several theories have been postulated for the cause of the increased periferal vascular tone that eventually leads to hypertension. Among these are the blood volume and imbalance of electrolytes theories. Blood Volume Theory Coleman and Guyton (1969) and Douglas et al. (1964) have postulated that excess water and salt loading causes an increase in extracellular fluid volume and a resultant in- crease in blood volume. The increased blood volume presum- ably increases mean systolic blood pressure and in turn the venous return. The increased venous return, in accordance 18 with the Frank-Starling law of the heart, increases cardiac output. An increased cardiac output elevates arterial pres- sure which induces a baroreceptor reflex to reduce heart rate and total peripheral resistance. The validity of this theory depends on proof of long term autoregulation which is theorized to slowly increase total peripheral resistance or vascular tone and eventually reduce cardiac output. Both reduced cardiac output and increased vascular tone are evident artifacts in essential hypertension (Douglas gp_g;. 1964, Ledingham 1971). Imbalance of ElectroHytes TheOpy An imbalance of electrolytes in the vascular wall in salt-induced hypertension has been thought to increase vascular tone that eventually leads to hypertension (Koletsky 1958, 1961). The imbalance is presumably init- iated by retention and intracellular accumulation of sodium and replacement of potassium by sodium. Vascular edema resulting from accumulation of sodium and water may decrease the diameter of the blood vessel wall enough to lead to increased resistance (Koletsky 1961). Tobian (1960) referred to this phenomena as "water- logging" of the blood vessels. Mallov (1961) hypothesized the waterlogging was due to a change in vessel membrane permeability. The water and solutes imbibed by the vessel would increase vessel tension. Mallov observed that aortic strips from hypertensive rats, placed in hypertonic salt 19 solutions exhibited increased tension in contrast to be- havior of strips from normotensive rats. A second possible explanation for increased vascular tone found in hypertension is that an electrolyte imbalance causes cellular necrosis in the vessel. This is accom- plished by altering the actomyosin complex or interfering with the energy producing mechanism in the vessel wall. Both are influenced by concentration of alkaline ions (Koletsky 1958). Electrolytes in Tissues of Hypertensives Total Bo Sodium Schackow and Dahl (1950) noted a lack of gross ccumu- lation of sodium or depletion of potassium in whole body samples of rats fed either a high salt (8% NaCl) or a low salt (0.55% NaCl) diet. Since the animals fed the high salt diet developed hypertension, the authors concluded that salt-induced hypertension did not occur as a result of salt retention in the tissues. Greene and Sapirstien (1952) found total body sodium to be greatly increased in hypertensive rats while total body potassium remained at normal levels. Plasma sodium also re- mained within normal limits indicating to them that the sod- ium ion had either penetrated intracellularly or been de- posited in bone. 20 Tissue Sodium Several investigators (Schackow and Dahl 1950, Greene and Sapirstien 1952, Tobian 1960, Haight and Weller 1961) observed normal plasma sodium levels in rats made hyper- tensive with or without salt. Attention then focused on the electrolyte content of various individual tissues. In adult renal hypertensive rats the electrolyte composition of the brain, gut, heart, liver, skeletal muscle and skin was not significantly different from normal control rats (Tobian 1960). Haight and Weller (1961) found no change in sodium, potassium or chloride content of skeletal or heart muscle of rats fed diets containing 2.8%, 5.6%1and 8.4% sodium chloride despite the fact that blood pressure increased as the sodium chloride level increased. Arteriolar tissue has been implicated as a possible storage site for sodium since the rise in arterial hyper- tension is mainly due to a narrowing of the arteriole lumen (Tobian 1960, Meneely g§_2l. 1961, Haight and Weller 1961). Investigation has centered on the sodium and potassium con- tent of the aorta. Principally because the aorta is more easily accessable than arteriolar tissue. Haight and Weller (1961) found a progressive increase in sodium and potassium content of rat aorta as dietary NaCl progressed from 2.8% to 8.4%. Tobian (1960) injected rats with deoxycorticosterone and gave them a 1% NaCl drinking solution. Among rats in 21 which blood pressure remained normal, the sodium content of the aorta increased and potassium content decreased slightw 1y. In contrast, the aorta of rats that developed hyper- tension had a larger increase in sodium concentration but in addition, developed an increase in potassium rather than a decrease. Tobian reported that the level of potassium in the aorta correlated better with blood pressure than the level of sodium. In both mild and severe hypertension a decrease in blood pressure was accompanied by a drOp in potassium concentration of the aortic wall. Na:K Ratio and Electrolyte Concentration Meneely et al. (1961) studied three groups of rats fed a diet varying in the amount of sodium and potassium chlor- ide. The control group received a diet containing 1.1% NaCl and 0.66%»KCl; Na:K ratio was approximately one. The "high sodium" group were fed a diet containing 9.8% NaCl and 0.6% KCl; dietary ratio of sodium to potassium was thirteen. The "high sodium + potassium" group received a diet containing 9.8% NaCl and 6.1% K01; dietary ratio of sodium to potassium was one, Just as the control group. Rats fed the high sodium diet had more body sodium than did the controls or rats fed the high sodium + potassium. The high sodium group also had a higher blood pressure than the controls or those rats fed high sodium + potassium, in which case the Na:K ratio was restored to one. Further support of the importance of the Na:K ratio in hypertension was offered by Stone gt_§l. (1957). He 22 observed that rats fed a high salt diet developed hyper- tension, renal damage and a decreased life span. A longer life span and decreased incidence of hypertension resulted when dietary potassium was increased. Whole body analysis indicated a reduced sodium content in those rats given high levels of dietary potassium in conjunction with the high sodium when compared to rats given high amounts of sodium alone 0 INTRODUCTION Cardiovascular disease is augmented by the presence of elevated blood pressure. Concrete explanations for the common occurrence of hypertension are few and far between. Although hypertension is known to result from various dis- eases and stressful situations (Ledingham 1971), the major- ity of hypertensive individuals remain undiagnosed. Diet is one of the many factors that can be investigated in a search for elements contributing to hypertension. The laboratory rat is a common model used in studies involving hypertension. The use of animals in such studies permits rigid dietary and environmental control. Heredity is an important factor in the development of hypertension (Ledingham 1971, Dahl 1961) and control of this variable is possible through use of laboratory animals. A definate cause and effect relationship has been shown to exist between salt and hypertension in the laboratory rat (Dahl 1961, 1967, 1972, Sapirstien g£_gl, 1950). The effect of diets of variable composition on blood pressure is less pronounced. Kempner (19hh) decreased blood pressure in hypertensive humans through a high carbohydrate-low salt diet. Hatch 2.9.2.1.- (1954) elucidated further by adding variable amounts of fat and protein to Kempner's diet and observing no change in the therapeutic effect of the diet. 23 2h Sucrose has been implicated as a causative factor in the deve10pment of hypertension (Ahrens 197k, Yudkin 1972) however, for lack of concrete verification, this remains merely speculation. Studies by Hall and Hall (1966) have suggested that the form of carbohydrate in the diet is an important considera- tion when investigating hypertension. They found blood pressure to be higher in rats given a sucrose drinking solution as opposed to rats given a glucose solution. Rats fed diets high in fat become obese (Schemmel g£_gl. 1969, 1972) and exhibit higher blood pressures than "normal weight" rats of the same age (Beebe g§_gl. 197h abstract). Whether the elevated blood pressure is due to the obesity or the high fat diet has not been clarified. Obesity has been strongly correlated to hypertension in humans (Chiang gt_gl. 1969); this has not been proven in the laboratory rat. Since most blood pressure investigations have been conducted with rats fed a chow-type of diet, any investi- gation into the effect of dietary variation on blood pres- sure must also include a diet similar in composition to the chow-type diet for comparative reasons. Subsequently a grain diet was used as a control diet in the present study. The primary purpose of the study was to observe any change in blood pressure or pulse rate in rats fed diets which varied in the source of the primary energy component, i.e., fat, sucrose or cornstarch. The effect of a salt-diet interaction on these parameters was also of interest. In 25 an effort to explain the effect of diet and/or salt on blood pressure, growth rate, specific organ size and tissue electrolyte concentration were examined. METHODS E erimental Desi n Experiment I Sixty-four male Osborne Mendel rats were weaned between 21 and 2h days of age and divided into 4 groups of 16 animals each. Each group of rats was fed one of four dif- ferent diets detailed in Tables 1 and 2. The diets differed in the source of the primary energy component. A cornstarch diet was developed on the basis of 100 grams and contained 67 grams (67%) of cornstarch. Sucrose was the primary energy component of a second diet also based on 100 grams and containing 67 grams (67%) of sucrose. The sucrose ration contained 5.8 kcal./gm of diet based on A, A and 9 kcal/gm for protein, sucrose and fat, respectively (Guthrie 1967). The kcal.:protein diet ratio as calculated from kcal consumed/gm of dietary protein eaten was 19.0 for the sucrose diet. The cornstarch ration contained 5.5 kcal./gm of diet based on u, 5.6 and 9 kcal./ gm for protein, cornstarch and fat, respectively (Bowes and Church 1965). The kcal.:protein diet ratio was 17.5 for the cornstarch diet. A fat ration was deve10ped which contained 51.8% fat in the form of vegetable shortening and contained 6.0 kcal./gm based on values of 4, h and 9 for protein, carbohydrate and 26 27 fat, respectively. The kcal.:protein ratio was 20.0. Previous work by Schemmel g§_§l. (1970) indicated that rats fed a diet high in fat (60% Crisco) consumed fewer grams of diet (30% fewer) than rats fed a grain ration. The fourth diet was a grain ration which contained 55.5% carbohydrate, as calculated by difference, predomi- nantly from corn. The diet contained 5.4 kca1./gm (Schemmel g£_§l. 1972) and a kca1.:protein ratio of 1h.8. The grain ration was used to represent standard growth and deve10pment and to allow comparison with previous studies which dealt with hypertension in the rat. Much of the previous blood pressure investigations have been performed with rats fed a standard chow-type ration similar to the grain ration (Sapirstein g§_§l. 1950, Dahl g£_al. 1967, Weller 22.2;- unpublished data). Each group of 16 rats was further divided into 2 groups of 8 animals each. One of the 2 subgroups was allowed to drink distilled, deionized water ad libitum, while the second group received a 2% sodium chloride (NaCl) in dis- tilled deionized water solution ad libitum. Such a design facilitated comparison of dietary effects on blood pressure with and without sodium chloride interaction. Weekly blood pressure and pulse rates were determined for each rat at the same time of day and on the same day each week. This procedure was followed for 10 weeks when the rats were sacrificed. Various parameters other than blood pressure and pulse rate were measured individually 28 for each animal. These included weekly food and fluid in- takes throughout the course of the study, weekly body weight gain, body composition (percent fat, lean and moisture), specific organ weight (heart, right adrenal and right kid- ney) and sodium and potassium concentration of the serum, heart, kidney and bone (right hind femur). Experiment II A second study was undertaken in which 24 male Osborne Mendel rats were weaned at 21-24 days of age and divided into 4 groups of 6 rats each. They were fed the same diets used in Experiment I. The rats were allowed to drink dis- tilled deionized water ad libitum for the first 5 days post-weaning. A 1% NaCl and water drinking solution was substituted for the distilled deionized water on day 4 and was continued for 9 weeks. This was followed by substitu- tion of a 1.5% NaCl solution for an additional 9 weeks or until the experiment was terminated. Blood pressure, pulse rate, food and fluid intake and body weight were monitored weekly for individual animals and compared with data from Experiment I. Heart, right kidney and right adrenal gland weights were recorded at the time of sacrifice. Expgrimental Conditions All experimental animals were housed in a ventilated room where the temperature remained at 251100. Twelve hours of light and twelve hours of darkness were allowed in each 29 24 hour period. The animals were sheltered individually in screen bottomed metal cages 18 x 18 x 25 cm in size. Activity was neither promoted nor restrained. Food was available to the rat in porcelain cups and allowed ad libi- tum. A weekly record of food intake was kept for each rat throughout the experiment by weighing the food cup filled at the beginning of the week and subtracting from it the weight of the cup at the end of the week. Correction for spillage was made by retrieving as much of the spilled ration as possible from a paper towel placed beneath each cage and adding this amount to the cup weight at the end of the week. Either distilled deionized water or a solution of sod- ium chloride and water were allowed to each animal ad libi- tum through a cylindrical 100 ml graduated water bottle. The drinking spout was ball-tipped to reduce leakage and facilitate weekly water intakes throughout the study. Very little leakage did occur (< 1.0%) when the bottles were correctly placed in the holder. Minimum leakage was meas- ured in 5 sample bottles over a period of 7 days. Leakage consisted of 2 ml/100ml water. Blood Pressure and Pulse Rate Measurement Weekly determinations of blood pressure and pulse rate were considered adequate in frequency for establishing any trends in either of these parameters over time. Since emotional stress is known to temporarily raise 30 blood pressure and pulse rate (Ledingham 1971) the experi- mental conditions were standardized as much as possible. Determinations were carried out in the early hours of the day (8 a.m. to 1 p.m.) on the same day each week to minimize diurnal variation. All blood pressure and pulse rate deter- minations were made indirectly from the tail of the rat (Figure 1). This technique was employed because it allowed frequent determinations to be made without affecting the physical condition of the rat. The rat was placed into a plexiglass restraining device designed to restrict movement. This was found most satis- factory in restraining the rat such that anesthetic was not necessary. The use of this device eliminated any influence anesthetic might have had on blood pressure and pulse rate. Weanling animals were often too small to be completely restrained by the device used, in which case the unit was lined with foam rubber to accommodate the size_of the ani- mal. The unit plus the animal was then placed on a heating pad set such that surface temperature was maintained at 551130. The heating pad was located on the floor of a large wooden box with a glass top‘. This set-up was a mod- ification of a more elaborate and expensive device offered by Narco Biosystemsa. Each rat remained inside the re- strainer in the box for 10 minutes prior to actual 1Courtesy of Dr. Collings Associate Chairman, Depart- ment of Physiology, Michigan State University. 2Life Science Instrumentation, Houston, Texas. 51 measurement of blood pressure and pulse rate. The 10 min- ute accommodation period was allowed for two reasons; (1) to allow the rat to relax and adjust to the new surroundings and (2) to raise the body temperature of the animal such that blood flowed freely through the tail. If the animal were not heated, blood flow through the tail would not be strong enough to be detected by the instrument used. A temperature range of 56.5-39.09C produces dilation of the tail vessels of the rat without appreciably altering blood pressure (Geddes 1970). .Measuring blood pressure and pulse rate involved the use of a Desk Model Physiograph3 with a plug-in module for recording these parameters. The tail of the animal was in- serted through a rubber lined metal blood pressure cuff 1.1 cm in diameter and 5.5 cm in length. The cuff was posi- tioned as close as possible to the point of attachment of the rat tail to the body. Because the tail of the rat is long and tapered, there is a pressure gradient along the tail. This necessitates that the cuff be placed at the root of the tail (Geddes 1970). Rubber tubing connected the cuff to the Electrosphygmograph Coupler4 where a hand bulb for expanding the cuff was located. 3The Desk Model Physiograph DMP-4B was obtained from garco Biosystems Inc., Life Science Instrumentation, Houston, 01380 #Type 7211 is a plug-in module of the Physiograph Solid State Channel Amplifier/Coupler System designed for the det- ermination of indirect blood pressure. This coupler combines a pressure transducer and amplifier to produce single channel recordings of occluding cuff pressure and superimposed Kor- otkoff sounds of humans and animals. 52 A pneumatic pulse sensor was attached lightly with masking tape to the tail of the rat directly posterior to the blood pressure cuff. The sensor was attached through a short length of flexible tubing to a Pneumatic Pulse Trans- ducer5 which was connected via rubber tubing to the coupler. A single channel recording was produced to show pulsations of blood passing through the rat's tail. The number of pulses per minute was determined by multiplying the number of pulsations recorded in 5 seconds by 20. This number was established as the pulse rate. Recording blood pressure required certain standard con- ditions. These are listed in Appendix A. Squeezing the hand bulb connected to the coupler expanded the pressure cuff lining and restricted blood flow through the tail. The single channel recording indicated the precise point of oc- cluding cuff pressure by the absence of pulsations. Pres- sure in the hand bulb was slowly released until pulsations were again recorded. Blood pressure was determined by measuring the distance from the base line to the point where pulsations appeared on the recording paper. Each millimeter on the paper represented 4 mmHg blood pressure (Pressure range switch set at 0-100 mmHg; maximum sensitivity of 20 mmHg/cm). Since the PhysiOgraph was capable of recording only the initial return of blood flow through the tail, 5This is a sensitive piezoelectric transducer for detection of pulsatile volume changes. 33 diastolic blood pressure was not recorded by this technique. Recorded blood pressures represent systolic pressure only. So as to establish accuracy and precision in blood pressure and pulse rate determinations a series of deter- minations were recorded consecutively at one minute inter- vals until at least 5 readings were identical to within 4 mmHg. Sometimes this would involve several measurements (10-12). Once the blood pressure and pulse rate were det- ermined the rat was removed from the restraining device and returned to the appropriate cage. emov r ans an a epo 3 Each experimental animal was anesthetized with ether on the day of sacrifice. Approximately % inch of the tail was removed at the tip with a razor blade. Blood was collected from the tail into a 5 ml test tube and spun in a centrifuge for 10 minutes at 5200 rpm5. The serum was removed into a second test tube, covered with parafilm and frozen. The animal was then killed with an overdose of ether. Immed- iately following death an incision was made down the midline of the rat on the ventral side. The right inguinal fat pad lying Just under the skin was removed and the abdominal wall opened with an incision. The right testicular and perirenal- retroperitoneal fat pads were removed and each fat pad was weighed immediately to the third place. 6International Clinical Centrifuge Model Cl. 34 The right kidney, right adrenal gland and the heart were removed and weighed to the nearest milligram immed- iately before being placed into individual self-sealing plastic bags and frozen. The right hind leg was removed, skinned, placed in a self-sealing plastic bag and frozen. Stomach, intestinal and cecal contents were removed before these organs and the remaining carcass were deposited into the glass jar, weighed and frozen. Body Composition Analyses Moisture Content Each carcass was thawed to room temperature in the glass Jar before autoclaving for 15 minutes under 15 pounds per square inch pressure (1209C) to soften the bones (Mick- elsen and Anderson 1959). After autoclaving, the carcass and Jar were reweighed and any water lost during autoclaving was replaced by the addition of distilled deionized water. The carcass was emptied into a 1 gallon Waring Commercial Blender7 and an amount of deionized water approximating the carcass weight was used to rinse the Jar and added to the blender. The mixture was blended for 5 minutes at high speed (15,500 rpm w/64 oz. water). Approximately 10 grams of the homogenate was weighed into a small dry, tared alum- inum pan. Duplicate samples were taken from each carcass. 7Model 013-6, vari Products Service Center, New Hartford, Connecticut 0 057. 35 Samples were placed in a drying oven for 24 hours at 70°C before being transferred to a vacuum oven. Following drying in the vacuum oven for 48 hours at 7000 the samples were cooled in a dessicator and weighed to a constant weight. Percentage moisture in the carcass was calculated using the following formulas: Percent dry weight of sample = dr wei ht of sam 1e x 100 we we g of samp e Percent dry 3 %'dry wt. x wt. carcass8 + wt. added water wt. of carcass of sample weight of’carcassC Percent moisture in carcag. ' 100% -‘%.dry weight of carcass Body Fat and Lean The 10 gram samples used for moisture determination were also used for determination of body fat. Samples were extracted with anhydrous ethyl ether for 6% hours on a Goldfisch fat extraction apparatus. This length of time was found sufficient to remove fat from the samples since re- extraction of some samples failed to result in additional extracted fat. After extraction, the extraction flasks were dried in a 70°C oven for 15 minutes, cooled in a dessicator and weighed. The following calculations were used to det- ermine body fat: Percent fat in sample 3 wt of fat in sam 16 x 100 y w . o samp e Percent fat in carcass = % fat in sample x % dry wt. of carcass IOO 8Refers to weight of carcass before freezing. 36 Grams fat in carcass a %»fat in carcass x carcass weight. The total amount of lean body mass present in the rat carcass was determined by difference using the following calculations: Percent lean body mass (LBM) = 100% - % fat in carcass Grams of LBM = % LBM x carcass weight. Preparation of Samples for Flame Emmission Spectrophotometry §_e.1:u_a All analyses were performed in duplicate. Frozen serum samples were thawed at room temperature before 0.1 ml of serum was diluted with 9.9 ml of distilled deionized water. The diluted serum was stored in an acid-washed9 100 ml poly- ethylene bottle until the sodium and potassium content of the sample was determined using flame emmission spectrOpho- tometry. Heart and Kidney Both heart and kidney samples were treated identically for analyses of sodium and potassium. The organ was thawed at room temperature and homogenized on a Polytron 9Acid wash consisted of 1 part concentrated hydro- chloric' acid to 2 parts distil ed deionized water. Glass- ware and bottles were soaked in the wash for 20 minutes followed by a 20 minute soak in deionized water and then rinsed with deionized water 5 times (Ku, Pao. 1974 personal communication). 37 Homogenizer‘o. Homogenizer tubes were acid-washed. The homogenate contained 4 parts distilled deionized water to 1 part tissue. A 1 ml aliquot of homagenate was weighed into an acid washed phillips beaker for digestion. The digestion procedure was a modification of the wet digestion procedure for minerals found in The Official Methods of Analysis of the Association of Official Analytical Chemists (Horwitz 1970). The modification involved the use of smaller amounts of reagents and was therefore more economical and achieved the same results‘]. The sample of tissue was digested with 60 ml of conc- entrated nitric acid under a hood specifically designed for work with perchlorates‘a. When the digestion mixture was reduced to 5-5 ml the mixture was cooled before 7 ml of concentrated perchloric acid was added. Continued digestion required covering the beaker with a watch glass until the most reactive phase of digestion had passed. When the mix- ture was reduced to 5-5 ml the tared weight of the beaker was brought to 20 grams with distilled deionized water. The solution was transferred to an acid-washed polyethylene bottle for storage prior to analysis by flame emmission spectrophotometry. 10Brinkmann Polytron Model PT 10-55, Brinkmann Instru- ments Inc., Cantiague, Westbury, N.Y. 11590 11Dr. Duane Ullrey, personal communication (1974). 1aThe hood was designed such that a continuous stream of water flowed down the back and roof of the hood. This allowed the perchlorate vapors to solubilize in the water and be carried away rather than accumulate in the hood where they are extremely explosive. 38 B993 Analyses for sodium and potassium in the bone of the right hind leg required that the bone be perfectly free of any flesh. This required removal of the flesh by a tech- nique more efficient and precise than mechanically possible. Therefore, a Dermested Beatle colony was used to eat away the flesh and leave clean bone‘B. Each bone was labeled with a cardboard tag attached to the joint and marked with waterproof ink. The entire process took approximately 2 weeks. Once the bones were flesh free, the femur was removed, cut into several pieces and weighed. The bone fragments were wrapped in filter paper and inserted into a fat extrac- tion tube. Fat was extracted from the bone for 6 hours so that mineral content could be expressed on a fat-free dry basis. The bone was extracted with absolute alcohol for 5 of the 6 hours on a Goldfisch fat extraction apparatus. Alcohol extraction was followed by extraction with anhydrous ethyl ether for the remaining 5 hours. The combined alcohol- ether extraction is more efficient for extracting total lipids than is ether alone (Joslyn 1970). Following extraction, the bones were dried in a llODF oven for 4 hours and ashed overnight (18 hours) in a muffle furnace set at lZOOPFB The bone was ground to a fine powder 13Obtained from Mr. Larry Bowdre, Assistant Museum Curator, Michigan State University. 39 with a mortar and pestle and transferred to an acid-washed polyethylene bottle. The powdered ash was dissolved with 5 ml of 6N hydrochloric acid and then diluted to 100 ml with deionized water and stored for analysis. Diet Rations Duplicate samples (0.25 gm each) of each ration were digested by the same wet ashing technique used for heart and kidney samples. Sodium and potassium contents of the rat- ions were determined using flame emmission spectrOphotometry. Flame Emmission Spgctrophotometry All sodium and potassium determinations were performed using a Flame Emmission SpectrOphotometerlu. A fuel-oxidant mixture was used for flame emmission; air being the oxidant and acetylene the fuel. Wavelengths were set at 589 nm for sodium and 766.5 nm for potassium. Standards were evaluated on the same day as the samples. Experimental samples were often too concentrated and therefore were diluted to within the optimum detection range of the instrument. Concentra- tion of elements in the samples was determined by the fol- lowing calculation: ppm or ugm/ml = (concentration in 22%) (final volume) samp e we g 1“An Instrumentation Laboratories 455 Atomic Absorption Emmission Spectrophotometer. 40 Analysis of Data Blood pressure and pulse rate were recorded weekly for each rat therefore the change in these values over time was of interest. Means and standard deviations were determined for all groups of rats after 1, 5, 5, 7, 9 and 10 weeks on the dietary regimes. Significance of diet, salt and time were determined by a 2-way repeated measures analysis (Sokal and Rohlf 1969). The repeated measures test observed trends in blood pressure and pulse rate throughout the experimental period. Actual calculation of the analysis was performed by the 5600 Controlled Data Corporation (CDC) computer, Michigan State University. Students t-test (Sokal and Rohlf 1969) was used to com- pare final mean blood pressure and pulse rate. Data from animals fed the three semi-purified diets was compared to data from grain-fed rats. In addition, within diet groups data was compared with and without the salt drinking solu- tion. Data from Experiment II was not compared statisti- cally with data from Experiment I. Means and standard deviations were calculated for body weight at weekly intervals. Final body weight of rats fed the various diets was compared with final body weight of grainpfed rats. Means and standard deviations were estab- lished for body composition data (percent fat, lean and moisture), heart, kidney and adrenal gland weights and com- pared via students t-test (Sokal and Rohlf 1969). 1+1 Sodium and potassium data for serum, bone, heart and kidney were analyzed with a students t-test after calcu- lation of means and standard deviations were performed. RESULTS AND DISCUSSION Egeriment I Diet and Kcal Consumption Rats that received the grain ration and distilled deionized water consumed a mean total of 1498;140 gm of diet over the 10 week feeding period from weaning to 13 weeks of age. Rats fed the high fat, high sucrose and high corn- starch diets in conjunction with distilled deionized water consumed 930:99, 16341113 and 1605:161 gms of diet respect- ively for the same period of time (Table 3). The caloric density of the diets varied due to varia- tion in the energy value of the primary dietary components in each diet. The grain ration contained 3.# kcal/gm (Table 2) while the high fat, high sucrose and high cornstarch rations contained a calculated 6.0, 3.8 and 3.5 kcal/gm respectively (Table 1). When food intakes were adjusted for differences in caloric value the gap in intake between rats fed the various diets diminished. Rats fed the grain diet consumed 509azfi76 kcal in 10 weeks whereas rats fed either the high fat, high sucrose or high cornstarch diets consumed 5578:594, 62103A29 and 6099;612 kcal respectively (Table 3). When rats received a 2% NaCl and water drinking solu- tion instead of distilled deionized water, food consumption 42 #3 decreased regardless of diet. Lenel gt_gl, (1948) noted a similar decrease in food consumption in chicks fed a stan- dard chow-type diet in conjunction with a 1% NaCl drinking solution. The decrease in food consumption was least pronounced in rats fed the grain ration. They consumed 40591622 kcal for the 10 week period which was a 20% reduction when com- pared to rats fed the grain ration and the distilled deion- ized water (Table 3). Rats fed the high cornstarch diet in conjunction with the 2% NaCl drinking solution consumed #522z927.kcal (Table 3). This was a 26% decrease in energy consumption when compared to rats fed cornstarch and dist- illed deionized water. Rats fed the high fat diet and the 2% NaCl drinking solution reduced caloric consumption to 3453;552 kcal (Table 3). This was a 38% decrease in energy intake however, this was still not as severe as the 58% decrease observed in rats fed the high sucrose diet in conjunction with the 2% NaCl solution. Rats in the latter group consumed 2605;894 kcal for the 10 week experimental period (Table 3). Variation in the quantity of salt in a diet has been implicated as changing osmolarity of the diet (Miller and Czajka 1967). Hypertonic solutions in the stomach slow the rate of gastric emptying and contribute to a feeling of fullness. Whether the 2% NaCl drinking solution contributed to a high stomach osmolarity in those rats given the solu- tion remains to be clarified. If such were the case the 1+4 rats would have experienced a slower rate of stomach empty- ing and the resultant full feeling may have led to a de- creased desire for food. Miller and CzaJka (1967) force-fed hypertonic glucose solutions to infant rats and observed decreased rates of stomach emptying and increased mortality as the osmolarity of the solutions increased. Since rats fed the high sucrose diet and the 2% NaCl solution exhibited the most severe reduction in food consumed, perhaps the sucrose molecule was exerting an osmotic pressure in the stomach which was com- pounded by the osmolarity of the sodium chloride solution. Yudkin (1972) has found that sucrose contributes more than any other constituent to the osmotic pressure of food. The smallest decrease in food consumption when drinking the 2%.NaCl drinking solution occurred in rats fed the grain ration. This suggests that the grain diet somehow allev- iates the feeling of fulness caused by the sodium chloride. Gastric emptying time may not be decreased in the grain-fed rats, on the contrary, it may be increased. Fluid and Sodium Consumption The'intake of distilled deionized water by rats fed either grain, high fat, high sucrose or high cornstarch diets was 2306;360, 24661A68, 2120;195 and 2394;136 ml respectively for the 10 week experimental period (Table 3). Because the drinking solution did not contain sodium, the total sodium intake was based on the amount of sodium #5 consumed in the diet (See Appendix B for actual sample calculation). Rats fed the grain diet received 7.5:0.7 grams of sodium for the 10 week period. In contrast, rats fed the high fat, high sucrose and high cornstarCh diets consumed 7.0:0.7, 8.2:0.6 and 8.0;0.8 grams respectively (Table n). When sodium values are converted to grams consumed per 100 grams body weight, again each group of rats received similar amounts of sodium; i.e., 2.0;0.2, 1.7;0.2, 1.9;0.2 and 2.130.} gm/IOO gm body weight for rats fed grain, high fat, high sucrose and high cornstarch diets respectively (Table h). Rats that received the 2%»NaCl drinking solution in- creased fluid consumption by an average of 170% regardless of the type of diet. Rats fed grain in conjunction with the 2%.NaCl solution consumed 6180:1480 ml of fluid for the 10 week.experimental period (Table A). In contrast, rats fed a diet of high fat, high sucrose or high cornstarch content consumed 6326;78, 6307:1117 and 6582;4hu ml respectively (Table 4). Sapirstien.gg_g;. (1950) observed a similar in- crease in the fluid consumption of rats offered a 2% NaCl drinking solution. The hypertonicity of the solution appears to stimulate thirst. Pitts (1971) has suggested the existence of a hypothalamic integrative mechanism responding to changes in osmolarity by stimulating thirst. Since rats receiving the 2% NaCl drinking solution consumed fewer grams of food than their dietary counterparts 46 given distilled deionized water, it follows that they re- ceived less sodium from the diet. When the total amount of sodium ingested for the 10 week period was calculated, the major portion was derived from the drinking solution. Rats fed the grain ration in conjunction with the 2% NaCl solu- tion consumed a total of 53.9111 gm of sodium (Table 4). Similar amounts of sodium were consumed by rats fed either the high fat, high sucrose or high cornstarch diet; i.e., 53.4;1.3. 52.417.5 and 57.0;3.7 gm respectively (Table 4). However, when the total sodium intake was converted to gm per 100 gm body weight, marked differences in sodium intake were observed. Grain-fed rats consumed 32.6;22.4 gm/Ioo gm body weight whereas high fat, high sucrose and high corn- starch fed rats consumed 26.5;0.9, 53.410 and 29.9:9.9 gm sodium/100 gm body weight respectively (Table 4). Egowth Rates The body weight of rats given distilled deionized water increased linearly throughout the 10 week feeding period regardless of the diet fed (Figure 2). Rats fed the high sucrose and high fat rations maintained similar weight gains from the third experimental week until termination of the experiment. These were also the largest weight gains. At the time of sacrifice (13 weeks of age) the high sucrose-fed rats weighed 21% more than grain-fed rats, whereas rats fed the high fat diet weighed 9% more than grain-fed rats. Rats fed the high cornstarch diet gained weight less rapidly 47 throughout the 10 week period (Figure 2). At the time of sacrifice rats fed the high cornstarch diet had a body weight that was 5% greater than rats fed grain. Winnie gt_gl. (abstract 1973) observed that male Osborne Mendel rats fed a sucrose diet gained significantly more body weight than rats fed either high cornstarch or grain diets. Similarly, Schemmel §£_§l. (1972) demonstrated that rats fed a diet high in fat maintained greater body weights than rats fed a grain ration. It was concluded from the study that the energy of the diet was easily transferred to body fat by the animals fed the high fat diet. Rats that consumed the 2% NaCl drinking solution ex- hibited a marked decrease in rate of growth when compared to dietary counterparts given distilled deionized water. By the second experimental week all rats in the 2% NaCl group had lower mean body weights regardless of diet. As time progressed the difference in mean body weight between the two groups of rats became progressively larger until at 13 weeks of age these rats given the 2%,NaCl solution weighed up to 50%lless than rats given distilled deionized water to drink (Figure 3). Animals given the 2% RaCl solution and fed the high sucrose diet maintained a slower rate of growth than rats fed either grain, high fat or high cornstarch diets with the 2% NaCl drinking solution (Figure 3). This smaller, less rapid gain in weight persisted throughout the experiment until at 13 weeks of age the high sucrose-fed rats weighed #8 34%lless than those rats fed the grain ration. The growth rate of grain-fed rats appeared least retarded when compared to rats fed the high fat, high sucrose or high cornstarch diets (Figure 3) which probably reflected food intake. Body weight of all groups of rats, regardless of drinking solution, appeared to reflect caloric consumption. An exception was the group of rats fed cornstarch. they consumed more calories than rats fed the high fat diet but maintained a lower mean body weight throughout the experi- ment. As in the case of Schemmel g§_§l. (1972) this sugr gests that rats fed the high fat diet are probably more efficient in depositing body fat than are rats fed the high cornstarch diet. As suggested earlier, rats given the 2% NaCl drinking solution may have experienced a "fullness" due to an osmotic effect which consequently reduced food consumption and in turn body weight. This effect was more pronounced in rats fed the high sucrose diet. Blgod Prgssure Blood pressure increased linearly during the first 4 weeks of observation in those rats which received distilled deionized water regardless of dietary treatment (Figure 4). Rats fed the high sucrose diet reacted slightly differently in that blood pressure increased markedly during the second week and then decreased in the third week (Figure 4). Heymann and Salehar (1949) observed that blood pressure #9 increased rapidly with age in growing Long-Evans rats within the first 3 months and up to a weight of 200 grams. Al- though blood pressure of rats given distilled deionized water began to plateau after 7 weeks of age, this time period corresponded to a mean body weight of approximately 200 grams. Blood pressure of all rats given the distilled deion- ized water remained in the normotensive range (<2140 mmHg) throughout the experiment. However, distinct patterns of changes in blood pressure over time were observed between diet groups. Grain-fed rats maintained the lowest blood pressures from week.2 until the experiment was terminated (Figure 4). In contrast the rats fed the high sucrose diet exhibited the highest weekly blood pressures with exception of weeks 3, 4 and 5. Rats fed the high cornstarch diet maintained blood pressures slightly below rats fed the high sucrose diet, with exception of weeks 3, 4 and 5 when blood pressure rose slightly above that of the high sucrose-fed rats (Figure 4). Rate fed the high fat diet developed blood pressures that were greater than grain-fed rats but less than blood pressures of rats fed either of the high carbo- hydrate diets. Final blood pressure reflected the trend that had occurred for the entire 10 week experimental period. Grain- fed rats had a blood pressure of 109:13 mmHg in contrast to #2:? '"T‘!_ "v‘v'fiWmA “1‘“‘7 - ‘ “ 114;:4, 12719 and ”missus for rats fed the high fat, high sucrose or high cornstarch diets respectively (Table 5). 50 Results of Students t-test for comparison of mean blood pressures of rats fed the various diets indicate that rats fed the high sucrose diet had a significantly higher blood pressure than rats fed the grain ration (P<.01). Blood pressure of rats fed either the high fat or high cornstarch diets was not significantly different from blood pressure of grain-fed rats. Animals given the 2% NaCl drinking solution exhibited an extremely labile blood pressure pattern with time (Figure 5). Rate fed the grain diet showed the greatest amount of stability and had the lowest blood pressure at the end of 10 weeks (127+20 mmHg) (Table 5, Figure 5). Rats in this group remained normotensive throughout the experiment. Rats fed the high fat, high sucrose and high cornstarch diets exhibited a weekly fluctuation in blood pressure and even- tually became hypertensive. With exception of weeks 6 and 10, those rats fed the cornstarch diet maintained the highest blood pressure throughout the experiment (Figure 5). Blood pressure of these rats reached hypertensive levels during week 5 and peaked at 179 mmHg during week.8. This was followed by a progressive decline in blood pressure until week 10 when blood pressure was lower than that of rats fed the high sucrose diet (Figure 5). Rate fed the high sucrose diet showed a progressive but variable rise in blood pressure throughout the experiment (Figure 5). In all reality blood pressure of these rats 51 reached the hypertensive level during week 6 but declined to normal in weeks 7, 8 and 9 before catapulting to 178 mmHg in week.10. Meanwhile, rats fed the high fat diet exhibited con- siderable variability in blood pressure until week 7 when blood pressure began to rise at a steady rate (Figure 5). Blood pressure surpassed the hypertensive level during week 8 (154 mmHg) and continued to rise until the experiment was terminated. Comparison of final mean blood pressure between dif— ferent diet groups given the 2% NaCl drinking solution indicated that once again the rats fed the high sucrose diet attained a significantly higher blood pressure (P< .02) than grain-fed rats. High fat and high cornstarch-fed rats achieved higher blood pressures than grain-fed rats but they were not statistically different. A Students t-test (Sokal and Rohlf 1969) was used to com- pare mean blood pressure between rats fed the same diet but given either the distilled deionized water or the 2% NaCl drinking solution. Results showed that in the group of rats fed the grain diet there was no significant difference in blood pressure regardless of which drinking solution was consumed. However, in each of the 3 remaining diets there was a significant difference in blood pressure between those rats given the distilled deionized water and those given the 2% NaCl solution. The difference was most significant in rats fed the high sucrose diet (P< .001). Animals fed the 52 high fat and high cornstarch diets developed blood pressures significantly different at the same level (P< .01). Sapirstien.gt;gl, (1950) observed considerable variab- ility in blood pressure of rats given a 2% NaCl drinking solution and fed a standard chow-type diet. He found that those rats which showed large amounts of variability in blood pressure were the rats which eventually became hyper- tensive. Perhaps this pattern of labile blood pressure is a prerequisite for eventual hypertension and therefore could be used as a warning signal of the ensuing disease. Since grain-fed rats given the 2% NaCl drinking solution showed the least amount of variability in blood pressure and were the only group receiving salt that remained normotensive, this suggestion seems noteworthy. Reference should be made to the fact that data repre- senting the high fat-fed and high sucrose-fed rats given the 2% NaCl drinking solution were obtained from 2 rats per group and should therefore be examined with caution. An unexplained high mortality rate occurred in rats under these two dietary regimes. However, this should not lessen the impact of the results since supportive data is explained in Experiment II (to be discussed later). Dalderup‘g§_§l. (1969) noted a decreased life span and early onset of glomerulonephritis in Wistar Albino rats when dietary sucrose was increased from 15%.to 30%»of the diet. Coupled with a hypertonic drinking solution, the effect of sucrose on life span seems more severe. Casual 55 observation of the rats that died suggests diarrhea may have been an important contributing factor. However, diarrhea was not evident in those rats fed the high fat diet in conjunction with the hypertonic salt solution. The ability of excessive salt consumption to provoke hypertension in rats is well documented. The effect of a salt-diet interaction has not been as well established. Hall gt_g;. (1966) observed higher blood pressures in rats given a 5%.sucrose-1%.NaCl drinking solution than in rats given a 5%.glucose-1%»NaCl solution. It has been suggested that the fructose molecule is the offender in such cases (Ahrens 1974). Sucrose elevated blood pressure regardless if excess salt was consumed or not although the presence of salt accentuated the elevation. The cornstarch diet also elevated blood pressure when compared with the grain diet, but not to the same extent as sucrose. Such results would agree with data by Hall. Rats fed the high fat diet developed a higher blood pressure than grain-fed rate. This is difficult to explain since rats are resistant to atherosclerosis (Bragdon and Mickelsen 1955) eliminating the possibility of athero- sclerotic plaques narrowing the vessel lumen and subse- quently elevating blood pressure. Obesity cannot be assoc- iated with the rise in blood pressure since no significant difference in body weight was observed between grain-fed and high fat-fed rats. When rats are fed a 60% hydrogenated fat 54 diet for 63 weeks a higher blood pressure is observed when compared to rats fed a grain diet for the same length of time (Beebe 333;. abstract 1974). The possibility exists that the percentage of body fat might be better correlated to blood pressure than body weight. Pulse Rate Final mean pulse rates can be found in Table 5. Pulse rate trends with time are illustrated in Figures 6 and 7. Mean pulse rates of rats given distilled deionized water followed a trend similar to that of blood pressure in the same diet group. Rats fed the high sucrose diet assumed the highest pulse rate throughout most of the experiment while rats fed the grain diet assumed the lowest. In con- trast, rats fed the high cornstarch diet developed a pulse rate that was lower than rats fed the high sucrose diet but higher than rats fed the high fat diet. Rats given the 2% NaCl drinking solution demonstrated as much variability in pulse rate with time as they had shown with blood pressure. Although considerable variation was observed between dietary groups at several points in time, there was no significant difference between pulse rates in the final experimental week. According to Farris and Griffith (1949) normal pulse rate in the anesthetized rat is 300 beats/minute. Pulse rate can be influenced by several factors, one of which is temperature. When body temperature rises, heart rate 55 increases but blood vessels dilate and blood pressure remains unchanged or lowered (Ganong 1971). The fact that the rats were heated during pulse rate measurement may ex- plain the overall higher pulse rates recorded that are in opposition to Farris and Griffiths' data. In addition, the rats were not anesthetized which may or may not have in- fluenced pulse rate. Geddes (1950) has identified an aver- age pulse rate of 420 beats/minute as normal in the unan- esthetized rat. Body Composition Body fat and lean body mass expressed in grams and as a percentage of body weight are presented in Table 6. Total body water and the amount of water in lean tissue expressed as percent are also presented in Table 6. Body Eat The percentage of body fat in each group of rats, regardless of diet or drinking solution, reflected mean body weight. A linear increase in body fat with increasing body weight has been established in rats fed a high fat diet and in rats fed a grain diet up through a body weight of 450 gm (Schemmel et al. 1969). Regardless of diet, rats given the 2% NaCl drinking solution had less body fat in terms of both total grams and percent of body weight than did rats given distilled deion- ized water. Body fat in rats of the former group ranged 56 from 10 gm (6% body weight) in rats fed the high sucrose diet to 22 gm (9% body weight) in rats fed the high corn- starch diet. Of the rats given distilled deionized water, body fat ranged from 30 gm (9% body weight) in grain-fed rats to 86 gm (24%.body weight) in rats fed the high fat diet. Individual comparison of diets and the effect on body fat accretion showed that grain-fed rats were least affected by the hypertonic drinking solution. Body fat in these rats was 9:3%.when given distilled deionized water and 733%.when given the 2% NaCl solution. Despite the fact that rats fed the grain diet in conjunction with the 2%1NaCl solution consumed large amounts of sodium, they managed to maintain body weight and body fat closest to the weight and fat con- tent observed in rats given the distilled deionized water. Body fat in rats fed the high fat diet in conjunction with distilled deionized water was 24;4%1as compared to 8:1% in high fat-fed rats given the 2% NaCl solution. Rats fed the high sucrose diet exhibited a similar reduction in body fat when given the 2% NaCl drinking solution; 6:5% in con- trast to 20:5% when given the distilled deionized water. Rats fed the high cornstarch diet fared slightly better in that body fat was reduced to a smaller extent by the salt solution; from 15:4% in rats given the distilled deionized water to 9:7% in rats given the 2% NaCl solution. 5? Lean Body Mass Since the percentage of body fat in rats given dis- tilled deionized water was always greater than the percen- tage of body fat in rats given the 2% NaCl solution, it follows that the percentage of lean in the former group was always less than in the latter. However, grams of body lean were higher in rats given distilled deionized water (Table 6). For instance, the bodies of rats fed the high fat diet and distilled deionized water contained 280344 gm of lean tissue which represented only 76;4%.of body weight. In contrast, rats fed the same diet but given the 2% NaCl drinking solution contained 163:15 gm of lean tissue which represented 92;1% of body weight. All rats given distilled deionized water, regardless of diet, had comparable amounts of lean body mass when ex» pressed as total grams rather than percent, i.e., 296:28, 280344, 314:25 and 298:42 gm for rats fed grain, high fat, high sucrose and high cornstarch diets respectively (Table 6). The opposite affect occurred in rats given the 2% NaCl solution. Comparable amounts of lean body mass were ob- served in rats of this group when lean body mass was ex- pressed as percent of body weight rather than total grams. The reason being, that rats given the 2% NaCl solution had less variability in body fat than rats given the distilled deionized water. 58 Body water Compared to rats given distilled deionized water, total body water was greater in these rats given the 2%lNaCl drinking solution regardless of diet. Since rats in the latter group had a smaller percentage of body fat total body water would be expected to be greater. Progressive expan- sion of the fat stores of the body results in little in- crease in water content. Leanness is associated with a high body water fraction; obesity with a low (Pitts 1972). Fer example, rats fed the high fat diet and distilled deionized water had a total mean body water content of 5414% and a body fat content of 24:4%. In contrast, rats fed the same diet but given the 2% NaCl solution had a body water content of 67;4%.and a fat content of 6;5%. When total body water was expressed as percent of lean there was no difference in water content of the rats fed any diet or drinking solution. This indicated that tissues of animals given the hypertonic drinking solution were not retaining excessive quantities of fluid. Conversely, neither were the tissues dehydrated. Organ Weights Fresh weights of the heart, right kidney and right adrenal gland from individual rate can be found in Table 7. Weights are expressed as milligrams and milligrams per 100 grams of body weight. 59 £222.12 Heart weight was greater in rats given distilled deion- ized water than in rats given the 2%.NaCl drinking solution. Since the former group maintained higher body weights, they may be expected to also have heavier hearts in view of the constancy of the relationship between body weight and heart weight (Grommet, unpublished data). For example, rats fed the high sucrose diet in conjunction with distilled deion- ized water had the largest body weight and consequently the largest heart weight (16261562 mgm). In contrast, rats fed the grain diet and distilled deionized water had the lowest body weight and the lowest heart weight (11301110 mgm). If rats fed the grain diet are of the same body weight as rats fed a high fat diet, it follows that heart weights are also equal (Grommet, unpublished data). This suggests that body weight is the critical factor influencing heart weight. However, heart weight was not simply a reflection of body weight when rats fed the high fat diet were com- pared to rats fed the high cornstarch diet. Rats given distilled deionized water and fed the high fat diet had a larger body weight than rats fed the high cornstarch diet, yet the rats fed the latter diet had heavier hearts, i.e., 14191233 mgm for rats fed the high cornstarch diet vs. 1128190 mgm in rats fed the high fat diet. When fed the high fat ration rats show an increased efficiency for accretion of body fat (Schemmel g§_g;. 1972). Perhaps calories are shunted towards fat stores 60 while muscular organs such as the heart are deprived of normal growth. On the other hand, since both sucrose and cornstarch diets produced the greatest hypertrOphy of the heart, the carbohydrate diets themselves may influence cardiac hypertrophy in some way. Hypertrophy of the heart was observed in all rats given the hypertonic drinking solution when heart weight was ex- I pressed as mgm/100 gm body weight. A cardiac hypertrophy of 25%.above expected.values, in addition to a consistent elevation of systolic blood pressure above 140 mmHg has been used as a criteria for evaluating hypertension in rats (Greene and Sapirstien 1952). If cardiac weight of grain- fed rats given distilled deionized water is used as the expected value, then rats fed the high fat, high sucrose and high cornstarch diets in conjunction with the 2%.NaCl solu- tion can be classified as hypertensive. Although grain-fed rats showed a cardiac hypertrophy above 25%.when given the hypertonic solution, they did not experience an elevated blood pressure above 140 mmHg. When examined on an individual basis of diet, rats fed the high sucrose diet and the 2% NaCl drinking solution ex- hibited the most severe cardiac hypertrophy. Hall and Hall (1966) also observed greater cardiac hypertrophy in rats given a 5% sucrose-1%rNaCl drinking solution as opposed to rats given a 5% glucose-1%1NaCl solution. 61 aim Fresh weight of the right kidney was less in rats given the 2% NaCl drinking solution than in rats given distilled, deionized water (Table 7). This was probably a reflection of the larger body weights in the latter group. However, when kidney weight was converted to mgm/100gm body weight, all groups of rats given the 2%.NaCl solution exhibited renal hypertrophy. Sapirstein g£_§;. (1950) observed renal enlargement relative to body weight in rats given a 2% NaCl drinking solution and fed a chow ration. When the effect of diet on kidney weight was examined, rats fed the high sucrose diet had the largest kidneys relative to body weight. This was true for those rats given either distilled deionized water or the 2% NaCl drinking solution. The right kidney of the latter group was 30% larger than the kidney taken from rats fed either high fat, grain or high cornstarch diets. If compared to kidneys of rats consuming the distilled deionized water , the size of the kidney from high sucrose-fed-2% HaCl solution group of rats was 100% larger than the kidneys from any group of rats regardless of diet. Since absorption of glucose in the kidney requires work and therefore increases oxygen consumption in the kidney, it follows that renal hypertrophy may be a compensitory mecha- nism in the rats fed the high sucrose diet. However, this should be equally as true for the glucose derived from starch. Yet rats fed the high cornstarch diet did not 62 experience as much hypertrophy as did rats fed the high sucrose diet. There is a possibility that the enlargement of the kidneys in rats fed the high sucrose diet may have been due to an accumulation of fat. This is based on the observation of Winnie g£_§;. (abstract 1974) that rats fed a sucrose diet had a higher percentage of fat in the kidney than did rats fed cornstarch or grain. Overfeeding has been shown to produce renal lesions and renal hypertrOphy in rats (Kennedy 1960). It is possible that fat stored in the kidney has pathological effects on renal tissue. A e l Gland Right adrenal glands taken from rats given the 2% NaCl drinking solution weighed less than adrenal glands from rats given the distilled deionized water with exception of rats fed the high fat diet. When these weights were converted to mgm/100gm body weight those rats given the 2% NaCl solution showed adrenal hypertrophy ranging from 25% to 114% above dietary counterparts given distilled deionized water. Rats fed the grain diet and the 2% NaCl solution had adrenal glands that were 25% heavier than those receiving distilled deionized water. Rats fed either of the high carbohydrate diets increased adrenal size by 50% over dietary counters parts given distilled deionized water. Adrenal hypertrophy was most severe in rats fed the high fat diet and the 2% NaCl solution. Adrenal weight increased 114% above high fat-fed rats given distilled deionized water. 65 It has been suggested that salt hypertension is pro- duced through a synergism between large quantities of salt and endogenous secretions of the adrenal cortex (Sapirstein 31_§;. 1950). Constant stimulation of the adrenal gland may cause the gland to hypertroPhy in rats given the 2% NaCl solution. On the other hand, if the renin-angiotensin- aldosterone system were reacting normally to a salt load, the gland may exhibit atrophy due to lack of stimulation by angiotensin (Berman 1973). Sodium and Potassium Concentrations semi NOrmal serum levels of sodium and potassium were pre- sent in rats fed the grain ration and given distilled, de- ionized water to drink (Tables 8 and 9). However, all re- maining serum sodium values were below normal regardless of diet or drinking solution. Conversely, serum potassium concentrations fluctuated between normal and above normal. Rats fed the high sucrose diet showed exceptionally high potassium values of 8.013.4 and 10.610.4 meq/L for rate given distilled deionized water and the 2% NaCl solution respectively. Such values are beyond the physiological max- imum for normality of 6.0 meq/L. Several investigators (Schackow and Dahl 1950, Greene and Sapirstein 1952, Meneeley g;_;;. 1961, Height and Weller 1961) have failed to show a change in serum sodium or potas- sium concentration in rats which ingested large quantities of sodium chloride. 64 ass-.2 Nermal sodium concentration in the heart of the rat is approximately 39 meq/gm of fresh tissue (Spector 1956). Most of the rats maintained normal sodium concentration in the heart regardless of diet or drinking solution. Rats fed either the high sucrose or high cornstarch diet and given the 2% NaCl solution were the exception in that these rats had identical sodium values of 48 meq/gm which were 23% higher than the expected value (Table 8). Results of several observations has suggested that concentration of sodium does not increase in the hearts of rats given excess NaCl in the diet or drinking solution (Meyer 21_g;, 1950, Meneeley g£_§;. 1961, Haight and Weller 1961). Concentration of sodium in the hearts of animals fed the different diets did not reflect sodium intake and there- fore would support the work of previous investigators. Potassium concentration in the heart of rats from each experimental diet group was slightly higher than the expected value of 84 meq/gm of fresh heart tissue (Spector 1956) (Table 9). Any differences in potassiim concentration between rats fed the various diets or given either drinking solution were slight and nonsignificant. Haight and Weller (1961) failed to observe any change in heart potassium concentration in rats given excess dietary sodium. Concentration of sodium and potassium in the aorta has been shown to increase linearly with increasing quantities of NaCl in the diet of rats (Tobian 1960, Haight and Weller 65 1961). In addition, concentration of these electrolytes was greater in hypertensive rats and became progressively greater as blood pressure increased. Kidney Since normal concentrations of sodium and potassium in the kidney of rats could not be obtained from the literature values for the rabbit were used as a guideline for expected values. Sodium concentration in the kidney of rats was slightly greater than expected from data for the rabbit which denotes 109 meq sodium/gm of fresh tissue as being normal (Spector 1956). Sodium concentrations in the kidney of rats given distilled deionized water were similar regardless of diet fed; i.e., 124110, 122110, 125118 and 126116 meq/gm of fresh kidney for rats fed grain, high fat, high sucrose or high cornstarch diets respectively (Table 8). In contrast, rats given the 2%.NaCl solution exhibited a marked increase in the amount of sodium retained in the kidney. Rats fed the grain, high fat and high sucrose diets increased kidney sodium concentration to 152139, 165149 and 165112 meq/gm of fresh tissue respectively (Table 8). Each of these values was significantly greater (P< .05) than values for rate given the distilled deionized water and fed the same diet. Sodium concentration of the kidney in rats fed the corn- starch diet and the 2% HaCl solution.was 197147 meq/gm (Table 8). This was a highly significant increase (P<:.O1) 66 when compared to concentration in kidneys of rats given the distilled deionized water. Interestingly, concentration of sodium in the kidney appears to reflect the total amount of sodium consumed for the 10 week period. Rats that consumed distilled deionized water consumed approximately equal amounts of sodium (Table 4) and subsequently had similar concentrations of sodium in the kidney (Table 8). Rats given the 2% NaCl solution in- gested similar amounts of sodium for the 10 week period with exception of rats fed the high cornstarch diet which con- sumed slightly but not significantly more sodium (Table 4). Concentration of sodium in the kidneys of these rats ex- hibited the same pattern. Rats fed the high fat, grain and high sucrose diets had similar concentrations of sodium in the kidney whereas rats fed the high cornstarch diet had slightly but not significantly more sodium in the kidney (Table 8). Meyer §£_g;. (1950) failed to observe any change in the concentration of sodium in the kidney when rats were fed 5% sodium in the diet. These results suggest that the admini- stration of sodium through the drinking solution has more marked effects on the retention of sodium in the kidney. The capacity of the kidney to excrete sodium in the urine may be impaired. Potassium concentrations observed in rats appear similar to the normal value for rabbits of 84 meq/gm fresh kidney (Spector 1956), Concentration of potassium in 67 kidneys of all groups of rats regardless of diet or drinking solution were not significantly different from each other with exception of rats fed the high fat diet and the 2% NaCl solution. Potassium concentration in these rats was 26% greater than the expected value of 84 meq/gm (Table 9). Meyer 21_§;. (1950) observed a significant increase in the concentration of potassium in the kidneys from rats fed a high sodium diet but he failed to offer an explanation. m For lack.of a value for bone sodium and potassium in the rat, values from horse femur were used as a guideline. Concentrations in the rat femur were within the same range as for the horse femur, 1.6., 130-169 meq sodium/gm dry fat- free bone (DFF) and 4.0-5.0 meq potassium/gm dry fat-free bone (Spector 1956). Sodium concentration of the right hind femur of rats given distilled deionized water and fed the grain, high fat, high sucrose and high cornstarch diets was 139121, 147156, 147125 and 156137 meq sodium/gm DFF bone respectively (Table 8). These values were not significantly different from each other which suggests no effect of diet on sodium concentra- tion in the bone. Sodium concentration in the femur of rats given the 2% NaCl solution were markedly higher than concentration in rats given distilled deionized water. Sodium concentration ranged from 50—136%»above values obtained in the latter group. 68 These results confirm speculation of Greene and Sapirstein (1952) that bone is a labile storage site for sodium. Sodium intake relative to body weight was similar in rats given.the 2% NaCl solution and fed either grain, high fat or high cornstarch diets (Table 4). Concentration of sodium in the femur of these rats waS‘ also relatively equivalent (Table 8). Only 1 femur sample could be attain— ed from rats fed the sucrose diet and given the 2% NaCl drinking solution. This rat consumed the largest amount of sodium relative to body weight (53.4 sm/100 gm body weight) and in turn had the highest concentration of sodium in the femur. If concentration of sodium in the bone increases pro- portionately with sodium intake then perhaps the bone is a labile storage site for sodium. Variable quantities of sodium could be stored by the bone which would play an imp- ortant role in maintaining constant plasma and tissue levels of sodium. Regardless of diet, the concentration of potassium in the femur of rats given distilled deionized water was rela- tively the same and near the normal value of 5.0 meq potass- ium/gm DFF bone (Table 9). In contrast rats given the 2% NaCl solution had lower than normal potassium values with exception of the high sucrose-fed rat (Table 9). Potassium concentration may be expected to decrease in the bone if it is assumed that sodium ions replace potassium ions when the rat is on a high sodium diet. 0n the other 69 hand, potassium ions may move into the bone in an attempt to maintain a constant sodium:potassium ratio. This could explain the high potassium content in the femur of the rat fed the sucrose diet. EXPERIMENT II A second study was conducted in an effort to accumu- late additional data concerned with the effect of diet and salt consumption on blood pressure. Since the 2% NaCl drinking solution appeared toxic to rats fed the high sucrose and high fat diets, it was hypothesized that a reduction in concentration of the solution might decrease mortality and provide comparable results. The same diets used in Experiment I were used in the second study. The experimental time period was extended to 18 weeks post weaning. During the first 9 weeks rats were given a 1% NaCl solution followed by a second 9 weeks of a 1.5% NaCl solution. During the sixth week of the experi- ment, a 2% drinking solution was substituted for the 1% sol- ution; within 24 hours 2 high sucrose and 1 high fat-fed rat died. This necessitated a return to the 1%.NaCl drinking sol- ntione DietI Kcal, and Sodium Consumption Total grams of diet consumed in 18 weeks for rats fed grain, high fat, high sucrose and high cornstarch diets were 2875199, 1684186, 25771l60 and 31681270 respectively (Table 10). The lower intake in rats fed the high fat diet was due to the fact that this diet was calorically more dense than the other diets. When grams of diet were converted to kca1., food consumption equalized between diet groups. Rats fed 70 71 the grain, high fat, high sucrose and high cornstarch diets consumed 97751335, 10,104156, 97931608 and 12,03811026 kcal. respectively for the 18 week experimental period (Table 10). Total fluid intake was relatively equal for all rats regardless of diet; i.e., 85651600, 896911026, 10,46911214 and 90551l470 ml. for rats fed grain, high fat, high sucrose and high cornstarch diets respectively (Table 10). Rats fed the high sucrose diet drank slightly more of the salt solu- tion than did rats fed the three remaining diets but the dif- ferences were not significant. Once again if sucrose prod- uced hyperosmolarity, thirst would be stimulated as a compen- sitory mechanism to return osmolarity to normal. Because the rats fed the high sucrose diet ingested larger amounts of the salt solution, total sodium intake was greatest in this group of rats. However, there was no stat- istical difference in total sodium intake between rats con- suming the different diets. Rats fed the grain, high fat, high sucrose and high cornstarch diets consumed S6.715.8, 56.419o9, 66.71l5.9 and 61.51,l§.1 gm of sodium respectively (Table 10). Total sodium intake represents sodium from both the diet and drinking solution. Sodium from the drinking solution constituted proportionately more of the total intake than did any specific diet (Table 11). If total sodium intake was expressed as gm sodium/100 gm body weight the same pattern occurred since body weights were relatively equal among rats regardless of diet. Rats fed the grain, high fat, high sucrose and high cornstarch 72 diets consumed 12.91103, 1107:201, 1502:304 and 11"}:3‘1" gm sodium/100 gm body weight (Table 11). Growth Rate All rats gained body weight at the same rate over the 18 week period regardless of diet (Figure 8). The severely depressed rate of growth observed in rats given the 2% NaCl drinking solution in Experiment I was not evident in rats given the 1-1.§% NaCl solution. In fact, if growth rates of rats in the first 10 weeks of Experiment II are compared with rats of Experiment I the growth curves are nearly iden- tical with exception of rats fed the high sucrose and high fat diets (Figures 8 and 2). Slightly less than expected growth rates for rats fed either of these diets was evident from the sixth experimental week. However, the depressed growth rate was not as severe as in rats given the 2% NaCl solution in EXperiment I (Figure 3). W Mean pulse rates followed a trend that was similar to pulse rates observed in rats given distilled deionized water in Experiment I. Rats fed the grain, high fat, and high cornstarch diets experienced a rapid decline in pulse rate occurring in the first 3 experimental weeks. Pulse rate then proceeded to level off with a certain degree of weekly variability (Figure 9). Rats fed the high sucrose diet, maintained the highest pulse rate throughout the experiment. A multivariate analysis of variance of pulse rate from rats of Experiment I and II through the first 10 experimental 73 weeks indicated an effect of both diet and time on pulse rats (Table 12). The greatest effect of time on pulse rate was seen post-weaning when pulse rate decreased in the first 3 weeks. The effect of diet on pulse rate was probably due to the higher values exhibited by rats fed the high sucrose diet. The physiological significance of a higher than normal pulse rate would be manifested in a greater work load for the heart (Opie 1965). Blood Pressure Mean systolic blood pressure increased linearly and at similar rates in all rats during the first 3 experimental weeks regardless of the diet fed (Figure 10). During the remaining period of time, blood pressure was more labile, however, not to the same extent as in rats fed the 2% NaCl solution in Experiment I. During the 4th week, rats fed the high sucrose diet exhibited a greater blood pressure than rats fed either grain, high fat, or high cornstarch diets. Rats fed the high sucrose diet maintained the highest blood pressures throughout the remaining 14 weeks of the experiment (Figure 10). Animals fed the grain, high fat, and high cornstarch diets eventually achieved approximately the same blood pres- sure as rats fed the high sucrose diet. There was no sig- nigicant difference in blood pressure of the different groups in the 18th week. By the 18th week rats fed the high sucrose diet deve10p- ed a mean systolic blood pressure of 140 mmHg which was the 74 only value in the hypertensive range. Blood pressure was climbing in all rats when the experiment was terminated which may indicate that definate hypertension may have eventually evolved with time. When data from both Experiments I and II are examined for equivalent lengths of time (first 10 weeks), a multi- variate analysis of variance indicates an effect of a diet- salt interaction on blood pressure (Table 13). The effect of salt is obviated in the fact that blood pressure was elevated in all animals that received a NaCl drinking solu- tion. The effect of diet appears to center around the high sucrose ration. Rats fed the high sucrose diet exhibited the highest mean blood pressure regardless if a salt solu- tion was consumed or not (Figure 10). It is apparant that the mechanism responsible for the development of hypertension is accelerated by the presence of the high sucrose diet. Furthermore, the addition of sodium chloride further augmented the blood pressure raising effect of sucrose. 75 TABLE 1.--Composition of Rations. Dietsg(%) Ingredients Fat Cornstarch Sucrose Proteina 29.9 20.0 20.0 Fat: Crisco 51.8 -- -- Corn oil -- 3.0 3.0 Carbohydrate: Sucrose 2.2 -- 67.7 Cornstarch 2.2 67.7 -- Mineralsb 7.5 5.0 5.0 Sodiumc 0.68 0.62 0.50 PotassiumC 0.59 0.52 0.49' Fiberd 4.5 3.0 3.0 Vitaminse 1.5 1.0 1.0 Dl-Methioninef 0.37 0.25 0.25 kcal/8mg 600 3.8 3.8 aCasein, purchased from General Biochemicals, Chagrin Falls, Ohio. bRogers and Harper salt mix, purchased from General Bio- chemicals, Chagrin Falls, Ohio. cValues obtained from flame emmission spectrophotometry, Model 453, Instrumentation Laboratories. dCellulose type, purchased from General Biochemicals, Chagrin Falls, Ohio. eA.0.A.C. Vitamin mix, purchased from General Biochemical Chagrin Falls, Ohio. Supplied the following (gm/kgm diet): p-aminobenzoic acid, 0.10; B1 , (0.1% in mannitol), 0.03; biotin, 0.0004; calcium pantoihenate, 0.04; choline, free base, 2.0; folic acid, 0.002; l-inositol, 0.10; menadione, 0.005; Niacin, 0.04; pyridoxine HCl, 0.04; riboflavin, 0.008; thiamine HCl, 0.005; dextrose, anhydrous, q.s.; (units/kgm) Vitamin A, 20,000.00; Vitamin D2, 2,000.00; Vitamin E acetate, 100.00. fPurchased from General Biochemicals, Chagrin Falls, Ohio. SValues used for calculating kcalories were 4, 4, and 9 for 1 gm of protein, carbohydrate and fat, respectively. 76 TABLE 2.--Composition of Grain Ration. Ingredientsa Percent Protein 23.4 Fat 3.0 Carbohydrate (by difference) 53.5 Fiber 508 ASh 603 Moisture 10.0 kilocalories/gmb 3.4 aThe grain diet contained (in %): ground corn, 60.7; soybean meal (50% protein), 28.0; alfalfa meal (17% protein), 2.0; fish meal (62.5% protein), 2.5; dried whey (67% lac- tose), 2.5; limestone (38% calcium), 1.6; dicalcium phos- phate (18.5% P, 23.4% Ca), 1.75; and iodized salt, 0.5. The following were also added: (in mg/kgm feed) Mn, 121; Fe, 95; Cu, 7; Zn, 4; I2, 4; Co, 2; choline chloride, 400; Ca panto- thenate, 6; riboflavin, 3; niacin, 33; menadione, 2; D1- Methionine, 500; penicillin, 2; streptomycin, 8; arsanilic acid 968; (in ug/kgm feed) vitamin B12, 7; and (in IU/kgm feed vitamin A, 8010; vitamin D2, 750; and vitamin E, 5. bSchemmel, R. et al. 1972 7. 7 .coapmfi>mu camcsMpw H ammZo .msoam some :fi mama mo ampsszn .vofinem meme op m nm>o umpmH52500gm :eafimwmw ammflmmms samuom_. m.m “av Hoez am + seeeemeeoo ensueomm m.muamom _e_uwom_ w.m Amy neeeaeeeeo 2:483 eawumoem @038 me Q 8% am + .883. mm_uom_m mmauo_mm mP—usmm_ m.n Amy emceesm manommm mmnummem moflonm 0.0 Amy Hoez am + new mmiflomem emnnwamm monomm 0.6 Amy new ow:_now_m mmeflmmo: mw_u:m__ e.m may Hoez am + names noonueonm cesiummom eo:.uwme. a.n paws eaeew Ha amok 11111. aw aexeeea eaeam eexeeea seam node was neeeeeeea .mxeos.o— sou Hoez &N no seams ceufinofioc coaaapmac mo soausaom wsfixnaac m sea: cosponsnsoo ma peas nonspmsuoo swan so awesome swag .pmm amen .eaeem e see when fleece: eeeopeo ones so message eaaae use peas eaaeeasaso-u.m mamas 78 .coapmfiemn camusmpm H swmzo .qsoam some ca massage mo ampszzn .voasma moms OF m smeo mxmpca m>HumH5ssom a.mnm.mm s.nuo.am mm.ui..m m._uo.o Ass Hoez am + neeeemeeeo m.ou_.m w.ouo.w 1- m.ouo.w va aeaeeeaaoo Qua.mm m.uue.mm m.nnm.wa m.aue.m Amv Hoez am + eeoeesm N.0Hm.— m.ofim.w 11 m.Qflm.w va mmouosm m.qum.mm m._ua.mm m.oua.m: a.oum.: Amv Hoez am + see m.oua.. a.ouo.a -1 a.ouo.a Amy new ¢.nmum.mm o.__um.mm m._.no.we a.ouo.m Aav Hoez am.+ eaeew em.quo.m ea.oum.a 1- ea.qum.a pawv eaeew hamemoo.\am am am am mssavom Heuoa shapes song «2 mvoom song «2 . vsospmoua .mxoew 0. non Homz RN no sense ueuwsoaec moaafiu Imus mo soausaom weakness s and: soaposSnmoo ca peas nonmpmcaoo swan no emouosm swan .pmm swam .samnm m smmufim com mums Hensoz onuonmo cams mo okays“ ssficom o>aueasssonn.¢ wands 79 TABLE 5.--Fina1 blood pressure and pulse rate of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a drinking solution of distilled deionized water or 2% NaCl for 10 weeks. Treatment ngodmifigssure Pulsgpgate Grain (8)a 109114b 595161b Grain + 2% NaCl (7) 127120 357171 Fat (8) 114113 585164 Fat + 2% NaCl (2) 15418° 410146 Sucrose (8) 12719 435146 Sucrose + 2% NaCl (2) 178114° 310171 Cornstarch (8 ) 1 1717 433318 Cornstarch + 2% NaCl (4) 150120° 395177 aNumber of rats in each group. bMean 1 standard deviation. cConsidered hypertensive (>140 mmHg). 8O .coa»ma>me vamusmum H smmzo .asOAM some qH mums mo gonads ow mammmmo .um>osms msmmso mom copmznvm no: pgmfims anon o>HAm an; sumo aflm mouse. sum mmumm mauopm t: Sez am + reassessoo :Hmm nun—m :Hmm musmm sHmF omHmm mmHomm A3 noscumssoo an: swam muse enumm; one :8. :59 Amy Sea am + 8225 an: QR anon mmuim muom mmumm 03? RV eoososm hoe are fine Dune. me one. :umom 3V Sea am + new mfla swam sump ssflowm swam omuem Runs: :3 sea mun. muse nuns smug. nus Sum. Dfimmm EV Ssz am .. 53o emu? amuse emua ommummm ammo cmuom emmuman as: 525 smmH HLWOM & am & am am sees; seem seem asom ens seem a; seeseeesa .mxoos 0. Mom Hoez &N so have: soufi20Hec coaafiumau mo soapsflom wsaxsaac s new: soaposSnsoo as peas noumumsnoe swan no mmouosm swan .pmm swag .sfimnw s new mass House: canonmo cams mo soapamomsoo been one pnwwos muom11.m mqm<9 81 .msosm some as mums mo senses o» mammmm n .moa»ma>ou unducmpm H_cm62m Twas... Tofimém mo.Hmmm smmumz. as”; Tmumom as 82 am + senescence a.mumfi 0030mm omnasm mamusmmi mofimam mmmumtz 8V sofisnssoo m.sum.m_ 93.4; 53$ mmmflmsop Euwmm momflmmw Amy Ssz am + $225 imum;— s.m_um.mm ERR amines. Rfimmm mmmummm. E 36.35 astH. 2.0.2.3 tennis 8.38. 88% 02.85 Amy Sez am + cos 0.3.02. o.a....o.om omuosm 03.8mm. omuomm omuwm: a: sea _.num.2 043.5 minim Ennis: min—Moss semuma AS Sez am + eases o.m...o.w oéfloém. osuosm 838m. onuoR Eamon: as: cases wwmnwmmma ems PWWOflWmms awe wwwnmwmms Ema musmfiw Hmsosug mwmscwx meadow usospmous .mxooe or you Homz &N no hopes wonasoaou coaaaumfic mo soapsaom wsfixsauc u made soapossnsoo ea peas sonmumssoo swan so omonoam swan .umu swag .swmaw s new mass House: osuonmo mama sou magmas: madam ascends pmman use hosuax unmfiu .pnmom11.n mqmda 82 .maco pea P Scum «use mpsmwonmomo .Ammnv osmme new coke may we vmwmmumxm .soaumfi>mu osmvsmuw H :mmzu .azosw some ma mama mo nonszZo p .osmmfip among mm vmmmmumxmm amusmm 3.3.9 3: whom a: Sez am + seasonseoo R33 SUN. 3% mmumop a: senescence commas Tune. sums .nummp AS Ssz am + $826 amuse. TNT an: mafimm. at $685 smmusmm 3me mums fume. AS Ssz am . sea on”; ofimm. mumm mmHg :3 use Elam—m Rum? mums 2.30. as Sez am + eases cannon. sofism. smug emnums. 033 cases nsmnvos swmcos newness Humos s=Som m mscfix psmom annom namepmoua .mxmos 0— now Homz &N no popes seaflsofioc uoHHHumHu mo soapsaom msfixsfiun 6 mad: soapossnooo ma page soumumsnoo swam no omouosm swam .umm swan .swmnm a new mass House: osuonmo mass Mo assay scam «swan use hoscHx pnmfis .mhdo: .ssnom mo accuses ssfivom11.w mqmds 83 .maso pen _ 509m #00 musmmmnmemm soapmw>ou vumcsmum H smmzv .mmosm some cw mums mo amnesZo .Ahmav enema» has comm new we 00mmmggNMp .osmmfiu momma mm commosaxmm ERA 0H5 mnmm 023$ 30 Sez am + possesses Tonia when 0..+.00 0.0u0.: $0 seeseossoo e030 $.00 flaw itowed, Amy Sez am + eeesesm m.0H0.m 03mm $.00 {9.0.0 2.0 oneness s.0.+.0.m 0.32 5me 3N0 Amy Sez am + use 0._Hw.m mHnw mew o.aHm.m va pom EMMA ~4wa 0......00 fiaumfi EV Sea am + eases smirk snows smnma sense 0:: 52s ammmwms mmmmmmm am we Hmuos .x psmom aspen unmanneaa .mxmos 0. you Homz &N so hope: veuasoaou emaaapmau «0 soapsflom msaxsaus m new: soapossn 1:00 ca peas nosmpmsuoo swan no.0mososm mm“: .»m« swan .sawuw a can mean Hesse: osuopmo mass no nssmm 0nd: unmas use hosofix ammuu .pndon .ssnom mo penance ssfimmmpom11.m mamas 84 TABLE 10.--Cumulative dietary and fluid intake of male Osborne Mendel rats fed a grain, high fat, high sucrose or high cornstarch diet in conjunction with a 1-1.5% NaCl drinking solution for 18 weeks. Treatment Food Intakea Fluid Intakea gm kcalie m1 Grain (6 )b 2875;99c 9775:535" 8565:555" Fat (5) 1684186 101041516 896911026 Sucrose (5) 25771160 97931608 1046911214 Cornstarch (6) 31681270 1203811026 905511470 a‘Cumulative for 18 weeks. bNumber of rats in each group. cMean1 standard deviation. .soapm«>ov vasechm H smmzo .msouw some ea mums mo sonssz s .mxeoe mp mom o>apmasssom 5 I ' l 8 4.00.: .555 04.0.3 {:05 $0 assesses 06.3.9 0.23.00 a.mfioém 0.0.3.2 RV omososm imum... 0.0usem a.mflafie 0.0.3.2 30 see om._um.m. omfiufism 00.0402 00.0.243— s$0 £20 .03 neon am am smoo_\sm 111. am mvfisam scum swoon scum assauom Hmpoe szfivom season psosamone .mxoos mp mom soausaom weakness H002 &m..1. a spa: soapossnsoo ma pods nonmpmsnoo swan no cmonosm : an .pmm swan .sfimsm 5 com mp0s House: osnonmo cams Ho oxmpsw ssficom eeflpmasssou1.__ mgm Mo momzamsd oumflsm>HpH5211.mp mamas 1.10000. 00 s 0 .00:.s Apnea x sense seasoesessH *cepooo. cm a N nmno.mn noommo pHmm 111m_00. 00 s m w000.m pseudo none Mw.1 .1 .1 1. 1. 1. 1. 1. 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Fmp a mp .mmm.o Anoae x eanV soanomuonsH 1.._000. mm a s 000m.m0 Aosssv scenes censuses scseesem uvm Bonomhm «0 owpmfinmpm seaaasononm essence a .onsmmona ocean so peas and name .osan mo noommo1nmosdmmoz wonmemom mom cosmwnm> no momhamsd onmanm>anH5211.mp mqmda 88 .msnmnmmmm o . 1-. 1,... nun emHsm use endomonm vooam11.p mmbon c... \‘n 89 .wsasmosunmom uses: 0. now sense emuasoamv soaafipmae mo soapsaom wsassfisv m spas scanossfi:00 sH news ADV soumnmsaoo swas no AOV mmouosm swan .Aiv nmw swan .Aev :fimsw m 00H mums Houses osuonmo mass mo uswfios moon smozuu.m mmwth sownm>somn0 no see; awmsmw_ 0 OF h p d d “'0‘ db” qu- q +1 .r 00— "w 9 be u .1 com me be “M m. 1. 00m S U. 1- w n. 00: mv 90 .wswsmosunmoa msmms 0. new moansaom wsfissaun Homz RN m spas soanossnsoo as nofiv ADV soumnmsuoo swas so qu mmonosm swan any new swan .Aev sfimsw m 00% mums House: masonmo made we nswaoa huon sm0211.m mmwam sownm>somn0 no see; O— o w h w m J m N p o n 1*. u u k fl » 1n v w \h e \ . .111 11.1\ P00. 0 \Il\\. w" \ a u .\ I 111111, .1OON mun ‘11 0. AA “M m. 4100M on u. 4 .m .03 m .1000 91 1801 170‘P 160-- 150-- “40-11- 1305- I I I a 120- e x E ‘I‘IIIIIIMV' 41llll!|.. - 110-- = e p 100“' 90"' Mean systolic blood pressure (mmHg) 80%- V\ W 1 1 1 1 1 - 1 11 o 1 2 5 t1 5 6 7 8 Week of Observation \0-1. 10 FIGURE 4.--Mean systolic blood pressure of male Osborne Mendel rats fed a grain (0), high fat (1), high sucrose (0) or high cornstarch (D) diet in conjunction with a drinking solution of distilled deionized water for 10 weeks post- weaning. 92 1801L 1704' 160" 1505- 1405b I a 130-1 120-11- . I I . ’ . 1' ”0.. «4“. Mean systolic blood pressure (mmHg) 100“” 901- Q 80”- 70- 55 0123351676910 Week of Observation FIGURE 5.--Mean systolic blood pressure of male Osborne Mendel rats fed a grain (O), high'fat (no, high sucrose (£0 or high cornstarch.(cn diet in conjunction with a 2%.NaCl drinking solution for 10 weeks postdweaning. 93 .wsanmoslnmom enema 0— non Hones vouanoaou eoaaan 1ma0 mo soapsaom wnansase m spas noanonsnnoo sa peas ADV noumnmssoo swan no on omonosm swan .Atv new swan .Aev samsw m sea mush amuse: canonmo mass no money omasm s00211.w mmbwah soanm>aemno no new: 0.. .a 1.... .a a. a _ o . . . . . . . i i n -roomw P u m JuomMW w q 9 103.. mu 9 9 1. :Refl m .u 94 .wsasmesanmom once: 0— uon soapsaom wsansanu aomz &m m spas soanossnsoo sa peas Ana nonunmsuoo swan no on mmonosm swan .va nan swan .Aev samsw m new mass Hesse: osuonmo mass no money smash smeznn.m mmbwah soanm>somno no new: o— m w m w m s m N p o 1. a u a a u. .1 T i a 100mm 0 s u m .1ommm “a U 1- 9 n 100:) a. 9 9 m. e I nuomsw .\ u /\ e 95 .wnasmmslnmon mnoms w. now soanmaom wsansauu aomz xm.p1— m spas noanossnsoo sa peas ADV nonmnmsuoo nwan no qu mmonosm swan .Anv sen smas .aev case» n sea ends asses: osnosuo case no ssmaea sees ssez11.w masoas soanmeuomno no new; m— o— m w u m m J m N — o i i n A i a .1 47+ .1 1. 1:189 "N a m nTOONnd O by m L100mm“... u. a...- 181m irOOm 96 .wsasmosunmom enema w. now soapsaom weassanu aomz Rm._1_ m spas soanossnsoo sa peas euv sonsnmsnoo swan no QUV omonosm swan .owv new swan .Aev samnw m new mass amuse: osnonmo mass no means amass sm0211.m mmpwam soanm>nomno no new: e a .. a a. m m .2 m a, 0 100mm m m 10%» w A... 9 1:00J) 0.. 9 P m / m 97 1801- 1704- 160‘- 150-- Ill-0'“- , . 9 o a 1301- 1" 120-11- 9 A, {l 110-- 1 4.!" /7"’ ” 100‘” 904t Mean systolic blood pressure (mmHg) 80H- 70“- I L L J l 1 l l l I I l | : 9 1 0 4 5 6 7 9 1 Week of Observation q.sqp Nui- \N FIGURE 10.--Mean systolic blood pressure of male Osborne Mendel rats fed a grain (0), high fat (i), high sucrose (0), or high cornstarch (U) diet in conjunction with a 1-1.5% NaCl drinking solution for 18 weeks post-weaning. CONCLUSIONS Blood pressure was elevated in rats given the hyper- tonic drinking solutions. As concentration of the solution increased, mean arterial blood pressure increased and became more labile. In rats fed the high fat, high sucrose and high cornstarch diets blood pressure reached hypertensive levels when the rats were given the 2% NaCl drinking solu- tion. Grain-fed rats did not become hypertensive when given the 2% NaCl solution. When given a 1-1.5% NaCl drinking solution only rats fed the high sucrose diet became hyper- tensive; rats fed the high fat, grain and high cornstarch diets had not reached hypertensive levels at the end of the 18 week feeding period but they were approaching hyperten- sion. When maintained on distilled deionized water blood pressure was highest in rats fed the high sucrose diet, yet all blood pressures remained in the normotensive range. Pulse rates were highest in rats fed the high sucrose diet regardless of which drinking solution was consumed. The presence of either hypertonic drinking solution did not significantly alter pulse rate over time regardless of the type of diet consumed. However, rats given the 2%1Na01 solution exhibited a more labile pulse rate with time than did rats given the 1-1.fi% solution or distilled deionized water. Growth rate was significantly depressed in rats given 98 99 the 2% NaCl solution. Rate of growth was not depressed in rats given the 1-1.5%1NaCl solution. The retarded growth pattern observed in the former group of rats reflected the marked depression in food intake. Growth rate was most ’ severely depressed in rats fed the high sucrose diet and least in rats fed the grain diet. Relative weight of the heart, right kidney and right adrenal gland increased in rats given the 2% NaCl solution. Rats fed the high sucrose diet exhibited the largest in- crease in relative weight of each organ with exception of the adrenal gland which was hypertrophied to the greatest degree in rats fed the high fat diet in conjunction with the 2%»NaCl drinking solution. Concentration of sodium and potassium remained un- changed in the serum and heart of rats given the 2% NaCl solution when compared to rats given the distilled deionized water. Sodium concentration in the kidney and right hind femur reflected total sodium intake and consequently in- creased in rats given the 2% NaCl solution. Potassium concentration remained unaltered in the kidney of rats given the 2% NaCl solution. In contrast, concentration of potas- sium in the femur was decreased in these rats. SUGGESTIONS FOR FURTHER STUDY .Sodium Chloride Absorption When compared to rats fed the high fat, high sucrose and high cornstarch diets and consuming the 2% NaCl drinking solution, animals fed the grain diet had the lowest mort- ality and retained the smallest amounts of sodium in tissues of the body. Blood pressure of these rats was not consid- ered to be in the hypertensive range despite the fact that these rats received as much sodium chloride as rats fed the semi-purified diets. It is well established that a 2% NaCl drinking solution produces hypertension in rats (Sapirstien 21_g1. 1950). This raises the question of whether sodium was absorbed by animals fed the grain ration. Further study might include 24 hour feces and urine analysis for sodium and potassium concentrations. This procedure would shed some light on the pathway of sodium once it enters the body of the animal. Although the amounts of fiber and protein in the diets were nearly equal, the type of fiber or protein may have some influence on the absorption of sodium and therefore warrants consideration for investi- gation as well. 100 101 Concentration of Sodium in Kidney and Bone Concentration.of sodium in kidney and bone increased in rats given the 2% NaCl solution. It appears that the concentration of sodium in the kidney and bone reflects total sodium intake. Further study may involve administra- tion of salt solutions of various concentrations to rats and measurement of sodium and potassium in kidney and bone of these animals. The effect of a follow-up period of salt restriction would verify whether the bone was actually a labile source of sodium. Consideration might be given to the effect of a high concentration of sodium in the bone on red blood cell formation. Simple procedures may include blood hematocrits and hemoglobins. Fructose and Blood Pressure Since the high sucrose diet appeared most detrimental to health of the rats, some light may be shed on the reason through the addition of a high fructose diet to the experi- mental design. Fructose has been implicated as the culprit in the sucrose effect on blood pressure (Yudkin 1973) but substantial evidence in support of this fact has not been produced. Appgtite Depression Miller and Czajka (1967) have decreased gastric emptying time in rats force-fed hypertonic glucose solutions. 102 The osmotic effect of hypertonic diets in addition to a hypertonic drinking solution may be completely different. Pursuit of gastric emptying time in rats given the hyper- tonic sodium chloride solutions and fed the various diets is worth consideration. Appetite was definitely depressed in rats given the 2% NaCl drinking solution yet the reason is obscure. 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APPENDIX A OPERATION OF ESG COUPLER FOR MEASUREMENT OF BLOOD PRESSURE AND PULSE RATE POWER switch ON RECORD SWitch OFF VARIABLE control set at 2 OmV/cm POLARITY to positive (+) FILTER switch to 10 Hz position MV/CM switch to 100 mV/cm position Adjust baseline with POSITION control Set ESG coupler PRESSURE range switch to 0-100 mmHg (maximum sensitivity of 20 mmHg/cm with full-scale deflection of 5 cm) . RECORD BWitch ON 111 APPENDIX B PROCEDURE FOR CALCULATING TOTAL SODIUM INTAKE Since the atomic weight of sodium (Na) is 23 and the atomic weight of chlorine (Cl) is 35.5 it follows that the atomic weight of sodium chloride (NaC13 is 58.5. Each am of NaCl consumed contains 23/58.5 or 39.3% sodium (Na§f To obtain total sodium intake from fluid intake the total quantity of fluid consumed (ml) was multiplied by the concentration of the NaCl solution. This number was then multiplied by 0.393 to obtain grams of sodium. Example: 6000 ml fluid x 0.02 x 0.393 = 47.16 gms Na Since the grain, high sucrose and high cornstarch diets contained 0.5% sodium (Na) and the high fat diet contained 0.75% sodium, total sodium intake from the diet was calcu- lated by multiplying total grams of diet consumed by either 0.005 or 0.0075 depending on the diet in question. Example: 1400 gm grain diet x 0.005 = 7.0 gm Na Total sodium intake was the sum of the sodium ingested from both the diet and drinking solution. 47e16 8m '1' 7e0 813 = 5he16 tOtal gm Na 112 MICHIGAN smTE UNIV. LIBRRRIES 1|HIWNWWNIIIIWll”NIIIHIIMHIWIWWI 31293103192740