-L" ' ILII3£\Alt‘l{ madam Michigan State U132 ’vCl‘Sity This is to certify that the thesis entitled The Effect of Obesity in the Rat on Cardiovascular Dynamics, Cardiac Anatomy and Biochemistry, and Body Composition presented‘by Janet Kolmer Grommet has been accepted towards fulfillment of the requirements for face 4 AM Major professor Date_ September 15, 1978 0-7639 THE EFFECT OF OBESITY IN THE RAT 0N CARDIOVASCULAR DYNAMICS, CARDIAC ANATOMY AND BIOCHEMISTRY, AND BODY COMPOSITION By Janet Kolmer Grommet A DISSERTATION Submitted to Michigan State University in partial fuifiliment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1978 ABSTRACT THE EFFECT OF OBESITY IN THE RAT ON CARDIOVASCULAR DYNAMICS, CARDIAC ANATOMY AND BIOCHEMISTRY, AND BODY COMPOSITION By Janet Kolmer Grommet This study assessed biophysical functions of the cardiovascular system in obesity. Hemodynamic measurements included blood pressure, cardiac output, heart rate, stroke volume, peripheral resistance, blood volume, and cardiac work. In addition, histological measurements were made of myocardial fibers and capillaries as well as cardiac gross anatomy and biochemical measurements. To produce obesity, a high fat ration was fed post-weaning to Osborne-Mendel rats, a strain of rats susceptible to dietary obesity. Both growth-onset and adult-onset forms of obesity were modeled as well as weight reduction and compared to normal weight rats. At 36 weeks of age, the normal weight rats weighed 503 gm, and the weight reduced rats weighed slightly more, 562 gm. The rats with adult-onset and growth-onset obesity weighed 726 gm and 867 gm, respectively, and had three times as much fat as the normal weight and weight reduced rats. Cardiovascular Dynamics Systolic blood pressure, recorded directly from the carotid artery, was significantly elevated to 150 mm Hg in both the rats with growth-onset and adult-onset obesity compared to 128 mm Hg in the Janet Kolmer Grommet .normal weight rats. Although body weight of the weight reduced rats approached that of the normal weight rats, systolic blood pressure remained high, 144 mm Hg. Neither diastolic pressure, pulse pressure, nor mean blood pressure were significantly influenced by body weight. Cardiac output, measured by the Stewart-Hamilton method using indocyanine green dye, increased with increasing body weight from 90 ml/min in the normal weight rats to 150 ml/min in the rats with growth-onset obesity. Heart rate was not affected by obesity and thus output increased as a result of increased stroke volume. On a per kilogram basis, however, output was constant. The hearts of the obese rats expended significantly more work compared to the normal weight and weight reduced rats which was accomplished by the increased stroke volume as opposed to increased mean blood pressure and was hypothesized to result from perfusing an enlarged body mass. Cardiac Anatomy Heart weight increased with increasing body weight from lSlO mg in the normal weight rats to 2228 mg in the rats with growth-onset obesity, an increase of nearly 50%. Similarly, the heart volume, length, and diameter increased with increasing body weight. Obesity did not significantly affect the right ventricle wall or septum width, but relative to the normal weight rats the left ventricle wall was enlarged in the rats with growth-onset obesity, 4.8 mm compared to only 4.0 mm. This enlargement was attributed to cellular hypertrophy as the myocardial fiber width was significantly greater in the rats with growth-onset obesity. The fiber number index, an estimate of the total number of myocardial fibers in the heart, Janet Kolmer Grommet tended to increase as heart weight increased although not significantly. Thus, hypertrophy more than hyperplasia accounted for the increased heart size associated with obesity. Although heart size increased with obesity, it apparently did not influence the number of capillaries in the heart. Thus, the density of capillaries in the left ventricle decreased and may perhaps be a contributing factor to myocardial ischemia. Cardiac Biochemistry The increase in cardiac protein from l50.50 mg in the normal weight rats to 237.29 mg in the rats with growth-onset obesity probably accounted for the hypertrophy noted in the obese rats and the resulting increase in heart weight. Cardiac fat content increased with increasing heart weight, but the percentage remained at 5-6% apparently not being affected by obesity. Quantification of the DNA content of the cardiac tissue indicated that obesity tended to increase the number of cardiac cells although not significantly but did result in an increase in cardiac cell size which was consistent with the histological fiber number index and fiber width measurements, respectively. To my swam Ba/Lba/La Chauotte Chaxla Mamianne Mambfiyn ii TABLE OF CONTENTS Chapter Page I. INTRODUCTION Need for Study ..................... 1 Purpose of Study .................... 2 Statement of Hypotheses ................ 3 Theory ......................... 3 Overview of Dissertation ................ 5 II. REVIEW OF LITERATURE Introduction ...................... 6 Determinants of Obesity ................ 7 Heredity Factors .................. 7 Inactivity ..................... 8 Appetite Disturbance ................ 10 Early Nutrition ................... 11 Meal Patterns . . . . ................ 13 Prevalence of Obesity ................. 14 Mortality and Disease in Obesity ............ 16 Excess Mortality and Obesity ............ 16 Cardiovascular Disease and Obesity ......... 18 Cardiovascular Dynamics in Obesity ........... 21 Blood Pressure ................... 22 Cardiac Output ............ - ....... 24 Blood Volume .................... 26 Cardiac Anatomy in Obesity ............... 26 Gross Anatomy .................... 26 Microsc0pic Anatomy ................. 29 Related Studies ................... 30 Cardiac Biochemistry in Obesity ............ 32 Protein ....................... 32 Fat ......................... 33 Deoxyribonucleic Acid (DNA) ............. 34 III. METHODOLOGY Experimental Animals .................. 36 Source ....................... 36 Housing ....................... 36 Experimental Rations .................. 37 Experimental Design .................. 37 Cardiovascular Dynamic Techniques ........... 39 Blood Volume and Hematocrits ............ 39 Blood Pressures and Heart Rate ........... 40 Cardiac Output ................... 44 Chapter Page III. METHODOLOGY, continued Stroke Volume and Vascular Resistance ........ 47 Cardiac Work .................... 47 Cardiac Anatomy . . . ................. 47 Gross Anatomy .................... 47 Cardiac Tissue Processing .............. 50 Microscope Slide Preparation ............ 3D Photomicrograph Preparation ............. 52 Microsc0pic Anatomy ................. 52 Body Composition Procedures .............. 57 Homogenate ..................... 57 Moisture Determination ............... 57 Total Fat (triglycerides) .............. 53 Lean Body Mass .............. . ..... 53 Cardiac Biochemical Procedures ............. 59 Protein ....................... 59 Fat (by acid hydrolysis) .............. 50 Deoxyribonucleic Acid ................ 52 Moisture Determination ............... 55 Data Analysis ..................... 55 IV. RESULTS AND DISCUSSION Introduction ...................... 55 Body Composition .................... 68 Body Height ..................... 68 Body Fat ...................... 7] Lean Body Mass ................... 7l Cardiovascular Dynamics ................ 73 Blood Pressure ................... 73 Cardiac Output ................... 78 Heart Rate and Stroke Volume ............ 80 Peripheral Resistance ................ 82 Blood Volume .................... 84 Total Body Hater .................. 88 Hematocrit ..................... 90 Respiration Rate .................. 92 Cardiac Work .................... 92 Cardiac Anatomy .................... 95 Gross Anatomy .................... 95 Microsc0pic Anatomy ................. lOO Cardiac Biochemistry .................. 105 Protein ....................... 105 Fat ......................... lO7 Moisture ...................... lO9 Dry Matter ..................... lO9 Deoxyribonucleic Acid (DNA) ............. ll2 Biochemistry Correlations .............. ll5 V. SUMMARY AND CONCLUSIONS Body Composition ........ . ............ ll8 Cardiovascular Dynamics ................ llB Cardiac Anatomy .................... 121 Gross Anatomy .................... 12] iv Chapter Page V. SUMMARY AND CONCLUSIONS, continued Microsc0pic Anatomy . . . . . . . . . . . . . . . . . 123 Cardiac Biochemistry .................. 124 Conclusions ...................... 126 Body Composition .................. 126 Cardiovascular Dynamics ............... 126 Cardiac Anatomy ................... 12? Cardiac Biochemistry ................ 129 APPENDICES ........................... 130 LITERATURE CITED ........................ 166 LIST OF TABLES Body weight and body composition of 36 week old male Osborne-Mendel rats ................. Systolic, diastolic, pulse, and mean blood pressure of 36 week old male Osborne-Mendel rats ....... Grouped frequency distribution of systolic blood pressure of 36 week old male Osborne-Mendel rats Cardiac output, heart rate, and stroke volume of 36 week old male Osborne-Mendel rats .......... Peripheral resistance in 36 week old male Osborne- Mendel rats ..................... Erythrocyte. plasma, and blood volume of 36 week old male Osborne-Mendel rats .............. Distribution of blood volume between body fat and lean body mass in 36 week old male Osborne-Mendel rats ........................ Total body water of 36 week old male Osborne-Mendel rats ........................ Hematocrit values for 36 week old male Osborne-Mendel rats ........................ 4.10 Respiration rate in 36 week old male Osborne-Mendel 4.11 Cardiac work in 36 week old male Osborne-Mendel rats . rats ........................ 4.12 Height and volume measurements of hearts of 36 week old male Osborne-Mendel rats ............ 4.13 Length and width measurements of hearts of 36 week old male Osborne-Mendel rats ............ 4.14 Myocardial fiber measurements of 36 week old male Osborne-Mendel rats . . . . . . . . . . . . . . . . . vi Page 70 74 75 79 83 Table Page 4.15 Myocardial capillary measurements of 36 week old male Osborne-Mendel rats .................. 102 4.16 Protein content of cardiac tissue of 36 week old male Osborne-Mendel rats ................. 105 4.17 Fat content (by acid hydrolysis) of cardiac tissue of 36 week old male Osborne-Mendel rats ........ 103 4.18 Moisture content of cardiac tissue of 36 week old male Osborne-Mendel rats .............. 110 4.19 Dry matter of cardiac tissue of 36 week old male Osborne-Mendel rats ................. 1]] 4.20 DNA content of cardiac tissue of 36 week old male Osborne-Mendel rats ................. 113 4.21 Correlation coefficients of biochemical measures and heart weight of 36 week old male Osborne-Mendel rats ........................ 116 LIST OF FIGURES Experimental design: randomized block ......... Instrumentation diagram for determining blood pres— sure(s), heart rate, and cardiac output ........ Example of polygraph recording giving blood pressure(s) and heart rate from cannulated carotid artery of male Osborne-Mendel rat .................. Circulation of indocyanine green from site of injection to withdrawal ..................... Dissection of the rat heart to produce consistent tissue samples for histological work (A,B) and biochemical analyses (C,D) .................... Positioning of tissue slices in paraffin blocks so the interphase of slices A and B are the faces of the blocks ........................ Photomicrograph of rat cardiac tissue stained with Gomori's one-step trichrome stain (560 X) ....... Diagram of photomicrograph mapped for data quanti- fication ....................... Time plot conducted at 90°C for determining environment for DNA extraction .................. Growth curves for male Osborne—Mendel rats ....... Body weight and body composition of 36 week old male Osborne-Mendel rats .................. Scatter plot of systolic blood pressure and body weight coordinates of 36 week old male Osborne-Mendel rats r=O.29 ....................... Plot of stroke volume and cardiac output coordinates of 36 week old male Osborne-Mendel rats (r=0.94) ..... viii Page 38 42 43 46 49 53 53 55 64 69 72 77 81 Figure Page C1 Simplified diagram of injection and withdrawal sites in cardiac output determination ............. 132 C2 Chart recordings of indocyanine green concentration as a function of time for determination of cardiac output in male Osborne-Mendel rats ............. 133 ~ ix APPENDICES Appendix Page A Composition of Rations ............... 130 B Procedure for Diluting Indocyanine Green for Use in Determining Cardiac Output in rats ..... 131 C Derivation of Cardiac Output Equation ....... 132 0 Conversion of Blood Pressure from Millimeters Mercury (mm Hg) to Meters Water (M H20) ...... 136 E Protocol for Gomori's One-Step Trichrome Stain . . . 137 F Summary of Procedure for Protein Determination . . . 139 G Summary of Procedure for Fat Determination ..... 141 H Summary of Procedure for DNA Determination ..... 142 I Calculations for Analysis of Variance and Tukey's w-Procedure .................... 144 J Critical Values for Correlation Coefficients . . . . 16S ABBREVIATIONS Body weight Heart weight Blood pressure Systolic pressure Diastolic pressure Pulse pressure Mean blood pressure Cardiac output Stroke volume Heart rate Peripheral resistance units Total body water Lean body mass Venous hematocrit Body hematocrit Deoxyribonucleic acid xi BW HN Ps Pd Pp MBP Q Vs HR PRU TBN LBM HCtvenous DNA CHAPTER I INTRODUCTION Need for Study Despite floods and droughts as well as inflation and recessions, Americans continue to be the best fed p0pulation in the world. In fact, they spend pr0portionate1y less of their disposable income on food compared to other countries of the developed World (U. S. Depart- ment of Agriculture, July 1974). Americans, for instance, spend only 16% of their income for food compared to 53% spent by Russians or 25% spent by Britishers.. Such expose may be difficult to "stomach" since the consumer price index for food rose 66% between 1970 and 1976 (U. S. Department of Agriculture, 1977); but nevertheless relative to other countries, food is still a good buy in the United States. This availability of food along with a sedentary life-style, however, fosters the development of obesity (Mayer, 1968). Obesity, perhaps a blessing of yesteryears when it indicated that an individual was prOSperous enough to over-indulge or when it tided a person through an infectious disease epidemic or a food shortage, has become every- thing of a curse in modern society (Christakis and Plumb, 1966). Nowadays prosperity is indicated by material possessions and lithe physiques rather than obesity, and contagious disease and severe food shortages have been nearly eradicated in the developed countries. In place of contagious diseases, chronic diseases such as cardiovascular disease have surfaced; and obesity is alleged as predisposing an in- dividual to cardiovascular disease. The need then for studying obesity arises from its prevalence and its association with cardiovascular disease, the number one killer in the United States. The reason for obese individuals being at risk to cardiovascular disease is still being deciphered. Purpose of Study This study concentrates on the cardiovascular physiology of obesity. The purpose is to evaluate the hemodynamic changes in the cardiovascular system caused by obesity and in turn to assess the effect of these hemodynamic alternations on selected anatomical and biochemical parameters of the heart. Thus, the study is to determine the effect of obesity on the biophysical functioning of the cardio- vascular system. Cardiovascular dynamic measurements included cardiac output, blood pressure, blood volume, and cardiac work. Cardiac anatomy measurements included weight, volume, length, and width, and microscopic measurements of myocardial fibers and capillaries. And cardiac biochemical measurements included quantification of cardiac protein, fat, moisture, and DNA content. Statement of Hypothesis For each phase of the study (cardiovascular dynamics, cardiac anatomy, and cardiac biochemistry), one broad hypothesis is stated as follows: 1. Obesity alters the hemodynamics of the cardiovascular system as measured by cardiac output, blood pressure, and blood volume. 2. Obesity alters the number and size of cardiac muscle fibers as well as the fiber to capillary ratio and diffusion distance as measured by histopathological techniques. 3. Obesity alters the protein, fat, moisture, and DNA content of the cardiac muscle as measured by biochemical procedures. Theory Epidemiological data suggest that obesity is a risk factor in the development of cardiovascular disease (Report of Inter-Society Commission for Heart Disease Resources, 1970). This theory has been given further support by the statistics compiled by life insurance companies, particularly the Build and Blood Pressure Study (Society of Actuaries, 1959), in which overweight policy holders who were 40-69 years old had 142% mortality ratio due to cardiovascular- renal diseases compared to 89% mortality ratio in policy holders in the same age category but who were slightly underweight or of average body weight. Despite years of research, the relationship between obesity and cardiovascular disease remains vague since cardiovascular dis- ease is a complex state, the end-result of the interaction of many factors. Neither is the relationship of other risk factors to cardiovascular disease (e.g., hypertension, hypercholesterolemia, hypertriglyceridemia, cigarette smoking, genetics, stress, lack of exercise, age, gendure, hyperuricemia, diabetes mellitus, and diet composition) clearly delineated (Doyle et a1., 1964; Rosenman et al., 1970; Keys and Blackburn, 1963; Kannel et al., 1971). At the present level of understanding, obesity along with these other factors are considered as risk factors rather than causes of cardiovascular disease. This means that with the presence of these factors, the risk or susceptibility to cardiovascular disease is increased. The risk of cardiovascular disease is greater when a number of the factors are expressed. To further the understanding of the relationship of obesity and cardiovascular disease, more needs to be known about obesity. Obesity with all of its ramifications is equally complex as cardio- vascular disease. Thus, various aspects of it should be studied in elucidating its relationship to cardiovascular disease, e.g., psychological or behavioral aspects, glucose and lipid metabolism, cardiovascular physiology. In this study, emphasis is on the latter. In what way does obesity, or adiposity, create an unfavorable environment for the cardiovascular system which leads to cardiovascular disease? Overview of Dissertation In the next chapter, Chapter II, pertinent literature is ‘ reviewed including background studies on obesity and previous studies on hemodynamics and cardiovascular anatomy and biochemistry. In Chapter III the methodology of the study is presented including the laboratory methods as well as a description of the experimental ani- mals, rations, study design, and data analysis procedures. Results of the study and accompanying discussion are contained in Chapter IV with a summary of the findings and conclusions in Chapter V. CHAPTER II REVIEW OF LITERATURE Introduction The cause of obesity appears to be simple: an energy intake which exceeds expenditure. But the determinants of such a positive caloric imbalance are complex because they may originate from meta- bolic, physiological, or psychological aberrations. Literature reviewed in this first section deals with some possible determinants of obesity including heredity, inactivity, appetite disturbance, early nutrition, and meal patterns. In reality, obesity may stem from several of these factors rather than a single factor thus being multi-causal in origin. Subsequent sections report data on the prevalence of obesity as determined by recent surveys, the mortality attributed to obesity, and the incidence of cardiovascular disease associated with obesity. Although the topic of obesity and mortality is frequently addressed, actuarial data reported in the literature are limited. Three components of obesity/cardiovascular physiology research are then reviewed. Considered first are studies of cardiovascular dynamics and obesity which emphasize assessment of blood pressure, cardiac output, and blood volume. Secondly, studies of cardiac anatomy are reviewed including both gross and microscopic pathological changes attributed to obesity. Lastly, studies of obesity and cardiac biochemistry, specifically the quantification of cardiac protein, fat, and deoxyribonucleic acid are reviewed. Determinants of Obesity HereditaryiFactors Seltzer and Mayer (1964) investigated if favorable circum- stances predisposed some people to developing obesity because of their constitutional make-up and related genetic endowment. Based on somato- typing of 180 obese adolescent girls (Y'= 46% overweight and 15.04 years), they determined a predominance of endomorphy, a high incidence of mesomorphy, and an extremely low incidence of ectomorphy among the obese compared to non-obese girls of the same age. Although the obese subjects were not somatotypically homogeneous, they were more homo- geneous (i.e., smaller standard deviation) compared to the non-obese girls. For instance, the absence of ectomorphy among the obese sug- jects indicated a tendency toward a smaller range of somatotypes. Obesity is more than just a random sample of physical types with ex- cess body fat. Seltzer and Mayer (ibid.) concluded: Since the somatotype is a reflection of the morphological constitution of the individual, it would then follow that there are constitutional factors operating in the predis- position to obesity. While results in no way detract from the concept that caloric intake in excess of caloric expen- diture is the immediate cause of obesity, a recognition of the constitutional individuality of the person may well give a greater understanding of the common observation that, under similar environmental circumstances, some of us be- come obese and others do not. In the now classical observations of Gurney (1936), the body build of the parents as well as the progeny of 61 "stout" women and 55 controls was reported. A higher incidence of overweight parents as well as overweight children was reported by the "stout" women compared to the controls. In cases where both spouses were overweight. 73% of the progeny were overweight; and when one parent was overweight only 41% of the children were overweight. In contrast, when both parents were normal weight, only 9% of the children were overweight. Gurney (ibid.) suggested that a hereditary factor in obesity may ex- plain why two people may experience the same environmental influences (e.g., age or number of pregnancies) and yet react differenctly in so far as developing obesity. Although certain individuals may be predisposed to obesity, Garn andBailey'(1976) were cautious about endorsing the genetic in- heritance theory of obesity. They correlated skinfold measurements in adoptive parent-child pairs as well as in biological parent-child pairs and reported similar correlations. For example, in children 15-18 years old, r = 0.39 in adoptive parents vs. children and r = 0.32 in biological parents vs. children. So although obesity may 'run' in some families and although the data may fit a genetic hypothesis, Garn (ibid.) stated: ...it is also possible that fatness is learned in family- line context and socially, rather than genetically inher- ited. Inactivity Obesity and inactivity are undoubtedly related, but inactivity is probably linked more to the perpetuation of obesity than to the pathogenesis of obesity. Using sophisticated methodology, Bullen, Reed, and Mayer (1964) studied 109 obese girls (176.5 pounds, 15.2 years) and 72 normal weight girls (118 pounds, 14.5 years) to test the hypothesis that inactivity contributes to obesity. Motion picture photography provided a direct method for assessing the body motion of the two groups as they engaged in recreational activities (swimming, volleyball, tennis) in summer camps. Pictures were supplemented by a questionnaire designed to reveal the girl's perception of her behavior and feelings about physical activity. Based on a method adapted from industrial situations, pictures were analyzed for the type of locomo- tion and the intensity of performance. Striking differences were ap- parent between the obese and non-obese groups of girls. On the aver- age only 9% of the obese group compared to 55% of the non-obese were actually swimming at a given time. And both in tennis and volleyball, the obese girls spent significantly more time standing or sitting as opposed to other forms of locomotion involving displacement of body weight. Consequently, in estimating caloric expenditure the obese girls were associated with caloric level 1 (less than 2 Kcal/min) and the non-obese with the four higher levels of 2, 3, 4, and 5 (2 Kcal/ min to 5 or more Kcal/min). Results of the questionnaire revealed that most of the obese and non-obese girls professed favorable atti- tudes toward physical activity. But the obese girls reported spending more of their leisure time in sedentary pursuits such as quiet hobbies, telephoning, and watching television compared to the non-obese. In a study of obese and normal weight adults, Bloom and Eidex 10 (1967a) reported that the obese subjects spend significantly more time in bed each day. And when out of bed, they spend less time standing compared to the normal weight individuals. In a related study involv- ing energy expenditure and obesity, Bloom and Eidex (1967b) reported that the obese individuals expended more calories in performing a given task compared to the normal weight individuals. Thus, they con- cluded that the obese individuals "store more energy" not because they are more efficient in performing physical tasks but because of their decreased level of activity at a given level of caloric intake. Appetite Disturbance Gastineau (1972) considered overeating a prerequisite for the development of obesity and summarized his views as follows: It seems almost unnecessary to argue that the obese person has eaten more than he requires; the obvious storage of fat provides prima facie evidence of this. The person who is 100 pounds overweight has approximately one-third of a million calories of fuel stored in his excess adipose tissue. However, overeating as related to obesity is more complex than a mat- ter of self-indulgence or gluttony, but rather it may involve a distur- bance in appetite. One theory proposes that obese subjects are more responsive to external than internal food cues. Thus, they eat because the hands of the clock indicate that its mealtime or because they noticed food in a shop window (external cues) rather than necessarily eating in response to hunger (internal cues). Nisbett (1962) reported that obese sub- jects when offered three sandwiches ate more than normal weight 11 subjects (2.32 compared to 1.88 sandwiches). However, when only one sandwich was visible but additional sandwiches were available but out of sight in a nearby refrigerator, the obese subjects consumed fewer sandwiches than the normal weight subjects (1.48 compared to 1.96 sandwiches). Interestingly, normal weight and underweight subjects ate the same number of sandwiches regardless if they were initially offered one or three sandwiches. Thus, Nisbett (ibid.) concluded that the obese subjects responded to the presence of the sandwiches, i.e., external cue, more than to hunger, i.e., internal cue. Another form of appetite disturbance involves an individual's use of food as a means of "coping" as described by Kaplan and Kaplan (1957) in their synthesis of the research dealing with psychosomatic aspects of obesity. Kaplan and Kaplan (ibid.) hypothesized that a person's habitual use of food to cope with anxiety, conflict, or bore- dom may lead to overeating which in turn will lead to obesity. And an environment associated with affluence, plentiful food supplies, and sedentary life-styles may perpetuate obesity rather than counteract excess consumption. Early Nutrition Widdowson and McCance (1960) compared the somatic development in rats reared in small litters of three pups, "fast-growing" animals, to rats reared in litters of fifteen to twenty, "slow-growing." They reported an accelerated growth rate in those reared in the small lit- ters such that at 21 days these animals were two to four times heavier than those from the larger litters. Although all rats were fed the 12 . stock diet ad libitum, the difference in size continued into adulthood. At_5 months of age the rats from the small litters were still approx- imately 100 gm heavier than those from the large litters. In general, the weight of the kidneys, adrenals, liver, stom- ach, and small intestine varied with the weight of the animal and not with the chronological age. All animals gained fat during suckling and lost it afterwards, but the "fast-growing" animals gained signi- ficantly more fat than the "slow-growing" rats. The percentage of body fat was the same in both groups by the time they reached the weight_of 170 gm. At each age, however, the "fast-growing" rats had significantly more body fat than the "slow-growing" rats. This sug- gested that overfeeding in infancy may lead to excess body fat. Based on a similar study of manipulating litter size to vary caloric intake, Knittle and Hirsch (1968) reported that early feeding experiences profoundly affected the total cell number and cell size of the epididymal fat pad. At all ages studied (5, 10, 15, 20 weeks), Sprague-Dawley rats reared in small litters had more and larger adipose cells than rats raised in large litters. The differences in cell number were statistically significant at all time intervals and the increased cell size was significantat 10 weeks of age and older. This study was consistent with the data reported from human subcuta- neous adipose tiSsue by Hirsch, Knittle, and Salans (1966) in which non-obese adults had 0.6055 pg lipid per cell and 26.83X1O9 adipose cells whereas cell lipid of obese adults was slightly increased at 0.9088 ug but cell number was three times greater with 77.02XlO9 cells. These authors suggested that maximal values for cell number and size 13 were not reached until adolescence or early adult life. Once estab- lished, however, only cell number remained fixed and cell size varied with weight changes. However, when caloric intake was manipulated by varying the composition of the dams' milk rather than litter size, the effect of early overnutrition was not sustained as reported by Schemmel, Mickelsen, and Fisher (1973). Nursing dams were fed either a calorically dense, high fat ration or a grain ration. Pups nursed by dams fed the high fat ration weighed significantly more at weaning than those nursed by dams fed the grain ration although at 42, 84, and 168 days this difference no longer existed. Meal Patterns Recognizing that many obese individuals consume more of their food within a relatively short period of time each day, e.g., at one large meal, Hollifield and Parson (1962) studied metabolic adaptations in rats which had access to food only between 8:00 and 10:00 A.M. daily compared to rats fed ad libitum. As a result of the "stuff and starve" feeding program, rats gained more weight (383 gm compared to 287 gm at 10 weeks of age). The increase in body weight appeared to be due to increased fat deposition and was associated with 5 to 15% increase in food intake. The rats allowed food for only two hours per day had higher rates of acetate-l-C14 incorporation into lipids in adipose tissue in vitro, low free fatty acid levels in adipose tissue, and a moderate increase in liver glycogen. Hollifield and Parson (ibid.) suggested that persons who consume most of their food 14 in a short period of time may have enzymatic adaptations which perpet- uate obesity and thus make weight loss difficult. Stunkard, Grace, and Wolff (1953) noted that in a group of 25 obese patients who experienced great difficulty in losing weight, 16 of them were characterized by their food patterns in which a quarter of the daily caloric intake was ingested following the evening meal. Stunkard (ibid.) referred to this as the "night-eating syndrome" which consisted of 1) nocturnal hyperphagia, 2) insomnia, and 3) morning anorexia. After counseling the patients over a six-month period, the investigators concluded that the syndrome represented a response to stress. Emotional problems of many of the subjects could "set-off“ the night-eating syndrome resulting in a period of positive caloric balance that negated the results of weeks of dieting. Mayer (1959) described a group of obese women under study and noted that they derived the same proportion of calories from fat (37%) and carbohydrate (49%) compared to a control group of normal weight women. Also, both groups of women snacked frequently, but meal pat- terns distinguished the two groups. Consistent with Stunkard's (op. cit.) observations, Mayer noted that the obese women shifted their food consumption to the later hours of the day. Prevalence of Obesity The Ten-State Nutrition Survey 1968-1970 (U. S. Department of Health, Education, and Welfare, 1972) defined obesity in adults as a skinfold measurement greater than the 85th_percentile of the data 15 collected from young white adults. Thus, males 18 years of age and older were classified as obese if they had a skinfold thickness of 18.6 mm or greater; and females 18 years of age and older with a skin- fold or 25.1 mm or greater were classified as obese. Based on these criteria the survey reported that 23.9% of the 50 year old white males and 13.2% of the 50 year old black males were obese. In 50 year old females, 41.9% of the white females and 52.7% of the black females 'were obese. In general, in all age groups the frequency of obesity was greater in white males than in black males whereas more black females than white females were obese. Overall a higher percentage of obesity was reported in women than in men. The Health and Nutrition Examination Survey - HANES (National Center for Health Statistics, 1975), a probability sample of the United States' population, reported trends in obesity similar to the Ten- State Nutrition Study. The actual percentages of obesity were lower in the HANES study, however, since the Ten-State Study concentrated on a low-income sample and the prevalence of obesity is generally greater in low socio-economic groups. In the 45-74 year old category of the HANES study, 13.4% of the white males were obese compared to only 7.7% of the black males. But obesity was more prevalent in women than men with 24.7% of the white women and 32.4% of the black women being obese. 16 Mortality and Disease in Obesity Excess Mortality and Obesity The association of obesity with excess mortality gained impetus from life insurance mortality studies. Even the earliest such studies conducted at the turn of the century linked obesity with early death (Actuarial Society of America, 1903; Association of Life Insurance Medical Directors and Actuarial Society of America; 1912-1914). The Build and Blood Pressure Study (Society of Actuaries, 1959) is the most recent intercompany life insurance study reporting variations in mortality according to body build. The standard distribution of cause of death was based on data contributed by nine large companies and represented 4,900,000 (ordinary) insurance policies. The study included policy holders age 15 to 69 during 1935 through 1953 traced to policy anniversaries in 1954 and excluded war-related deaths. Five body build groups were used in analyzing the actuarial data: group I marked underweights, group II slight underweights and average weights, group III slight overweights, group IV moderate overweights, and group V marked overweights. Data were expressed as mortality ratios, i.e., the ratio or percentage of actual to expected mortality. For men and women in groups I-III, mortality ratios decreased or remained un- changed with duration of the policy whereas for men and women in groups IV-V mortality ratios began at a higher level (than the former groups) and tended to increase with the duration of the policy. For example, 40 to 69 year old men in group I who had a policy for l to 5 years experienced a 96% mortality ratio; for those with a policy for l7 16 to 19 years, the mortality ratio decreased slightly to 94%. In contrast, 40 to 69 year old men in group V who had a policy for l to 5 years experienced a mortality ratio of 120%, a higher initial mor- tality ratio than the men of group I; the mortality ratio further increased to 153% for those who had a policy for 16 to 19 years. Insurance company studies of actuarial data have been based on the simplest of body build classifications, the assignment of individ- uals to height-weight categories. This placed emphasis on the concept of underweight and overweight rather than differentiating between in- dividual variations in body build and body composition. According to Seltzer (1966), height-weight categories may be useful to insurance companies for broad screening of applicants, but for medical purposes these data need re-evaluation. Using the ponderal index, a measure of body shape or build determined by dividing height (inches) by the cube root of body weight (pounds), Seltzer (ibid.) re-evaluated the data. The rationale for the ponderal index is that weight is a measure of volume and volume increases according to the cube of the linear dimen- sions, and thus it is a better index of linearity and laterality than height-weight categories. Instead of increasing weight for height plotting a positive straight line relationship with mortality as indi- cated by the insurance company studies, ponderal index plotted against mortality indicated a curvilinear response. That is, mortality did not significantly increase until extremes in body weight were reached. At this point the mortality ratio increased in an almost geometric progression. Seltzer (ibid.) noted that this sharp upward trend in mortality was due primarily to extreme endomorphs who were also 18 classified by the insurance companies as group V marked overweightS> (30% or more excess body weight) while extreme ectomorphs had the most favorable mortality experience. The simplification in actuarial studies of classifying physique by only height and weight tended to obscure the relationship between body build type and mortality. According to Seltzer (ibid.): It may well be that body build type makes people more prone to disease, and particularly so in obese people. Cardiovascular Disease and Obesity In the Build and Blood Pressure Study (Society of Actuaries, 1959), mortality ratios progressively increased from underweight to overweight policy holders for each of the following causes of death: heart and circulatory system diseases, vascular lesions of the central nervous system, digestive diseases, and nephritis. In underweight and overweight men, the primary cause of policy termination, however, was heart and circulatory disease. Nearly 50% of the policies of over- weight policy holders were terminated by this cause compared to only half that number in underweight policy holders. For women termination of the policy due to heart and circulatory disease was second to malig- nant neoplasms. As a spin-off from the Muscogee County, Georgia, community survey, Comstock, Kendrick, and Livesay (1966) classified 24,000 sub- jects by degree of subcutaneous fat (based on measurement of the tra- pezium site from roentgenograms) and related this classification to mortality during the subsequent 15 years. The sample population 19 included both black and white males and females who were 15 years of age or older in 1946. The mortality ratio from all causes was highest in the group with the most subcutaneous fat: 98% mortality in sub- jects with a fat layer of O to 4 mm; 96% mortality in subjects with a fat layer of 6 to 9 mm; and 112% mortality in subjects with a fat layer of 10 mm or more. In partitioning the mortality data into speci- fic causes (i.e., cardiovascular-renal, diabetes, accidents, cancer, tuberculosis), deaths from diabetes and secondly deaths due to coro- nary heart disease had marked positive correlations with body fat. In this latter correlation, only the classification with the thickest fat layer of subcutaneous fat showed a significant increase in mortal- ity: 91% mortality ratio with 0 to 4 mm fat, 89% mortality ratio with 5 to 9 mm fat, and 137% mortality with 10 mm or more fat. Further partitioning of the body fat and mortality data into age, sex, and race classifications revealed that the most marked association of subcutaneous body fat thickness with excess mortality occurred among young adult white males: 349% mortality ratio with 10 mm or more sub- cutaneous fat in the 15 to 34 year olds as opposed to 99% mortality ratio with 10 mm or more subcutaneous fat in 55 year olds and older. The absence of excess mortality in the fattest, oldest white males raised questions about the relationship of obesity to mortality. Appreciable differences in the mortality of fat and lean young people may have effected the median thickness of subcutaneous fat in the older age groUps. Unfortunately, body weight data were not available for Comstock's (ibid.) evaluation of the roentgenograms so differences in 20 interpretation of the data based on body weight could not be noted. Sanders (1950), however, matched a group of 48 patients with uncompli- cated coronary-artery disease to a group of controls from private practice; matching was based on sex, age, nationality, domicile, and religion. Although there was no difference in relative body weight between the two groups, the groups with coronary-artery disease had significantly more body fat as determined by skinfold measurements. The heart disease epidemiology study of the National Heart Institute (Kannel et al., 1967) studied a sample of over 5,000 men and women age 30 to 62 from Framingham, Massachusetts, for a period of 12 years beginning in 1949. The study identified various factors of risk for coronary heart disease and attempted to assess the inde- pendent contribution of each of the various interrelated factors. The independent contribution of body weight to the development of manifestations of coronary heart disease (e.g., angina pectoris, my- ocardial infarction) were explored. In the 12 years of observation, 252 men and 128 women developed coronary heart disease with the pre- dominant manifestation in women being uncomplicated angina pectoris and in men the development of more serious and lethal forms of coronary heart disease. In both men and women the risk of developing coronary heart disease increased with increasing initial relative body weight (i.e., body weight relative to median of the population to which the subject belongs with respect to height and sex). The association between initial relative weight and development of myocardial infarc- tion was not significant whereas the association between relative body weight and development of angina pectoris was related. 21 Furthermore, subjects with relative body weight greater than 20% of the median had an excess of sudden death due to coronary heart disease. Because of the complicating effect of elevated blood pressure and serum cholesterol, the relationship of relative weight to development of manifestations of coronary heart disease was examined at specified levels of blood pressure and serum cholesterol. Persons with elevated blood and/or cholesterol were compared to those without these eleva- tions after subdivision into obese and non-obese groups. Even among men who had neither elevated blood pressure nor elevated cholesterol, the obese group developed an excess of angina pectoris and sudden death (but not myocardial infarction). Obese subjects with increased blood pressure and serum cholesterol had further increased risk. Kannel (ibid.) stated: This would not be if adiposity was only acting through an association with elevation of blood pressure and cholesterol level. On the other hand, the incidence of myocardial infarction although not related to adiposity had a distinct excess in any weight category with elevated blood pressure and cholesterol levels. Cardiovascular Dynamics in Obesity Cardiac dynamics have been studied under a variety of circum- stances including high altitude, exercise, and pharmaceutics, but emphasis is given here to cardiac dynamics in obesity. The particular parameters of cardiac dynamics which are reviewed include blood pres- sure, cardiac output or blood flow, and blood volume. Changes in 22 these parameters are integral to the physics of the circulatory system. Blood Pressure Undoubtedly blood pressure has been assessed more frequently than any other hemodynamic parameter in obesity. A pitfall exists, however, in many of the studies; due to the generally increased arm circumference of the obese subject, the brachial artery may not be adequately compressed which may result in erroneously high blood pressure readings. In studies designed to assess changes in blood pressure sub- sequent to weight reduction, this pitfall is compounded. With weight reduction the arm circumference of the obese individual may decrease resulting in a more accurate and actually lower blood pressure read- ing. Unfortunately, such decreases in blood pressure have been at- tributed solely to weight reduction without regard to the initially high but inaccurate blood pressure reading. According to Vaughan and Roa (1973) this pitfall can be cor- rected by using a larger blood pressure cuff, 38 X 18.5 cm, compared to the standard cuff, 23 X 12.5 cm. They employed a general rule of thumb in which the inflatable arm bag was approximately 20% wider than the arm diameter of the subject. Thus, the standard cuff was used for subjects with an arm circumference less than 50 cm at the brachium, and the larger cuff was used for those with a circumference greater than 50 cm. In regard to blood pressure in obesity, the consensus of Opinion was perhaps best stated by Chiang, Perlman, and Epstein (1969) 23 who reviewed the t0pic of overweight and hypertension: Hypertension is more common among the obese than among the non-obese and, conversely, a significantly proportion of hypertensive persons in the population are over- weight. Obese hypertensive subjects experience a greater risk of coronary heart disease than the non-obese, and mortality rates for obese hypertensive persons are higher than for those with obesity alone or hyperten- sion alone. Weight reduction has been shown to lower blood pressure, and it may bring about a more favorable prognosis in obese hypertensive persons. Alexander (1963) reported perhaps the most accurate blood pressure values on obese humans in that he measured blood pressure directly, that is intra-arterially, and thus avoided complications due to arm circumference. In a group of obese subjects whose average body weight was 300 pounds, or approximately twice their ideal body weight, he reported slight or moderate increases in blood pressure in 50% of the subjects and severe increases ()200/120 mm Hg) in 10% of them. The remaining 40% of the obese individuals were nor- motensive. Alexander (ibid.) concluded, therefore, that even extreme obesity does not invariably lead to hypertension. Furthermore, he found no correlation between the level of systemic blood pressure and the amount of excess weight or total body weight. In a study on the cardiovascular effects of weight reduction, Alexander and Peterson (1972) reported that mean blood pressure aver- aged 102 mm Hg in nine markedly obese subjects. But after a weight loss of 39 to 84 kg (or 24 to 55% of their initial body weight), blood pressure decreased in eight of the nine subjects and remained un- changed in one. Mean blood pressure following weight reduction averaged 87 mm Hg (P<0.05). 24 Martin (1952) studied blood pressure changes subsequent to weight reduction in both hypertensive obese subject and normotensive obese subjects. He recognized a decrease in blood pressure as clinically significant if systolic pressure decreased 20 mm Hg or if diastolic pressure decreased 15 mm Hg. Under these conditions, blood pressure fell in four normotensive obese subjects and in seven hyper- 'tensive obese subjects with weight reduction. Martin (ibid.) reported that there was no significant difference between the two groups of obese subjects or between the males and females. Neither was there a correlation with age or initial body weight. Overall, systolic blood pressure was reduced 3.5 mm Hg for every 10 pounds of weight lost. Cardiac Output Alexander et a1. (1962-63) reported that cardiac output tended to rise with increasing amounts of excess body weight. With an ex- cess body weight of 100 kg, cardiac output wasltlL/min or approximate- ly double the value predicted for ideal body weight. In studying the distribution of the blood in this high output state, he reported no difference in cerebral blood flow compared to the predicted normal values (58 compared to 54 m1/min), a slight reduction in renal blood flow (741 compared to 911 ml/min), and an increase in splanchnic blood flow (1451 compared to 1145 ml/min). The increase in splanchnic blood, however, in no way accounted for the increased cardiac output in the obese subjects. Assuming that the blood flow to resting mus- cle tissue would be approximately the same in both the obese and 25 normal weight individuals, Alexander (ibid.) stated: It has, therefore, been concluded by this process of exclusion that the high output state of extreme obesity is largely accounted for by a greatly increased flow to fat tissue depots. In a related study, Alexander (1963) reported that cardiac output, as well as body oxygen consumption, increased as much as three times in a group of obese subjects whose average body weight was 300 pounds. The increase in cardiac output was well correlated with the excess body weight, and systemic arterio-venous oxygen difference in these subjects usually remained normal (4.5 volumes %) suggesting a normal circulation time despite the increased cardiac output. In ex- _ pressing cardiac output per unit body weight, Alexander (ibid.) noted that it was only about 60% of the predicted value (3.1 L/min/M2 body surface area) for ideal body weight. Backman et a1. (1973) in studying cardiovascular function in 19 extremely obese subjects (body weight 108 to 172 kg) also noted increased cardiac output with obesity. They reported, however, that cardiac output was normal when linearly related to oxygen consumption (P:(0.01). That is, in the obese subjects the regression line of cardiac output (average = 8.2 L/min) and oxygen consumption (average = 368 ml STPD/min) was not significantly different from that of normal weight individuals. Apparently, cardiac output increased in obesity to meet the oxygen demands of the increased body size. Thus, Backman et a1. (ibid.) concluded: . . .the circulatory dimensions and the cardiac output reflect the adequate adaptation to the metabolic demands laid upon the circulation in obesity. The elevated fill- ing pressures of the ventricles and the high systemic and pulmonary vascular resistance, however, suggest that obesity is not without deleterious effect on car- diovascular function. 26 Blood Volume Calculating blood volume from total hemoglobin and hemoglobin concentration, Backman et a1. (1973) reported an average blood volume of 51.1 ml/kg body weight (or 7.6 L total volume) in five obese males and 50.5 ml/kg body weight (or 6.8 L total volume) in fourteen obese females. Blood volume in milliliters per kilogram was low compared to approximately 74 ml/kg in normal weight subjects. Thus, there was a negative correlation between the percentage overweight and the milli- liters of blood per kilogram body weight. Expressed in milliliters blood per kilogram ideal body weight, blood volume was 94.3 m1/kg for the obese males and 99.2 ml/kg for the obese females. Backman (ibid.), however, reported a positive relationship between oxygen uptake at rest and blood volume (P<’0.001). Alexander (1959) reported that circulating blood volume in 40 obese subjects at rest was substantially increased compared to pre- dicted values for ideal body weight. The increase was well correlated with their excess body weight. With a body weight 100% greater than ideal, blood volume (and cardiac output) increased 50%. Cardiac AnatomyirIObesity Gross Anatomy Several reports in the literature indicate that obesity is accompanied by increased heart size and pathological changes. Amad, Brennan, and Alexander (1965) quantified macroscopic changes in the hearts of six obese adult men (average body weight = 150 kg) and six 27 obese adult women (average = 136 kg) who were free of complications from hypertension (‘< 150/90 mm Hg) and coronary arteriosclero- sis. The average heart weight of 651 gm for the men and 499 gm far the women was roughly twice that predicted for their ideal body weight. Thus, heart weight per kilogram predicted body weight was approximate- ly twice the value for the heart weight per kilogram of actual body weight. In men these values were 9.39 gm/kg predicted body weight compared to only 4.34 gm/kg actual body weight. Both values were slightly less in the women. Thus, in obesity there was substantially less cardiac tissue per unit body weight. The average left ventricle width of 18 mm was outside the normal range of 8 to 15 mm. In a much earlier study, Smith and Willius (1933) conducted postmortem examinations on the hearts of 136 obese patients whose average body weight was 45% greater than ideal and whose average age was 52 years. Data from these obese subjects were compared to values Smith (1928) collected from 1,000 Mayo Clinic patients of normal weight. In 52 of the 136 obese patients (average body weight 43% greater than ideal and average age 49 years), there was no evidence of heart disease or hypertension and, therefore, Smith and Willius (op. cit.) referred to them as the control group of obese patients. In this sub-group there was no evidence of change in the coronary arter- ies. The ratio of cardiac weight to body weight averaged 0.38% (0.41% in the men and 0.35% in the women) which was less than the normal ratios of 0.43% for men and 0.40% for women. Average cardiac weight for the male patients was 444 gm, an increase of 150 gm compared to normal weight males; and average cardiac weight for the females was 28 345 gm, or 95 gm greater than normal weight values. The heart weight tended to parallel the increase in body weight to approximately 104.5 kg, but thereafter the increase in cardiac weight was less than the proportion in normal weight individuals. In regard to this observa- tion, Smith and Willius (op.cit.) stated: The data clearly demonstrate that the weights of the hearts of an appreciable proportion of obese persons is less than that demanded by the height and weight of the body. This fact in itself may be responsible to a considerable degree for the fairly common apparent circulatory inadequacy of obese persons. Nine of the 136 obese patients studied by Smith and Willius (0p.cit.) did not have evidence of heart disease but had varying degrees of cardiac insufficiency. Interestingly, excess body weight was 60% in this group compared to 45% excess body weight in the entire group of obese patients. The average ratio of cardiac weight to body weight was 0.39% which was slightly higher than the group of 52 patients who had no evidence of heart disease or hypertension (the control group of obese patients), but it was still lower than the normal ratio. Average cardiac weight was 450 gm, or 74 gm more than the control group of obese patients. Smith and Willius (op.cit.) com- mented: This, we believe, indicates that cardiac enlargement beyond certain limits in obesity, as well as in other conditions, results in circulatory inadequacy leading to cardiac failure. Sixty of the obese patients had hypertension, average blood pressure 175/82nm1Hg. The average degree of excess body weight was 44% which was comparable to the entire group of 136 patients. The average ratio of cardiac weight to body weight was 0.45% which was 29 considerably more than the 0.38% in the control group of obese pa- tients. The average cardiac weight was 467 gm which was 91 gm more than the control group. In regard to this group Smith and Willius (0p.cit.) stated: These figures clearly illustrate the effect of hy- pertension on the heart in obesity, the cardiac weights unmistakably indicating cardiac hypertrophy of consider- able degree. This fact is further emphasized when the individual cardiac weights, as well as the average cardiac weight of this group, is compared with that in the control group. In contrast to the group of obese subjects without heart disease and to the group with cardiac insufficiency, no definite correlation was evident between cardiac weight and body weight in these obese patients with hypertension. Similarly, obesity studies using animal models have reported gross anatomical changes in cardiac tissue. For example, in 48 week old Osborne-Mendel rats fed a high fat ration since weaning and weighing nearly twice as much as ideal body weight, Grommet (1972) reported that heart weight was 2.2 gm or 41% greater than the heart weight of normal weight (i.e., grain-fed) littermates. Relative to body weight, however, cardiac tissue was decreased in the obese rats from 272 mg/lOO gm body weight in the normal weight rats to 204 mg/lOO gm body weight. Microscopic Anatomy, Amad, Brennan, and Alexander (1965) in studying cardiac patho- logy in obese subjects reported microscopic changes in the heart as well as the previously discussed organ weight changes. Examination of 3D the myocardium revealed diffuse muscular hypertrophy in all twelve subjects. In four of the cases, small foci of fibrosis in the myo- cardium could be detected microscopically but not macroscopically. Epicardial fat was estimated to be normal in nine of the twelve subjects, and gross infiltration of the myocardium with fat was not observed in any of the subjects suggesting that the increased heart weight was due to changes in muscle mass and not fat. The endocardium was histologically normal in all cases. The major coronary vessels were normal in eight of the subjects, and the aorta and pulmonary artery were normal in all twelve. These researchers concluded: The findings of this study have been interpreted as pro- viding further support for the proposition that manifes- tations of myocardial insufficiency do occur in very obese subjects without evidences of other heart dis- ease. . . . Related Studies Some studies reported in the literature, although not studies dealing with obesity, have implications for obesity. For example, as an adjunct to a study of cardiomegaly in anemia, Rakusan et a1. (1965) studied postnatal cardiogenesis in the rat in terms of cardiac muscle fibers and terminal capillary beds. He concluded that there were three periods of growth in the heart. In the first (birth to four weeks), the number and diameter of cardiac muscle fibers in- creased as well as the number of capillaries. The increase in the number of capillaries was greater, however, than the increase in fiber numbers and consequently the fiber to capillary ratio decreased from 4 to 1.5 during this period. Heart weight increased at the same rate 31 as body weight. In the second period (four to seven weeks postnatal), cardiac muscle fibers increased in diameter but not in number. The increased diameter resulted in an increased diffusion distance, i.e., the dis- tance between two capillaries. The number of capillaries continued to increase although the increase was slower than in the first period. In this period the fiber to capillary ratio dropped from 1.5 to 1.0, a ratio of one capillary to one cardiac muscle fiber. From this second period throughout the 90 days of the study, heart weight increased less rapidly than body weight. In the third period, the muscle fiber to capillary ratio remained constant at 1:1. And diffusion distance which had begun to increase in the second period continued to in- crease throughout the remainder of the study due to increased diameter of the fibers. Goldstein, Sordahl, and Schwartz (1972) examined ultrastruc- tural changes in the myocardium of rabbits with left ventricular hyper- tr0phy and failure. In this study, hypertrophy resulted from inser- tion of a clip around the aorta which created gradual aortic stenosis. With aortic constrictions of 50 to 55%, heart weight was 174 to 200% of normal and the following ultrastructural changes were noted: focal and complete widening of 2 discs, lengthening of intercalated discs, increased numbers of mitochondria, and increased amounts of glycogen. Goldstein and co-workers (ibid.) suggested that the in- creased Z substance might be essential for the production of new sar- comeres hithe myofibrils. Furthermore, the new sarcomeres might be responsible for the lengthened intercalated discs. 32 Cardiac Biochemistry in Obesity Protein Winick and Noble (1967) studied cellular response, including cellular-protein,las a result of increased feeding in neonatal rats. The caloric intake was increased by limiting the litter size to three pups. Compared to controls, heart weight was increased in the rats from small litters both as weanlings (0.44 gm compared to 0.36 gm) and as adults (1.74 gm compared to 1.42 gm), and the increased heart weight was accompanied by a pr0portional increase in total organ protein. Weanling rats from the small litters had 69.3 mg protein per heart compared to 58.1 mg, and adults had 310 mg protein per heart compared to 272 mg. Winick and Nobel (ibid.) concluded: Therefore, the increased net weight described previously is associated with an actual increase in protoplasmic mass and does not merely represent edema. The researchers, however, did not point out that the percentage of cardiac protein was the same in both the small litters and the con- trols. Since data from the rats reared in three-pup litters was not significantly different from that of six-pup litters (typical litter size), these data were pooled and comparisons in the study were made between the combined three and six pup-litters and the litters of twelve pups, or underfed litters. Winick and Noble (ibid.) referred to the twelve-pup litters as control and the combined three and six- pup litters as experimental which is somewhat misleading when the question concerned cellular response with increased feeding. 33 Gudbjarnason, Braasch, and Bing (1968) studied protein synthesis in cardiac hypertrophy. Although their work did not deal specifically with obesity, it perhaps has implications since obesity may foster the development of cardiac hypertrophy. In their study hypertrophy developed in rabbit hearts in response to increased outflow resis- tance, i.e., aortic stenosis. as a result of placing an adjustable constrictor around the aorta. The ratio of left ventricle weight to total body weight increased from 1.41 gm/kg to 2.32 gm/kg or 65% in the hypertrophied hearts. Protein synthesis, as measured by the in- corporation of glycine-Z-C-14 into protein of the heart muscle, in- creased 92% during the early stage of cardiac hypertrophy development. When protein synthesis was inhibited by injection of actinomycin D or a protein-free diet for two to three weeks prior to the production of aortic stenosis. the animals developed heart failure more fre- quently than controls. Thus, Gudbjarnason and co-workers (ibid.) stated: These observations indicated that the development of cardiac hypertrophy is a protective compensatory mechanism and when the development of this compensa- tory mechanism is interrupted or inhibited the myo- cardium fails. Their data, however, did not clearly indicate if the increased protein synthesis resulted in an increased concentration of protein in the hypertrophied cardiac muscle or merely an increase in amount of pro- tein. Smith andldillius(l933) determined the fat content of the left 34 ventricle in postmortem examinations of twelve patients. Fat content varied from 11.6 to 23.9% with an average of 15.8%. In sixteen sub- jects with normal body weight and presumably healthy hearts, fat con- tent varied from 12 to 20% with an average of 16.5%. Thus, the same variation in fat content of the heart occurred in both the normal weight and obese patients. In addition, microscopic examination showed no evidence of increased amounts of intracellular fat in the hearts from the obese patients. Smith and Willius (ibid.) noted, however, an increase in fat deposition on the epicardium or subepicar- dium, i.e., connective tissue beneath the epicardium, in 95% of their obese patients. In practically all cases this deposition of fat, or adiposity of the heart, was located over the right ventricle more than the left. In most instances a positive relationship existed between the excess of epicardial fat and degree of obesity. In a few extreme cases the epicardial fat penetrated through the wall of the right ventricle and was deposited even beneath the endocardium. Smith and Willius (ibid.) stated: It seems reasonable to believe that when fat is deposited in such quantities it is capable of interfering with the action of the heart. This extreme involvement, however, was not common. . . . We believe that the part played by adiposity of the heart in producing cardiac failure is, in most instances, that of adding a burden to some other disease, such as hypertension or coronary sclerosis, and that any cardiac disease is distinctly more serious if cardiac adiposity is present. In some instances, although rarely, cardiac adiposity in itself is responsible for cardiac failure. Deoxyribonucleic Acid(DNA) In addition to the cardiac protein data already reviewed, 35 Winick and Noble (1967) reported an increase in cardiac DNA in the rats reared in small litters, i.e., neonatal rats with increased caloric consumption. Both as weanlings and as adults, these rats had increased whole body and individual organ DNA. In weanlings cardiac DNA was 0.91 mg per heart for the rats reared in small litters com- pared to 0.62 mg for the rats reared in large litters; and in adults DNA was 1.98 mg per heart compared to 1.43 mg. The increase in DNA indicated that the heart contained more cells. Since cardiac weight, protein, and RNA increased proportionally, the ratios remained un- changed indicating that cell size was unaffected. In an abstract, Norman and Carter (1961) reported that DNA per heart, but not DNA concentration, was increased in enlarged hearts of rats. The cardiac enlargement in this study, however, was due to anemia not obesity so extrapolation to obesity is limited. Since the ratio of RNA/DNA was elevated, Norman and Carter (ibid.) concluded that the enlarged hearts were due predominantly to hypertrophy as opposed to hyperplasia. This conclusion assumed that the elevated DNA was not the result of ploidy or proliferation of blood vessels in the heart. Parenthetically, water content in the enlarged hearts was un- changed compared to hearts from control rats. CHAPTER III METHOLODOLGY Experimental Animals Source Osborne-Mendel rats for the study were from the colony of the Department of Food Science and Human Nutrition, Michigan State Univer- sity which is maintained under the direction of Dr. Rachel Schemmel. This colony was originally started from stock obtained from the Nation- al Institutes of Health (NIH), Washington, D. C. For consistency in pup weights, litters were reduced to 8 pups, a litter size which Osborne-Mendel dams can adequately nurse. Pups to be used in the study were weaned from the dams at 3 weeks of age. The study was limited to male rats. Housing Rats were individually housed in metal cages with wire screen bottoms (18 X 18 X 25 cm). Temperature of the animal room was thermo- statically controlled ca. 230C throughout the study; lighting was on a '12 hour light-dark cycle. 36 37 Experimental Rations To produce obesity, a high fat ration was fed to the rats post- weaning. This method of producing experimental obesity was relatively free of complications, since the rations could be fed to the rats on an ad libitum basis (Mickelsen, Takahashi, and Craig, 1955). This avoided the physiological aberrations of other experimental obesity techniques such as force feeding or surgery. Osborne-Mendel rats fed a grain-based ration ad libitum do not become obese (Schemmel, Mickelsen, and Tolgay, 1969): grain-fed rats, therefore, provided normal weight rats for comparison. The primary difference in the grain and high fat rations was in fat and carbohydrate content, i.e., energy components, and conse- quently Calorie content. On a weight for weight basis (w/w), the grain ration contained 3% fat, 54% carbohydrate, and 3.4 digestible Calories/gram ration; the high fat ration contained 60% fat, 8% car- bohydrate, and 6.7 digestible Calories/gram ration (Schemmel, Mickelsen, and Gill, 1970) or nearly twice the Calories per gram. Details of both rations are presentediriAppendix A. Ration and water were available ad libitum. And body weights were recorded every other week for 36 weeks. Experimental Design The study consisted of 4 groups of rats (treatments) and 8 rats (replications or blocks) per group as diagrammed in Figure 3.1. The 38 REPLICATIONS (BLOCKS) TREATMENTS FIGURE 3.1. 1 2 3 4 5 l 2 3 4 5 l 2 3 4 5 l 2 3 4 5 -- Experimental design: 6 7 8 6 7 8 6 7 8 6 7 8 randomized block. 39 8 rats of treatment A were littermates to the rats of treatment(s) B, C, and D: that is, the rats numbered 1 were littermates, the rats numbered 2 were littermates, etc. The design of the study was a randomized block design, i.e., the variation due to littermates was blocked (Cochran and Cox, 1957, p. 108). Treatment A rats were fed the grain-based ration from weaning to 36 weeks of age and served as the group of normal weight rats. As a model for growth-onset obesity, treatment 8 rats were fed the high fat ration from weaning to 36 weeks of age; whereas to model adult-onset obesity, treatment C rats were fed the grain ration from weaning to 18 weeks of age and the high fat ration from 18 to 36 weeks of age. Treatment 0 rats were fed the high fat ration from weaning to 18 weeks of age and the grain ration from 18 to 36 weeks of age to model weight reduction. Such groups can seldom be partitioned out in clinical studies. Cardiovascular Dynamic Techniques Blood Volume and Hematocrits Blood volume was measured using 5 pCi autologous 5ICr labeled1 erythrocytes (Sterling and Gray, 1950) and 5 pCi isologous 1251 labeled2 rat serum albumin (Greenwood, Hunter, and Glover, 1963). The isotopes were injected simultaneously into the femoral vein of the ether-anesthetized 1 51Cr: E. R. Squibb, 1925 Greenwood Avenue, Sharonsville, OH 45241. 2 125I: Cambridge Nuclear, Radiopharmaceutical Co., P. 0. 528, Princeton, NJ 08540. 4O rat and blood samples were taken from the Opposite femoral vein 10 and 15 minutes post-injection, i.e., after the isotOpes equilibrated in the circulatory system. Counts per minute of 5i‘Cr and 1251 in the blood sample were counted in a gamma counter3 and converted to milliliters of erythrocytes and plasma, respectively, and summed to yield milliliters of blood. Blood volume data were expressed on an absolute basis as well as relative to body weight. A portion of the blood sample was used to determine the venous hematocrit of each rat. Micro-capillary tubes were filled with blood, sealed, and centrifuged with a microhematocrit high speed centrifuge (11,500 rpm; 13,000 g) for 5 minutes. In addition, body hematocrit was determined for each rat by dividing the volume of erythrocytes as determined with 51Cr by the total volume of blood calculated by summing'the‘erythrocyte and plasma volume. The F 1 factor was then cel calculated for each rat by dividing body hematocrit by venous hematocrit. Blood Pressures and Heart Rate After blood volume was measured, blood pressure (systolic, diastolic, pulse) was recorded directly from the carotid artery. Direct or intra-arterial blood pressure required placement of a cannula4 3Counter: 1185 series, Automatic gamma counting system, Nuclear Chicago, a subsidiary of G. A. Searle & Co., 16445 West 12 Mile Road, Southfield, MI 48076. 4Cannula: Intramedic polyethylene tubing (1.0. = 0.023", 0.0. = 0.032"), Clay Adams, 141 East 25§h_Street, New York, NY 10010. 41 in the carotid. Rats were anesthetized with sodium pentobarbital5 and placed in a suspine position to "prep" for surgery. The tracheal area was shaved and a midline incision ca. 3 cm was made to expose the esophagus and trachea. With blunt dissection the carotid was isolated. The cannula was inserted into the artery and secured with ligatures. Instrumentation for the procedure including pressure transducer and polygraph are diagrammed in Figure 3.2. When the 3-way valve was opened to the pressure transducer,6 the diaphragm of the transducer was deformed by pulsations from the artery; the pulsations were amplified and charted by the polygraph.7 Systolic and diastolic pressure (mm Hg) were read directly from the chart as shown in Figure 3.3 with systolic being charted as the "mountains" and diatolic as the "valleys." Pulse pressure (Pp) pressures, i.e., Pp = PS - Pd, and mean blood pressure (MBP) as the arithmetic average of systolic and diastolic pressure. Mean blood pressure refers to the area mean of the blood pres- sure curve and is not necessarily the arithmetic average of systolic and diastolic blood pressure. In humans, for instance, it is calculated 5Sodium pentobarbital: Purchased as Nembutal, Abbott Labora- tories, North Chicago, IL 60064. Purchased anesthetic contained 50 mg sodium pentobarbital/l ml solution. A dosage of 50 m or 1 ml solution/kg body weight was administered intraperitoneally (i.p.). Generally, a half hour after injection, a second injection of half strength, i.e., 25 mg/kg, was administered. This procedure produced stage III anesthesia which was necessary for surgery. 6Pressure transducer: Statham pressure transducer 17074 P24AD, Hato Rey, Puerto Rico 00917. . . 7Polygraph: Grass Model 7 polygraph with low level D.C. pre- amplifier Model 7P1 and Grass polygraph 0.0. driver amplifier Model 70A C, Grass Medical Instruments, Quincy, MA 02169. 42 POLYGRAPH PRESSURE (pre- amp, amp, recorder) TRANSDUCER '— 3—WAY 1M VALVE ~ . AMPLIFIER Lil-£1, RAT ' DENSITOMETER RECORDER (cuvette, dye tracer) ch INFUSION/WITHDRAWAL PUMP FIGURE 3.2. -- Instrumentation diagram for determining blood pressure(s), heart rate, and cardiac output. 43 150 TEEEEEEEEEEEE ___ “1"1'111'1,“1111511111 lllliflilllll “7'34 1111111 - h {Un_ --Systolic h [Hi ' --Disatolic CALIBRATION (mm Hg) CHART SPEED (5mm/sec) FIGURE 3.3. -- Example of polygraph recording giving blood pressure(s) and heart rate from cannulated carotid artery of male Osborne-Mendel rat. 44 as P5 + 2Pd/3 (Burton, 1972) or as Pd + 1/3Pp (Henry and Meehan, 1971). The mean blood pressure equation was derived from the control group or normal weight rats in the study, and only coincidentally was the arithmetic mean also the area mean of the blood pressure curve. Heart rate was also measured from the tracing in Figure 3.3 by counting the number of systolic/diastolic cycles or beats per unit of time. The unit of time was dependent on the chart speed, e.g., 5 mm/sec as noted in Figure 3.3. These pressures and rate readings were recorded prior to determining cardiac output. Cardiac Output Measuring cardiac output (milliliters/minute) required the same surgical set-up as described for Blood Pressures and Heart Rate plus the isolation of a jugular vein. Actually, however, all surgery including the isolation of the jugular was accomplished at the onset to minimize contact with the intact cannula. Output was determined by the Stewart-Hamilton indicator-dilution method (Hamilton, 1962). In this method, 0.1 ml indocyanine green8 solution/500 gm body weight was rapidly injected as a bolus into the exposed jugular vein of the anesthetized rat. An explanation for diluting the dye for cardiac output determination is presented in Appendix B. 8Indocyanine green: Purchased as Cardin-Green disposable unit, Hyson, Westcott and Dunning, Inc., Charles and Chase Streets, Baltimore, MD 21201. 45 9 withdrew Following injection an infusion/withdrawal pump blood from the cannulated carotid artery at a constant rate of 4 ml/min. This withdrawal rate was rapid enough to yield a smoothly plotted dye concentration curve yet slow enough to avoid circulatory shock. As shown in Figure 3.2, the cannula carried the dye-laden blood through a dye densitometer.10 Electrical impulses from the densitometer were amplified and plotted on the recorder11 as a dye concentration curve. Following the plotting of the curve, the infusion/withdrawal pump was reversed to return withdrawn blood to the rat. The time lapse from injection of indocyanine green to plotting of the curve was a matter of only a few seconds. As diagrammed in Figure 3.4, the dye entered the cardiovascular system at the jugular vein, traversed the right heart, pulmonary system and left heart, and then was detected in the carotid artery (the first extracardial, anterior branch of the aorta). Cardiac output (0) was calculated from the dye concentration curve using the following equation: 0 (L/min) = dye injected (mg) area under dye concentratTon curve (mg/L X min). Derivation of this equation including a dye concentration curve from the study are presented in Appendix C. 9Infusion/withdrawal pump: Model 950, Harvard Apparatus, Co., Inc., 1150 Dover Road, Millis, MA 02054. With pump set at position #2 and using 10 ml syringe, withdrawal = 4.12 ml/min. 10Densitometer: Gilson-Medical Electronics, Inc., P. 0. Box 27, Middleton, WI 53562. ' - HRecorder: Esterline Angus graphic recording instrument, series E single point recorder (Model E llOlE), Esterline Angus Instruments Co., Inc., P. 0. Box 2400, Indianapolis, IN 46224. 46 l - WITHDRAWAL INJECTION —--- Jugular vein - I ,1\ ....... .-- From pulmonary circulation Vena cava ---- RA LA IN To -<~ - — - ‘lRV l LV l -- Carotid artery pulmonary 7 k Circulation f”,¢r”J .......... Aorta \/' FIGURE 3.4. -- Circulation of indocyanine green dye from site of injection to withdrawal. 47 Stroke Volume and Vascular Resistance Determination of these parameters was dependent on values for cardiac output, heart rate, and mean blood pressure. Since cardiac output (0) is the product of stroke volume (Vs) and heart rate (HR), stroke volume was determined from the following equation: Vs = Q + HR. The resistance of the peripheral vascular bed to the output of the heart, that is the total peripheral resistance (TPR), Was expressed in peripheral resistance units (PRU's) using the following equations (Lichstein, 1971): peripheral resistance units (PRU) = MBP (O in ml/min) peripheral resistance units (PRUTOO) = MBP + 100 gm 8W (0 in ml/min) Cardiac Work Cardiac work was calculated from the mean blood pressure (MBP) and stroke volume (Vs) data using the following euqtion (Folkow and Neil, 1971, p. 189): Cardiac work (gm-meter) = MBP (M H20) X Vs (ml). Calculations for converting mean blood pressure from millimeters of mercury (mm Hg) to meters of water (M H20) are summarized in Appendix 0. Cardiac Anatomy Gross Anatomy Following the study of cardiovascular dynamics, the rats were sacrificed by over-etherization. After the heart ceased beating, it was removed from the thoracic cavity, the atria were drained of blood, 48 and the vessels entering and leaving the heart were carefully trimmed to obtain consistent weighing results. The hearts were rinsed in isotonic saline to remove excess blood and blotted dry. 1 Prior to further dissection, the following measurements were recorded: 1) the organ was weighed (grams) on a tissue balance, 2) the base to apex length (millimeters) was measured with a ruler, and 3) the heart volume (milliliters) was determined from the volume of saline the heart displaced in a graduated cylinder. Heart weight was used in calculating 4) the percentage cardiomegaly or the difference in heart weight of an obese rat and a normal weight rat + heart weight of the normal weight rat and 5) the cardiac index or heart weight + body weight. Using a sharp razor blade, the heart was further dissected. Two adjacent transverse tissue slices (A and B) were cut from each side of a precisely measured transverse midline of the organ as diagrammed in Figure 3.5. When slicing the tissue, little pressure was applied to the blade to avoid compressing the fresh tissue. Each of the slices was 2-3 mm in width. From these fresh tissue slices, 6) heart width or diameter (millimeters), 7) left ventricle lateral wall width (millimeters), 8) septum width (millimeters), and 9) right ventricle lateral wall width (millimeters) were measured with a ruler. These gross anatomical procedures were done as rapidly as possible to avoid enzymatic degradation of the tissue which could affect subsequent analyses. However, removing the heart from the thoracic cavity before it had totally ceased beating was of no value in hastening the procedures. For desirable histological slices, the 49 heart must cease beating. When the heart ceases beating in vivo, it stops in systole (contracted) producing a firmer tissue for slicing. Each tissue slice plus 20 volume of formalin were placed in a closed container to preserve the tissue for histological analysis. Processing of the tissues is described in the following section. The remainder of the heart, labeled C and D in Figure 3.5 was frozen and subsequently used for biochemical analysis as described in a later section, Cardiac Biochemical Procedures. FIGURE 3.5. -- Dissection of . . the rat heart to produce consistent idllne tissue samples for histological work (A, B) and biochemical analyses (C, D). 1\ mg):- n 50 Cardiac Tissue Processing Each tissue slice was encapsuled in a perforated tissue con- tainer, identified with rat number and tissue slice number, and placed in 10% buffered neutral formalin for 2-3 weeks. (Tissues should be in formalin a minimum of 24 hours before further processing. Extended times in formalin are not detrimental to the tissue.) Buffered neu- tral formalin was prepared from the following: 100 m1 37-40% formal- dehyde, 900 m1 H20, 49m sodium phosphate monobasic, and 6.5 gm(anhyd) sodium phosphate dibasic (U. S. DHEW, 1972, pp. 6-7). Since formalin can leave deposits in tissue, tissues were rinsed in saline before moving them to the technicon.12 The 12 chambers of the technicon were filled as follows:]3 2-formalin, 1-80% alcohol, 1-95% alcohol, 4-100% alcohol, 2-xylene, and 2-paraffin Chambers. And tissue slices were gently agitated ("sloshed") in each chamber for 1 hour. In this process formalin fixed the tissue, i.e., preserved the tissue in its natural state by halting autolysis. The goal of tissue processing is to permeate the tissue with paraffin so it can be sliced for microscopic examination. Since paraffin is not miscible with water, the tissue was first dehydrated with alcohol. .To avoid distorting the tissue, the dehydration process was done gradually with progressively concentrated alcohol. The tissues were then "sloshed" 12Technicon: Technicon Tissue Processor, Model 2A, Technicon Co., Chauncey, NY. 13Arrived at this technicon schedule after consulting with Dr. Vance Sanger, Department of Pathology, Michigan State University, December 10, 1973. 51 in xylene which is miscible in both water and paraffin. Finally, the dehydrated, xylene-treated tissues were susceptible to paraffin.14 To insure consistent paraffin infiltration, tissue slices were moved from the technicon to the paraffin infiltrator, a paraffin bath equipped with a vacuum pump which pulled air from the tissue to aid complete paraffin infiltration. The infiltrated tissues were then embedded in paraffin blocks such that the adjacent faces of the tissue slices A and B in Figure 3.5 were the faces of the paraffin blocks diagrammed in Figure 3.6. Thus, if the first paraffin block was damaged and consequently the second block was needed for slide preparation, the sections of the second block would be adjacent to those of the first block. Microscope Slide Preparation Paraffin blocks were chilled a minimum of 2 hours to facilitate facing and sectioning the tissues with the microtome.15 Periodically the knife of the microtome was also chilled. The ribbon of tissue (6 p thick) sectioned from the microtome was floated onto a water bath (45°C), affixed to the slide (pre-washed in alcohol) using.slide adhe- sive,16 drained of water, and placed on the slide warmer and 14Paraffin: Paraplast tissue embedding medium (Boiling point= 56-570C), Biological Research, Inc. 15Microtome: A. 0. Spencer, Model 820, American Optical, In- struments Division, Buffalo, NY 14240.‘ 16Slide adhesive: Tissue-Tac, Dade Division, American Hospital Supply Corporation, Miami, FA 33101. 52 subsequently overnight in the drying oven to assure adherence of the tissue section to the slide. Four slides were made from each paraffin block; that is, four slides were made per rat. One slide from each set of four was stained with Gomori's one-step trichrome stain in which the cardiac muscle fibers were red, collagen was green, and nuclei were blue to black (Gomori, 1950). An example of the stained tissue is shown in Figure 3.7, and details of the staining procedure are presented in Appendix E. The extra micro- scope slides were stored in case they were needed for additional his- tological examination at a later date. In order to stain the tissues, they needed to be free of the paraffin which had given it structure for sectioning and needed to be hydrated. Thus, the first steps of staining were the reverse of tis- sue processing. First the slides were submerged in xylene (in which paraffin is miscible) and then gradually hydrated by submerging in decreasing concentrations of alcohols. After the staining steps, the slides were again dehydrated (increasing concentrations of alcohol, xylene) and then cover slipped using Permount.17 Photomicrograph Preparation Using the stained slides, fields of cross-sectional cardiac muscle fibers in the left ventricle were photographed through a micro- scope (photomicrographs). From each microscope slide, four to five 17Permount: Permount histological mounting medium, Fisher Scientific Co., Chemical Manufacturing Div., Fair Lawn, NJ 07410. 53 Slice A Slice B Face Tissue fl Paraffin - Base FIGURE 3.6. -- Positioning of tissue slices in paraffin blocks so the interphase of slices A and B are the faces of the blocks. FIGURE 3.7. -- Photomicrograph of rat cardiac tissue stained with Gomori's one-step trichrome stain (560 X). ..U ,r u at . . o a. ..i. I . . .9. I I I D . .. . n 5 IL lb. , . . . .Hl . o x a D a . ft. m x .. .u r V . a a}. .71: ' .n I » o . T. I “I. end N5 . '\ .i. r) t «J . O .«t J, 09.1.. -. .I. . . ' ..I~ x. . . ~ n O p- 8‘ P. 0‘ ~. . n p 4 .f— 54 photomicrographs were prepared for each rat using a Leitz 4" X 5" camera-microscope18 and Ektapan film.19 Photomicrographs were taken using the customary 10X ocular, the 40X objective, and a minimum camera bellows extension of 28. Based on the Leitz magnification formula of ocular X objective X 1.25 X (bellows extension/25), cardiac tissue was magnified 560x. Prints were made directly from the 4" X 5" negatives so data quantification was not complicated with calculations for enlarge- ment. Microscopic Anatomy Microscopic anatomy data were derived from quantification of the photomicrographs using a double-blind technique so the treatment group of the rat was not known during data quantification. Since the magnification was 560x, each 560 mm2 area on a photomicrograph (e.g., a rectangle 28 mm X 20 mm) was equivalent to 1 mm2 of cardiac tissue. An area 84 mm X 100 mm was mapped off on the photomicrograph and subsequent evaluations were made in this area. As shown in Figure 3.8 the area 84 mm x 106 mm was selected as it was a multiple of 560 (8400 mm2 = 15) representing 15 individual 560 mm2 area on a photomicrograph or the equivalent of 15 mm2 of cardiac tissue. Furthermore, the area 84 mm X 100 mm nearly filled the 4" X 5" photomicrograph. ‘8 4" X 5" Leitz automatic camera and Leitz Wetzlar ortholux microscope: Distributed by Donald Main and Co., Scientific Instruments, 510 North Dearborn Street, Chicago, IL 60610. l9£ktapan film: 4 x 5 inch, 4162 thick, Eastman Kodak Co., Rochester, NY 14650. 3'19?! ‘, V -._...' ...so- 1...... fl “9 o ( I." . 9’: 7.3:"? 't 55 acoooe___.e em mmzucv e 100 milimeters 5 inches -- Diagram of photomicrograph mapped for on 80 cowl 3t 3 EC R.ql Uf G...- It F" a u q a t a d 56 In the photomicrographs capillary muclei appeared as solid black ellipsoids generally adjacent to cardiac muscle fibers as op- posed to the centrally located nuclei of the muscle fibers or the gray dots of erythrocytes. Capillaries were quantified in the 84 mm X 100 mm area by counting the number of capillary nuclei. The total number of capillaries in the area was then divided by 15 to determine the num- ber of capillaries per square millimeter cardiac tissue. As an indi- cation of the absolute number of capillaries in the heart, capillary number per square millimeter cardiac tissue was multiplied by the heart weight; this estimate of the absolute number was referred to as the capillary number index. Capillary data for each rat were reported as the average number in the four to five phOtomicrographs. Similarly, cardiac muscle fibers were counted in the 84 mm X 100 mm area and divided by 15 to yield the number of fibers per square millimeter cardiac tissue. The absolute number of fibers in the heart was estimated by multiplying the number per square millimeter by the heart weight; this estimate of the absolute number was referred to as the fiber number index. As with the capillary data, muscle fiber data for each rat were reported as the average number in four to five photo- micrographs. Muscle fiber width was measured in millimeters by measuring the width of fibers touching the 84 mm leg of the 84 mm X 100 mm area. Width measurements of the fibers were made parallel to the 84 mm leg and at the widest diameter of the fiber. Fiber width was reported as the average of the measured fibers. 57 Body Composition Procedures Homogenate Composition of the rat carcasses (fat, lean, moisture) was determined in duplicate, as outlined by Mickelsen and Anderson (1959). Contents of the gastrointestinal tract were removed since technically this is not part of the carcass composition. The carcasses were weighed, placed in wide-mouth Mason jars, and autoclaved for 15 minutes at 15 pounds of pressure. To facilitate homogenizing each carcass in the blender,20 a measure of water equal to the weight of the carcass was added. A portion of the homogenate was reserved for composition analysis. Moisture Determination Samples of the homogenate weighing approximately 10 gm were dispensed to tared aluminum pans and weighed. Samples were dried 3p yggpp_(70°C) until they were a constant weight. The difference in wet and dry weight was assumed to be moisture. The percentage of moisture in the carcass and total body water (TBW) were calculated from the fol- lowing set of equations: % dry weight of sample = dry weight of sample (grams) X 100 wet weight of sample (gramE) 20Blender: Waring Commercial Blendor, Model CB6 Capacity 1 gallon, Waring Products Div., Dynamics Corp. of America, New Hartford, CT 06057. 58 % dry weight of carcass = carcass (grams) + water (grams)X % dry weight of sample carcass (grams) % moisture in carcass - 100% - % dry weight of carcass TBW (milliliters) = % moisture in carcass X carcass (grams) Total Fat (triglycerides) The dried sample from the moisture determinations were ex- tracted on a Goldfisch extraction apparatu52] with ethyl ether for 7 hours. To insure that all fat (triglycerides) was extracted in this time span, several samples were re-extracted. No fat was found in the re-extraction. Fat from the extraction was collected in dried, tared flasks. Carcass fat, expressed as a percentage of the carcass weight and as grams of fat, was calculated from the following set of equations: % fat in dry sample = fat in sample (grams) X 100 dry weight of sample (grams) % fat in carcass = % fat in dry sample X % dry weight of carcass X 100 fat in carcass (grams) = % fat in carcass X carcass weight (grams) Lean Body Mass Lean body mass (LBM) was then determined by difference: % LBM = 100% - % fat in carcass LBM (grams)= % LBM X carcass weight (grams) 2lGoldfisch extraction apparatus: Model 35001 serial 4017, Laboratory Construction Company (manufacturer), 8811 Prospect, Kansas City, MO 63031. '59 ,3. , fat in carcass (grams) + LBM (grams) = carcass weight (grams) Cardiac Biochemical Procedures Sections C and D of the heart as labeled in Figure 3.5 had been frozen at the time of dissection. (The balance of the heart had been used for histological procedures previously described in Cardiac Anatomical Work.) The frozen sections C and D were pooled and homoge- nized (1:10) on ice with cold water since theldeoxyribonucleic acid procedure required a chilled sample to inhibit autolysis. As they were needed, aliquots of the chilled homogenates were used for the biochemical procedures. Protein Cardiac protein content was analyzed according to Lowry's (1951) method for insoluable protein as modified by Miller (1959). A summary of the procedure including method, standard, and reagents is present hTAppendix F. In this procedure the protein in 1 ml of cardiac tissue homo- genate was precipitated with trichloroacetic acid (TCA) and then dis- solved in NaOH. The addition of copper reagent to the alkaline solution resulted in chelation of the copper by the protein; formation of the Cu-protein complex was nearly complete in 5-10 minutes at room 60 temperature. With the addition of the Folin reagent,22 the cupric ions (Cu++) of the Cu-protein complex reduced the Folin reagent and yielded a blue color which was read at 750 nm in the spectrophotome- ter.23 Heating accelerated the development of this color. Likewise, the standard prepared from bovine serum albumin (BSA)24 was pretreated with alkaline and copper reagent before reaction with the Folin reagent. After reading the standard at 750 nm, an ex- tinction coefficient was calculated: extinction coefficient = absorbance (nm) concentration (pg/ml) Protein quantity in the cardiac samples was then calculated from the following general equation: protein (mg/gm) = absorbance ;- extinction coefficient X 1000 dilution factor X volume homogenate (m1) é- heart weight (gm) % protein (mg) = % protein X heart weight (gm) Fat (by acid hydrolysis) Fat content of the rat hearts was determined by the Association of Official Analytical Chemists' (AOAC) method for fat by acid hydrolysis 22Folin reagent: Phenol reagent 2N solution (Folin-Ciocalteau) Fisher Scientific Co., Chemical Manufacturing Div., Fair Lawn, NJ 07410. 23Spectrophotometer: Beckman DB, Beckman Instruments, Inc., Fullerton, CA. 24BSA: Bovine crystalline, (ICN) Nutritional Biochemical Inc., 26201 Miles Road, Cleveland, OH 44128. '61 (Horwitz, 1970). A summary of the method is presented in Appen- dix G. Approximately 3 gm samples of the cardiac homogenate were de- livered to tared Mojonnier fat extraction tubes25 and weights were re- corded. After the addition of HCl, the tubes were vigorously shaken and boiled in a water bath. Ethyl and petroleum ether were added to the acid-treated samples, shaken, and decanted to a dried, tared beaker. Both ethyl and petroleum ether were used; since the petroleum ether is more polar, it more clearly separated the organic or ether phase from the water phase. The ether extraction as described was repeated twice each time decanting the ether phase into the beaker. Ether in the beaker was allowed to evaporate, the beaker was dried to constant weight, and the weight of fat in the tared beaker was recorded. In an ether extraction, triglycerides but not phospholipids are partitioned into the organic phase. In cardiac tissue, phOSpholi- pids are more abundant than triglycerides ocCurring in a ratio of 4:3 (Widdowson and McCance, 1955). With acid and heat treatment, the P bonds of the phOSpholipids were hydrolyzed and the phospholipid “skeleton" was then soluable in the organic phase. Thus, in deter- mining fat by acid hydrolysis, triglyceride and phospholipid content in combination was measured and results should be reported as fat content by acid hydrolysis. 25Mojonnier fat extraction tubes: Monjonnier flask Model G3, Mojonnier Brothers Co., 4601 West Ohio St., Chicago, IL 60604. 62 Equations for calculating the fat content by acid hydrolysis follow: sample weight = heart weight (grams) X homogenate weight (grams) (grams total volume homogenate (milliliters) % fat = fat weight (grams)X 100 sample weight (grams) total fat = % fat X heart weight (grams) Deoxyribonucleic Acid Deoxyribonucleic acid (DNA) content in the cardiac tissue was determined by the method of Schneider (1945). A summary of the proce- dure is presented in Appendix H. Keeping the sample chilled is of utmost importance until the DNA is isolated; chilling reduces the activity of the DNA-ase enzyme. Therefore, the frozen tissue was homogenized with gpld_H20. Addition of gpld_10% trichloroacetic acid (TCA) precipitated both the protein and DNA in the cardiac tissue homogenate. After centrifuging the acidified sample, the supernatant was discarded and the precipitate was dissolved in 5% TCA which precipitated only the protein. Thus, the supernatant (which contained the DNA) was saved for analysis. Additional 5% TCA was added to the supernatant, and it was incubated in a water bath to facilitate isolation of the DNA. After incubation the sample was centrifuged to precipitate interferring agents from the DNA. The time and temperature of the water bath incubation were crucial in the DNA analysis. Too high a temperature or too long an 63 incubation time could "damage” the DNA. If the DNA structure were altered, e.g., if the DNA uncoiled, the DNA would not undergo the final reaction of the analysis. On the other hand, if the time and tempera- ture of incubation were too mild, not all the DNA in the tissue would be isolated. According to Lovtrup and R005 (1961) proper incubation time is dependent on the particular tissue being anaylzed. To determine the proper incubation environment for quantifying DNA in rat cardiac tissue, samples were incubated at 90°C for time periods ranging from 10 to 120 minutes and graphed in Figure 3.9. From these data an incubation time of 30 minutes was selected since this yielded maximum recovery. Apparently, prior to 30 minutes not all the DNA was extracted from the tissue; whereas at incubation times longer than 30 minutes, destruction occurred resulting in lower yields. After incubation in the water bath, fresh diphenylamine - acet- aldehyde solution26 was added to an aliquot of the DNA extract, mixed, and incubated overnight at room temperature as described by Burton (1956). Samples were read in the spectrophotometer27 at 600 nm. Using the following general equation, the spectrophotometer reading for the DNA was converted to pg DNA: pg DNA/mg tissue = absorbance (nm) X dilution factor extinction coefficient tissue weight (mg) 26Diphenylamine: No. D-882 diphenylamine free base, Sigma Chem- ical Co., P. 0. Box 14508, St. Louis, MO 63178. Glacial acetic ac1d: 2501, 995%, Mallinckrodt Chemical Works, St. Louis, MO 63160. Acetal- dehyde: 2401, Mallinckrodt Chemical Works, St. Louis, MO 63160. 7Spectrophotometer: Beckman DB, Beckman Instruments Inc., Fullerton, CA 80566. 64 15 ‘. ‘g I) '. " 1. S. 2’10 ' c Q) E 2" c: 5 Z :3 30 60 90 Time (minutes) FIGURE 3.9. -- Time plot conducted at 90°C for determining environment for DNA extraction. 65 Moisture Determination Moisture content of the heart was determined in the same manner as was moisture content of the carcass. (Refer to Body Composition Procedures - Moisture Determination.) An aliquot of the homogenized tissue was dispensed to a tared aluminum pan and dried in vacuo (70°C) until it was a constant weight. The difference in weight before and after drying was considered moisture content. Data Analysis The randomized block design of the study was analyzed as a single classification (or one-way) analysis of variance, ANOVA. Analyses were performed on the Control Data Corporation (CDC) 6500 computer, Michigan State University using Jeremy D. Finn's multivariance program as outlined by Scheifley and Schmidt (1973). The program included univariate and multivariate analysis of variance and covariance. Furthermore, calculation of statistical significance between means, as opposed to the overall significance reported in the computer output, was performed according to Tukey's w-procedure, an a posteriori test for multiple comparison among means (Sokal and Rohlf, 1969). A Canon Canola F-20P calculator with statistic function keys was used to compute correlation coefficients. Both the Tukey's test and correlation coeffi- cients were performed at alpha levels of 0.05 and 0.01. Thus, any statistical significance discussed is as a minimum PS0.05 unless other- wise noted. The ANOVA tables and Tukey's test calculations are detailed in Appendix I. Similarly, the critical values for determining significant correlation coefficients (r) are presented in Appendix J. CHAPTER IV RESULTS AND DISCUSSION Introduction This study concentrated on assessing the impact of obesity on cardiovascular dynamics. By employing various treatment groups, the study examined the effect of two primary types of obesity. These in- cluded growth-onset obesity which manifested itself early in life as well as adult-onset obesity which expressed itself only after maturity. In addition, the effect of weight loss on cardiovascular functions was studied. Thus, a total of four groups of experimental rats were used in the study: normal weight rats,1 rats with growth-onset obesity,2 3 rats with adult-onset obesity, and weight-reduced rats.4 The cardiovascular dynamic parameters which were assessed 1Normal weight: Rats were fed the grain ration from weaning to 36 weeks of age. 2Growth-onset obesity: Rats were fed the high fat ration from weaning to 36 weeks of age. 3Adult-onset obesity: Rats were fed the grain ration from weaning to 18 weeks of age and the high fat ration from 18 to 36 weeks of age. 4Weight reduced: Rats were fed the high fat ration from wean- ing to 18 weeks of age and the grain ration from 18 weeks to 36 weeks of age. 66 67 included blood pressure, cardiac output, heart rate, stroke volume, peripheral resistance, blood volume, total body water, hematocrit, respiration rate, and cardiac work. I To assist in interpreting the Changes noted in cardiovascular dynamics, cardiac anatomical and biochemical parameters were also as- sessed. The cardiac anatomy component of the study included measure- ments of heart weight, cardiomegaly, cardiac index, heart (displace- ment) volume, heart length and width, left and right ventricle wall width, septum width, fiber width, fiber density, fiber number index, capillary density, capillary number index, and capillary to fiber ratio. The biochemical component of the study included quantification of cardiac protein, fat, and moisture content as well as quantification of deoxyribonucleic acid (DNA) which was expressed as cardiac cell number, concentration, and cell size. Futhermore, in order to quantify obesity more specifically than in terms of body weight, body composition including body fat and lean body mass was determined. Thus, the study of the impact of obesity on cardiovascular function is presented in four parts: 0 Body composition 0 Cardiovascular dynamics 0 Cardiac anatomy 0 Cardiac biochemistry 68 Boderomposition Body Weight Body weight data for the four treatment groups are plotted as growth curves in Figure 4.1. Body weight for the normal weight rats plotted as a typical growth curve, i.e., an initial rapid weight gain followed by a gradual plateau in body weight. Rats with growth-onset obesity had an extended period of rapid weight gain and thus the growth curve was accentuated compared to that of the normal weight rats. Beginning at 10 weeks of age, rats with growth-onset obesity (367 gm) weighed significantly more than normal weight rats (307 gm). THe growth curve for the rats with adult-onset obesity was similar to that of the normal weight rats until 18 weeks of age. At that time these rats were changed from the grain ration to the high fat ration and the slope of the curve began to increase to parallel that of the rats with growth-onset obesity. The growth curve of the weight-reduced rats was similar to that of the rats with growth-onset obesity until 18 weeks. At that time these rats were changed from the high fat ration to the grain ration and the slope began to.drop to parallel that of the normal weight rats. Body weight data at 36 weeks of age, the time of sacrifice, are summarized in Table 4.1. The normal weight rats had the lowest body weight (503 gm), followed by the weight reduced rats (562 gm), the rats with adult-onset obesity (726 gm), and the rats with growth- onset obesity (867 gm). Body weight of the normal weight and weight reduced rats was not significantly different; but the rats with 69 800_ ‘ 4 ‘ ‘ 4‘,‘L <3 C) <3 " C) A‘ o :55 600r A o ; ‘AagaAaAAA .5.” A O . o o g o o 0 Q) . . . 3 400 l 0 a " A ' g 0 Ollormal weight A ' AGrowth-onset obesity 200‘ . O Adult-onset obesity . CSWeight reduction 0 o 1 l I I O 10 20 30 40 Age (weeks) FIGURE 4.1. -- Growth curves for male Osborne-Mendel rats. 70 cowuow>mu unaccoumwcoms “cosuowcu can mums m a U .ucmo; mcw>oemg one young pmzpumma ucwocumom m:_>uasm Leave um:_ELmumu mo: :oeu_mog5ou mocwm agape; mace on Ezm >P_memmom: ac: on mmoa anon new. uco ace xuom "whoz mn_m Funeme mama Genes. seawem cameoseoc oes.o= enmm emem.e Rape emnoom mopneme »o_aoao someo-o_=e< seem eeawee ease esnmmm e~_neem »o_aoae nonee-eozoce mama Nmnmme new. epnpe Nenmom agape; Peace: Axe NV Asa. Azm NV Ase. flee. games avoa coma some seem. apnoea: xuom emucmsucmch $9 mums PavemZTmccoamo m_oe cpo xmmz om eeae_moQEeo soon see Sem_oz seem -- ._.e “seep 71 growth-onset obesity weighed significantly more than the rats with adult-onset obesity. Rats with growth-onset and adult-onset obesity weighed significantly more than either normal weight or weight reduced rats. Body Fat Body fat data at 36 weeks of age are summarized in Table 4.1 and followed much the same pattern as body weight. The normal weight rats weighed the least and had the least amount of body fat (61 gm or 12%) followed by the weight reduced rats (108 gm or 19%), the rats with adult-onset obesity (300 gm or 41%), and the rats with growth-onset obesity which weighed the most and had the most body fat (393 gm or 46%). Body fat content of the normal weight and weight reduced rats was not significantly different. But the rats with growth-onset obesity had significantly more fat than rats with adult-onset obesity. In general, the rats with growth-onset and adult-onset obesity had three times as much body fat as the normal weight and weight reduced rats. Body fat as a component of body weight is illustrated in Figure 4.2. Lean Body Mass Lean body mass data at 36 weeks of age are summarized in Table 4.1. Whereas the body weight and body fat content of both groups of obese rats was significantly more compared to the normal weight and weight reduced rats, lean body mass was approximately the same among the four groups: normal weight rats (425 gm), rats with growth-onset obesity 72 E Body fat .Lean body mass 800.. 50° ” 45% 4174 Body weight (grams) pd N ha] ‘. ‘ . r35: ‘ *8 U .92": i , y 1 , . ‘a- .1.“ ’ I- . . '. f, .33? f . ,f '1 _i "I" ,r.. ~ - .Qvlf .. “a ' v.. . .. ‘7‘. ‘5 ‘V ('2: . . . .- ’ 9 o t“ 'A“\ , 840 4‘3’5 r. o '3‘, -‘ ’«lhn' . '. “ 't P“ :9 a 4, n . 9" T P. :‘ '.' m at... h .. . .., '~ .--. . '- ‘T ...g' ."‘ n' A ' 3"“ t J ‘1 - . -. - if", =- ' - " aha. o r.” .o- I . V. ~ one. ...c - .- .‘fi..oo ,-;."~ ~ ' '5..; I V “ ..019_"" . n." ’ c, .53" . l_'m . "r“ ‘ " 3 I. I a. ‘- e. ‘-0.'.'4 \::-. Z. l'-" T 'r' ..‘0 deem. ; 2 i‘i- «~ ‘ fl ‘ um.“ . 5.." P " 0' - v‘ . D's ' ’ ' ., .- o .. , . a" l ' Q" .. .. 8855 . rivals-f.:t 1v -. ’3 ‘ J ”aux, '\A¢‘f a” ._ , o I f . a . ' .h 9 50‘ "fi: F T ' . >- .u’a 4;: Of... 4,‘ . ‘ ‘ P - ., " ‘fio_"\ " ~ I I. 'w - 2.5.1“ ' i i, .p’q,“ -.- 73".“ '., q. 1 ‘2‘“, f. , 1, 1. _ . .,.‘ . .', . .:£. . 3 _ I, I . .1 ‘ .'..'. . ‘ 3?:- " .' . .3. ‘ . :‘V'm . 5' ‘~ If... KL" 4% .x-f ; T r 3' ‘4 {‘45 '.,J,v.a~ I ’ Growth-onset Adult-onset obesity Weight reduction obesity FIGURE 4. 2 -- Body weight and body composition of 36 week old male Osborne-Mendel rats. 73 (462 gm), rats with adult-onset obesity (418), and weight reduced rats (436 gm). Since body weight and body fat followed similar patterns but lean body mass remained approximately the same among the four groups, the correlation of body weight with body fat (r=a 0.97) was consider- ably higher than the correlation of body weight with lean body mass (r = 0.55). Lean body mass as a percentage of body weight was inverse- 1y related to the body weight as shown in Figure 4.2. Normal weight rats had the lowest body weight (503 gm) but the highest percentage of lean (88%); and rats with growth-onset obesity were the heaviest (867 gm) but had the lowest percentage of lean (54%). Cardiovascular Dynamics Blood Pressure Blood pressure data from the four treatment groups are summarized in Table 4.2. Blood pressure data included systolic, diastolic, pulse, and mean pressure. Systolicypressure--Systolic blood pressure was lowest in the normal weight rats (128 mm Hg), intermediate in the weight reduced rats (144 mm Hg), and highest in the rats with growth-onset and adult- onset obesity (150 mm Hg). Pressure of the obese rats was significantly greater compared to the normal weight rats (P< 0.10). In Table 4.3 systolic blood pressure data are presented as a grouped frequency distribution to emphasize that the pressure of normal weight rats clustered in the lowest range, that the pressure of rats with growth-onset and adult-onset obesity clustered in the highest 74 :o_pow>mu acouceumncmms unmanned“ can mace a Q o e_nomp a new “_ne__ e_nee_ eo_ooseoc oem_oz «_nem_ __Amm m_nm~_ omwomp so_moee nomeo-e_=e< mpnwmp opeem m_nm__ omnomp »e_moeo “once-eozecw m_nm__ m_ne~ mpa~o_ m_nmm_ oem_oz _eeLoz am cam: «mp—5 ozoumma umpoumxm Umucgmeh. 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