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I . .0! .4 4 u . o 4 4 a D! 4 0.4. i o n _ 4. . .0 t. .0 . . 4 . ...f . 1 I I 4 4' l 4. 4 ' o ’ o... v 4 . . r .. . I- 4 . . 4 .4 .o 4 . u _ 4 .v 40 o 4 4 v 4" I . . 4 I. 4. . . . 4 4o 4 l4 4 4 4 n ' I _ 4 r . a .4 o l4 4' . . o ..L . . .v .H 44 -. 4' 'n o n 4 4 4 4 4. 4 44.4 v 4 4 4. . . _ I a 4 I l- o .4. . ,4. . . ,. . .|(O.OO4 .4 . _ O .00 4, . 4 .4 . . . 4 .4 .440 4 rh. H£$I I4 I.” P . . o. 4 Fl . . . u . . IO! 0'4”“. . I'l’. .‘l '0 .Humyk‘ 4 o . II .I. L I B '3 A R Y h'liethigan State University air-”‘1' THESIS ABSTRACT ORAL CONTRACEPTIVE STEROIDS: EFFECTS ON FOOD CONSUMPTION, DIGESTIBILITIES OF VARIOUS NUTRIENTS, BODY COMPOSITION, AND LIFE SPAN IN RATS BY Kanagavalli Manoharan Effects of oral contraceptive steroids on food con— sumption, digestibilities, and retentions of various nutri- ents, body composition and life Span were studied in female rats fed a basal grain ration and the contraceptive ster- oids norethynodrel, a progestine and mestranol, an estrogen. The results from these rats were compared with those from control rats fed just the basal grain ration. Food con— sumption and body weights were measured. Urine and feces were collected twice; 22 days and 173 days after steroid treatment to study the digestibility and retention of nu- trients. Four groups of rats were killed at different time intervals to study the changes in body composition and to determine whether the changes are reversible. Two groups were sacrificed at 4 weeks and 25 weeks and 3 days 1 Kanagavalli Manoharan respectively after continuously feeding the steroid mixed diet. The other two groups were sacrificed after refeeding the control diet for 6 weeks following 4 weeks and 25 weeks and 5 days of steroid treatment. Food consumption and body weights were signifi- cantly lower for the steroid treated rats° Analysis of urine and feces revealed that the digestibilities of pro— tein, fat, sodium, and potassium were not affected during the short—term or long-term feeding of the steroids. A significant difference occurred in the retentions of the dietary nitrogen and sodium between the treated and control rats. After 22 days of steroid treatment, the control rats retained significantly (P < 0.05) more nitrogen than the treated rats. Dietary sodium was retained significantly more (P < 0.05) by the treated rats than by the control rats at this period. At 173 days of steroid treatment the treated rats retained significantly more (P < 0.01) dietary nitrogen than the control ones. No difference was noticed in the retention of potassium between the two groups either at 22 days or at 173 days after steroid treatment. Body composition analyses revealed that percent moisture in the carcasses of the treated rats was higher 2 Kanagavalli Manoharan in all four groups. However, it was significant (P < 0.05) between the treated and the control rats only in the first group which was sacrificed after 4 weeks of steroid treat- ment. Percent moisture in the lean body masses was almost the same for the treated and the control groups. Absolute amounts of dry body weights were significantly higher for the control rats both after 4 weeks and 25 weeks and 3 days of steroid treatment. Significant difference occurred in lean body masses between the treated and the control groups after 25 weeks and 3 days of steroid treatment. Refeeding the control diet for 6 weeks did not bring the dry body weights and lean body masses comparable to control rats. Treatment with steroids suppressed the gain in body fat in treated rats. This happened both in short and long- term feeding of steroids. Refeeding the control diet helped the treated rats to gain more fat. Nitrogen as a percent of either wet or dry lean body mass was not signif- icantly different between the treated and control groups throughout the experiment. However, a significant differ— ence was noticed between the two groups in the absolute amounts of nitrogen and protein after 25 weeks and 3 days of steroid treatment. Refeeding the control diet for 6 3 Kanagavalli Manoharan weeks did not change difference in body nitrogen and pro- tein between the treated and control groups. Sodium and potassium per 100 gm. of wet and dry lean body masses were almost the same for both groups. Life Span study revealed that there were no sig- nificant differences in the life spans of the treated and control rats° However an increased incidence of mammary tumors was observed in the treated group. ORAL CONTRACEPTIVE STEROIDS: EFFECTS ON FOOD CONSUMPTION. DIGESTIBILITIES OF VARIOUS NUTRIENTS, BODY COMPOSITION, AND LIFE SPAN IN RATS BY Kanagavalli Manoharan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition 1969 ACKNOWLEDGMENTS My sincere thanks and gratitude to the following: Dr. Modesto G. Yang Dr. Dr. for his advice, guidance and encouragement throughout the study. Dena C. Cederquist, Dr. Harold D. Hafs, Olaf Mickelsen, and Dr. William W. Wells for their valuable suggestions. Dr. Duane E. Ullrey for his assistance in the use of flame emission spectrOphotometer. Dr. Vance L. Sanger for his help in histological and patholog- ical studies of tumors. My husband for his patience, help, and encouragement. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . . . . . . . . LIST OF APPENDICES . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . REVIEW OF THE LITERATURE . . . . . . . . . . . . . History of the Oral Contraceptive Pill . . . . Structure of Progestins and Estrogens. . . . . Mechanism of Action. . . . . . . . . . . . . . Biological Properties of Oral Contraceptives . Effects of Oral Contraceptives on Metabolisms. MATERIALS AND METHODS. . . . . . . . . . . . . . . Animals. . . . . . . . . . . . . . . . . . . . Contraceptive Steroids . . . . . . . . . . . . Digestibility and Balance Studies. . . . . . . Body Composition . . . . . . . . . . . . . . . Life Span Study. . . . . . . . . . . . . . . . Page ii viii ix 10 l3 17 38 38 39 4O 41 43 Table of Contents.——Cont. Page Preparation of Rats for Analysis . . . . . . . . 43 Analysis of Various Components . . . . . . . . . 44 Statistical Analysis . . . . . . . . . . . . . . 46 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 47 Food Consumption and Body Weight Gain. . . . . . 47 Digestibility and Nutrient Balance . . . . . . . 52 Body Composition . . . . . . . . . . . . . . . . 62 Life Span Study. . . . . . . . . . . . . . . . . 99' SUMMARY. . . . . . . . . . . . . . . . . . . . . . . 104 LITERATURE CITED . . . . . . . . . . . . . . . . . . 107 APPENDICES . . . . . . . . . . . . . . . . . . . . . 115 iv Table LIST OF TABLES Nitrogen and Fat Digestibilities after 22 Days of Steroid Treatment . . . . . . . . . . . . Percentage Retention of Dietary Nitrogen in Rats after 22 Days of Steroid Treatment. . . Nitrogen and Fat Digestibilities after 24 Weeks and 5 Days of Steroid Treatment. . . . Percent Nitrogen Retention in Rats Calculated on the Basis of Four Days of Dietary Nitro— gen and 4 Days of Urinary and Fecal Nitrogen after 24 Weeks and 5 Days of Steroid Treatment. . . . . . . . . . . . . . . . . . Percent Digestibilities of Sodium and Potassium During the Two Collection Periods. Percent Retentions of Sodium and Potassium During Two Collection Periods. . . . . . . . Body Weights of Rats Killed at Different Lengths of Time of Feeding Steroids (first and third parts of table) and after Refeed- ing of Control Diets for 6 Weeks (second and fourth parts of table) (Values are in gms.). . . . . . . . . . . . . . . . . . . . Moisture in the Whole and Lean Body Masses and Absolute Amounts of Dry Body Weight and Lean Body Masses after Four Weeks of Steroid Treatment. . . . . . . . . . . . . . . . . . Page 54 55 57 58 61 63 64 66 List of Tables-~Cont. Table 9. 10. 11. 12. 13. 14. 15. 16. Per Cent and Absolute Amounts of Nitrogen and Fat in Rats after 4 Weeks of Steroid Treatment. . . . . . . . . . . . . . . . . . Amounts of Sodium and Potassium in the Whole Body and Per 100 gm. of Wet and Dry Lean Body Masses of Rats Killed after 4 Weeks of Steroid Treatment . . . . . . . . . . . . Moisture in the Whole and Lean Body Masses and Absolute Amount of Dry Weight and Lean Body Masses in Control and Refed Rats placed on Control Diet for 6 Weeks after 4 Weeks of Steroid Treatment . . . . . . . . Per Cent and Absolute Amounts of Nitrogen and Fat in Control Rats and in Rats Refed the Control Diet for 6 Weeks after 4 Weeks of Steroid Treatment. . . . . . . . . . . . . . Amounts of Sodium and Potassium Present in the Whole Body and Per 100 gm. of Wet and Dry Lean Body Masses in Refed Rats which were Placed on Control Diet for 6 Weeks and Control Rats after 4 Weeks of Steroid Treatment. . . . . . . . . . . . . . . . . . Per Cent Moisture in the Whole and Lean Body Masses and Absolute Amount of Water, Dry Weight, Wet and Dry Lean Body Masses in Rats after 25 Weeks and 3 Days of Steroid Treatment. . . . . . . . . . . . . . . . . . Per Cent and Absolute Amounts of Nitrogen and Fat in Rats after 25 Weeks and 3 Days of Steroid Treatment. . . . . . . . . . . . . . Amounts of Sodium and Potassium Present in the Whole Rats and the Amount Present Per 100 gm. of Wet and Dry Lean Body Masses after 25 Weeks and 3 Days of Steroid Treatment. . . . vi Page 69 74 76 79 81 85 88 9O List of Tables——Cont. Table Page 17. Per Cent Moisture in the Whole and Lean Body Masses and Absolute Amounts of Water, Dry Weight, Wet and Dry Lean Body Masses in Control Rats and in Rats Refed the Control Diet for 6 Weeks after 25 Weeks and 5 Days of Steroid Treatment. . . . . . . . . . . . . 94 18. Per Cent and Absolute Amounts of Nitrogen and Fat in Control Rats and in Rats which were Placed on Control Diet for 6 Weeks after 25 Weeks and 5 Days of Steroid Treatment . . . . 98 19. Amounts of Sodium and Potassium in the Whole Body and Per 100 gm. of Wet and Dry Lean Body Masses in Refed Rats which were Placed on Control Diet for 6 Weeks and in Control Rats after 25 Weeks and 5 Days of Steroid Treatment . . . . . . . . . . . . . . . . 100 vii LIST OF FIGURES Figure Page 1. Structures of two of the progestins and the estrogens used in oral contraceptives . 9 2. Body weights (upper curves) and weekly food consumption (lower curves) of the control and treated rats. . . . . . . . . . . . . . 48 viii L IST OF APPENDICES Appendix Page I. Composition of Grain Ration (in %) . . . . . 116 II. Per Cent Nitrogen Digestibility. . . . . . . 117 Per Cent Nitrogen Retention. . . . . . . . . 117 Table 1. Average Food Consumption and Body Weights of Rats. . . . . . . . . . . . . . . . . . 118 2. Feed Efficiency in the Treated and Control Rats . . . . . . . . . . . . . . . . . . . 119 3. Absolute Amount of Fat Per Gram of Nitrogen in the Body of the Rats Killed at Various Time Intervals . . . . . . . . . . . . . . 120 4. Life Span of Rats Treated with Steroid Mixed Diet and Rats Fed the Control Diet . 121 ix INTRODUCTION During the past ten years, a revolution has occurred in the field of contraception. The revolution was brought about by the discovery of the oral contraceptive pill. In 1960 the oral contraceptive pill was licensed for general use in the United States. Now the use of the pill has be— come a common practice among women. It was estimated in 1965 that 3.8 million women under 45 and living with hus- bands were using the oral contraceptive pill in the U.S.A. and an additional 4.7 million indicated that they might use it in the future. The percentages of women now using the oral contraception vary positively and strongly with the amount of education they have. The widespread use of this medication for the control of conception or for various other reasons by healthy fertile women for many years im— plicates concern for the safety with which these prepara- tions can be used. Clinical data from observations and research on thousands of women pointed out no apparent re— lationship between contraceptive therapy and any other major abnormalities. This made the FDA panel declare the 1 pill "not unsafe" for human use. However, there are some women who are very susceptible to changes brought about by oral contraceptive therapy. Special interest attaches to the physiological actions of the oral contraceptives because these prepara- tions contain two hormones which are the synthetic counter- parts of natural estrogen and progesterone. Thus, one could expect these hormones to have some effects on their target organs such as the uterus, ovary, and pituitary. They might also have endocrine effects characteristic of the ovarian estrogen and progesterone. Recently there have been many reports on the oral contraceptive pills and their action on pitutitary and adrenocortical secretions and me- tabolisms of various nutrients. There have been some con— troversies whether the pill really changes the secretion rate of adrenal cortex and thyroid and causes liver damage. But there is no doubt that it may cause many changes in carbohydrate, fat, protein, mineral, and various other me— tabolisms associated with its hormonal action. Seemingly, it brings changes even in cholesterol metabolism which plays a central role in the synthesis of all body steroids. Many questions have to be answered concerning the long-term and intermediate-term safety of these preparations. Regarding long—term safety, the most fre- quent question concerns the possible increased occurrence of malignancies as a result of the use of these endocrine preparations. Concerning the intermediate safety, the questions relate particularly to possible deleterious ef- fects on the liver, adrenals, and pituitary. Additional side chain like l7—alky1ation which is not part of the natural estrogen and progesterone but is present in the synthetic counterparts may also bring about some delete- rious effects on various organs. In the view of nutrition, changes in body composi— tions of various compartments imposed by the changes in various metabolisms are very important. Since contracep— tive preparations are taken through oral route, digesti- bilities of various nutrients may be affected. Estrogen which is one of the component of the pill is known for its action on 'appetite' and 'weight gain' eSpecially in lab- oratory animals (Meites, 1949; Sullivan and Smith, 1957). Results of the few experiments thus far published clearly demonstrate that the nutritional significance of the oral contraceptives should be studied in greater depth. The present experiment was undertaken to study the extent and in which compartments of the body these antiovulatory steroids bring changes, whether digestibil- ities of various nutrients like fat, proteins, carbohydrate and some minerals are affected and to see whether any changes occur in the balances of various major nutrients. In addition to the above mentioned aSpects of the experi- ments, long—term study was initiated to determine the life span and various other abnormalities, if any, like cancer, tumor, etc. Food intakes and body weight gains were also studied. REVIEW OF THE LITERATURE History of Oral Contraceptive Pill Various types of contraceptive methods were being used for many years. But none of these methods proved to be completely effective. Only in the past few years, there has been a change in the medical attitudes toward contra—' ceptive techniques. In 1960, a new approach was made in the field of contraception with the availability of the contraceptive pill which proved to be nearly 100% effective' in controlling conception. The develOpment of the contra- ceptive pill has really been an interesting research ad- venture. Synthetic sex hormones, estrogen, and progester- one are the two active components of the oral contraceptive_ pill. The very idea of controlling conception with pro- gesterone was initiated by Beard (1897) who postulated that the corpus luteum of the Ovary which secretes progesterone was responsible for the inhibition of ovulation during (pregnancy. Then, when progesterone was isolated, Makepeace gt_§l, (1937) administered this hormone into rabbits and 5 found that this inhibited the ovulation. Later on Pincus (1956) reported that the oral administration of natural progesterone was not suitable for contraceptive purposes probably because of the variable absorption from the gastro—intestinal tract. So research was turned toward synthetic steroid hormones having both the properties of progesterone and high oral activity. Norethynodrel, the progestin of the first approved oral contraceptive pill was prepared by Frank Colton at the Searle Laboratories. Its contraceptive effectiveness was tested and proved by Rock gt a1, (1956). Even though norethynodrel proved its effectiveness in the control of conception, the incidence of spotting and breakthrough bleeding was high. Estrogen was added to the progestin to control the occurrence of spotting and it was proved successful. Later on, estrogen was added to other pro- gestins in order to improve endometrial support. In addi- tion to norethynodrel, several other progesterone prepara— tions, all with added estrogen, have been tried including norethindrone, norethindrone acetate, medroxyprogesterone acetate, ethynodiol diacetate and chlormadinone acetate. All have been effective in various dosage combinations. Thus an effective biochemical control of conception was attained by means of oral contraceptive pills. Many oral contraceptive pills are now available under various brand names in various countries. Some of them are Enovid, Orthonovum, Anovlar, Norlestrin, Lyndiol, Ovulen, Provest, Ovex, Aconcen, etc. Their composition is presented below. Product Progesterone Estrogen Enovid Norethynodrel Mestranol Orthonovum Norethindrone Mestranol Anovlar Norethindrone acetate Ethynylestradiol Norlestrin Norethindrone acetate Ethynylestradiol Lyndiol Lynestrenol Mestranol Ovulen Ethynodiol diacetate Mestranol Provest Medroxyprogesterone acetate Ethynylestradiol Ovex Megestrol acetate Ethynylestradiol Aconcen chlormadinone acetate Mestranol Common preparations currently available commercially con- sist of a progestin in combination with an estrogen throughout the treatment period or the so-called "com— bined therapy." In sequential oral contraception, estrogen alone is given for 15 days of therapy, followed by the com— bination of estrogen and progesterone for 5 days. CAQuens and Oracon are the first "sequential" oral contraceptives to be marketed. One of the major problems with the use of sequential therapy is the failure of occurrence of menses following the withdrawal of the therapy and, in addition, it is not as effective as the other therapy. Structure of Progestins and Estrogens The chemical structures of two of the progestins used in the oral contraceptives are shown in Figure l. Norethynodrel, norethindrone, and several other compounds have an ethynyl group at the 17 position of the steroid molecule; it is apparently this group with the nor struc— ture that gives these compounds their high oral activity. Compounds in which the 19—methy1 group in the steroid mole- cule has been replaced by a hydrogen atom are referred to as l9-nor compounds. Each of these compounds has a carbon- carbon double bond in the basic steroid molecule. Norethy- nodrel, a progestin which is used in Enovid, has this double bond at the 5(10) position. This double bond at the 5(10) x .--./ T 0/’ /\ .\@_' gummy. ”‘2 = .. - .. ‘ 3 a u ._ ‘ 1.x , .‘ '7: rl‘ I" . . . u"; a I: 4 . . I. rqtl‘ \r‘ a. )7 .3. L’ V "i ‘4! I: Viz-TI; ‘LJ 3 5-343 mi {2:8 {4’ . ¢ Or '3 P31. .- ,. : ‘1’.) Q {5‘ I“ C. ‘ ":3 r n: F. i; h (41(- E'“.." i i \ a "wfi L 1» \‘5 Eve.) H 0 E? tag-'1'; ,z.:: Kittie! Fig. l.--Structures of two of the progestins and the estrogens used in oral contraceptives. 10 position is biologically significant, for in addition to being progestational, it makes norethynodrel estrogenic and devoid of androgenic effects in both animals and men (Drill, 1966). In other substances like norethindrone, the double bond is at the 4(5) position. Some authors, thus, classify norethisterone as a derivative of 19- nortestosterone. But norethynodrel cannot be classified as a testosterone derivative because the double bond is in the 5(10) position rather than the 4(5) position. The two estrogens presently used in all oral contraceptive preparations are either mestranol or ethynylestradiol. Their structures are also shown in Figure 1. Both com- pounds contain a l7—ethynyl group which imparts high oral potency. Mechanism of Action The mechanism by which contraception is assured has been a subject of some discussion. The high degree of ef- fectiveness has been attributed to inhibition of ovulation. However, two other mechanisms have also been suggested. One is the alteration of the cervical mucous so that the 11 sperm penetration is inhibited, and the other is the alter- ation in the endometrium so that it becomes unsuitable for nidation. However, the inhibition of ovulation is un- doubtedly the most important mechanism involved. The ad- ministration of oral contraceptives, which are the combi- nation of estrogen and progesterone, mimic natural estro- gen and progesterone. They prevent ovulation much like the natural hormones which prevent ovulation during preg- nancy. By studies on animals, it has been established that oral contraceptives achieve ovulation control by inhibit- ing the secretion of gonadotropins from the pituitary gland. This was shown by Saunders and Drill (1958) who demonstra- ted that norethynodrel. the progestin in Enovid, decreases the gonadotropin content of the pituitary gland in the ovariectomized rat. Other studies have also demonstrated the effectiveness of norethynodrel (Saunders, 1964) and various other progestins in inhibiting the secretion of the pituitary gonadotropins in the rat. Estrogens are also quite effective in inhibiting pituitary gonadotropin secre- tion in the rat and, in terms of dosage, they are much more potent than the progestins (lbid.). 12 Venning (1962) found evidence for the suppressed follicle growth as well as ovulation with norethynodrel which would indicate that Follicle Stimulating Hormone and Leutinizing Hormone were decreased. Further evidence that ovulation was suppressed came from the work of Shearman (1965). He demonstrated two cycles in patients aged 26 years. The first was without treatment which showed the normal pattern of excretion observed in ovulatory menstru- ation with a peak of estrogen excretion at about the 14th day associated with ovulation and a luteal phase rise in estrogen and pregnanediol followed by regression before the start of menstruation. On day 5 of the second cycle, treatment with Anovlar was started. There was a progres— sive fall in estrogen excretion and no evidence of ovula— tion or corpus luteum activity. When Anovlar was withdrawn in the next month, the pattern of normal ovulation was produced. These assays were repeated after a further six months treatment and the same results were obtained. 13 Biological Properties of Oral Contraceptives The Adrenal Cortex and the Pituitary Pregnancy or the administration of estrogens in— creases protein-bound cortisol in the plasma and, thereby, the total plasma cortisol. A decreased excretion of 17— keto steroids (17KS) and l7-ketogenic steroids (17KGS) has been observed by Starup gt_al. (1966), who treated women with 5 mg. of megestrol acetate and 0.1 mg. of mestranol for a period of 3-74 weeks. This suppression might have been caused by an inhibition of the production or release of ACTH. However, the authors found a normal response to metyrapone or ACTH during treatment in all patients studied, indicating a normal adrenal cortical function during treat— ment with megestrol acetate plus mestranol. The authors concluded that the pituitary-adrenocortical feedback me- chanism was undisturbed. Since they found increased plasma protein—bound cortisol during treatment they also concluded that the protein-binding of plasma cortisol made it less available for conjugation by the liver and renal excretion. Even though it has been shown that protein-bound cortisol is physiologically inactive, there is a possibility that 14 the increased circulating corticosteroids may cause di- minished carbohydrate tolerance in certain subjects taking oral contraceptives. In contrast to the work of Starup §E_§1, (1966), Enovid has been reported to decrease the response to me- tyrapone (Mestman g£_al,, 1963) but since the response to ACTH is not affected, the changes in the excretion of 17KS and 17KGS appear to be related more*to inhibition of pi- tuitary responsiveness than to altered adrenal cortical response (Leach §t_§1,, 1965). Reduced ACTH reserves noted in some Of the Enovid users with reduced glucose tolerance (Waine gt_al., 1963) suggests a direct action of steroids especially estrogen on adrenocortical secretion. A direct corticoid action of medroxyprogesterone acetate has been demonstrated in adrenalectomized and hypophysectomized sub- jects (Cumanni §§_§1,, 1963). Certain oral contraceptives have also been found to change the secretion rate of mineralocorticoid hormones like aldosterone from the adrenocortex (Singer gt $1., 1963; Laidlaw gt 31., 1962); 15 Liver Function Whether oral contraceptives cause liver damage has been a subject of controversy. Finnish investigators Eisalo §t_gl. (1964) have reported reversible elevations of serum transaminase and Bromosulfonphthalein (BSP) in a series of 12 menOpausal women. On the other hand, such contraceptives have not been found to cause liver damage by other investigators (Linthorst, 1964; Tyler, 1964). Linthorst treated 52 women with lynestrenol for a period of 14-43 months. No laboratory or clinical indication of hepatic dysfunction was noticed. Borglin (1965) treated 36 women with lynestrenol or lynestrenol + mestranol for a considerable period. Even though the frequency of ab- normal liver function tests was fairly low, in a few cases, the values obtained did deviate from norma1——name1y an in- crease in the transaminase activity and a slight increase in the BSP retention. Serum Glutamic Pyruvic Transaminase was also increased in two of the patients treated with lynestrenol. However, the alkaline phosphatase values, bilirubin values, and the thymol turbidity tests were normal. The work of Datta gt_a1, (1965) with rabbits using two anovulatory steroids, noracyclin and orthonovum, showed 16 that no hepatic damage was associated with these steroids by estimating the serum Glutamic Oxaloacetic Transaminase level in the serum. Thyroid Function Measuring the protein bound iodine (PBI) and the red cell uptake of radioactive triiodothyronine are mea— sures of thyroid functions. The free thyroxine factor may also be used to assess thyroid status in women taking oral contraceptives. Goolden g£_al, (1967) measured PBI and the uptake of 131I-triiodothyronine in a group of women who were taking oral contraceptives and in a control group of healthy women. Free thyroxine factor which is prOpor- tional to the free thyroxine in the serum was calculated. The data suggested that thyroid status was normal in the women taking estrogen and progestational compounds. How— ever, in subjects studied before and after 3 months of use of oral contraceptives, there was a tendency to a reduced uptake of radioiodine by the thyroid of the users of 2mg. Ovulen but not by users of Enovid or 1 mg. Ovulen (Pincus, 1965). 17 Effects of Oral Contraceptives on Metabolisms Oral contraceptives contain estrogens and proges- tational compounds and thus are likely to exhibit biolog- ical activities characteristic of natural ovarian hormones. In fact many of the metabolic actions of oral contracep- tives mimic the effects of estrogen administration. There have been many reports during the past few years which point out the estrogenic or progestational or combined effects of oral contraceptives on carbohydrate, fat, pro- tein, and mineral metabolisms. Various changes in metab— olisms may be brought about by alterations in the secre- tion and functional rates of endocrine glands. In addition they may also alter the binding power of plasma proteins and thus bring changes in metabolism. Since the estrogen and progesterone present in oral contraceptive pills are synthetic and have additional modification like 17- alkylation, these steroids may have some other deleterious effects which are not associated with natural estrogen and progesterone. l8 Carbohydrate Metabolism Oral contraceptives seem to produce alterations in glucose metabolism. An impairment of oral glucose toler— ance have been noted in a group of women receiving Enovid (Gershberg §£_a1,, 1964). Ten percent of 59 women receiving Enovid had elevated fasting levels and 20% had elevated one hour levels and 46% had elevated 2 hour levels. The inci- dence of abnormal glucose tolerance appeared to be greater in women with a family history of diabetes than those with-_ out. The observation of abnormal glucose tolerance, espe- cially with diabetic women, is in agreement with the work of Cochran and Pote (1963). They found fifteen of a group of 30 menOpausal women under Enovid therapy for 21 months, to have abnormal glucose tolerances. Wynn and Doar (1966) investigated a number of aspects of carbohydrate metabolism in women taking various oral contraceptives. They found abnormalities of oral and intravenous glucose tolerance, plasma nonesterified free fatty acid (N.E.F.A.) and blood pyruvate values. But the mean fasting—plasma-glucose was not significantly different from that of a control group. A striking metabolic abnormality was an increased fasting- blood—pyruvate level or an increased maximum pyruvate increment following glucose administration or both. 19 This occurred in about 20% of women in the test group. These abnormalities were similar to those found in steroid diabetes. The elevated plasma N.E.F.A.s, shown in the test group, might be a cause of impaired glucose assimilation and pyruvate oxidation (Randle, 33 31., 1963). The strik— ing elevation of blood pyruvate level might be due to an excess of glucocorticoid action, even though it has been reported by Plager §E_§l, (1964) that deSpite elevated levels of bound and unbound plasma cortisol in the estrogen- treated subject, the total amount of cortisol in the tis- sues may be equivalent to that found in the control subject since there may be a higher level of transcortin present outside the vascular compartment in the estrogen-treated subjects. Another study was undertaken by Spellacy and Carl- son (1966) to explore the area of carbohydrate metabolism in subjects receiving an oral contraceptive pill by measur— ing both plasma insulin and blood glucose before and after drug therapy. This was done in a fasting state and after an intravenous glucose stimulus. Twenty-five subjects were tested before and after one cycle of oral Enovid treatment. The results of this study showed both the glucose and 20 insulin levels were higher in the drug-treated group. These authors postulated that the rise in insulin level could be due to the prolonged stimulation of the pancreas by these oral contraceptive drugs. It has been suggested that the estrogen component of the contraceptive drugs is apparently responsible for the changes in carbohydrate metabolism, because treatment with a progestational agent alone does not alter glucose tolerance (Puchulu §E_§l,, 1967). A short-term study of the effect of sequential treatment with ethynylestradiol and megestrol acetate on glucose tolerance revealed that estrogen was more involved in producing abnormal glucose tolerance than progesterone (Pyarala 23 31., 1967). The results of this study indicated that glucose tolerance de- creased during the estrogen phase of sequential treatment and then was slightly impaired during the following phase of combined estrogen—progesterone treatment. Puchulu £3 a1. (1967) have suggested that ethynylestradiol has less effect on glucose tolerance than mestranol, which has been employed as an estrogen component in contraceptive drugs. Another study by Buchler and Warren (1966) suggests that the estro- gen effects are related to changes in delayed absorption 21 of glucose rather than any diabetogenic effect, since normal intravenous glucose tolerance was observed in pa— tients treated with diethylstilbestrol. Lipid Metabolism The involvement of gonadal hormones in fat meta— bolism has been a subject of study. It has been reported that when estrogen therapy, such as ethynylestradiol or dioxydiethylstilbestrol, is combined with orally active synthetic androgens, such as methyl testosterone, it caused a sharp increase in low density lipoproteins (L.D.L.). This was followed by a decrease in high density lipopro— teins (H.D. L.)(Russ gt al., 1955). It is possible that the chemical structure of progestin in oral contraceptives can be close to various synthetic androgens. Oral contra- ceptives can then be expected to cause changes in lipid metabolisms and tranSport. The work of Aurell §E_§1, (1966) proves this to a certain extent. They studied the effect of the oral contraceptive Anovlar containing 50lyg. of 17- ethynylestradiol and 4 mg. of norethisterone. Serum lipids and serum lipoprotein fractions were measured before ad- ministration and at regular intervals during one year. After a one year administration there was a significant 22 rise in serum-lipids, especially in serum low density lipo-l proteins. The low density lipoprotein reached levels typ- ical for the post-menopausal women. This effect seems in favor either of an androgen like activity of norethisterone or that the prolonged medication leads to an inhibition of normal ovarian increment of estrogen. Pincus (1965) studied the effect of Enovid which contains norethynodrel and mestranol. The study covered a whole year to rule out the seasonal influence on the serum lipid levels. In pre-menopausal women, no signifi? cant changes were found in either blood cholesterol or s— lip0protein levels following Enovid therapy. Elevation of serum triglyceride, cholesterol, and ‘ low density and very low density lipoprotein levels have been described in a group of 102 women receiving cyclical oral contraceptives (Wynn gghgl., 1966). Thirty-one pere cent of the women had fasting-serum—triglyceride levels above 131 mg. per 100 m1., which is the greatest value ob- served in a control group of 75 women. The authors attri—i bute the changes in serum lipid and serum lipoprotein patterns to the combined effect of the estrogen with a progestational steroid sharing the metabolic effect of a 17-aé—a1ky1ated androgen . 23 Andrews 33 a1. (1949), testing the effect of stil- bestrol and testosterone on growth and fattening of black face wether feeder lambs, showeduthat implants of stil- bestrol increase the rate of gain and feed efficiency. The lambs were divided into groups and were given five different treatments for a period of 68 days. The re— sults of their experiment showed that subcutaneous im- plantation of 12 or 24 mg. of stilbestrol or 10 mg. of testosterone significantly increased the gains of wether lambs during the 68—day feeding period. All hormone— treated groups required significantly less feed per pound of gain than the control lambs. However, no measurements of skeletal growth were made. Moreover, the data on ,carcass grades suggest that the lambs which received stilbestrol were characterized by somewhat less finish and more growth than the controls and that the lambs which received testosterone were fatter and less growthy than the stilbestrol groups. The study of Jordan et a1. (1956) revealed the combined effect of an estrogen and a progesterone. They treated lambs with the implants of various hormone combi— nations. Stilbestrol was implanted in one group. 24 Testosterone and estradiol combination and progesterone and estradiol combination were implanted in two different groups. The results of this trial showed that implants of stilbestrol and the combinations of testosterone and estradiol significantly increased the average daily gain. The combination of progesterone and estradiol increased the daily gain slightly. but the increase was not suffi- cient to be significant. The authors suggested that the progesterone inhibited the growth acceleration from the estradiol to a certain extent. The work of Day 32-31, (1960) is in agreement with the above mentioned work. They used forty-eight Poland China barrows in their exper— iment to study the influence of subcutaneous progesterone- estradiol (high and low levels) implants on average daily gain and carcass composition. The hormone implants had no significant effect on gain although the high level of progesterone-estradiol combination tended to reduce the growth rate. The most pronounced difference between the control group and the progesterone-estradiol implanted barrows was that the treated animals had a significantly decreased average back fat. Carcasses of the control and treated barrows were similar; however, those from the 25 barrows implanted with high level of progesterone and estradiol showed a trend toward increased leanness. Stimulation of body weight gain or increase in food utilization efficiency, which occurred with lambs, treated with estrogens, did not occur with rats. The experiment conducted by Meites (1949) with diethylstil— bestrol and by Sullivan and Smith (1957) with estradiol, showed growth depression in rats. However, in both ex- periments, the pair fed control groups paralled the growth of animals treated with estrogen. But the work of Yang §£_al, (1969) revealed that even though steroid treated animals and their pair fed controls consumed equal quan- tities of diet, the control rats had greater body weights. The effect of prolonged treatment with norethynodrel on body weight was found to depend on the age of the animals (Holmes and Mandl, 1962). The treatment reduced the growth of rats aged 55—66 days; and caused a slight reduction in the weight of the fully grown animals aged 148 days. Sim- ilar observations were found with animals of intermediate age, 89—102 days treated for a period of up to 86 days. The study of Bakker and Dightman (1966) showed that women taking norethynodrel did not reveal a signif- icant trend in the direction of weight gain or weight loss. 26 There was no evidence to support the hypothesis that nor- ethynodrel leads to weight gain due to increased deposition of fat. The variation among the different reports concern- ing the weight changes in women is quite wide, and so it is difficult to reach a general conclusion regarding the oral contraceptives and changes in body weight. Protein Metabolism Not much work has been done on the effects of oral contraceptives on protein metabolism and nitrogen reten- tion. Whitehair gt 31. (1953) treated lambs with 24 mg. of stilbestrol implanted in the neck region. Marked in- creases in calcium, phosphorous, and nitrogen retentions in the stilbestrol treated lambs were observed. Landau and Lugibihl (1963) said that protein catabolism was in- duced by progesterone which was associated with a decline in plasma amino nitrogen. In four studies in normal sub- jects, all amino acids measurable except phenylalanine participated in the lowering of the concentration of plasma amino acids. Fasting concentration of most of the amino acids were 12-34% lower than control values. Since when protein cataboliSm was accelerated by progesterone the amino acid levels drOp, enhancement of the utilization of 27 the amino acids and their conversion to urea by liver was the indicated mechanism. Moreover, the authors also men— tioned that urinary amino acid nitrogen was unaffected. However, the work of Adams (1966) indicated that 19—nor- progestin or l7—acetoxy progestin caused a gain of lean tissues and a loss of fat without gain in body weight and positive nitrogen, potassium, and sodium balances in women compared to premedication period. The changes in lean body mass and in the amount of protein retained were more uniform with the l7—acetoxy progestins than with the l9-nor progestins. This study was conducted for a short period of three weeks only. It should be pointed out that no genuine anabolic effect can be demonstrated with any of the preparations in current use. Fluid Retention Mahan (1962) treated 100 women with 5 mg. of Enovid. He reported that there were minor side effects in approxi— mately 10% of the cases. One of them was weight gain, as— sociated with this medication, due to fluid retention. Ringrose (1963) reported that out of 115 persons who were taking orthonovum, four noticed swelling of ankles in eight of the 31 cycles. In the study of Street (1960) with 10 28 persons taking 10 mg. Enovid, four complained of edema of the fingers and ankles during the middle to the last part of the menstrual periods. The above mentioned cases of edema were all judged by subjective means. Mineral Metabolism The hormones in the oral contraceptive pills have been reported to change the secretion rate of mineralo— corticoids from the adrenal. This change in secretion rate of mineralocorticoids would be expected to change the mi-_ neral metabolism of those who are taking these preparations. It was shown by Ehrlich §t_gl. (1960) that proges- terone inhibits the salt retaining effect of aldosterone. Therefore progesterone must be intimately involved in the hormonal control of renal sodium excretion during pregnancy. In contrast to the effect of progesterone described here, it was shown by Eugenia gt _1. (1961) that progesterone failed to elicit any influence on electrolyte excretion caused by deoxycorticosterone. According to Singer §E_§l, (1963) in intact male rats, and hypophysectomized‘rats fed a normal diet, the subcutaneous administration of progesterone for 4.5 days and 5.5 days respectively resulted in a significant increase in the aldosterone secretion rate. 29 This suggested the fact that the mechanism, by which this increase in aldosterone secretion was brought about, prob— ably did not involve the pituitary gland. The authors discussed the possibility that injected progesterone served as a precursor to aldosterone. Another possible mechanism suggested was that the progesterone was inhibiting the effect of endogenous aldosterone on the kidney, which would lead to an increase in the aldosterone production. This mechanism was also suggested by Laidlaw £2 31. (1962) who explained that the anti-aldosterone action of proges-I terone was overcome by hypersecretion of aldosterone and the sodium balance was restored in four men who received 50-200 mg. progesterone daily intramuscularly. The effect of estrogen may also account for the altered metabolism of aldosterone, both in pregnancy and in the treatment with oral contraceptives. While the mechanism of action of progesterone on sodium balance is obscure, many cases of hypertensions have been reported by Woods (1967) and Laragh §t_al, (1967) in patients who were taking oral contraceptive pills. They also reported marked improvement in most of the patients after the drug treatment was stopped. In the study of 30 Laragh gt a1, (1967) nine out of ten patients who were on the pill had very striking and sustained increases in the concentration of renin—substrate in the serum and there was an increase in renin activity in a few patients. How- ever, in none of the patients can the increased serum renin activity be attributed to a state of sodium depletion be— cause the range of urinary sodium excretion together with the absence of clinical edema provided evidence for normal sodium metabolism. The authors thus concluded that the administration of pharmacological doses of estrogen and progesterone required for a contraceptive action were found to produce a number of abnormalities in the renin— angiotensin-aldosterone system. Only a few balance studies on calcium and phOSpho— rous have been done. Womack §t_al, (1950) treated a four month old male infant with estrogen who was suffering from severe osteoporosis. The baby was given a weighed milk diet with vitamin supplements considered adequate to meet his fundamental nutritional requirements. This control period comprised of a total of 18 days. Balance studies were done by collecting urine and stool after the patient had been on the experimental diet. In addition one m1. of 31 Progynon B, an estrogenic substance, was given intramus— cularly for 30 days, while the diet and other conditions remaining unaltered. The result showed that the average daily calcium retention was 0.154 gm. for the control pe— riod and 0.153 gm. for the estrogen-treated period. Less phosphorous was retained during the estrogen treatment. The average daily retentions were 0.234 gm. and 0.142 gm. reSpectively for the control and estrogen periods. Roent— genograms revealed no change in the degree of osteoporosis. In another study Ackermann §£_al, (1954) determined the . nitrogen and calcium balances in six elderly women before and during estrogen therapy to reinduce menstruation. No effect was noted in nitrogen balance on any of these sub- jects, all of whom were initially in positive nitrogen balance. There was little effect on the calcium balance in subjects initially in positive balance, but some in- creased retention in subjects initially in negative bal- ance. Treatment with progesterone had less effect. The work of Adams (1966) showed an interesting fea- ture regarding phosphorous balance. Eight female subjects of 21-29 years were initially stabilized on estrogen with mestranol for fourteen days. Then the test progestins 32 norethynodrel and chlormadinone acetate were added to the mestranol and they were continued for another three weeks. The result showed a rather uniform negative phOSphorous balance which ensued when the patients were placed on pro— gestin therapy. There was no significant difference in the route of phosphorous loss from the body for any of the drugs used. Much less has been done to study the effect of hor- mones like estrogen and progesterone on other minerals like potassium, magnesium, chloride, etc. A study done by Sele lers (1951) with a small number of dairy cows, given large doses of diethylstilbestrol in late pregnancy and later followed over the parturition period, showed no appreciable or consistent changes in blood levels of sodium, potassium, magnesium, or chloride following either estrogen adminis- tration or parturition. Few papers have been published recently which are concerned with the effect of estrogen and oral contraceptives on c0pper metabolism. German §t_§l, (1961) worked with 11 patients with Wilson's disease to whom ethynylestradiol was administered. In four pa- tients the serum c0pper and ceruloplasmin concentrations and the urinary copper excretion increased. In six 33 patients there was an increase in the serum copper concen- tration, three of whom showed cupruresis. The levels of serum copper and ceruloplasmin and the urinary copper ex- cretion showed no change in one patient. One patient im— proved clinically, four were unchanged, and six deterior- ated during the estrogen administration. The increase in serum concentration of copper has also been demonstrated with the administration of the oral contraceptive pill, Enovid (O'Leary and Spellacy, 1968). Fourteen female subjects, more than six weeks after partue rition, were tested with 10 mg. Enovid daily for 21 days. Prior to treatment, the mean concentration of copper in the serum was 142 ug. per 100 ml. After one cycle of ad— ministration of oral contraceptive, the mean rose to 241 pg. per 100 ml. The change was statistically significant. The long—term effects of this alteration in serum copper content, especially in those people taking contraceptives for a long period, remain to be determined. Bone Metabolism Sex hormones have been shown to have an effect on bone growth. To a certain extent, they regulate the morph— ogenesis of the skeleton and may in part control the extent 34 of skeletal growth. It has been shown by Abdul-Karim _£_§l, (1968) that the estrogens are necessary for the orderly process of endochondrial ossification in the fetal rabbit. However, large amounts of estrogen inhibit the growth of cartilage and hence longitudinal osseous growth. It has been shown by Whitelaw gt_al, (1963, 1967) that estrogens hasten the bone maturation and epiphyseal closure in prepubertal girls. Breibart gt a1, (1963) have shown that progestational compounds also increase the bone age. of the infants when they are given to pregnant mothers. Gedalia §t_§l, (1964) conducted an experiment with 20—day old female rats. Estradiol benzoate (0.4 mg.) was injected subcutaneously twice a week and the rats were sacrificed after 2, 4, and 6 weeks. The results showed that the mean specific gravity and the breaking strength of the dry defatted femur was significantly higher in rats after two weeks of estrogen treatment as compared with control rats (p < 0.01). After six weeks, the differences between the experimental and control groups were no longer present. No significant increase in the mean calcium and fluoride contents of the femur ash of the estrogen-treated rats as compared with the control rats occurred. No 35 difference was detected between the mean phOSphorous con— tent of the femur ash of the estrogen-treated rats and controls. Parenteral administration of large doses of estro- gens to mice result in the deposition of new bone in the marrow cavities. This change is most pronounced in the long bones. Here the new bone is first laid down at both extremities on the trabeculae of the spongiosa and then becoming more compact, it extends along the longitudinal axis of the shaft toward the center of the marrow cavity. Barker and Crossley (1962) conducted research with mice. There were ten male and seven female pairs and each had its litter—mate control. All mice were kept under ident- ical conditions and fed a standard diet. Both mice in each pair were given weekly subcutaneous injections of 0.1 mgm. estradiol monobenzoate. Treatment was begun when the mice were between 21 and 28 days old and continued for periods ranging from 4 to 20 weeks. On completion of the course of the injections the pairs together with their controls were killed. X-rays were then taken of the femurs, which were then decalcified, cut into longitudinal sections, stained with haematoxylin and eosin and mounted. The 36 radiographs of the femurs of the mice treated with estra- diol showed an increase in the amount and density of the bone, first at the distal and later at the proximal epi- physis. This is the characteristic initial response of the mouse skeleton to estrogens. The amount of bone for- mation increased with the length of the course of the injections. The effect of an estrogen and progesterone combi- nation on bones of the adult and immature female rats has been shown by Yang §t_§l, (1969). They treated adult rats with 0.1911 and 0.0028 mg. of norethynodrel and mestranol respectively or 0.0967 mg. of norethynodrel and 0.0014 mg. of mestranol per kilogram of body weight per day. Both the levels were effective. Immature rats were treated with 0.128 mg. of norethynodrel and 0.0019 mg. of mestranol per kg. per day. The results of this experiment showed no significant difference in the lengths of the femurs of the adult rats when compared to their pair fed control rats. However, the lengths of the femurs of the immature rats were significantly shorter when compared to their pair fed immature controls. Cross sectional growths of the tibias were significantly different between control and 37 experimental group of the adult rats, 100 microns and 83 microns respectively. With immature rats there was no difference in the cross section of the tibias betweenthe control and experimental groups. Tetracycline labeling was used to measure the cross section of the bones. MATERIALS AND METHODS Animals One hundred and fifty female Sprague-Dawley rats, 3 weeks old, were obtained from a local supplier. They were fed a grain ration until 11 weeks old. The composi- tion of this diet is shown in Appendix I (Campbell gt 31., 1966). At that time they were divided into groups. The. first group comprised of 20 rats out of which 10 served as control rats were continued on the same grain ration. The remaining 10 were fed the grain ration plus the con- traceptive steroids. The average weight of these rats was 257 gms. at the start of the experiment. These 20 rats served as representatives of the entire 150 rats. Food intake, body weight, and digestibility studies were conducted with these rats. The second group consisted of 80 rats, 40 each of control and experimental. These rats were used for the determination of body compositional changes during steroid therapy. Finally the third group of 22 control and 28 experimental rats was used to study 38 39 the long—term effect of the steroid therapy. All rats were housed in individual suspended wire cages and were main- tained in an air—conditioned room at a constant temperature of 27°C and with 12 hours each of light and darkness. The rats of the long—term study were kept at the Veterinary Barn in contrast to all the rest that were kept at the animal room in the Home Economics building. Contraceptive Steroids The contraceptive steroids used in this experiment were norethynodrel and mestranol, the progestin and estro- gen of the first oral contraceptive pill Enovid. The do— sage used to feed the rats was equivalent to that used by women (0.1 mg. norethynodrel and 0.0015 mg. mestranol per Kilogram per day). The steroids were fed to the rats by first dissolving in 70% ethyl alcohol and then mixing thoroughly with the grain ration by means of a food mixer. Preparation and Feeding of Diet The concentration of the steroids in the diet varied slightly and was adjusted according to the changes 40 in body weights and food intake of the 10 rats which repre— sented the whole experimental group. The experimental diet was adjusted and prepared every week. However, eight weeks after the beginning of the eXperiment, when the weights of the rats and their food-intakes were stabilized, the diet was prepared once in two weeks. Both the experimental and control diets were fed on an ad libitum basis. Water was also given free choice. Digestibility and Balance Studies Digestibility and balance studies were done by collecting urine and feces for a known period of time. Two collections were done. The first collection was done 22 days after feeding the experimental diet with the 20 representative rats. The second collection was started after 24 weeks and 5 days of steroid therapy. For this collection 20 rats of the long-term study, which were housed at the Veterinary Barn, were used since the 20 representative rats, originally used for the first collec— tion, came down with reSpiratory infection. The collection periods were 4 days in both the trials. 41 Feces were dried in a force-air oven at 90°C and then ground in a Wiley-Mill to a fine powder. These pow- dered feces were analyzed for nitrogen, fat, sodium, and potassium. After removing the food spilled in the urine by filtering, urine samples were made into a known volume by adding deionized water. Aliquots were taken and anal— yzed for nitrogen, sodium, and potassium. The grain ra- tion, portions of which were saved during the collection period, was also analyzed for nitrogen, fat, sodium, and potassium. The quantities of nitrogen, sodium, and potassium consumed and the total excreted in the feces and the urine were used to calculate the balances and digestibilities. With fat, only digestibility was determined. Percent di— gestibilities and retentions of various nutrients were calculated using the formulas shown in Appendix II. Body Composition After treating the rats with steroids, they were sacrificed at four different intervals for body composi- tion analysis. 42 First Group Ten steroid-treated and 10 control rats were killed after they were fed for four weeks. This was immediately after the first urine and feces collection. Second Group This group consisted of 10 treated rats, 10 control rats. They were sacrificed 10 weeks after the start of the experiment. The experimental rats were on steroid treat— ment for the first four weeks and were fed just the control grain ration for the rest of the six weeks. This was to see whether there is any reversibility in the body composi- tional changes after the steroids were removed from the diet. Third Group Eight steroid-treated and eight control rats were killed after the experimental ones were fed with steroid- mixed diet for 25 weeks and 3 days continuously. This sacrifice was made one day after the second collection. Fourth Group The treated rats were on the steroid diet for 25 weeks and 5 days and then they were placed on control diet 43 for six more weeks. After this they and 7 control rats were killed. This was 31 weeks and 5 days after the ini— tiation of the experiment. All the above mentioned rats were picked at random. The first two groups of rats were killed by a blow on their heads and the next two groups were killed by over etheriza- tion. Life Span Study For this study, the animals were kept in the Veter- inary Barn and were weighed only 3 times to avoid disturb- ing them unduly. The life span of each one of the treated and control rats was noted when it died. Tumor growths and other incidences were also recorded. Preparation of Rats for Analysis The gastro-intestinal tracts of the rats were re— moved and their contents washed off. The carcasses and the G.I. tracts were placed in tared jars. The jars con- taining the entire carcasses were then autoclaved at 15 44 lbs. pressure for 20 minutes. The rats were then homogen- ized in a Waring blender with water equaling approximately to the body weights of the rats. This homogenate was found to be too soft in consistency. It was discovered by trial and error that 100 ml. for rats that weighed 200-300 gms. and 150 ml. for the rats that weighed above 300 gms. worked best. Analysis of Various Components Moisture Aliquots of carcasses were analyzed for moisture by the method described by Mickelsen and Anderson (1959). Three aliquots were analyzed for each carcass sample. Nitrogen Nitrogen in the feces, urine, and the carcasses was determined by Kjeldahl method. Carcass homogenate was weighed in a piece of wax paper and together drOpped in a Kjeldahl flask since the consistency of the homogenate was quite thick. Nitrogen was converted into protein by applying a factor of 6.25. 45 Fat Pat in feces, grain ration, and carcasses were analyzed by ether extraction in a goldfisch extractor. Three dried aliquots were analyzed for each carcass sample. §gdium and Potassium Carcass, feces, and grain ration‘were ashed for the determination of minerals. Urine was used as such. Sodium and potassiumwwere determined by atomic absorption and flame emission spectroeCOpic methods respectively. For these determinations, the feces and diet samples were weighed (0.15-0.35 gm.) and ashed in a muffle furnace at 475°C for approximately eight hours.' Wet carcass samples were weighed (4-7 gms.) in Vicor crucibles and dried in the force-air oven at 90°C overnight, and then ashed in a muffle furnace for approximately 10 hours. ‘The ashed samples were dissolved in 1 ml. of 50%.HC1 and made into known volumes. Appropriate dilutions of these original ash solutions were taken in duplicates and analyzed for sodium and potassium.by comparing with.known standard; solutions of sodium and potassium. Urine samples were only diluted with deionized water and the minerals were determined as the ash solutions. 46 Statistical Analysis All data were analyzed by analysis of variance (Dixon and Massey, 1957). RESULT S AND DISCUSS ION Food Consumppion and Body Weight Gain Food consumption and body weight gains were signif— icantly lower for the steroid—treated rats, Figure 2. The average food consumption and body weights of the steroid- treated and control rats for 26 weeks are presented in Table 1 (Appendix). The results of the present studies are in agreement with the works of Meites (1949) and Sulli- van and Smith (1957) who showed that estrogen treatment caused a reduction in food intake and body weight gain in rats. Depression in body growth and appetite has also been noted by Husain and Pincus (1965) who treated rats with the same steroids used in the present experiment. They fed female rats Enovid and noted that the treated rats had decreased food intake and body weight gains throughout a six-month period. Water consumption was also lower for the treated rats near the end of the treatment. In the present experiments food intake during the first 19 weeks was measured with the 20 representative 47 48 01 C 00.. ’I.‘~.‘ 3eo~ ........ a I" A ’O.s. I 3&30 .. I..- l': ...... ‘\\ A '.,I’ /°’.\, ”/9 ’0’ ’0 l./' /—“\'\ o A/ o ./a . “...." 260 j ./ . / cm . .01- 0 -— ~'/ ‘ . r~:‘: the-'7 I- ql‘sv ' T'st-:'.‘ I.--) . - A Cu} 1 F101. RA: :3 LINE SPAN " o I R 5.53? ED RAT S STU DY ZOOF O C ..... x a.“ . I.‘ ..--~-‘d I \'?.\ \ ' e .0 I - I ‘00 " / ‘\./ "" \ "'37 \WJ-e. ... .... \e‘. fig/l. v \ ..... v\ ' \ o \ I ‘ I \I L__._.... L... J "6 l0 l2 1473 1:3 20 22 24 2c; 6 W 35 HE’- 031 E33358}; iEPJT WElGHT ‘IN GMS L_.- -.3 ..-—1... l. 4......— L- Fig. 2.--Body weights (upper curves) and weekly food consumption (lower curves) of the control and treated rats. ‘K 49 rats. After this period it was not possible to continue the food intake measurement without the addition of a few more control and treated rats. The addition came from the group which was to be used for body composition an- alysis. This was necessary because some of the rats died from respiratory infections. Thus, from the 20 to 26 weeks of the experiment, the measurement was continued with rats from the representative group as well as those from the groups originally planned for the determination of body compositional changes. For about 7 weeks the control rats continued on an average of 2-2.5 gms. more food per day than the treated rats, but consumption was the same for both groups from the 7th to 9th weeks. This was the time when the infection occurred. After this time, the control rats began to consume a little more food but again the food consumption ofboth groups went down far below normal until the 15th week. During the 12th and 13th weeks, the average food intake of the treated rats was only 8.6 gms. per day and during the 15th week the control rats' average food consumption was 8 gms. per day. From the 16th week onwards the food consumption of the control rats grad- ually went up and remained between 15 and 17 gms. while 50 the consumption of the treated rats remained at 13—15 gms. throughout the experiment. From the growth curves it is obvious that the growth rates of the treated rats were always below the growth rates of the control rats. The treated rats also lost weight during the first week of the treatment itself. It took another week for them to regain the lost weight and then they stayed in the same average weight for one more week; after that they were gaining weight at a slower rate than the control rats. Food consumption and body weight went down for both groups when they became sick at the 7th week. It should be pointed out that the body weights went down very drastically for both groups when they consumed only an average of 8 gms. of food per day. The treated rats were consuming an average of 8.6 gms. of food per day during the 12th week, which is far below the average intake of 13 to 15 gms. per day. For that par- ticular week, the average body weight went down from 276 gms. to 242 gms. The control rats consumed about 9.5 gms. of food per day during the 14th and 15th weeks, at which period they lost weight from 316 gms. to 292 gms. The treated rats lost more weight; however, they recovered 51 faster. Though the sickness came into picture for a while, it did not change the usual pattern of food consumption and body weight gain between the control and treated rats, since they continued to exist throughout the experimental period. In order to show that the growths of the two groups were not appreciably changed subsequent to the in- fection and that they were similar to those of rats not affected by the infection, the body weights of the life- Span—study rats have also been included in Figure 2. How— ever, these rats have been weighed only three times: at. the beginning, at 14 weeks, and at 25 weeks of steroid treatment. It has been shown by Meites (1949) that the growth— inhibiting effect of estrogen was due mainly to its ability to depress appetite. Sullivan and Smith (1957) also proved that the restriction of food intake in the control rats equal the quantity consumed by the treated animals dupli- cated the effects of estrogen on depression of body weight. However, Glasser (1954) showed that the daily injection of 0.1 mg. of stilbestrol for 21 days to adult male rats re— ceiving a diet containing 18% casein resulted in a marked loss of body weight which exceeded that of the pair fed 52 controls. The author claimed that the difference was suf- ficient to suggest some direct effect of the hormone on body weight. Since the rats of the present experiment were fed on an ad libitum basis, the exact effect of estrogen and progesterone could not be determined. The mechanism by which these hormones depress body weight, apart from the restriction of food intake, has never been clarified. It has been suggested that the estrogen may reduce the secretion of pituitary growth hormone (Richards and Kueter, 1941) and thyrotropic hormone (Meites and Turner, 1948) or increase in adrenocorticotropin (Baker, 1949), any one of which would depress growth. Another hypothesis sug- gested by Meites and Turner (1948) was that deficiency of B vitamins might be created by the administration of arti- ficial estrogen like hexestrol, which would result in growth depression. Digestibility and Nutrient Balance The digestibilities of protein, fat, sodium and potassium were not significantly different between the steroid—treated and control rats either after 22 or nearly 53 173 days after feeding the steroids. Table 1 shows the percent digestibilities of nitrogen and fat for the first collection. During this period the percent digestibility of nitrogen was not significantly different between the treated and control rats; however, the control rats re— tained significantly (P < 0.05), more dietary nitrogen in their body than the treated rats, Table 2. This was due to a lesser quantity of urinary nitrogen expressed as a percent of what was absorbed. Until the first collection period, the food intake of the treated rats was 2-2.5 gms. less per day as compared to the control rats. The de- creased food intake might have resulted in increased di- gestion and absorption of nitrogen. However, in spite of the fact the treated rats ate less food and digested it efficiently, they excreted as much urinary nitrogen as did the control rats. Urinary nitrogens excreted in the four day period for the treated and control rats were 1.53 and 1.57 gms. respectively. The difference was in the amount of food consumed. The voluntary restriction of food in- take without conserving body nitrogen of the treated ani- mals may be one possible reason for the decreased nitro— gen retention of the treated animals. 54 Table l Nitrogen and Fat Digestibilities after 22 Days of Steroid Treatment % Digestibility of % Digestibility of Nitrogen Fat Treated Control* Treated Control 82.2 77.5 85.8 87.7 82 l 82 3 96 4 83 2 81.3 81.1 78.3 88.9 81.4 80.8 88.5 88.1 83.8 83.2 76.8 87.1 82.1 79.8 85.1 81.3 80.8 82.8 83.1 80.0 82.0 Mean 82.4 81.0 85.2 87.0 S.D. 1 3 1.8 8 0 2.2 *Only 9 rats are included in control group for nitrogen digestibility since one rat was sick at the time of col- lection. .umu mom ammo o How mum mmoam>+ .COHuomHHoo map mCH IHSU MUHm mm3 OH mo poo H dozen .msonm Houucoo mflu How UoUDHUCA mum mums m >Hc0ee .mumu Umummwu onu auHBLXN.m mo pony cone mHoE Amo.o V mv haucmoHMHcmfim we tossed soap IomHHoo mmmo snow was mcflnso moms Homecou QuHB cofiucmumu cmmonuflc Xm.HH some mafia 55 ¢.¢ om.o ¢H.o ¢.N om.o mo.o .Q.m m.HH hm.a mm.o N.m mm.H om.o cow: m.oa h.H mv.o H.0H n.H mo.o o.m o.m no.0 osma m.H ov.o h.m m.H mm.o m.HH m.H mm.o b.0H m.o mH.o m.> ©.H vo.o m.h n.H H¢.o m.ha o.a Ho.o ©.m w.H mm.o v.0 o.H Ho.o m.m m.H mm.o m.¢a m.H m¢.o m.OH N.H om.o o.mH m.H om.o H.v n.H o¢.o o.m N.H mm.o m.m m.H om.o “.5 79:3 79:3 ex; 79:3 79:3 acoflucmumm comonufiz comouuflz «coflucmumm cmmouuflz cmmouufiz cmmouufiz mumcflnb Hmumm cmmouufiz wannauo Hmoom ¥¥HOH¥COU UmummHB +ucwEummuB peonwum mo when NN Houmm mumm CH cmmOMUHzlmHmumHQ mo coeucmumm ucmonwm N magma 56 For the second collection also, there were no significant differences in the digestibilities of nitro— gen between the treated and control groups, Table 3. How— ever the nitrogen balance study during the second period revealed that the treated rats retained significantly (P < 0.01) more dietary nitrogen than the control rats, Table 4. The average quantity of urinary nitrogen of the control rats was not much altered in the second time as compared to the first time. They were 1.502 and 1.57 gms. reSpectively. But the total urinary nitrogen and the urinary nitrogen as a percentage of what was absorbed were decreased with treated rats in the second collection period. The average total urinary nitrogen was 1.35 gm. during the second collection compared to 1.53 gm. average during the first time. There was not much difference in the food consumption between the two groups during the second col- lection period. The averages were 15.0 gms. per day for the controls and 14.5 gms. per day for the treated rats. The rats used in the second collection were placed in metabolism cages three days before the collection was started. For this reason, both the treated and control rats lost an average of 14 gms. of body weight. However, 57 Table 3 Nitrogen and Fat Digestibilities after 24 Weeks and 5 Days of Steroid Treatment % Digestibility of %.Digestibi1ity of Nitrogen Fat Treated Control Treated Control 79.6 82.5 84.3 82.8 82.0 82.1 77.7 70.3 82.1 80.1 88.0 75.2 82.2 81.1 86.1 85.1 83.0 84.0 63.2 79.0 84.5 83.1 84.1 81.8 86.6 82.5 86.9 82.6 85.7 82.6 Mean 83.7 82.2 80.0 78.5 S.D. 2.3 1.0 10.0 6.0 58 .muuu Howucoo on» mo cofiucouou Km.h may cosy AHo.o v mv nmflmflfl >HDCMUAMHcmHm me upon omumouu may AuHB coflucmumn comouuflc Xma coma mseee o.m no.0 go.o m.¢ Ha. no.0 .Q.m m.n m.H om.o o.mH mm.H mm.o one: ©.m m.H mm.o m.ma ~.H hm.o ~.oa m.H nm.o o.mH m.H mm.o m.HH >.H No.0 5.0H m.H mm.o m.oH m.H em.o H.m ~.H m~.o m.m m.H mm.o m.mH ¢.H om.o H.n m.H Hm.o H.HH m.H om.o H.ma m.H m¢.o m.¢a m.H o¢.o m.m| o.a mm.o ¢.ha m.H mm.o v.m m.H hm.o m.mH ~.H om.o H.v m.H om.o m.hH v.H mv.o A3 79:3 723 A3 79:3 79:3 COHucouom ammonuflz comonuflz coflucmuom cmmowuflz ammonuflz eecmmouuflz mumcflnb Hmomm eecmmouuflz hnmcflnb Hmomm Houucoo omummua ucmEumoue ofloumum mo when m ocm wxmmz om umumm comonuflz Hmomm pom MHMCHHD mo when Hoom com cmmouuflz mumumfln mo mama Hoom mo mammm exp co owumHoonu mumm cH coflucmumm cmmouufiz ucmouom a magma 59 when the collection was started they were beginning to gain weight. .During the 4 days of the first collection period the average weight gain of the control rats was 5 gms. and that of the treated ones was 3.4 gms. But during the second collection the average weight gain of the treated rats for 4 days was 6.2 gms. versus 3.8 gms. for the con- trol rats. This slow gain in body weight and decreased food consumption of the control rats at the second collec- tion revealed that the rats of the 2 groups might have re- acted differntially to the new environment. It could also be true that the treated rats had adapted to the steroid in the long range and thus begun to conserve more nitrogen in order to compensate for the loss at the beginning of the steroid treatment. It has been pointed out by Leathem (1956) that more urinary nitrogen is excreted by the ani- mal with well-filled protein stores than by the depleted animals. Even though the treated rats were not depleted, they had less protein in their body as compared to the control ones according to analysis of body composition at this time. This is another possible explanation for the increased nitrogen—retention of the treated rats. 6O Feed efficiency was calculated by dividing the average body weight gain per rat per day in grams by the average food consumption per day in grams, Table 2 (Ap- pendix). During the first collection it was 0.05 gm. weight gain per gram of food per day for the treated rats and 0.1 gm. of weight gain for the control rats. During the second collection they were respectively .12 gm. and 0.06 gm. Percent digestibilities of fat for the first and second collections were not significantly different be— tween the treated and control groups, Tables 1 and 3. During the first collection the average percent of fat digestibilities were 85 and 87 respectively with the treated and control rats. At the time of the second col— lection these values were 80%.and 79% respectively. Percent digestibilities of sodium and potassium for the treated and control rats at both collections were not significantly different between the treated and con— trol rats, Table 5. The percent retention of dietary so— dium was significantly higher (P < 0.05) with the treated rats than with the control rats during the first collec- tion period; however, there was no difference in potassium 61 Table 5 Percent Digestibilities of Sodium and Potassium During the Two Collection Periods At 22 Days of Steroid Treatment At 24 Weeks and 5 Days of Steroid Treatment Treated Control Treated Control % Digestibility of Sodium 96.5 89.8 96.8 98.1 93.6 92.0 82.4 97.1 94.8 94.4 97.1 98.5 96.9 95.1 97.1 92.0 98.1 96.2 97.0 98.7 97.4 98.1 98.5 97.7 95.4 97 2 99.5 93.6 97.3 96.5 99.1 95.6 97.7 95.5 92.8 97.4 94.1 94.8 99.0 93.4 Mean 96.2 95.0 95.9 96.2 S.D. 11.6 2.5 5.1 2.4 % Digestibility of Potassium 95.1 82.7 96.6 97.0 91.3 89.8 87.2 96.0 86.8 90.9 97.5 97.4 94.3 92.1 96.8 94.1 97.0 94.8 94.5 96.9 95.7 95.6 96.6 96.0 94.4 94.0 99.0 92.6 94.8 92.1 98.3 95.4 95.5 95.0 87.4 95.8 89.4 92.4 97.4 89.4 Mean 93.4 91.9 95.1 95.0 S.D. 3.2 3.7 4.3 2.5 62 retention between the two groups, Table 6. The percent retentions of dietary sodium and potassium, during the second collection were not significantly different be- tween the two groups, Table 6. It seems that the steroids may have their effect on sodium retention during the ini- tial stages of treatment. Later on, the rats may become adapted to the steroids and they exert no more effect on sodium excretion. The steroids do not seem to have any significant effect on potassium retentions. Body Composition The present study on body composition of rats re- vealed some interesting changes that occurred in various components such as water, dry body weight, fat, and nitro— gen. First Gropp During steroid feeding, the treated rats did not gain as much weight as the control rats (Table 7). The average weight gain of the treated rats during the 4 weeks 'was 12 gms. while the control rats gained an average of 32 gms. during that period. Analysis of the carcasses 63 Table 6 Percent Retentions of Sodium and Potassium During Two Collection Periods % Rentention of Sodium %.Retention of Potassium Treated* Control* Treated Control At 22 Days of Steroid Treatment 24.1 17.1 29.5 16.1 28.9 26.8 17.1 24.6 19.9 30.9 22.5 24.7 28.3 26.2 26.7 20.2 39.4 23.9 26.5 19.4 32.7 27.5 21.5 33.7 42.5 17.2 43.5 26.7 35.6 26.9 21.8 24.3 41.5 31.2 26.8 31.0 27.9 23.2 21.5 21.1 Mean 32.1 25.1 25.7 24.2 S.D. 7.6 4.9 7.2 5.3 At 24 Weeks and 5 Days of Steroid Treatment —45.6 56.3 39.1 31.0 3.0 18.7 27.1 16.0 -ll.0 30.9 31.8 27.9 45.5 -10.1 31.8 33.8 62.2 59.0 27.5 34.4 59.4 54.3 39.1 31.9 83.2 37.2 27.8 32.0 60.2 35.1 33.1 30.7 67.6 54.8 34.1 32.8 81.7 62.2 38.1 26.8 Mean 40.6 39.8 32.9 29.7 S.D. 43.4 22.7 4.7 5.4 *The mean 32.1%.sodium retention of the treated rats is sig- nificantly higher (P < 0.05) than the mean of 25.1% sodium retention with control rats. 64 Tafle7 Body Weights of Rats Killed at Different Lengths of Time of Feeding Steroids (first and third parts of table) and after Refeeding of (Values are in gms.) Control Diet for 6 Weeks (second and fourth parts of table) Treated Control Treated Control Treated Control Treated Control Start 261 263 Start 239 254 Start 260 254 Start 252 255 4 Weeks 1 wk 2 wks 3 wks 4 wks 252 258 261 273 272 283 292 295 10 Weeks 5 wks 6 wks 7 wks 8 wks 10 wks 258 260 258 269 275 287 291 290 302 303 25 Weeks + 3 Days 24 Weeks 25 Weeks 296 299 335 331 31 Weeks + 5 Days EEEEEEEBE 266 276 279 287 292 296 308 307 310 305 312 317 320 331 339 343 344 353 65 revealed that the control rats had significantly more dry body weight (P < 0.05) when compared to the treated rats, Table 8. The treated rats had significantly more (Pr< 0.05) water expressed as %.moisture of the body than the control rats, Table 8. Even though the treated rats weighed less, they had almost equal quantity of water as the control rats at the end of 4 weeks of steroid treatment. It would be appropriate to mention here that the sodium balance study done by collecting urine and feces after 22 days of steroid treatment revealed that the treated rats retained signifi— cantly more sodium (P < 0.05) than the control rats. This result is in accord with the phenomenon of an increased water retention by the treated rats during this particular time. The increased water retention by the treated rats at the initial stages of steroid treatment was not signif- icant after 25 weeks of therapy. Complaints about "fluid retention" in women receiving contraceptive steroids may be true during the initial periods but for later periods, the occurrence of fluid retention may be doubtful. Since there are reports (Adams, 1966) that indicate a gain in the lean body mass in women during the treatment with contraceptive steroids, lean body masses were 66 m.m m.ma o.ma m.va n.m m.m .o.m o.>w m.>mm o.HoH m.moa v.Hh H.mo one: m.o> m.mmm m.©OH m.m©H m.Ob N.Ho on v.on m.mmm m.mma m.HmH v.05 h.mm mo o.mo H.0Hm m.mm H.mva m.o> o.mw mo m.mm N.Hmm v.mm «.mma m.oe m.mo .no m.Hm h.oom m.mw v.mea v.0n m.mm we v.0h o.oom o.moa m.onH w.oe m.mo mo h.m> n.0mm n.0HH m.mmH m.ae v.00 om o.mo m.oam h.am m.ama H.oe e.Hw mo H.mo m.on o.mm v.aea H.m> n.mm mo o.ao m.nom m.vOH m.ova m.on v.mm Ho mumm twosome 793 79:3 793393 72333: $392: $768 .02 mmmz woom mum: zoom who no .ufim mo usooad hoom coma waosz ca smog who smog um: ouoHOmnd opoaomnd ca ououmfloz owoumfloz pom possumoua ofloumum mo mxmwz Moon Hmumm momma: atom coon can named: mpom who mo mu2508< musHomnd pom momma: woom coma pom oaonz may as ousumfloz m magma 67 .mumn powwow“ ca .mem o.aoa sme ecu soap Amo.o v my Hoover >HDGMOHMHcmHm we mums Homecoo cH uzmflo3 who ousHOQO .mEm H.NHH some o£B«« .muow Homecoo CH who» ImHOE wm.mm some one coco Motown Amo.o v mv maucmoflmwcmfim ma mums ooummuu GH wusumflofi wa.mm some 0:84 0.0 H.Nh v.m© m.wh m.m® h.vh o.¢© H.mo ©.vh 0.0m b.mw m.mH m.mmm ©.m¢m m.mmm m.HmN o.vmm m.th m.NNN v.0NN m.©mm m.©mH 0.0mm 0.0H H.NHH m.®HH m.HNH m.HHH O.MHH w.NNH w.woa ©.HOH v.NNH ©.Hm H.0HH b.HH o.¢©a ©.HBH h.¢®H o.vha o.moa m.NmH m.mmH m.m©H m.mmH m.mma m.H©H mumm Houucou v.0h n.0h ¢.m© m.o> O.Hh N.Hh N.Hh m.on m.o> N.Oh m.mm m.hm o.H© m.mm m.mm m.mm ®.H® m.mm 0.0m m.mm IQOm com: om mm me he mm mm on Mb Nb an .pcoonn.m manna 68 determined for both control and treated rats, Table 8. There were no significant differences between these two groups in the absolute amount of wet and dry lean body masses after 4 weeks of steroid treatment. The average wet lean body mass of control rats was 235.5 gms. whereas that of the treated rats was 227.3 gms. The average total amount of dry lean body masses were 69 and 67 gms. respec- tively for the control and treated rats. The percent mois- ture in the lean body mass was also not significantly dif- ferent between the treated and control groups. The aver- ages were 71.4% and 70.7% for the treated and controls respectively, Table 8. Percent nitrogen in wet and dry lean body masses was not significantly different between the two groups, Table 9. Absolute amounts of nitrogen and protein were also not significantly different between treated and con- trol rats after 4 weeks of steroid treatment. The control rats had an average of 0.6 gm. nitrogen and an average of 4 gms. protein more than the treated rats; however, they were not statistically significant. Body fat as a percent of either wet or dry body weight was significantly more (P < 0.05) for the control 69 m.o v.m e.H m.v oe.o m.o H.o .o.m o.mv m.mm m.ma m.am ma.m a.~a e.m cams :.ae m.mm m.ma m.mm m.m m.ma o.m om «.mm m.ma e.wa :.vm m.m m.~H m.m me m.am m.am ~.NH m.oo e.m :.NH m.m we e.ae m.mm o.ma m.mm m.m m.ma :.m :e H.ma m.mm e.ma m.mm a.m m.~a o.m we m.vv N.Hv p.04 :.Hm m.m m.~H :.m m: «.mm m.am a.ma v.mm m.m m.~a e.m a: m.:v o.mm >.ma m.mm o.m m.~a e.m m: o.mm m.mm e.ma «.mv m.a m.~a :.m we a.Hv 6.5m ~.ma m.mm m.m m.ma o.m an mumm Houucoo k.m m.a m.m m.m om.o H.H m.o .o.m a.am m.mm o.mH o.am 6H.m «.ma o.m cams m.om o.am «.ma H.oa a.m H.m a.m on m.oa m.km «.ma m.mm o.m m.NH e.m mo m.em o.0m m.aa o.ma m.» m.~a e.m mm m.em m.mm H.Ha o.Hm ~.m a.ma e.m :6 m.o~ a.mm m.m o.ma m.: «.ma o.m so 6.Hm 0.4m 6.HH m.mm m.m «.ma o.m mo o.oa a.mm m.ma m.em m.m m.ma o.m as :.m~ m.am 5.4H :.Hm m.m H.ma m.m mo m.mm m.mm «.ma o.mm m.m m.~a m.m mm m.ma m.aa m.:a «.ma m.: o.ma m.m Hm mumm ooumoua ”.mempa awe Lav a.m:mv x.msmv mmmzawwom mmmzawwom .oz musHOmna «.ummamz seem .namamz soom ammuoum mo ammouuae mo ammo mun ammo um: am: one a :a use “we :a one pea mam use was :a ammouuaz :H cmmoupaz ucofiumoue oflouopm mo mxooz v “mums mumm ca new one comouuflz mo mucooad muoaomnd pom unmouom mmfifia 70 . .mumn topmowu ca mew om mo oon> some mop coco Amo.o v mv seamen m: mums Homecoo ca pom m0 .m5@ me some oceeee . .mpmn omumouu cH mm mm mo moam> some one can» Amo.o v my Hmsmflz we mums Houucoo CH wm.mm mo moam> some mseea . .Amo.o v my mums women one mo wo ma moam> news ecu coco Hermes wauchAMHcmHm m: msoum Honucoo ecu mo wm.ma moam> ewes mow: 71 rats than for the treated ones, Table 9. Absolute amount of fat was also significantly more (P < 0.05) in control rats than in treated rats. There were no significant dif- ferences in the amount of body fat per gram of body ni- trogen between the treated and control groups, Table 3 (Appendix). However, when the amount of fat per gram of food eaten was calculated, the treated rats were less efficient.- The marked loss of body weight and decreased body weight gain that occurred in estrogen treated rats (Meites, 1949; Sullivan and Smith, 1957; and Glasser, 1954), and in estrogen and progesterone treated rats (Husain and Pincus, 1965) are in agreement with the pres- ent work. Furthermore, the voluntary restriction of food intake may be another reason for the decreased amount of body fat in the treated rats. The control rats consumed more food and thus could have converted "extra".calories into body fat. It has been shown by many investigators that women who were treated with contraceptive steroids had decreased glucose tolerance (Gershberg §£_§1,, 1964; Pydrald, gp_al,, 1967; and Wynn and Doar, 1966). It has also been shown by Wynn and Doar that the plasma nonest— erified fatty acid was elevated in patients taking oral 72 contraceptive pills. This increased release of fatty acids has been suggested to be because of impaired glucose util— ization (Randle pp 31., 1963) and, thus, may also be a cause for the decreased amount of body fat observed in the treated rats. This work and the work of Bakker and Dightman (1966) with women using norethynodrel do not support the hypothesis that these steroids lead to weight gains due to increased deposition of fat despite the complaints of some women. However, according to Pincus (1966) exceptional weight gain has been attributed to some but not all oral contraceptives. The percentage of women who were losing weight was more than the percentage of women who gained weight, taking the oral contraceptive pills Enovid and Ovulen. Analyses of carcasses of treated and control rats for sodium and potassium after 4 weeks of steroid treatment revealed that there were no significant differences in the amount of sodium and potassium in the body between the treated and control group rats. The absolute amounts of sodium and potassium in the whole body of rats and the amounts present per 100 gms. of wet and dry lean body 73 masses are presented in Table 10. The treated rats had an average of 288 mgm. of sodium and the control 303 mgm. These values were not significantly different. Since there were no significant differences in the amount of wet and dry lean body masses between the two groups of rats, this sort of result is expected for sodium. The average abso- lute amounts of potassium were 850 mgm. and 864 mgm. for treated and control respectively. They were not signifi- cantly different from each other. Sodium and potassium per 100 gms. of wet and dry lean body masses were also not significantly different for the treated and control groups. Since from the time the experiment was started the treated rats were eating less food per day than the control rats, but have the same amount of sodium in their bodies, the treated rats must have significantly retained more of the dietary sodium than did the control rats. This was re- vealed during the first collection period. Second Group The body composition of the second group of rats which were fed the control diet for 6 weeks after 4 weeks of steroid treatment revealed that the sickness which has already been mentioned came into picture when these rats 74 n.mm ¢.mm v.mh o.ma o.o h.mm .Q.m m.mmma m.hom o.vwm m.mmv o.mma o.mom coo: m.mmHH o.mvm H.mmm o.mmv N.mNH v.NHm om H.0mva «.mav mqmnm m.amv m.mma m.mHm on H.vmaa o.mom h.ham w.mov N.mNH m.vam we m.moaa w.mmm v.5mh h.mmv m.mma o.oam he m.mvma H.Nom m.mmm m.mmv N.hma m.hmm on N.mmmH «.mnm o.amm o.vmv o.NNH v.Hhm me o.moma m.bem h.mmm m.mmv H.mma 0.5mm on >.ooma m.omm v.0vm w.amv v.ama 5.0mm me m.omma m.hmm v.am> o.mm¢ m.mma m.Hmm Nb m.aoma 0.5mm o.mmm m.ovv m.ama h.m0m an mumm Homecoo m.ooa h.mm m.mo H.0m o.m m.mm .Q.m H.momH «.mhm m.0mw m.amv OHBNH m.wmm cmmz n.0HmH H.mmm v.5mm m.mov m.oma «.mmm on v.oomH m.mnm o.mwm o.moo m.oma w.hmm mo m.moma m.hom H.m>e m.vmv m.mma m.mom mm m.ooma m.vhm H.mmm v.vmv m.oma v.mh~ no «.mmma m.mmm H.0mh v.mmv «.mma o.mmm om >.moma o.mmm m.ovm m.mo¢ m.hHH m.mmm mm m.mmHH m.mvm m.mmm v.mmv v.mma 5.0Hm om m.onma o.mom N.oom o.mmv «.5NH m.mhm mo m.ooma H.mem m.o~m v.mmv m.bma >.mhm No H.mmmH m.mmv m.mmm b.0hv N.ova m.omm Ho mumm ooumoue A.anv A.EoEv Aeamav A.Eosv mum: A.Eoev mmmz A.Emav mom: woom mum: moom woom smog woom good some who come uoz DMMSMHMMZ who .80 no: .80 uMMuommcz .oz 80 ooa.uom Em ooa.uom M mo .psd OOH mom 00H mom oz mo .uad pom M m0 95: M m0 95 oz mo deg mz mo 9:4 m muoHOmnd ouoaomnd ouoaomnd ouoaomnd muoaomnd ouoao no il ucoEumouB oflououm mo mxomz v Houmm ooHHHM mumm mo memos: zoom coma use one um: mo .mEm 00H mom one moom oaosz one :a Eowmmmuom one sbfloom mo musdofid OH magma 75 were going on their control diets for the second week. However, the sickness affected the control and treated rats to the same extent. This could be seen from the. weight gain data of these rats, Table 7. The carcass analyses for moisture, dry body weight, and lean body masses of refed and control rats are presented in Table 11. Even though the average %.moisutre in the body is slightly higher with the refed rats, it was not significantly dif- ferent from that of the control group. The calculated absolute amount of water was higher (P < 0.05) with control rats since they weighed more. Total absolute amount of dry body weights were also significantly more (P < 0.05) in control rats than in treated rats. The average % mois- ture in the lean body mass of the refed rats was 71 as compared to 69.7 of the control rats. Even though the mean %.moisture in the lean body mass of the refed rats was significantly higher (P < 0.05) than that in control rats, both the 69.7%.moisture in control rats and 71% moisture in refed rats were within the normal range of moisture in the lean body mass. The absolute amounts of wet and dry lean body masses were significantly higher in control rats than in refed rats. There was essentially 76 m.m v.mH m.m v.m 0.0 z.m .m.m o.mo H.0HN m.aom m.mma o.az m.oo one: H.N> m.mvm m.mHH v.vzH m.oz m.om OH m.mo m.vom m.mHH «.mom p.00 v.om m H.om m.vam z.ooa m.oma N.oz o.mm m m.hm H.mom m.om N.zoa m.Hh m.H© z w.mo m.mom m.vm ~.zvm m.on o.ao o H.mm o.mmm z.ooa 0.0mm H.Hz 0.00 m v.mo m.on H.mm e.mma m.az m.vo w m.vo m.zam m.ooa h.NmH m.oz o.mm m H.mm N.mNN m.mm H.mmH m.Hh o.mw N m.oo m.mmm v.moa o.mmm m.mz H.Ho H mumm owmom x.m:ev a.m:mc A.m:mv...zeom x.m:mc..umumz awe .mmmz Awe zoom .02 +mmm2 zoom #mmmz zoom ca .u3 zum mo ucooam zoom comm macs; CH comm zum comm pox wusHomom ouoaomom cm onoumfloz ououmfloz pom ucoaomona oHowopm mo mxwoB o nouns mxom3 o How poem Houucoo co ooomam mpmm oomom ocm Homecou CH mommmz zoom comm ocm pcmfloz zum mo ucooam muoaomnm osm mommmz zoom comm osm maocz ecu ca mnoumfloz HH mange 77 .mo>oa Amo.o v my um ucmommwcmmm mm moflm3 mmmfi zoom comm zHo mHoE omm mum“ monuc00+ .Ho>om Amo.o v mv mm ucmoflmmcmmm mm pH .mmmE zoom comm uo3 whoa oom moon Houuc00# .mumu oommonu cm pomp comm Amo.o v m0 Hommmm zHDGMOHmmcmmm mm mums monucoo mmH3 .m3 zHo mo .mEm muomommm««« .mumu oouoouu CH some some Amo.o v mv mommam zmucmommmcmmm mm mums monocoo cm Homes 00 ucoosm musHOmmm some om9:« .mumu monocoo CH wz.00 mo smog emu comp mmo.o v m0 Hom0mm zmucmommmcmmm mm memo mouucoo emu oomou mums emu mo mmmE zoom comm emu om wuoumflofi man coma omee 0.h m.05 0.vh 0.0m 0.05 0.00 0.00 0.v0 m.vh m.0m m.mh H.0N m.mom H.00N H.m5N m.m0m H.m0m N.mNN 0.0mm m.zvm m.v0N m.avm 0.0a 0.0mm 0.0NH 0.0mm m.ova 0.h0H N.v0H v.00 N.0HH 0.0m 0.0mm m.0H 0.H H.N .Q.m 0.00a 5.00 h.mm com: m.mza H.0h N.mm Ho N.m0a 0.0h N.0m 0m 0.0mm 5.05 0.0m 0m m.oha h.m0 0.00 mm N.0mH m.00 0.00 0m v.HmH H.0h H.H0 mm 0.NhH 0.00 N.0m om H.moa 0.Hh 0.00 mm 0.00H 0.05. m.hm mm mumm Houusou .ucooun.mm magma 78 no increase in the dry weight of the refed rats during the 6 weeks period of refeeding of the control diet compared to the treated rats of the first group. But at this pe- riod, the control rats gained an average of 4.7 gms. of dry weight. The refed rats of the second group lost about 8 gms. of wet lean body mass when compared to the treated rats of the first group whereas during this same period the control rats gained 8 gms. In the same way, the refed rats lost 3 gms. of dry lean body mass due to sickness while the control gained an average of 1 gm. of dry lean- body mass. It seemed that the treated rats were more severely affected by the infection than were the control rats. Neither the body nitrogen nor the protein was re- sponsible for the differences that occurred in the total absolute amounts of wet and dry lean body masses between the control and the refed groups since the % nitrogen in the wet and dry lean masses and absolute amount of nitro- gen and protein in the total carcasses, were not signif— icantly different between the refed and control groups, Table 12. Differences in the lean body masses were caused by some changes which were not identified at this time. 79 0.00 0.0 0.0 0.0 m.a 0.0 0.0 .0.0 0.0g 0.00 0.0m 0.00 00.0 0.0a 5.0 coo: H.H0 0.0V 0.5m 0.50 0.0 0.00 5.0 av 0.00 0.00 0.5m 0.00 0.0 v.~m 0.0 00 0.00 0.00 5.00 v.50 0.0 5.0m 0.¢ 00 0.00 0.00 0.00 0.50 0.5 0.0m 0.0 50 0.00 5.00 0.00 0.00 v.0 H.NH 5.0 00 0.H0 0.00 0.00 0.00 0.0 «.mm 5.0 00 v.0v 0.50 0.00 0.00 0.0 0.0a 0.0 V0 0.00 0.00 0.0m 0.50 0.5 0.0a 5.0 00 «.00 v.00 H.0H 0.00 H.0 0.0m 0.0 00 muom mouucou 5.0 0.0 0.0 0.0 v.0 v.0 H.0 .m.m 0.00 H.50 0.0m N.H0 0H.0 0.0m 5.0 coo: 0.00 0.00 0.0m 0.50 0.0 0.0a 5.0 0H 0.50 H.0o 0.H~ 0.0V 0.5 5.00 0.0 0 0.00 0.00 0.0m 0.00 0.0 0.00 5.0 0 0.00 0.00 0.0m H.0v 0.5 0.0a 0.0 5 0.H0 0.00 H.0H 0.00 0.0 0.0a 0.0 0 0.mv 0.00 0.0m 0.00 0.0 0.0a 0.0 0 0.00 0.00 0.0m v.00 H.0 0.0m 5.0 v 0.00 0.00 0.0m 0.00 m.0 0.00 5.0 0 5.00 0.00 0.HH m.v0 5.0 0.0a 0.0 N m.mv 0.00 0.0m 0.00 0.0 0.0a 5.0 H mumm oommm . 3:3 7:3 A E00 pom mwv A00 smououm co0ouuaz may moo: A00 moo: .02 mo .usm unmmmz zoom promo: zoom mo .usm mo .uam smog zum comm um: umm ouomommm zum cm pom uoz cm mom ouomommm ouomommm cm somouumz cm comoummz ucofiuooma omouomm mo mxooz v Houmo mxooz 0 How poem Houucoo omu oomom muom am one muom monocoo cm mom oco co0oummz mo mucoofim omDHOmmm ooo mcoouom NH onoB 80 Neither the percentage nor the absolute amount of fat was significantly different between the control and treated groups, Table 12. From the body weight data (Table 7), it was obvious that the refeeding of the con- trol diet for 6 weeks helped in promoting faster weight gains even though the gain was not comparable to the con— trol rats. DeSpite the facts that the body weights were different, the absolute amounts of fat in the body of the control and refed rats were not significantly different. Two possible reasons for this can be suggested. One is that the withdrawal of the steroids resulted in corrected fat metabolism. The other one is that the appetite might have increased and thus indirectly affected body fat by increasing it. The amount of body fat per gram of nitrogen in the body was calculated for this 2nd group of rats and has been presented in Table 3 (Appendix). There were no significant differences between the refed and control rats in the amount of body fat per gm. of nitrogen in the body. The average absolute amount of sodium in the car- cass was 272 mgm. in refed rats and 326 mgm. in control rats. The control rats had significantly more sodium (P < 0.01) than the treated ones, Table 13. The average 81 0.50 0.00 0.00 0.50 0.0 0.00 .0.0 5.0000 0.550 0.000 0.000 0.000 0.050 coo: 0.0000 0.050 0.000 0.000 5.000 0.000 00 0.0000 0.000 0.005 0.000 0.500 0.000 0 0.0000 0.000 0.000 5.000 0.000 0.000 0 0.0500 5.000 0.005 0.000 0.500 0.000 5 0.0000 0.000 0.000 0.000 5.000 0.000 0 0.0000 0.050 0.000 5.000 0.000 0.000 0 0.0000 0.500 0.000 0.000 0.000 0.050 0 0.0000 0.000 0.500 0.000 0.000 0.000 0 0.0500 0.000 0.000 5.000 0.000 0.050 0 0.0000 0.000 0.005 0.000 0.000 0.000 0 a A.E0EV A.E0Ev moo: A.E0Ev0ezoom 0.8080 0002 0.50EV«««mmoz 0.8080 zoom zum mo doom pox mo o0om3 omm doom zum mo doom poz 00 «zoom o0om3 .oz .580 mom .50 com mom :0 x mo .00 80 mom .20 03 mom 90. :0 mz mo :9: 0 00 9:0 M 00 9:0 9:0 0.8.0:. 02 mo ...::0 «.2 00 9:0 9:0 030.: ucoeuooua o0ouopm mo mxooz 0 momma muom 0omusoo oom mxooz 0 Mom po00 0ouucoo co oooo0m omos mo0m3 muom oomom o0 mommoz zoom soom zmm oco moB mo .80 000 mom oco zoom o0om3 om» :0 usomosm 650000000 oco Ed0oom mo mmooofi0 00 onoB 82 .mxoo3 0 com momo 00mmcoo omm oomom mmom c0 mcomomm .808 0.000 00 coo8 omm comm 000.0 v my mom00m z0mcoo0m0c00m m0 mmom 0ommcoo c0 mmo8 zoom coo0 mo3 mo .80 000 mom .808 0.000 00 mc508o coo8 omB«a« .mmom oomom c0 850omomom no .808 0.000 00 coo8 omm comm 000.0 v 00 mom00m z0mcoomm0c00m m0 mmom 0ommcoo c0 8o0mmomom no .808 0.000 00 mc508o 0omom coo8 omB«« .mmom oomom c0 850oOm 00 .808 0.050 00 coo8 omm comm A00.0 v my mom00m z0mcoo0m0c00m m0 mmom 0ommcoo c0 .808 0.000 8:0oom mo mc508o 0omom coo8 omee 0.000 0.0000 5.0000 0.0500 0.0500 5.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.00 0.050 0.500 0.050 0.050 0.000 0.000 0.000 0.000 0.000 0.050 0.000 0.000 0.000 0.0000 0.0000 0.000 0.000 5.005 0.000 0.005 0.000 0.00 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 mmom 0ommcou 0.0 0.000 0.000 5.000 0.000 0.000 5.000 5.000 0.000 0.000 5.500 0.00 0.000 0.000 0.500 0.000 0.000 0.500 0.000 0.000 0.000 5.000 00 00 00 50 00 00 00 00 00 .mGOUII.00 o0moB 83 absolute amounts of potassium in control and refed rats were 827 and 907 mgms. respectively. Control rats had significantly more potassium (P < 0.05) than the refed ones. Increased amounts of sodium and potassium in the carcasses of the control rats are expected since they had significantly more lean body masses. However, the calcu- lated amounts of potassium per 100 gms. of wet and dry lean body masses were not different for the refed and con- trol groups. Similarly, the amounts of sodium per 100 gms. of dry lean body mass for the refed and control groups were not significantly different, but the amounts per 100 gms. of wet lean body mass was significantly more (P < 0.05) in control rats than in refed rats. However, the differ- ence was only 10 mgm. Comparisons of the first group which was sacrificed after 4 weeks of steroid treatment and the second group which was sacrificed following 6 weeks of refeeding the control diet after 4 weeks of steroid treatment revealed that there were no significant difference in body nitrogen. The increase in nitrogen between the treated rats of the first group and the refed rats of the second group during 6 weeks period was 0.03 gm. The increase in nitrogen 84 between the control rats of the first and second group was 0.04 gm. at this period. There was a marked increase in the amount of body fat in rats refed the control diet. The average increase was 4 gms. of fat between the treated rats of the first group and the refed rats of the second group during this six weeks period. At this period the average increase of fat was only 3.6 gms. between the control rats of the two groups. Third Group The third group of 16 rats was killed after 25 weeks and 3 days of steroid treatment. Their body weight data are shown in Table 7. Moisture content in the body and lean body, absolute amounts of dry body weights, wet and dry lean body masses are presented in Table 14. This group of 16 rats weighted almost the same during the com- mencement of the experiment, but they had different weights when they finished their 25 weeks and 3 days of steroid treatment. The mean average body weights were 260 and 254 gms. respectively for the treated and control rats at the start of the experiment. However, they respectively weighed 299 and 331 gms. at the time of the sacrifice. The treated rats had a slightly higher percentage of moisture in their 85 0.0 0.0 0.00 0.5 5.0 0.0 .0.0 0.05 0.000 0.000 0.000 0.00 5.00 8002 0.05 0.000 0.000 0.000 0.05 0.50 5 0.05 5.000 0.50 0.000 0.00 0.00 0 0.05 0.000 5.000 0.050 0.05 0.00 0 0.05 0.000 0.00 0.000 0.00 0.00 0 0.05 0.000 0.000 0.000 0.00 0.00 00 0.05 0.000 0.000 5.000 0.05 0.00 00 0.55 0.000 0.000 0.000 0.00 5.00 00 0.05 0.000 0.000 5.000 0.00 0.00 00 0000 0000089 1.0.83 023 7.0.83 .03 8.983 2: $02 :3 8000 .oz 0002 >000 0002 >000 >80 00 .080 80003 no .080 >000 8000 00003 00 0000 >80 0:000 003 «00500000 000500000 :0 08500002 08500002 000 000800088 0008000 00 0>00 0 000 00003 00 80000 0000 00 000002 >000 8000 >80 080 003 .000003 >80 t80003 00 005080 00500000 000 000002 >000 0000 080 00003 000 :0 08500002 0000800 00 0000B .0008 0000080 0003 .080 0.000 00 0008 000 0000 000.0v 00 800000 >000000m00000 00 0008 0080000 0003 0008 0000 003 00 .080 0.00m 00 0500> 0008 00B .0008 0000080 0003 000003 >80 00 .080 0.000 00 0500> 0008 000 86 0000 000.0 v 00 800000 >0000o0m00000 00 0008 0080000 0003 000003 >80 00 .080 0.000 00 0500> 0008 009* .0008 0000080 000 00 .080 0.000 00 0008 000 0000 000.0 v 00 800000 >000000000000 00 0008 0080000 00 .080 0.000 80003 00 005080 00500000 0008 009* 0.0 0.00 0.00 0.00 0.0 5.0 .0.0 0.05 0.000 0.000 0.000 0.05 5.00 0002 0.05 0.000 0.0m0 0.500 0.05 0.00 ON 0.00 0.000 0.000 m.00N 0.05 0.00 00 5.05 0.000 5.000 0.000 0.05 5.50 50 0.05 0.000 5.000 5.000 0.00 0.00 00 0.05 0.000 0.000 0.000 5.00 0.00 000 0.05 0.000 0.000 0.000 0.00 5.00 000 0.00 0.500 o.mm0 0.000 5.00 0.00 000 0.00 0.050 0.000 0.000 0.00 0.00 M00 0000 0080000 .0coouu.vH 00000 87 whole body than the control rats, but it was not statis- tically significant. There was no significant difference between the control and treated groups in the percent mois- tures of the lean body masses° However, the average abso- lute amount of water in the body of the control rats was significantly higher (P < 0.05) than that of the treated rats since the former had more body weight. Absolute amount of dry body weight was also significantly higher in control rats (P < 0.01). Control rats had significantly more wet lean body mass (P < 0.05). There was no significant dif— ference in the dry lean body masses of the control and experimental groups. Averages were 74 gms. in the treated rats and 78 gms. in control group. Percentages of nitrogen in wet and dry lean body masses were not significantly dif- ferent for the treated and control groups. Percent nitro— gen of the lean body masses, absolute amounts of nitrogen and protein, percent fat of wet and dry carcasses and ab- solute amounts of fat are presented in Table 15. It is obvious from this table that even though the dry lean body masses between the two groups were not significantly dif— ferent at this period, absolute amounts of nitrogen and protein in the body were significantly different for these 88 .Amo.o v my mum» omummuu 0:» :H ucmmmum umm mo .msm «.mm mo unseen map swap Magma: MaucmoflMHcmflm ma mumn Honucoo Ga ucmmwum you no .80 m.Hm mo unflucwsw wmmum>m one @ .wmm mEmm man no mumu omummuu on» :mnu “mamas Amo.o v mv %HucmoflMHcmHm mum mumu Houucoo on» no mommmoumo map aw cflmuoum 0cm.:mmouufin mo ucsoem muDHOmnm# j h.va m.> ¢.v ~.¢ 5.0 m.o H.o .o.m m.am N.mm ¢.wH H.No m.m m.NH m.m cam: m.hm m.mv m.ba v.vm m.oa m.ma m.m om v.mv m.mm H.ma m.hm m.oa H.NH m.m ma o.H> H.0m N.HN m.vm m.oa m.wa m.m ha m.m> m.om m.vm m.mm H.m w.NH m.m Ha «.mm m.om m.HH o.Ho m.m v.ma m.m wao m.hm n.vm o.va «.mm m.m v.ma w.m mHo H.mm «.mm m.oa H.vm m.oa h.ma m.m wao H.0v m.mm m.NH m.mo H.0H o.ma h.m mHo mumm Houuaou h.NH ~.m o.v m.N m.o m.o H.o .o.m m.mm o.Nm w.mH H.5m m.m m.NH m.m cow: m.mm o.mv m.mH n.wm H.m h.ma m.m h h.vm m.mm ©.m m.mm m.m m.HH h.m m m.mv m.ov m.oa 0.0m H.m m.ma h.m v m.mH b.0m v.> H.0m o.m ¢.NH m.m N m.mv m.mm m.ma h.mm m.m o.NH b.m mo N.Hm m.mm m.oa «.mm m.m h.ma m.m v0 @.mm v.mm h.m m.mm v.m o.mH m.m mo H.mm m.vm m.ma n.5m N.m v.ma m.m Ho mumm omummua 723 camp 3; 3; A 9.3 A 93 3; 3; . #Cflwuoum #:mmouuflz oz mo undead upmflmz atom unmflmz zoom mo .uad mo .ue¢ mmmz upom smog mum: woom smog umm muDHOmn¢ who CH umm umz cfl #mm musHomnd musHomad who cw cmmOHUfiz pmz CH cmmouuflz ucmfiummue @Houwum mo whoa m cam mxmmz mm Hmumw mumm ca umm cam cmmouuwz mo mpcsosd musaomnd was unmoumm ma GHQMB 89 two groups. The control rats had mean values of 9.9 gms. of nitrogen and 62.1 gms. of protein. These values are significantly higher than the mean values for the treated rats which were 9.2 gms. of nitrogen and 57.1 gms. of pro- tein. Expressing fat as a percentage of wet body weight or as a percentage of dry body weight revealed that there was no significant difference between the control and treated groups. This is thus a significant improvement in this group of treated rats sacrificed at 25 weeks and 3 days of steroid treatment as compared to the treated rats sacrificed at 4 weeks of steroid treatment which had a significantly lesser percentage of fat (P < 0.05) in both wet and dry carcasses. However, a significant dif- ference was found in the absolute amounts of fat in the control and treated groups. The control rats had a mean value of 52 gms. of fat which was significantly higher (P < 0.05) than the mean value of 36 gms. of fat in the treated rats. Table 16, presents the total amounts of sodium and potassium present in the carcasses and the amounts of sodium and potassium present in 100 gms. of wet and dry lean body masses. The treated rats had an average of 355 mgm. of sodium and the control rats had an 9O h.mm m.m H.OOH O.mm v.0 m.mm .O.w O.mOwH O.nHv OeNmOH w.mhv v.HvH m.Ohm cam: H.55vH O.hmv H.OmHH m.>mv m.mvH m.Omm Om m.movH m.Omv h.mOmH m.mmv m.OMH O.NHv OH m.mOOH m.mmq O.vMHH w.va NomvH H.mmm 8H m.mHMH v.mov m.mmm h.mv¢ h.mMH v.0mm HH m.mmmH H.HH¢ m.mOOH Oommv m.HmH m.~mm OHO moHOMH H.OHv H.50m «.mvv m.hmH H.mHm mHO m.hmMH m.HHv m.mmOH O.mmv h.NMH m.vmm «HO m.vmmH m.mHv m.¢mHH 0.0nv m.wvH h.Hov MHO mumm Houucoo m.mvH m.mv H.vHH w.mm m.mH m.Hv .O.m H.vbmH O.mHv 0.0HOH m.Om¢ H.OvH m.mmm cmmz 0.0VMH m.mmm n.¢om N.OHm m.HmH h.vom h m.nOOH m.mmm m.mmm m.¢mm m.OHH O.mbm m v.8wvH m.vvv «.mhh m.mmv H.mvH H.wmm v m.mmmH 0.0mv O.vHHH m.HOm n.va O.hmm m H.vaH m.mov m.thH O.vmm h.mmH «.mmm mO v.HmvH v.0mv O.thH m.mmv 0.0¢H O.mnm vO O.vva O.mm¢ v.mmHH m.omv m.mmH O.mom mO m.mmvH H.mmv m.OmOH b.05v H.mVH H.vmm HO mumm topmoue whammmwom whammmwom A.EOEV mwmmmmwom mwmmmmwom A.Emav boom 23 atom 2? .oz smog who mo cmmq um: mo smog wuo mo smog um: mo Ca M mo ca mz mo umm .Em OOH mom .Eb OOH mom .uEd.Hmuoa .EO OOH Mom .EO OOH Mom .aE¢ Hmuoa M mo .92 M mo AER m2 mo 55 mz «.0 .22 ucmsumwue OHOHmum mo whoa m can mxmmz mm nmumw momma: zoom cmmH who cam umz .EO OOH Mom ucmmmum pascad may mam mumm mHosz may GH ucwmmum EsHmmmuom cam EdHcom mo mucsoad OH mHQMB 91 average of 371 mgm. in the whole carcasses. These values are not significantly different from each other though the control rats had significantly more wet lean body masses. Quantities of sodium per 100 gms. of wet and dry lean body masses were almost the same for the control and treated rats. Similarly, the total amounts of potassium present in the whole carcasses of treated and control groups did not show any significant difference. Furthermore, there was no difference in the amounts of potassium per 100 gms. of wet and dry lean body masses between the treated and control groups. Results on the amounts of sodium in the carcasses of the treated rats indicate support for the reports of Woods (1967) and Laragh gt a1. (1967) about hypertension in women taking oral contraceptive pills. We can compare the first and third groups of rats which were killed at 4 weeks or 25 weeks and 3 days of steroid treatment in order to know the long-term effect of the pill. If we take the treated rats of the first and third groups and compare them with each other for their absolute amounts of dry weight, it is seen that the in— crease in dry body weight from the 4th to the 25th week 92 is only 9.1 gms. while the corresponding increase for the control groups for the same interval was 17.5 gms. The increase in the wet lean body mass was 16 gms. between the two treated groups while it was 26 gms. for the control groups. However, the increase in the dry lean body masses was approximately the same during this 21 weeks interval. The differences were 7 gms. for the treated groups and 9 gms. between the control groups. Similarly there was not much difference in the gain of nitrogen between the treated and control groups. This difference between the two treated groups was 1.04 gms. and between the control groups, it was 1.12 gms. Less improvement occurred in the fat content of the treated rats belonging to the third group in 21 weeks interval. The difference in the abso- lute amount of fat between the first and third group treated rats was only 2 gms. while at this period the con- trol rats had a mean increase of 8.5 gms. Body fat per gram of food was still lower in the treated rats than in the control rats. However there was no difference between the control and treated groups in the amount of fat present per gram of nitrogen in the body, Table 3 (Appendix). From the above mentioned results, it is clear that the steroid 93 treatment definitely exerts some effects on many components of the body. Fourth Group The last or the 4th group of 14 rats were killed immediately following 6 weeks of refeeding the control diet iafter 25 weeks and 5 days of steroid treatment. These rats were placed on the control diet two days after the 3rd group of rats were sacrificed. Their body weight data are presented in Table 7. Their percent moisture in the whole body and the lean body and the absolute amounts of water, dry body weight, wet and dry lean body masses are presented in Table 17. Refeeding the control diet again did not bring the percent moisture of the whole body of treated rats to the value of the control rats. Percent moisture in the lean body masses of the treated and control rats were almost the same. The absolute amount of water in the treated rats was 174.5 gms. and in the control rats 190 gms. This was because the control rats weighed more. How— ever, the absolute amounts of water in the refed and con— trol rats were not statistically different. But it was approaching statistical significance. The absolute dry body weight was still higher (P < 0.05) with control rats 94 O.m m.mH O.H~ H.mH v.O N.m .Q.m m.mm n.mnm H.m¢H m.OOH m.OO N.hm Cmmz h.vn m.mvm O.HNH 0.0hH m.OO m.mm mm v.mm 0.0nm v.mOH N.>OH «.mO H.mm OHH m.mm H.OON v.OmH O.HON 0.00 O.HO Om 0.0m >.me v.mMH m.hOH O.mO v.mm OO H.5m m.mmm m.OOH H.OON m.Oh m.mm Om m.Oh N.mmm H.NNH v.OhH b.mO H.mm mv m.vm 0.0bm %.mOH O.NOH m.mO h.mm MHH mumm HouuCou v.m m.ON N.OH H.mH 0.0 m.m .O.m O.m> m.Ovm 0.0HH m.vhH 0.00 >.Om mez N.OO N.Hmm m.VHH O.NOH H.Oh O.mm OH H.N> v.Omm v.0m ¢.vOH m.mO m.¢O OH h.m> v.ovm v.OO 5.5OH m.OO m.MO OH m.mn O.Hmm h.mmH O.th m.wO O.mm mH O.mm v.mmm m.mMH m.OOm h.O> «.OO wH m.m> O.vvm N.ONH O.HbH 0.00 O.hm NH O.mn 0.0vm m.OMH h.th v.05 v.5m HH mumm umwmm 3.93 @723 12.93 793363 E 3...: 3; zoom .02 mmmz hpom mmmz wcom uanmz aha mo .uad awom CmmH mHonz CH umm CmmH who CmmH umS muCHOmQC muCHomnfl CH mnsumHoz musumHoz quEummuB OHOHmpm mo mama m UCm mxmmz mm kumm mxmmz O How umHQ HOHuCOU may comma mumm CH OCm mumm HOHuCOU CH mmmmmz hwom CmmH who UCm umz .usmHmz mum .Hmpmz mo muCnoad muCHomnd UCm mommmz >00m CmmH OCm mHOCS map CH mudumHoz quonm 5H mHQMB 95 .mxmm3 O Mom umHO HouuCoo Co OmUMHm mumz Coan much Ommmu OCH CH Umnmmfioo mm mommms moon CmmH Am0.0 v my wnw OCm um3 mHQE >HquoHMHCmHm m>ms mumu HOHuCOUO .mpmu 6mmmu may no .mem m.mHH umnu can» Am0.0 v my HmamHC mHuCMOHmHCmHm mH mum“ HouuCoo mo HCOHOB who no .Em H.m¢H mCHm> muCHomnm Cams mCB* 96 than with the treated ones. Six weeks of refeeding the control diet did not have much effect in increasing the dry body weights of the treated rats compared to the con- trol rats. The mean difference in the increase in dry body weight was 8.6 gms. between the treated rats of the 3rd and the refed rats of the 4th group while the corresponding difference was 13.5 gms. between the control rats of the 3rd group and the 4th group. Calculated amounts of wet and dry lean body masses were also significantly higher (P < 0.05) with the control rats than with the treated rats. On comparing the wet and dry lean body masses of the 3rd and 4th group, we can see that the mean wet lean body mass of the treated rats of the 3rd group was 244 gms. and it was 262 gms. with the control rats. The mean dry lean body mass of the treated rats belonging to the 3rd group was 74 gms. and control rats 78 gms. After refeed— ing the control diet for 6 weeks to the fourth group of rats, the mean wet lean body mass increased to 250 gms. with the treated rats and it was 274 gms. with the control rats. The difference was 6 and 12 gms. for the treated- refed and control-control rats respectively. Similarly the increase in mean dry lean body mass was 1 gm. between 97 the treated-refed rats and 6 gms. between the control— control rats. Refeeding the control diet for 6 weeks did not increase the lean body masses of the treated rats as it did with the rats fed continuously the control diet. Percent nitrogen in the lean body masses, absolute amounts of nitrogen and protein in the whole carcasses, percent fat of the wet and dry weight of the carcasses, and the absolute amount of fat appear in Table 18. Percent ni- trogen in the wet and dry lean body masses of the control and treated rats were almost the same. However, the total absolute amounts of nitrogen and protein in the carcasses of the control rats were higher (P < 0.05) than the treated rats. In essence, there was not much change in the abso— lute amounts of nitrogen and protein among the treated rats of the 3rd group and the refed rats of the 4th group which were refed with control diet for 6 weeks subsequent to feeding on the experimental diet. But during this 6 weeks period, the difference in nitrogen and protein were 0.5 and 3.1 gms. respectively between the control rats of the 3rd and 4th groups. Feeding the control diet for 6 weeks did help the treated rats to gain more fat. During this 6 weeks period of feeding the control diet, the mean 98 . .mxmw3 O How umHO HouuCoo Co OmomHm mnm3 COHC3 mumu Ommmu CH CmCu AmO.O v my mumu Hoanoo CH HmCmHC mum CHmuoum OCm ComouuHC mo muCCOEm quHowQ<# 0O.mH OO.h OO.v OH.v O0.0 ON.O no.0 .O.m h.mm m.ov O.>H «.mO v.OH m.mH O.m mez m.Ov N.Om h.mH m.mm v.m O.NH O.m mm O.mm O.mv m.mm 0.00 m.OH O.~H O.m OHH N.Om m.Om O.HH 0.00 O.HH m.NH O.m Om v.Ov m.vm m.mH m.hO 0.0H H.NH O.m OO «.mh b.nv m.Hm O.>O 0.0H v.mH >.m Om «.mv O.nm m.mH 0.0m O.m m.NH O.v mg m.OO n.mv m.mm m.nO m.OH O.NH O.m MHH mumm HouuCou O¢.OH O¢.O OO.v Om.¢ m>.O mm.O m0.0 .O.m O.mv n.mm h.vH m.mm v.m m.mH m.m Cmmz H.mv m.mm m.OH m.mm 0.0 m.mH O.m OH v.OH m.Om m.b m.mm 0.0 m.NH h.m OH O.mm m.vm 0.0 n.mm 0.0 m.mH hum OH v.OO m.m¢ m.OH m.OO h.m v.mH gm.m OH H.Om 0.0m O.vH m.nO 0.0H h.NH >.m vH O.mm >.H¢ >.nH m.nm N.O O.NH O.m NH m.Om m.mv m.mH H.mm m.m O.NH m.m HH mpmm Ommmm A.msmvumm COO va H.583 stmv Aww Aw» . mo .uEC uCOHmz %Oom uCOHmz wcom #CHmonm #Cm wuqu mmmz Oom mmmz com oz musHomna mun CH umm ums Ca umm mo pea mo pea cmmq who may cmmq pas map umm . muCHOmQ4 quHOmQC CH Comousz CH Comouqu I, quEHmmHB OHOkum mo mama m OCC mxmmz mm Hmumm mxmmz O How umHQ HOHHCOU Co OmomHm wumz CUHCB mpmm CH OCm mumm HouuCOU CH Ham OCm Comouqu mo mpCCOE¢ quHomnd OCm pCmonm OH mHQMB 99 increase in fat of the refed rats was 7.6 gms. which is much higher than the 2 gms. fat gained by the treated rats of the 3rd group in 21 weeks of eating the steroid diet. Among the 4th group of rats, there was no significant dif- ferences between the refed and control groups in the per- centages of fat of the wet and dry carcasses and in the absolute amount of fat in the carcasses. The total amounts of sodium and potassium in the whole carcasses and per 100 gms. of wet and dry lean body masses were calculated and presented in Table 19. None of the above values differed significantly between the refed and control rats. There was not much difference in the amounts of sodium and potassium both in the whole and lean body masses after refeeding the control diet for 6 weeks. It is important to note that the treated rats had significantly lesser (P < 0.05) wet and dry lean body masses but still had sodium and potassium close to the control values. Life Span Study Finally, mention must be made of the rats which were kept for studying the long-term effect of the pill. 100 0.0NH O.mm .m.NHH N.mv O.MH H.Om .Q.O H.ONMH N.¢O¢ v.mOHH ¢.OO¢ m.OvH O.¢Om Cmmz 0.0h¢H 0.0¢v m.MOHH ¢.Ov¢ 0.0MH O.mmm Om m.¢OOH m.vmm O.¢OO m.OOm mohHH ¢.hHm OHH ¢.mOMH m.¢Hv v.NONH m.OOm N.NmH %.va Om H.OOmH O.VNV m.hHNH 0.0vv h.OMH O.hOm OO O.nOmH v.OOv m.HOHH m.OO¢ 0.0MH H.HO¢ Om 0.0MMH O.mO¢ H.5NOH O.vO¢ H.H¢H N.hmm mq O.mONH N.¢Om O.HOOH O.NNm m.OOH O.¢¢v MHH mumm HOHHCOU %.mm O.mH 0.00 O.NH ¢.m ¢.¢m .Q.m 0.00MH m.mHv h.OMOH O.mO¢ N.O¢H H.Omm Cmmz 0.0mmH H.¢O¢ N.¢MO 0.00¢ 0.0MH O.NNm OH O.HH¢H m.Om¢ m.hHOH O.mmv n.OMH O.th OH v.¢m¢H O.mmv «.vaH N.OO¢ O.H¢H H.Omm OH m.OONH 0.00¢ 0.000H N.mO¢ O.¢¢H m.vOm mH H.vaH m.OHv N.ONNH O.NO¢ m.va H.NN¢ OH 0.0VMH m.mow n.HOO N.Om¢ H.5MH %.mmm NH m.OOMH m.vHv N.¢mOH h.Omv O.mmH 0.0mm HH mumm Umwwm A.EOEV A.EOEV A.EOEV hwom A.EOEV A.EOEV A.EmEv %Uom Mme meq mmmz CMWH CH & mo Mmmz CMWH mmmz CMWH CH m2 mo .02 HQ mo EU umz mo Em .HEO Hmuoe HQ mo EU um: mo EU .Hfim Hmuoe umm OOH OH .Os< OOH OH .OEO OOH OH .Hea OOH OH .OEO quEummHB UHoumHm mo mmmn m UCm mxmmz mN Hmumm mumm HouuCoo CH OCm mxwmz O How umHQ HOHHCOU Co OwumHm mum3 £0H£3 mumm Ummmm CH mmmmmz %Uom CmmH wan UCm HOB mo .80 OOH Hmm OCm mwom mHonz may CH ECHmmmuom OCm ECHOom mo mquoE< OH mHQMB lOl Their growth rates were similar to the rest of the animals used for the other studies, Figure 2. After they had eaten diet containing steroids for about nine months, two out of these 28 rats developed rapidly growing mammary gland tumors. The tumors were examined at the Veterinary Diag- nostic Laboratories of Michigan State University by Dr. V. Sanger. The tumor in one of them was in the inguinal re- gion and measured 3 x 2 x 1.5 inches. The tissue was com— posed entirely of connective tissue stroma and small dis- crete ducts. Most ducts were intact and lined with small epithelial cells. A few groups of these cells were present which were not confined to the arrangement of a duct but were bunched in the stroma. Mitotic figures were scarce. Evidence for malignancy was sparse. The tumor in the second animal was in the axillary region. This tumor tissue was composed of a dense arrangement of mammary gland alveoli and cells which had escaped from the alveolar ar— rangement or had never formed a structure. The stroma was sparse. Much of the tissue appeared as well-organized alveoli with epithelial cells lining the alveolus and secretion filling the lumen. Some of the structure was large and Open. Others were small and partially collapsed 102 because of crowding. The epithelial cells were large, hyperchromic, and crowded. Large numbers of cells were present in the stroma and had not formed alveoli, Many mitotic figures were present. Most cells appeared to be relatively immature although some had secretory materials in the cytoplasm. This tissue presented the appearance of early malignancy. After 74 weeks of steroid treatment, two more treated rats developed mammary gland tumors. One control also had a small mass in the axillary region, and at the time of this report was still alive. However, during the 1 1/2 year period of the experiment, the occurrence of tumors in four out of 28 treated rats is more significant than in one out of 22 control rats. This result agrees with the finding that the prolonged administration of es— trogen to rats caused a high incidence of mammary tumors (Dunning and Curtis, 1952, 1954). According to the present experiment, estrogen and progesterone treatment resulted in a 4—fold incidence of tumors. Unfortunately, the total number of rats used may not be sufficient for a proper statistical evaluation, though the incidence is high enough to warrant further investigation of this aspect of oral contraceptives. 103 Life span of the treated and control rats during the 1 1/2 year period was calculated. .Twelve rats in each group were included for the statistical analysis of the life span. These groups did not include those which had tumors and those which were_sti11 alive. The life spans of the 12 control and 12 treated rats are given in Table 4 (Appendix) and their mean values were 60.9 weeks and 61.6 weeks respectively. Statistical analysis proved that these values were not significantly different. SUMMARY Contraceptive steroids norethynodrel and mestranol fed to 11 weeks old female rats on a body weight basis caused a reduction in food consumption and body weight gain. Treatment with steroids for a short or a long period did not affect the digestibilities of protein, fat, sodium, and potassium. However, feeding the steroids for 4 weeks resulted in different retentions of nitrogen and sodium between the control and treated grOUpS. Control rats re- tained significantly more dietary nitrogen (P < 0.05) than the treated rats and the treated rats retained more dietary sodium (P < 0.05) than the control rats. Feeding the steroids for 24 weeks and 5 days resulted in a higher re- tention (P < 0.01) of nitrogen by treated rats when com- pared to the control rats. No effect of steroids was ob- served in the retention of potassium either at short or long term feeding of steroids. Many changes occurred in the body composition of rats due to steroid treatment. Treated rats had less dry body weight throughout the experiment. Significant 104 105 differences occurred in the lean body masses of the treated and control rats. Treated rats had less lean body mass after 25 weeks and 3 days of steroid treatment. Refeeding the control diet for 6 weeks did not alter the situation. Percent nitrogen in wet and dry lean body masses was not altered between the treated and control groups. However, significantly less (P < 0.05) absolute amounts of body nitrogen and protein were observed with the treated rats after 25 weeks and 3 days of steroid treatment than the control rats. Refeeding of the control diet did not bring the values any closer to control rats. Significantly more (P < 0.05) percentage and absolute amounts of fat were ob- served with the control rats compared to the treated ones after 4 weeks of steroid treatment. Also, absolute amounts of body fat were significantly higher (P < 0.05) with the control rats than the treated rats after feeding the steroids for 25 weeks and 3 days. Refeeding the control diet for six weeks after each treatment helped the treated rats to gain more fat. Not much difference was observed in the amounts of sodium and potassium in the carcasses of the treated and control groups. 106 Life span was not affected due to steroid treat— ment. An increased incidence of mammary tumors was ob- served in the steroid treated group. Since there was not much difference in the percent digestibilities of various nutrients and in the percent— ages of various components of the body except the fat component between steroid treated and control groups, it may be concluded that the contraceptive steroids have no marked effect on the body composition of adult female rats. L ITERATURE C IT ED Abdul—Karim, R. W., J. T. Prior and R. E. L. 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Supplementary minerals and vitamins were added to provide per kg. of diet: (in mgl) Mn, 121; Fe, 95; Cu, 7; Zn, 4; I 4; Co, 2; Choline chloride, 400; Ca pantothenate, 2i 6; rifoflavin, 3; niacin, 33; menadione, 2; DL—methionine, 500; (in microgram) vitamin B 7; (in I.U.) vitamin A, 12' 8010; vitamin D 750; vitamin E, 5. 2' 116 APPENDIX II % NITROGEN* DIGESTIBILITY ‘% Nitrogen* _ Dietary Nitrogen - Nitrogen in the feces Digestibility — Dietary Nitrogen X 100 % NITROGEN* RETENTION % Nitrogen* _ Dietary Nitrogen — Nitrogen in urine + feces . — . . X 100 Retention Dietary Nitrogen *Other nutrients were substituted in the place of nitrogen in order to find out their digestibilities and retentions. 117 118 Table 1 Average Food Consumption and Body Weights of Rats Food Consumption* Body Weight* Treated Control Treated Control Weeks Weeks (gms.) (gms.) (gms.) (gms.) l 92 99 Start 257 257 2 106 133 1 248 268 3 105 120 2 255 278 4 ~109 118 3 255 278 5 101 126 4 260 288 6 96 115 5 266 296 7 105 107 6 268 301 8 111 111 7 274 297 9 105 111 8 277 304 10 106 116 10 276 307 ll 79 95 12 242 316 12 61 85 14 257 292 13 60 82 15 269 296 14 87 84 16 270 301 15 89 50 17 281 306 16 96 97 18 283 313 17 96 102 19 304 333 18 107 114 20 305 333 19 97 108 22 301 339 20 96 107 24 299 352 21 97 108 25 302 347 22 95 115 23 88 110 24 92 119 25 94 117 26 89 114 Mean 94.6 106.3 273.8 304.9 S.D. 12.7 16.9 19.4 25.2 *The food consumption and the body weights of control rats are significantly higher (P < 0.01) when compared to the treated rats. ‘ 119 Table 2 Feed Efficiency* of the Treated and Control Rats Treated Control At 22 Days of Steroid Treatment 0.23 0.08 0.06 0.17 0.06 0.13 -0.01 0.05 -0.01 0.08 0.14 -0.18 0.04 0.03 0.05 0.20 0.18 0.01 0.03 Mean 0.05 0.10 S.D. 0.12 0.06 At 173 Days of Steroid Treatment 0.14 0.00 -0.02 0.04 0.12 -0.16 0.12 0.05 0.13 0.14 0.19 0.19 0.21 0.00 0.25 0.13 0.06 0.18 0.02 0.08 Mean 0.12 0.06 S.D. 0.08 0.10 *Weight gain in gms./gm. of food/day. (-) Negative value indicates that rat had a negative weight gain. 120 Table 3 Absolute Amount of Fat Per Gram of Nitrogen in the Body of the Rats Killed at Various Time Intervals First Group Second Group Third Group Fourth Group (Treated) (Refed) (Treated) (Refed) Rat Amount No. Amount No. Amount No. Amount No. (gm.) (gm.) (gm.) (gm.) 61 5.48 1 5.12 01 4.23 11 5.98 62 3.96 2 3.54 03 2.83 12 5.72 63 3.47 3 4.90 04 3.14 14 4.61 64 5.37 4 3.20 05 5.14 15 6.20 65 3.69 5 5.18 2 2.10 16 2.65 66 2.80 6 3.93 4 5.44 18 2.08 67 3.38 7 4.19 5 2.89 19 4.80 68 3.51 8 5.04 7 5.94 ' 69 4.86 9 7.29 70 5.66 10 4.36 Mean 4.22 4.67 3.96 3.69 S.D. 1.02 1.19 1.42 3.35 (Control) (Control) (Control) (Control) 71 4.66 32 5.90 013 3.95 113 7.41 72 4.84 33 3.95 014 5.08 43 4.99 73 4.99 34 4.70 015 4.27 56 7.33 74 4.15 35 3.97 016 3.60 60 4.30 75 5.41 36 4.19 11 8.07 50 3.47 76 5.13 37 6.45 17 6.90 119 7.78 77 5.22 38 6.10 19 4.19 58 4.92 78 3.58 39 5.95 20 5.57 79 6.10 41 5.56 80 5.02 Mean 4.91 5.20 5.21 5.74 S.D. 0.69 0.98 1.57 1.73 1") ll llll‘.||| 121 Table 4 Life Span of Rats Treated with Steroid Mixed Diet and Rats Fed the Control Diet* Weeks the Rats Lived Rat No. Treated Rat No. Control 9 38 49 44.5 7 43.5 32 45.0 27 44.5 40 49 19 48 42 51.5 16 64 35 52‘ 14 64.5 36 53 25 66 30 53 3 71 39 67.5 20 71.5 43 74 12 72 37 74 5 77.5 50 80.5 4 79 29 87 Average 61.6 60.9 S.D. 14.3 14.8 *Remaining rats are still alive and will be allowed to continue on the trial. 6 3972 II III l l Ill III Ill II II I | ll I ll 31 mumw