ROLE or THE ADRENAL convex AND THYROID GLAND FUNCTION UPON SOME. PHYSICAL AND cnwm PROPERTIES or PORCINE MUSCLE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY David Glen queI 1965’ TH £515 This is to certify that the thesis entitled ROLE OF THE ADRENAL CORTEX AND THYROID GLAND FUNCTION UPON SOME PHYSICAL AND CHEMICAL PROPERTIES OF PORCINE MUSCLE presented by David Glen Topel has been accepted towards fulfillment of the requirements for I Ph.D. Food Science degree in ‘ 3 Major groleésér Date $38!: 9, 1965 0-169 ABSTRACT RDLE OF THE ADRENAL CORTEX AND THYROID GLAND FUNCTION UPON SOME PHYSICAL AND CHEMICAL PROPERTIES OF PORCINE MUSCLE By David Glen T0pel This study consisted of three separate investigations and included 148 pigs. In Part I, the influence of thiouracil and Tapazolelfeeding upon adrenal and thyroid weight, plasma l7-hydroxycorticosteroid (17 OHCS) levels, and some chemical and physical properties of the l, ggggi muscle was studied. Part II included the determination of plasma sodium, potassium and 17 OHCS levels, adrenal weights and extractability of muscle proteins and sodium and potassium content of the l, gg£§i_musc1e from normal pigs and those exhibiting slight and severe PSE musculature. The influence of exogenous adrenocortical-like steroids upon plasma l7 OHCS, sodium and potassium levels and several porcine muscle characteris- tics was studied in Part III. The goitrogens Tapazole and thiouracil were fed at various levels and for varying periods of time. Both drugs caused hypertrophy of the thyroid gland to approximately the same degree, but thiouracil had a more pronounced effect upon adrenal atrophy than Tapazole. ‘L, dogs; muscle pH, myofibrillar and sarcoplasmic protein extractability, non-protein nitrogen and sodium and potassium levels were quite similar when Tapazole and thiouracil treated pigs were compared to controls. Thiouracil treat- ment, however, produced a pale, soft exudative (PSE) condition in the ham muscles, esPecially the gluteus medius in some pigs; whereas, Tapazole treatment resulted in normal colored, firm appearing ham musculature. Plasma 17 OHCS levels were lower than control values in both the Tapazole and thiouracil treated pigs, but these differences were not significant. IWl-methyl-Z mercaptoimidazole David Glen Topel In Part II, significantly lower quantities of sarcoplasmic, myofi- brillar proteins and NPN were extracted from PSE musculature than normal muscle. Considerable variation in the quantity of extractable protein was found between muscle samples in the slight PSE grOUp. These extracta- bility data indicate that muscle protein extractability is not entirely consistent with the visible PSE muscle characteristics. Plasma 17 OHCS levels from pigs possessing severe PSE muscles were 3.3 7/100 ml lower than the normal group. This difference was approaching significance (P‘< .05). Considerable variation existed within the three groups for plasma l7 OHCS which was apparently due to the variation from one individual to another in disposition prior to collecting the blood sample. Muscle pH was significantly different between the normal (pH 5.46), slight PSE (pH 5.35) and severe PSE (pH 5.18) groups. IL,.Qg£§$ muscle area was significantly (P < .01) larger for the severe PSE group (4.64 sq. in.) than normal pigs. A highly significant correlation coeffi- cient (-.43) was obtained between 1, dogs; area and muscle firmness scores for the pigs in this study. Thus, the more muscular pigs probably are more prediSposed to PSE muscle than poorly muscled pigs. Administration of prednisolone or methyl prednisolone produced adrenal atrophy and lower levels of plasma l7 OHCS for each level and period of time studied in the experiments of Part III. Quantity of sarcoplasmic and myofibrillar proteins from glucocorticoid treated pigs, in all three experiments, 'was not significantly different from controls. Daily pred- nisolone injection (200 mg/day) for seven days resulted in significantly higher muscle NPN values. Methyl prednisolone at either 225 or 450 mg David Glen Topel per day for 21 and 25 days produced no significant differences in muscle NPN values. However, muscle samples were collected either 3 or 5 days after the last methyl prednisolone administration; whereas, muscle sam- ples were collected 24 hours after the last prednisolone administration. Marked differences in rate of post-mortem muscle pH fall were ob- tained between prednisolone treated and control pigs. Prednisolone was found to have a sodium retaining effect upon muscle while methyl predni- solone resulted in sodium dimunition. Neither drug significantly altered ‘muscle potassium level of the-l..dg£§i_muscle. Sodium and potassium levels in the plasma of pigs treated with prednisolone or methyl predni- solone were within the normal range and similar to values obtained for controls. ROLE OF THE ADRENAL CORTEX AND THYROID GLAND FUNCTION UPON SOME PHYSICAL AND CHEMICAL PROPERTIES OF PORCINE MUSCLE By DAVID GLEN TOPEL A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1965 ACKNOWLEDGMENT The author wishes to express his appreciation to his major professor, Dr. R. A. Merkel, for his guidance throughout this research and for his assistance in the preparation of the manuscript. The general counsel of Dr. J. Wismer-Pedersen in selecting the re- search topic, and the advice of Dr. G. D. Riegle in the hormone assay portion of the author's research are gratefully acknowledged. Appreciation is expressed to Dr. B. S. Schweigert, Chairman of the Department of Food Science, and Professor L. J. Bratzler for their in- terest and stfinulative encouragement. Special thanks are due to Dr. W. T. Magee and Mr. Ken Kemp for their advice and assistance with the statistical analyses. The author is indebted to Eli Lilly and Company, Indianapolis, Indiana, for furnishing the Tapazole and to The Upjohn Company, Kalamazoo, Michigan, for their generous c00peration in providing prednisolone and methyl prednisolone for this study. Lastly, the author expresses his appreciation to his wife, Jackie, and to his parents and family for their encouragement and assistance during the author's graduate study. ii TABLE OF CONTENTS INTRODUCTION 0 O O O O O O O O O O 0 O O O O O O O 0 REVIEW OF LITERATURE . O O O O O O O O O O O O O O 0 Role of the Thyroid Gland in Soft, Exudative Porcine Muscle Adrenal-Thyroid Relationship . . . . . . . . . Role of Adrenal Glucocorticoids in Muscle Disorders Hyperfunction of the adrenal cortex . . . Hypofunction of the adrenal cortex . . . . . . Role of Adrenal Cortex in Amino Acid Metabolism Inhibitory Action of Steroids on Adrenal Steroidogenesis . Natural steroids . . . . . . . . . . . . . Synthetic steroids . . . . . . . . . . . . Metabolism of Glucocorticoids . . . . . . . . . Species Differences in Plasma Glucocorticoids . Influence of Housing and Exercise on Plasma 17 OHCS and Adrenal weights . . . . . . . . . . . Influence of 17 OHCS on Na and K Metabolism . . Levels of Sodium and Potassium in Porcine Muscle Levels of Sodium and Potassium in Pig Serum . . Extractability of Muscle Proteins . . . . . . . Protein Extractability of Normal and Soft Exudative Factors Which Influence Meat Hydration . . . . Muscle proteins and water . . . . . . . . iii levels ‘Muscle ll 11 ll 11 12 13 13 15 15 16 18 21 21 Page Water-holding capacity as influenced by specie, age and sex differences . . . . . . . . . . . . . . 22 Muscle location and muscle hydration . . . . . . . . 22 Influence of color and hydrogen ion concentration on meat hydration . . . . . . . . . . . . . . . . 23 Temperature-pH relationship upon meat hydration . . 24 Histological factors associated with meat hydration 25 Preslaughter factors and methods of prevention of soft pork . . . . . . . . . . . . . . . . . . . 27 Effect of electrolyte content, fat and protein . . . 29 EXPERMNTAL WTHOD S O 0 0 O O O O O O O O O I O O O O O O O O 3 0 EXperimental Design and Pre-slaughter Treatment . . . . . 30 Part I . . . . . . . . . . . . . . . . . . . . . . . 30 Part II . . . . . . . . . . . . . . . . . . . . . . 31 Part III . . . . . . . . . . . . . . . . . . . . . . 31 Slaughter, Cutting and Sampling Procedure . . . . . . . . 32 Adrenal and Thyroid waights . . . . . . . . . . . . . . . 33 Carcass Measurements . . . . . . . . . . . . . . . . . . 33 Panel Evaluation of Muscle Color and Firmness . . . . . . 33 Muscle Sample Preparation . . . . . . . . . . . . . . . . 34 Muscle Protein Extraction Procedure . . . . . . . . . . . 34 smple preparation 0 O O O O O O O O O O O C O O O C 34 Protein extraction . . . . . . . . iv Page Sodium and Potassium Analysis . . . . . . . . . . . . . . . 35 Muscle sodium and potassium analysis . . . . . . . . . 35 Plasma sodiun and potassiun analysis . . . . . . . . . 36 l7-Hydroxycorticosteroid Assay of Porcine Plasma . . . . . 36 Extraction of 17 OHCS from porcine plasma . . . . . . 36 Spectrophotometric analysis for 17 OHCS . . . . . . . 37 Muscle pH . . . . . . . . . . . . . . . . . . . . . . . . . 38 Statistical Analysis . . . . . . . . . . . . . . . . . . . 38 RESULTS AND DISCUSSION Part I. The Influence of Hypothyroid Function Upon Pale, Soft, Exudative Porcine Muscle . . . . . . . . . . . . 39 Effect of goitrogen drugs upon porcine muscle proper- ties . . . . . . . . . . . . . . . . . . . . . . 39 Protein extractability . . . . . . . . . . . . . . . . 41 Thyroid-adrenal relationship . . . . . . . . . . . . . 44 Part II. The Relationship of Plasma 17 OHCS Levels to Some Chemical and Physical Properties of Porcine Muscle . . 48 Protein extractability . . . . . . . . . . . . . . . . 52 Ultimate pH . . . . . . . . . . . . . . . . . . . . . 55 Non-protein nitrogen . . . . . . . . . . . . . . . . . 55 Plasma 17 OHCS levels . . . . . . . . . . . . . . . . 57 Sodium and potassium levels . . . . . . . . . . . . . 6O Carcass muscling . . . . . . . . . . . . . . . . . . . 61 Part SUMMARY . III. The Influence of Exogenous Adrenocortical-like Steroids Upon the Adrenal Gland and Various Chemical and Physical Properties of Porcine Muscle . . . . . Adrenal gland atrophy . . . . . . . . . . . . . . . Plasma 17 OHCS levels . . . . . . . . . . . . . . . Muscle proteins . . . . . . . . . . . . . . . . . . Non-protein nitrogen . . . . . . . . . . . . . . . . Muscle pH . . . . . . . . . . . . . . . . . . . . . Plasma and muscle sodium and potassium content . . . BIBLIOGRAPIiY . Q 0 0 O O O O 0 O O O O I O O O I O O O O O 0 APPENDIX . vi Page 63 63 64 65 69 69 72 74 76 86 Table LIST OF TABLES Page Means for sarcoplasmic and myofibrillar protein fractions, pH and muscle color and firmness scores of pigs fed Tap3201e O O I O O O O O O O O O I O O O O O O O C O O O 40 'Means and standard deviations of muscle protein fractions, NPN, pH, ham and loin muscle firmness and color scores, and thyroid and adrenal gland weights of pigs fed thiouracil and Tapazole . . . . . . . . . . . . . . . . . . . . . . 42 Means and standard deviations of fatback thickness, % lean cuts, loin eye area, muscle sodium and potassium and plasma l7 OHCS of pigs fed thiouracil and Tapazole . . . . . . . 47 Means and standard deviations of some chemical and physical characteristics obtained from normal pigs and those exhibiting slight or severe PSE musculature . . . . . . . . . . . . 52 Means and standard deviations of muscle and plasma sodium and potassium levels and-1, dorsi area of pigs possessing . normal or slight and severe PSE muscle . . . . . . . . . 60 Means and standard deviations of chemical and physical characteristics from control pigs and those injected with prednisolone for 7 days . . . . . . . . . . . . . . . . . 66 Means and standard deviations of chemical and physical characteristics from control pigs and those injected with prednisolone or fed methyl prednisolone . . . . . . . . . 67 Means and standard deviations of chemical and physical characteristics from control pigs and those fed methyl prednisolone for 10 and 25 days . . . . . . . . . . . . . 68 vii Plate II III IV Figure LIST OF FIGURES AND PLATES Seve re PSE ham 0 C O O O O 0 I O O O O O O O O O O O O O 49 S 1 ight PSE ham 0 0 O O O O O O O O O O O O O O I O O O O 5 0 Normal ham . O O O O O O O O O O O O O I O O O I O I I O 5 1 Plate IV illustrates how PSE musculature can be detected in the split carcass . . . . . . . . . . . . . . . . . . 62 Post-mortem pH fall 0 O C C O O I O 0 C O O O O O O O O O 71 viii Appendix II III IV VI VII VIII IX LIST OF APPENDIX TABLES Page Thyroid Study I . . . . . . . . . . . . . . . . . . . 86 Thyroid Study II . . . . . . . . . . . . . . . . . . . 87 Study .11, Firm, Normal Group . . . . . . . . . . . . 90 Study II, Slight PSE Group . . . . . . . . . . . . . . 92 Study II, Severe PSE Group . . . . . . . . . . . . . . 94 Study III, Prednisolone-Experiment l . . . . . . . . . 96 Study III, Prednisolone-Experiment 2 . . . . . . . . . 98 Study III, Prednisolone-Experiment 3 . . . . . . . . . lOO Adrenal weights, myofibrillar and sarcoplasmic protein values and plasma 17 OHCS levels from animals possessing nomalmuscle....................102 ix Iii-I‘ll INTRODUCTION The subject of porcine quality has recently received considerable attention, especially among European and American researchers. The term porcine quality referred to in this manuscript is defined as those physi- cal, chemical and morphological factors which are related to palatability characteristics. While emphasis has been placed primarily upon measure- ment and improvement of the quantitative aspects of porcine carcasses such as degree of muscling, lean to fat ratio, etc. in the past two de- cades, the qualitative characteristics have received less attention. It had been assumed that quality was not a serious problem in porcine muscle probably because of the young slaughter age and processing methods employed. Research findings within the last six to eight years indicate a rela- tionship exists between the pork quality factors such as degree of firmness, color of muscle, intramuscular fat and the palatability factors. 'More recently the industry has become aware of the pale, soft, exudative (PSE) condition in porcine carcasses. This condition results in abnormally high shrinkage and poor water binding properties during processing. In addition, PSE pork has been shown to be less tender and juicy than normal pork. Work by Ludvigsen (1953), Briskey and Wismer-Pedersen (196la,b), Bendall and Wismer-Pedersen (1962) and GoldSpink and MCLoughlin (1964) showed a relationship between post~mortem pH and muscle temperature with the incidence of pale, soft, exudative pork. The PSE condition resulted when a rapid dr0p in pH occurred while muscle temperature remained above -1- -2- 37°C. Although data are available on various post-mortem chemical and physical changes in porcine muscle, the causal factors for the rapid pH drop remains largely unexplained. It is known that the thyroid and adrenal glands, directly or indirectly, influence Specific enzymes involved in oxidative and anaerobic pathways in muscle tissue (White gtflgl,, 1964). Little evidence is available con- cerning the influence of adrenal or thyroid hormones on physical or chemical prOperties of muscle and much of this work has been conducted with rats and dogs. Ludvigsen (1957) and Henry ggngl. (1958) reported data indicating that the thyroid and adrenal gland activity can alter physical and chemical properties of porcine muscle. No other data were found in the literature relating the activity of these two glands to ulti- mate physical and chemical properties of porcine muscles. After reviewing these facts, this study was undertaken with the following experimental objectives: 1. To determine if there is a possible relationship of the porcine thyroid and adrenal glands with specific post~mortem muscle charac- teristics associated with the PSE condition. 2. To study the relationship between goitrogenic activity, adrenal size and plasma glucocorticoid levels. In addition, specific chemi- cal and physical prOperties of the l, dgrgi were studied. 3. To investigate the influence of exogenous adrenal glucocorticoids upon various chemical, physical and morphological pr0perties of porcine muscle. REVIEW OF LITERATURE Role of the Thyroid Gland in Soft, Exudative Porcine Muscle Ludvigsen (1953) reported a condition in Danish Landrace pigs which he called "muscular degeneration" (MD). He described the condition as alterations of the musculature, appearing macrosc0pically as a discolor- ation, the altered musculature having a pale or graying color. Freshly cut muscle was juicy and had an extremely sour smell. Ludvigsen noted that the condition was especially prevalent in the 1, dogs; muscle, but that on occasion it was found in all muscles of a given animal (total MD). The pH of normal I, dgggiymuscle was 6.8 to 7.0 approximately 45 minutes postdmortem, while values as low as 5.3 to 5.5 were found when "muscular degeneration" was observed. Ludvigsen (1953) produced total MD by feeding 1 gram of methyl thiouracil daily for 10 days prior to slaughter. Likewise, if the same dose of methyl thiouracil was fed for 20 to 24 days, 2 to 2 1/2 months before slaughter, total MD will result as well as several secondary effects, such as loss of appetite, interruption of growth and exams . The author also reported that total MD muscle changes were counteracted when pigs were fed 2 grams of iodinated casein daily containing 2.7 to 3.0% free thyroxin for a period of 10 days, three weeks prior to slaughter. These experiments were conducted with pigs having total MD diagnosed by means of a muscular puncture. Briskey (1963) fed methyl thiouracil for 10 days prior to slaughter and reported that the ham musculature from these pigs was pale, soft, and exudative when compared to untreated controls. When pigs were exercised to reduce the glycogen level, quality was measurably improved. Terrill g£_§1, (1948, 1950) and Acevedo gtflgl. (1948) reported no differences in physical or chemical composition of the carcasses from thiouracil fed pigs, but weight of the thyroid gland was significantly increased by this goitrogen. Carcass firmness was one of the physical characteristics observed; however, the authors apparently were referring to firmness of fat rather than skeletal muscle. Thyroid-Adrenal Relationship There is considerable evidence suggesting an interrelationship be- tween the thyroid gland and adrenal cortex. Leblond and Hoff (1944) noted a decrease in adrenal gland size of rats receiving goitrogenic sulfa drugs or thiouracil. Baumann and Marine (1945) confirmed these findings and they noted involution of the adrenals to half their normal size in rats fed thiouracil for four months. Histological studies re- vealed adrenal involution involved all three zones of the cortex. The fasicular zone showed a greater amount of lipoid material than normally seen while the lipoid content of the remaining zones was reduced. The authors concluded that cortical involutionwas apparently a compensatory reSponse for loss of thyroid secretion. Maqsood (1950) reported high environmental temperature significantly decreased weight of the adrenal gland in male mice to about the same degree as thiouracil administration. Maqsood indicated the decrease in adrenal weight was probably due to a decrease in thyroid secretion rate which occurred during exposure to high environmental temperature. McCarthy'g£.§l, (1959) provided thiouracil (0.1%), Tapazole (Lilly 0.001%, 0.005% and 0.01%), potassium perchlorate 1% in drinking water and fed a low iodine diet, and incorporated p-aminosalicylic acid (1%, 2%, 4%), p-aminobenzoic acid (2%, 4%), and sulfaguanidine (2%, 4%) in the diet of rats. After 12 weeks of treatment, atrophy of the adrenal gland was evident in all treatments except p-aminosalicylic acid. McCarthy and'Murpheree (1960) later reported the possibility of direct action of p-aminobenzoic acid on the adrenal gland as a more likely mechanism for induction of adrenal atrOphy. The authors found an inhibi- tion of the thyroid gland as indicated by reduced 1131 uptake by the thy- roid. Thus, further evidence is presented to associate goitrogenic activity with ability to induce adrenal atrophy, although the possibility of direct action of p-aminobenzoic acid on the adrenal gland coupled with thyroid inhibition cannot be excluded. Lazoéwasem (1960) also reported that 0.3% thiouracil fed to rats for 3 weeks brought about adrenal atrophy accompanied by thyroid and pituitary enlargement as compared to non-thiouracil fed controls. Pituitary ACTH content of thiouracil fed rats was less than 1/3 that found in'controls. These data support the hypothesis that adrenal atrOphy following thiouracil is probably associated with lowered ACTH titers. Role of Adrenal Glucocorticoids in Muscle Disorders In reviewing the literature, one recognizes a paucity of information regarding the hyper and/or hypo affects of glucocorticoid levels upon skeletal muscle characteristics. Hyperfunction of the Adrenal Cortex. Hyperfunction of the adrenal cortex leads to hyperglycemic states in the human and other animals. This condition is usually referred to as hypercorticordism (Cushing's syndrome). Although the morphologic causes of hypercorticoidism is somewhat confused, therewas characteristically an augmented secretion of glucocorticoids (C-ll-oxysteroids). In complications of spontaneous Cushing's syndrome, muscle weakness appears as a complaint in one half to more than 80% of the patients (Charles E; 31,, 1952). On the other hand, the frequency of muscle weakness among patients receiving synthetic hydro- cortisone type steroids in pharmacologic dosages has not been established. Severe or striking examples of muscle weakness have, however, been des- cribed during treatment with cortisone, 9-alpha fluorohydrocortisone, prednisone, and triamcinalone (Perkoff gt 31,, 1959; Williams and Lond, 1959; MacLean and Schurr, 1959; and Harman, 1959). Slight atrophy of the triceps surae muscles of the rat was produced by immobilization of one hind limb in a plaster of Paris cast. However, disuse atr0phy was significantly aggravated when the immobilized animal was exposed to stressors and/or treated with cortisol (Bajusy, 1958). Faludi ggual. (1964) induced myOpathy in dogs by injecting large doses of anti-inflammatory steroids (cortisol, prednisolone, methyl pred- nisolone, hexamethasone and triamcinolone). Weight loss and muscle atro- phy occurred in all treated groups, but was most pronounced in the triam- cinolone treated dogs. Marked differences existed between the treated groups in the capacity to decrease muscle size. Cortisol reduced muscle size the least. Fiber thickness of the muscle changed least in the methyl-prednisolone treated group and was most pronounced in the triamci- nolone group. The authors indicate that further investigation is necessary to understand the pathogenesis of this myopathy. The mechanism responsible for hyperglycemia and muscle weakness caused by the glucocorticoids is in part explicable by the effect of these hormones Upon gluconeogenesis. This was first inferred by Long 2; 5Q? (1940) from their data with rats. They found that when either a potent adrenal cortical extract or one of the adrenal C-ll-oxysteroids was administered to fasted normal, hypophysectomized, or adrenalectomized rats, urinary nitrogen excretion increased. Analysis indicated that 53 to 65% of the protein catabolized was converted to glucose. Ingle (1941) force-fed rats a high carbohydrate diet and observed that adrenal corti- coid administration brought about a concommitant increase in urinary nitrogen along with hyperglycemia and glycosuria. Studies with radioactive isotOpes have clearly demonstrated the effect of adrenal C-ll-oxysteroids upon gluconeogenesis. Welt ggugl. (1952) injected 014 labeled glucose at a constant rate into rats and estimated gluconeogenesis by comparing activities of injected and excreted glucose. They found that cortisone administration resulted in a sevenfold increase in glucose production from non-carbohydrate material. The origin of steroid myOpathy from hypercorticoidism.remains speculative. One current hypothesis is that it is related to losses of cell protein by the great increase in the rate of gluconeogenesis, thus causing muscle weakness. Hypofunction of the Adrenal Cortex. In man atrophy of the adrenal cortex produces a syndrome known as Addison's disease. With few excep- tions, the symptoms involve extreme muscular weakness as well as other abnormalities (Turner, 1955). According to Ludvigsen (1957) hypofunction of the thyroid and adrenal cortex appears to be the actual cause of the muscular changes characteristic of PSE pork. From his observations he concluded that these muscle changes resulted from an insufficiency of thyroid hormone in the blood and a reduced corticotrOphin content of the anterior pituitary gland. The condition described by Ludvigsen in pigs may not at all be comparable to Addison's disease in man because death of the animal seldom occurs. Ludvigsen (1957) also reported reduced lactic acid in venous blood of MD pigs after exercise and increased constriction of the arterioles and capillaries in such muscle. He postulated that this constriction is reSponsible for lack of rise in lactic acid in the blood during exercise which is normally expected. Ludvigsen (1957) also suggested that adrenal cortical hormones have an influence upon vasomotor reactions. One of the symptoms he observed in MD pigs was vasoconstriction of the skeletal muscles during exercise, and since hydrocortisate has a striking vasodilating effect, Ludvigsen postulated the pituitary-adrenal cortex axis obviously plays an important role in the regulation of vasomotor reactions. Wimmer-Pedersen (1959) observed that the incidence of the condition described by Ludvigsen (1953, 1957) was very rare in Danish Landrace pigs and called the condition usually encountered "pale and watery." Henry 25.31. (1958) submitted 12 pigs to high nutritional levels and found theywere sensitive to such stress as evidenced by hypematrenia. The authors indicated low quality pork (exudative myopath18)was involved with a hyperproduction of aldosterone and somatotropic hormone (STH), with concommitant deficiency of ACTH and glucocorticoids. The muscles were loaded with sodium and a potassium deficiency ensues. There was progressive disappearance of striation in the fibrillae and a loss of muscle pigment. Eventually decalcification, polyuria and general asthemia resulted. Role of Adrenal Cortex in Amino Acid Metabolism Wool and weinshelbaum (1959) reported the function of cortisone and cortisol in addition to their participation in the regulation of protein metabolism, was the mobilization of endogenous protein. They incorporated C14 labeled amino acids into a protein fraction of diaphragms excised from adrenalectomized and normal rats and found less incorporation of 614- amino acids in the protein fraction from normal rats compared with adren- alectomized rats. Wool (1960) investigated this difference between normal and adrenal- ectomized rats by a rather indirect approach. It was found that cortisone decreased the rate of penetration into the muscle cell and decreased accumulation of Cl4-amino acids by the cells of isolated rat diaphragm. Therefore, less amino acids might be available for protein synthesis. Adrenalectomy did not effect amino acid tranSport into the diaphragm cells; -10- however, adrenalectomy increased the rate of incorporation of radioactivity from L-phenylalanine into protein without a simultaneous effect on the rate of penetration of that amino acid. Kaplan and Shimizu (1963) measured the effects of cortisol on the concentrations of free amino acids in muscle tissue. Administration of cortisol appeared to result in increased concentration of virtually all amino acids and urea in the muscle of both fasted and non-fasted rats. These findings indicate evidence against the hypothesis that cortisol exerts its effect at the muscle cell membrane. If cortisol decreases the permeability of muscle cells to amino acids which, in turn, results in diminution of the pool of amino acids available for activation and incor- poration into protein, then less amino acids should have been found in the muscle cells when cortisol was administered. Kostyo (1965) reported adrenal steroids caused an appreciable delay in the reSponse of the muscle amino acid transport process both in gigg and in yitrg, This suggests the interaction of the steroid with the muscle cell may be at some site other than the membrane transport system. Ryan (1963) found a variable effect on rat muscle amino acids depend- ing on length of administration of hydrocortisone. Twenty four hours after injection of hydrocortisone an increase in the free amino acids of plasma and muscle was found. These acids were decreased after 10 days of treatment with hydrocortisone. -11- Inhibitory Action of Steroids on Adrenal Steroidogenesis Natural Steroids. The studies of Ingle and co-workers (1937, 1938) showed that prolonged treatment with corticosteroids caused atrOphy and hypofunction of the adrenal cortex. The view has generally been accepted that the mechanism reSponsible for this action of corticosteroids is an inhibition of pituitary ACTH production (Boland and Headley, 1949). This concept of the mechanism of diminished adrenal function has found wide acceptance in the literature and the possibility of direct inhibition by corticosteroids has been raised only upon occasion. Synthetic Steroids. Peron g£_§1, (1960) found that corticosterone inhibited inugiggg Steroidogenesis and similar findings with glucocorti- coids by Fekete and Ggrgg (1963) suggests that a direct adrenal inhibitory mechanism plays a physiological and pharmacological role, in addition to the regulatory mechanism controlling adrenal steroid functiontnediated by the pituitary gland. Christy gtflgl. (1956) reported that prednisolone administered for periods of one to two weeks appeanuito be four or more times as effective as similarly administered cortisone in suppressing adrenocortical respon- siveness. Metabolism of Glucocorticoids Schapiro and Ratz (1959) injected 014 labeled hydrocortisone in rats and reported that the C14 activity was widely distributed in the tissues. -12- The results were variable from one experiment to another, but in most experiments radioactivity was higher in the posterior pituitary gland than in any other tissue. Activity in the anterior pituitary, liver and kidney was higher than that in muscle or-brain. Gold (1960) reported the biologic half-life of free ll-desoxycortisol in circulating plasma of man was 42 minutes (estimated as Porter-Silber chromogen). Species Differences in Plasma Glucocorticoids Kruger g£_§1, (1965) reported normal level for free glucocorticoids in human plasma was approximately 16 to 187r/100 ml. These authors pointed out, however, that method of measurement and the time of day the sample is collected may influence results. Bush (1953) studied the levels of corticosterone in the rat, rabbit, ferret, cat and Rhesus monkey. All species examined were found to secrete large amounts of l7-hydroxycorticosterone (17 OHCS) and/or corticosterone. The ratio of 17 OHCS to corticosterone secreted varied from.20 in the Rhesus monkey, but did not vary appreciably between members of any one Specie. Bush suggested that Specie differences observed in adrenocortical secretion are genetically determined and cannot at present be related to any known differences in adrenocortical function. Obser- vable physiological differences may, however, exist and caution should be taken in generalizing from ACTH results with rats. Lindner (1959) reported normal levels of 17 OHCS for sheep to be 0.5 to 1.0 ug/lOO ml plasma and Brush (1960) and Shaw st 31. (1960) re- -13- ported 3 to 4 ug/100 m1 plasma to be normal for cattle. No reported levels of 17 OHCS in pig plasma were found in the literature. Influence of Housing and Exercise on Plasma 17 OHCS Levels and Adrenal weights Barrett and Stockham (1963) reported that rats housed in single cages, undisturbed for 18 hours prior to bleeding showed reproducible levels of 5.5 ug/lOO ml plasma. Rats housed in groups of 20 under the same conditions exhibit mean levels of 9.5 ug/100 ml plasma. Non-Specific stimuli such as environmental change, noise, handling, weighing, etc., all produced marked increases in plasma corticosterone levels which re- mained supernormal for at least 2 hours. Cornil ggflgl. (1965) showed that muscular exercise caused a signi- ficant fall in plasma cortisol level. Addis 55‘31, (1965) studied the influence of environmental tempera- ture and humidity upon quality of the gluteus medius muscle and adrenal gland weight of the pig. No significant differences were reported for color, firmness scores and pH of the gluteus medius muscle or weight of the adrenal gland from these pigs. Influence of 17 OHCS on Na and K.Metabolism The adrenal cortex secretes a spectrum of steroids with differing biological properties. At one end of the Spectrum is cortisol, weak in its effects upon renal electrolyte function, but active in protein and carbohydrate metabolism. At the Opposite extreme is aldosterone, active -14- in electrolyte regulation but having a negligible effect upon carbohy- drate and protein metabolism in amounts normally secreted. Corticosterone is intermediate, sharing some of the biological activities of both the aforementioned steroids but less potent in both respects (Barger_e£.§1. 1958). The work of Seldin g£_§1, (1951) suggested that cortisol administered in very high doses induces sodium retention and increases potassium ex- cretion. Cortisol is the dominant secretion of the adrenals in man and other Species and it maintains a normal glomerular filtration rate (Men- delsohn and Pearson, 1955). However, it should be noted that cortisone action permits the organism to maintain the integrity of the regulatory mechanism so as to enable the nephron to adapt readily to changing salt loads (Ingle, 1952). Davis and Howell (1953) studied the sodium retaining activity of cortisone in adrenalectomized dogs having acites. Administration of cortisone in physiological doses led to natridresis either by affecting the glomerular filtration rate, the rate of tubular reaborption of sodium or both. Knowlton (1960) reported no impressive alterations in sodium or potassium content of the skeletal muscle from adrenalectomized rats in- jected with cortisone acetate in amounts sufficient to induce severe hypertension. In contrast, Skeletal muscle from adrenalectomized rats injected with comparable amounts of desoxycorticosterone showed a marked increase in muscle sodium and reduction in potassium content. -15- Faludi st 31. (1964) reported serum electrolytes were within normal limits throughout the experiment when cortisol, prenisolone, methyl pred- nisolone, dexamethasone and triamcinolone were administered to dogs at a dosage of 100 mg/day. Swingle 25,3}, (1958) indicated that adrenal steroids possessing potent glucocorticoid activity function as a homeostatic mechanism in the salt and water balance of the body by enabling the animal to freely transfer fluid and electrolyte from one body compartment to another. These authors indicated that aldosterone apparently will not function in this capacity. Levels of Sodium and Potassium in Porcine Muscle Kirton and Pearson (1963) reported a range of 1750-3070 ppm potassium and 350-470 ppm sodium (fresh basis) in samples of ground pork. Lawrie and Pomeroy (1963) and Gillette ££;§l: (1965) reported a smaller range for the 1, gggsi muscle, but these values were similar to those of Kirton and Pearson (1963). Levels of Sodium and Potassium in Pig Serum Bohstedt and Grummer (1954) reported a range of 314 to 336 mg % sodium in pig serum. Widdowson ggngl, (1956) reported normal values for serum sodium and potassium of the pig to be 331 i 10 mg % and 23.4 i 2.1 mg %, respectively. Similar findings were reported by Kornegay SE 31. (1964). -16- Extractability of Muscle Proteins One of the first studies reported concerning the extractability of muscle proteins was by Deuticke (1932). He found that muscles which had been fatigued by Stimulation, frozen and pulverized, imparted less pro- tein to an extracting solution than those freshly extracted. Bate-Smith (1934) studied the effects of a series of extracting solutions. He found that with ammonium and lithium chlorides of adequate strength, no differ- ences could be observed between the extractability of fresh and rigor muscle. Bailey (1954) pointed out that the most direct eXplanation of this early work was that stimulation and rigor involve a change of State which is reflected in a loss of muscle protein solubility in some salt solu- tions but not all. In light of recent knowledge, they probably involve the combination of myosin and actin to give a less soluble complex. In freshly minced relaxed muscle, the ATP acts as a Specific dissociating agent. In rigor or fatigued muscle, extraction is facilitated by salts which depolymerize the complex. Bailey concluded that, considering the large amount of recent work on the theory of contraction and rigor, this is probably an over simplified explanation. From present theories of rigor, it might be thought the disappear- ance of ATP from the muscle is largely responsible for an "in vivo" aggregation of myosin and actin which retards protein extractability. According to Bailey (1954), this is incorrect. 'While ATP hastens the rate of solution, it does not increase the final yield, except when the -17- extracting solution has an ionic strength above 0.5. Crepax (1951) indi- cated that the action of ATP upon extractability of the muscle proteins is that of strengthening the dissociating action of electrolytes on the binding forces which hold the proteins in place within the muscle. The characteristic decrease in extractability of contracted muscles is not due to the hydrolysis of ATP which accompanies these contractions. Bailey (1954) stated that extractability is not solely determined by solubility. He indicated that this is probably because the dissolution of F actin or F actomyosin threads, several microns long, is seriously impeded in a mechanical way by the insoluble conponents of muscle. The extractability of myosin and actin depends, in part, on the mutual com- bination of these proteins and the hindrance to diffusion by the surround- ing insoluble muscle structures. A relaxed muscle, freshly minced, will yield free myosin, even on coarse grinding, but further comminution and stronger salt solutions will bring out large amounts of actomyosin. Homogenization must be continued to mechanically break not only the sur- rounding structures but to disperse the concentrated actin gel. Considering the above facts, Bailey (1954) drew the following conclu- sions for muscle protein extractability: At any particular stage of rigor the extractability of the intracellular protein fraction appears to be determined by pH, ionic strength of extracting solution, type of extractant and by adequacy of grinding. Jacob (1947) studied the effect of pH on extractability of sarco- plasmic proteins. He found that pH 7.7 was Optimal for extraction of all ~18- muscle proteins within the phosphate buffer range. At all pH values, a precipitate formed on dialysis. The quantity of precipitate varied considerably above pH 7.8, was least at 7.6 and became more abundant at lower pH values; however, the precipitate formed above pH 7.1 was soluble in 0.5M KCl. Helander (1957) obtained the maximum protein solubility in the pH range of 6.5-9.0. Recovery was substantially the same within these limits. Therefore, pH 7.4 was selected for use in his studies. He also indicated that optimum ratio of solvent volume to tissue volume was 10:1. Saffle and Galbreath (1964) reported that fat had no effect upon the extractability of salt-soluble proteins. Muscle pH had a significant effect on the amount of salt-soluble protein which could be extracted. As pH increased, amount of protein extracted increased. The amount of salt-soluble protein was 50% greater in prerigor beef than 48 hours post- mortem. Borchert and Briskey (1965) found that liquid nitrogen freezing of prerigor muscle increased extractability of both sarc0plasmic and myo- fibrillar protein fractions as compared to controls which were chilled under normal conditions for 24 hours. Protein Extractability of Normal and Soft Exudative Muscle Bendall and Wismer-Pedersen (1962) showed that washed muscle fibrils obtained from PSE pork had a lower water retention at low ionic Strength and much lower extractability at high ionic strength than fibrils from normal pork. Washed fibrils from PSE pork showed a greater protein con- -19- tent than similar fibrils from normal pork. The authors concluded that the greater fibrillar protein content in the watery fibrils was probably caused by a layer of denatured sarcoplasmic protein which was firmly bound to the surface of the myofilaments. Hill (1962) reported values for the amount of sarcoplasmic, myofibril, stroma and NPN in porcine, bovine and ovine muscle. Results showed that stroma proteinmwashighest in bovine muscle and lowest in porcine. He also reported differences between individual muscles for extractability of the protein fractions. MCLoughlin (1963) concluded that solubility of the sarcoplasmic and fibrillar proteins from exudative muscle was reduced at low and high ionic strengths, which was in agreement with Bendall and Wismer-Pedersen (1962). 'McLoughlin and GoldSpink (1963a) and Goldspink and McLoughlin (1964) observed the effect of temperature and pH on the solubility of sarc0plasmic proteins. They concluded that color of post-rigor muscle could be maintained if the temperature of muscle was reduced to about 30°C before the pH approached 6.0. Solubilities of sarc0p1asmic and myofibrillar protein were deter- mined by Sayre and Briskey (1963) at the time of slaughter, onset of rigor mortis, completion of rigor mortis and 24 hours post-mortem in muscles exhibiting a wide range of physiological conditions during the post-mortem period. Muscle protein solubility was grossly altered by conditions of both temperature and pH which existed at onset of rigor mortis or during the first few hours after death. Sarc0plasmic protein -20.. solubility at 24 hours was decreased to 55% of that found at 0 hour in muscle groups exhibiting high temperature and low pH at the onset of rigor mortis. Conversely, only a 17% reduction of sarcoplasmic protein solubility was noted in groups with high pH at onset. Myofibrillar protein solubility ranged from no reduction during the first 24 hours after death when pH remained high at onset to 75% reduction in muscle with low pH and high temperature at the onset of rigor mortis. The 24 hour pH of muscle appeared to have only a minor influence on protein solubility. Sayre and Briskey (1963) concluded that protein solubility appeared to be one of the major factors affecting juice-retaining properties of muscle. Partmann (1963) measured extractability of the actomyosin fraction to determine the degree of denaturation of fish muscle during freezer stor- age. The extractability of the structural proteins of rosefish and cod decreased at higher storage temperatures and with advancing storage time. Scopes (1964) reported that sarcoplasmic proteins are denatured readily at pH values below 6.0 at 37°C and at higher temperatures independent of pH. He found that denaturation of sarc0plasmic protein in £153 is asso- ciated with decreased myofibrillar solubility in KCl. Starch-gel electro- phoresis indicated one major and several minor proteins were Specifically denatured in conditions of low pH and high temperature. The major protein has been identified as ATP-creatine phOSphotransferase. -21.. Factors Which Influence Meat Hydration Muscle Proteins and Water. A comprehensive review of the basic con- cepts of meat hydration has been reported by Hamm (1960). He stated that the "true hydration water" of muscle is the amount of water that attaches to protein by monomolecular and multimolecular adsorption. This water is bound directly to polar groups of proteins and makes up about 4 to 5% of the water in muscle. The physical prOperties of bound water are differ- ent from those of free water. Bound water has a lower vapor pressure and a lower dissolving power than normal water. Hamm (1960) further Stated that most of the water in muscle is free, chemically Speaking. Apparently free water is mechanically immobilized by the network of the cellular protein membranes and protein filaments. Hamm concluded that changes in water-holding capacity of meat caused by changes of protein charges (i.e. by pH, ions, etc.) are not due to changes of true hydration water fixed to the polar groups of meat proteins. He stated that the amount of free water "immobilized" within the tissue is influenced by the spatial structure of muscle. Tightening this spatial structure (network of proteins) decreases immobilized water and loosening the protein structure has the opposite effect. This so called "stereo effect" (Hamm, 1959) is extensively influenced by changes of pro- tein charges. The presence of certain ions or adjustment to certain pH values greatly affects the Spatial protein arrangement and consequently affects water-holding capacity. Hamm (1959) defines water-holding capacity of meat as the ability of meat to hold its own or added water during appli- cation of force (pressing, heating, grinding, etc.). -22- Water—Holding Capacity as Influenced by Specie, Age and Sex Differences. Schon and Stosiek (1958a,b) found an inherent difference in water-holding capacity between Specie, age, and sex of meat animals. The reasons for these differences have not been elucidated. They utilized the adductor and 1, gg£§3.muscles and found that pork has a greater water-holding capa- city than beef. The water-holding capacity of bovine muscle increased in the order of steer to heifer to cow; however, no sex differences were ob- served for porcine muscle. Muscle Location and Muscle Hydration. Swift and Berman (1959) reported considerable variation for glycogen content and buffering capacity among eight bovine muscles. However, all muscles Showed characteristic patterns for pH changes and water retention; even though they contained residual glycogen when the ultimate pH‘WaS attained. The relatively high charac- teristic pH of certain muscles could not be attributed to lack of glycogen alone. Urbin.g£_§l, (1962) reported variation within the cross-sectional area of the 13 §2£§i_muscle when free moisture determinations were made by a modification of the.Grau-Hamm procedure. The medial portion of the _1. ggggi muscle had Significantly lower free moisture values than the lateral portion. Variation in juice retaining prOperties both between and within selected pork muscles was reported by Tapel'g£;21, (1965). The rectus femoris had the greatest difference in juice retaining properties between proximal and distal sections of the ham muscles studied. -23- Influence of Color and Hydrogen Ion Concentration on Meat Hydration. Water holding capacity of porcine muscle is positively related to both muscle color intensity and pH (Judge 25.31., 1958; Bate-Smith, 1948; Briskey 25:31,, 1959, 1960; Lawrie, 1958; Wismer-Pedersen, 1959). Dark, firm muscle was higher in pH and lower in free water than pale, soft muscle. The influence of pH on meat hydration is a typical example of the importance of protein net charge. The isoelectric point of muscle pro- teins is approximately pH 5.0. At the isoelectric point, the net charge of muscle proteins is minimal. At this pH, meat hydration has a sharp minimum. Normal pH of meat is about 5.5 which is close to the isoelectric point, consequently water holding capacity is quite low (Hamm, 1959). According to Briskey g£_gl, (1959), there were no significant differ- ences in pH or the ratio of expressible water to total water among four muscle color classes of fresh hams. Hams were classified in four groups: 1) pale; 2) two-toned; 3) two-toned, normal and 4) dark color. Relative amounts of eXpressible water, however, increased Significantly during the chilling process. This increase was greatest from muscles which possessed high concentrations of glycogen at the time of slaughter. Briskey and'Wismer-Pedersen (1961a) recorded continuous pH and temperature changes during post-mortem chilling. Carcasses that had a sharp, significant decrease to about pH 5.1, 1 1/2 hours post-mortem and a subsequent elevation to pH 5.3 to 5.6 possessed pale, exudative tissue with soft, inferior structure. -24- Briskey and Wismer-Pedersen (1961b) concluded that the pH-time sequence and subsequent develOpment of watery or normal tissue was depen- dent on a number of factors. However, only the following three factors were emphasized: 1) amount of glycogen in muscle tissues at the time of slaughter; 2) the phoSPhorylase activity and state of glycogen in the tissue; and 3) the methylene-blue reduction activity of muscle tissues. Temperature-pH Relationship Uppn Meat Hydration. Wismer-Pedersen and Briskey (1961b) studied the effects of various combinations of temp- erature and acidity in relation to muscle structure. The authors suggested that pale, exudative pork could be produced by maintaining body temperature for an extended post-mortem period. Conversely, they indicated that rapid chilling of muscle samples, which had a low pH within 45 minutes of slaughter, prevented this development of soft, watery muscle. Bendall and Wismer-Pedersen (1962) concluded the immediate cause of wateriness in pork was the combined effect of high temperature (37°C) and low pH on the muscle proteins which is in agreement with the suggestion of Wismer-Pedersen and Briskey (1961b). Bendall and Wismer-Pedersen (1962) also concluded that the depression and elevation oflfli values which was reported by Briskey and Wismer-Pedersen (1961a) was a reversible phenome- non due to the effect of temperature on the pK values of charged groups of the fibrillar and sarcoplasmic proteins. They consequently refuted the suggestion of Wismer-Pedersen and Briskey (1961a) that the depression and elevation of pH was a factor in the development of soft, watery pork. Bendall ggugl. (1963) reported a further study on the rates of ATP turnover in relation to pH and onset of rigor mortis in muscle samples -25... removed immediately after slaughter and held at 37°C. Muscle which was eventually soft and watery showed a much faster rate of ATP turnover and a shorter lag period prior to the onset of rigor. The authors were unable to explain the differences in rates, which could not be altered by various pre-mortem treatments. The authors noted that muscle which was allowed to go into rigor at a constant temperature of 37°C became watery and pale. They concluded that the probable reason for soft, watery pork was a com- bination of high temperature and low pH and suggested that the condition could be prevented by cooling rapidly to 30°C or below. Sayre ggugl. (1964) concluded that when onset of rigor mortis occurred at pH values below 5.9 with temperatures above 35°C, the 1, g£3§i_muscle became pale and exudative. Conversely, the authors reported that if on— set of rigor mortis occurred when pH values remained above pH 6.0, the muscle was dark and firm. Bodwell (1964) held one side of pork carcasses at 37°C and normally chilled the opposite side. He reported the 37°C treatment rarely induced soft, watery muscle as had been expected from reports of previous workers. Bodwell concluded that a low pH at a high muscle temperature BEE §g_was not a causal factor in development of PSE pork. Histological Factors Associated with Meat Hydration. Histologically there are several unusual features and abnormalities related to muscle pH (Lawrie, 1958). Muscles with an ultimate pH of 5.3 had discernable cross- striation but the muscle fibers were frequently twisted and broken. At pH 5.1 the protein gel of more than half of the fibers appeared to have -26- coagulated. At pH 4.9, all fibers were abnormal: some showed no cross- striations but had longitudinal markings; still others had cross-striations, but were both twisted and finely corrugated. Wismer-Pedersen (1959) studied histological samples of porcine loin muscle possessing different pH values. No systematic differences in appearance of the fibers and cell structure were observed between muscles with high and low pH. Variations in the distribution of water in bovine-1. dorsi, semimem- branosus, serratus ventralis and rectus abdominus muscles were histologi- cally studied by Lockett g£_gl. (1962). It was reported that extracellular Space of muscle tissue was positively correlated with the water-protein ratio; whereas, intracellular water content was negatively correlated. The evidence indicates that, in muscles which characteristically contain a relatively high proportion of water to protein, the additional water is located in extracellular spaces. McLoughlin and GoldSpink (1963b) did not observe any histological changes characteristic of degeneration in the soft, watery muscles. Lawrie (1960) observed distinct histological differences between normal and exudative muscles in Landrace pigs. However, these differences appeared to be primarily related to the 24 hour post-mortem pH level rather than the soft, watery condition. Bendall and Wismer-Pedersen (1962) reported histological infbrmation to support their conclusion that washed fibrils from soft, watery pork showed a greater protein content than similar fibrils from normal pork. -27- The authors concluded that greater fibrillar protein content in the watery fibrils was probably caused by a layer of denatured sarcOplasmic protein, which was firmly bound to the surface of the myosin filaments. Cassens gtflgl. (1963) used electron microscopy to follow changes in porcine muscle during the 24 hour post-mortem chilling period. The effect of ante-mortem subjection to elevated temperatures or to elevated temperatures and then chilling upon the rate and magnitude of change in post-mortem muscle color, texture and water binding was studied. Normal muscle exhibited a gradual disruption of sarcoplasmic components with little if any change in the myofibrils. Muscle that went into rigor rapidly at a low pH and high temperature ultimately appeared soft, pale and watery and electron micrographs revealed a rapid disruption of sarco- plasmic components and some disorganization of the myofilaments. ‘Muscle that went into rigor rapidly at a high pH and a reduced temperature ulti- mately appeared dark, firm and dry and electron micrographs revealed a high degree of organization and preservation of myofibrillar structure. Preslaughter Factors and Methods of Prevention of Soft Pork. Briskey .g£.gl. (1959a,b,c; 1960) reported a decrease in glycogen content of ham muscles from pigs subjected to exhaustive anteemortem exercise. An in- verse relationship was reported between initial glycogen level and 24 hour pH, water-binding capacity and color. Sayre ggugl. (1961) subjected pigs to a 0 to 5°C environment for 30 to 40 minutes prior to slaughter. They reported that the treatment re- sulted in a decrease in initial muscle glycogen and post-mortem accunula- tion of lactic acid. Although color intensity of the chilled muscle -28... increased, water-binding capacity was not consistently affected. The authors concluded that factors other than initial glycogen content, 24 hour pH and rate of glycolysis were important in determining water- binding capacity. Short-term excitement and exercise immediately prior to slaughter resulted in muscle with inferior water-binding prOperties. Long and short-term ante-mortem sucrose feeding produced elevated initial glycogen levels and resulted in muscles that were slightly soft and pale (Sayre g£_§$,, 1963a). These authors further concluded that total phOSphorylase activity was not affected by pre-Slaughter treatment and did not appear to be associated with rate of postdmortem glycolysis or with ultimate muscle characteristics. Meyer $5431. (1962) studied 20 Poland China and Chester White pigs and concluded that glucose tolerance was not an absolute indicator of post~mortem changes in muscle characteristics, but that there was a definite trend for those animals with a high glucose tolerance to have higher initial muscle glycogen levels and an ultimately inferior muscle quality. Kastenschmidt 25:21, (1964) studied the effects of four ante-mortem environmental temperature treatments, which were as follows: 1) warm (42-45°C for 30-60 minutes); 2) cold (l-3°C for 30 minutes); 3) warm followed by cold treatment; and 4) cold followed by warm treatment. The warm treatment alone was reported to induce the development of extremely pale, soft and exudative muscle. The authors concluded that warm treat— :ment followed by cold treatment resulted in dark, dry firm muscle. The other two treatments resulted in less marked changes. -29- Borchert and Briskey (1964) removed the wholesale ham immediately post-mortem and immersed it in liquid nitrogen (-195°C) for various periods of time. It was concluded that immersion in liquid nitrogen with subsequent equilibration and thawing at either -18°C or 4°C pre- vented the development of pale, soft, exudative muscle. Effect of Electrolyte ContentLyFat and Protein. Swift and Berman (1959) reported Statistically Significant correlations between water retention and pH, fat content and ratio of moisture to protein. A highly significant correlation was found between water retention and zinc content, in contrast to an inverse relationship found between water retention and either calcium or magnesium content. This information indicated that zinc differs in an important aSpect from the two other ions. The possibility that zinc may participate in determining pH as a component of an enzyme systemwas pointed out. Sodium chloride will increase water holding capacity by the influence of chloride ions rather than sodium ions (Hamm, 1959). Salt cross-linkages between peptide chains may be Split off by binding of chloride ions and there is an increase of meat hydration of both the net charge and stereo effect (Hamm, 1959). Sherman (1961) and Hamm (1959) indicate that polyphOSphates and citrates influence water retention due to their relatively high ionic strength and to their influence on muscle pH. These salts function primarily to form strong complex compounds with alkaline earth metals. They eliminate bivalent cations in meat, especially magnesium ions. EXPERIMENTAL METHODS This study was divided into three separate investigations. Part I involved the influence of thiouracil and Tapazole administration upon adrenal and thyroid gland size, plasma 17 OHCS levels, and certain chemi- cal and physical properties of the 1, gg£§i_muscle. Part II includes the determination of plasma sodium and potassium content, 17 OHCS levels, adrenal weights, and pH, protein extractability, sodium and potassium content of the 1, g2£§i_muscle from normal pigs and those exhibiting slight and severe PSE muscle. Part III concerned the effect of exogenous hormone like adrenal steroids upon plasma l7 OHCS, sodiun and potassium content, and several porcine muscle characteristics. Experimental Design and Pre-slaughter Treatment Part I. Part I consisted of two separate experiments. In experi- ment 1, twelve Hampshire pigs were randomly assigned to two lots with three barrows and three gilts per lot. A normal finishing ration was fed to the pigs in Lot I (controls); those in Lot II were fed the finish- ing ration plus 35 gm Tapazolelper 100 pounds of feed from 160 to 210 lbs (slaughter weight). Thirty-five crossbred (Hampshire x Yorkshire) barrows and gilts were randomly divided into five lots for experiment 2. The pigs in Lot I (con- trols) were fed a normal finishing ration. Each of the pigs in the other four lots received the same finishing rations plus the following goitro- genic compounds: 1 1-methyl-2-mercaptoimidazole. -30- -31- Lot 11 - 0.5 gm Tapazole daily for 21 days Lot III - 1.0 gm thiouracil daily for 21 days Lot IV - 0.5 gm Tapazole daily for 10 days Lot V - 1.0 gm thiouracil daily for 10 days The animals ranged in weight from 190 to 210 lbs at slaughter. Part II. Sixty market weight barrows and gilts representing four breeds (Yorkshire, Poland China, Hampshire and Landrace) were included in this phase of the study. The pigs were obtained from the Michigan Swine Improvement Station (East Lansing) and several Michigan swine pro- ducers. Slaughter weight ranged from 200 to 230 pounds. Part III. Three eXperimentS were included in this phase. In ex- periment l, fifteen Hampshire barrows ranging from 208 to 219 pounds live weight were randomly divided among three lots. The pigs in Lot 1 served as controls, while those in Lots II and III were injected intra- muscularly daily with 100 and 200 mg of prednisolone (delta-l-hydrocor- tisone), respectively, for seven days. Experiment 2 included ten Hampshire and five Yorkshire pigs weighd ing 190 to 208 pounds. The pigs in Lot I served as controls; those in Lot II were injected intramuscularly with 100 mg of prednisolone daily for 10 days. Each pig in Lot III was fed 225 mg methyl prednisolone (6-methyl-delta-1-hydrocortisone) daily for 21 days. The methyl pred- nisolone was incorporated in 2 pounds of the finishing ration. Hater teas provided at libitum at all times while feed was provided at libitum (each day but was removed at night to insure consumption -32- of the 2 pounds of feed containing methyl prednisolone each morning. The pigs in Lot II were slaughtered 24 hours after the last prednisolone injection; those in Lot III were slaughtered 5 days after the last hor- mone feeding. Nine Yorkshire and nine Hampshire barrows and gilts ranging in weight from 180 to 215 lbs were randomly divided into three lots with 6 animals per lot in eXperiment 3. The pigs in Lot I served as controls while those in Lots II and III each received 450 mg methyl prednisolone daily for 10 and 25 days, respectively. The methyl prednisolone was fed with 2 lbs of finishing ration as previously described in experiment 2. These pigs were slaughtered 3 days after the last methyl prednisolone feeding. All pigs included in this study were held off feed approxi- mately 12 hours prior to being slaughtered in the Meat Laboratory. How- ever, water was provided ad libitum to all pigs. Slaughter, Cutting and Sampling Procedure The pigs were electrically stunned, bled and slaughtered in accor- dance with conventional procedures. Blood samples (250 ml) were collected in a glass centrifuge tube containing ammonium heparin immediately after sticking. The blood was immediately centrifuged for 40 minutes at 2600 rpm in a refrigerated centrifuge (4°C). The plasma was decanted into freezing jars, sealed, and stored at -30°C until assayed. All glassware was washed with sulfuric acid and rinsed six times with deionized water. Samples of the 1, gg£§i_muscle from the 5th or 6th lumbar region were excised from the left side of the uneviscerated carcass of pigs in -33- Lots I and II of experiment 2, in part III of the study. The samples were obtained approximately 40 minutes post-mortem for the initial pH determination. The carcasses were then eviscerated, Split and placed in 4°C coolers. Samples for pH determination were removed from the_1,.§g£§i muscle each half hour for 4 hours post-mortem. Ultimate pH determination was recorded 24 hours postdmortem. The carcasses from the other experi- ments were dressed in the usual manner and chilled for 24 hours at approximately 4°C. Adrenal and Thyroid weights The adrenal and thyroid glands were removed from the carcass during evisceration, immediately trimmed of adhering tissues and weighed to the nearest 0.01 gm on the Mettler balance. CarcassIMeasurements The cutting procedure and carcass measurements obtained in part 1 were essentially as described by the Pork Evaluation Committee at the 1952 Reciprocal Meat Conference. No cutout data were obtained from pigs in the other phases of this study except loin eye area was obtained from pigs in part II. Panel Evaluation of Muscle Color and Firmness .Muscle color and firmness characteristics of the right loin and ham from pigs in part I were subjectively evaluated 24 hours postdmortem by a five member panel. The panel visually rated each loin on a five point scale as follows: very dark (5), Slightly dark (4), grayish pink (3), -34- slightly pale (2), or very pale (l). The panel scored the light ham muscles either grayish pink (3), slightly pale (2) or very pale (l) and the dark muscles as very dark (5), slightly dark (4) or grayish pink (3). Firmness scores for both ham and loin muscles consisted of firm (3), slightly soft (2) and very soft (1). Muscle Sample Preparation The section of the right 1, dgggi muscle between the 10th and last thoracic vertebrae was excised from each carcass and trimmed of adhering tissue for protein extraction. The remaining sections of the 13‘Qgggi muscle were later excised, trimmed of adhering tissues and ground five times through a 2 mm plate. The ground sample was sealed in glass jars and frozen for subsequent sodium and potassium analyses. Muscle Protein Extraction Procedure Sample preparation. The muscle samples were ground once through a 2 mm plate in a prechilled grinder to minimize heat denaturation. Samples were ground into a beaker and immediately covered with parafilm to prevent evaporation. Protein extraction. Protein solubility of the.1..dg£§iymuscle was determined by a modified method of the procedure described by Helander (1957) and Lawrie (1961). Ten grams of muscle were homogenized for 1 minute with 30 m1 cold 0.03 M potassiun phOSphate buffer at pH 7.4. An additional 70 ml of 0.03 M buffer was added and the mixture was gently stirred for 30 minutes at 4°C. It was then centrifuged at 2600 rpm for -35- 20 minutes (4°C). The supernatant was retained. The centrifugate was resuspended in 100 m1 cold 0.03 M buffer solution, stirred and centri- fuged twice more as described above. Total soluble nitrogen designated fraction (A) was determined on the combined supernatants and soluble non-protein nitrogen (NPN) designated fraction (C) from the supernatants after precipitation of the protein by 20% trichloroacetic acid. The difference (A-C) represented sarcoplasmic protein nitrogen. The residues from extraction with 0.03 M buffer were susPended in 100 ml of a cold (4°C) mixture of KI (1.1 M) and potassium phosphate buffer (0.1 M) at pH 7.4. 'The mixture was stirred gently for one hour at 4°C and then centrifuged at 2600 rpm for 20 minutes (4°C). Extraction with the KI-phOSphate buffer and subsequent centrifugation was repeated two additional times. The combined supernatants from the KI buffer ex- traction were assayed for total myofibrillar nitrogen. Nitrogen deter- minations of each fraction were made by the micro Kjeldahl procedure as outlined by A.O.A.C. (1960). Nitrogen values were exPressed as mg of protein per gram of fresh tissue assuming a nitrogen content of 16.7% (Bailey, 1937). Sodium and Potassium Analysis Muscle sodium and potassium analysis. Sodium and potassium content of the 1, g23§1_muscle was determined by flame photometry utilizing the TCA extraction procedure of'Mounib and Evans (1957) as modified by Kirton and Pearson (1963). A Beckman DU Spectrophotometer with a model 9220 flame attaclunent was used for the analyses. The potassium and sodium -36- content was calculated from a standard curve determined by plotting the percent transmittance against the parts per million (ppm) of these elec- trolytes in the standard solution. The standards contained 3, 9, 15, 22.5, and 30 ppm potassium and 0.6, 1.8, 4.5, and 6.0 ppm sodium in a 2% TCA solution. These standards were run concurrently with each group of muscle samples analyzed. Sodium was read at 589 mu and potassium at 768 mu. Plasma sodium and potassium ana1ysis. Plasma sodium and potassium determinations were made by diluting 0.1 m1 of plasma to 10 ml with de- ionized water containing 0.02% nonionic tergitol. Both sodium and potassium analyses were made from the same dilution of plasma. The Standard solution contained sodium and potassium in a ratio of 10:1. The Standards contained 5.0, 15.0, 25.0, 37.5, and 50.0 ppm sodium and 0.5, 1.5, 2.5, 3.75, and 5.0 ppm potassium. Other steps in the analysis were the same as those described for muscle sodium and potassium. l7-Hydroxycorticosteroid Assay of Porcine Plasma Extraction of 17-hydroxycorticosteroids from_porcine plasma. The 17- hydroxycorticosteroids (170HCS) in porcine plasma were determined by a modified procedure of Peterson‘ggug1. (1957). Ten m1 of plasma was care- fully added to 50 m1 of Spectral grade methylene chloride (Merk and Co.) in a 500 ml separatory funnel. Extraction was carried out by gentle rota- tion for 10 minutes. The methylene chloride plus extracted hormones were transferred to glass Stappered centrifuge tubes. The sample was washed ‘with 4 ml of cold (4°C) 0.1 N sodium hydroxide by vigorous shaking for 15 to 20 seconds. The alkali layer is then removed by aspiration. -37- Two 10 ml methylene chloride-hormone aliquots (for unknown and blank) were transferred to separate 40 ml ground glass-st0ppered conical test tubes. To the unknown tubes 0.4 m1 of phenylhydrazine-sulfuric acid- ethyl alcohol reagent was added to the methylene chloride extract; for blanks, 0.4 m1 of blank reagent (Peterson's method) was added. The tubes were stoppered, Shaken vigorously for 15 to 20 seconds and allowed to Stand for 30 minutes. The supernatant methylene chloride phase was re- moved by aspiration and the phenylhydrazine-sulfuric acid-ethyl alcohol reagent was allowed to stand at room temperature for 10 hours for maximum color development. Spectrophotometric analysis for 17-0HCS. The contents of the glass- stoppered tubes were transferred to micro cuvettes (Beckman 3.5 x 12.8 x 46.6). Absorbance of the colored productS‘wasmeasured against the acid- alcohol blank at 410 mu in a Beckman DU Spectrophotometer. Ten ml of deionized water was run through the entire procedure to serve as reagent blank and 10 ml of water containing 8 7 cortisone acetate (United State Pharmac0peia) served as the standard. The ethyl alcohol used in the study was 200 proof, USP, Rossville Gold Shield (Commercial Solvents Corp., Terre Haute, Indiana). It was found that this alcohol gave better results than that obtained by the method of Peterson 2; El: (1957). All glassware was scrupulously cleaned with soap, water and concentrated sulfuric acid and then rinsed six times with deionized water. ~38- Muscle pH A muscle sample weighing approximately 2.5 gm was homogenized for 1 minute in a waring blendor containing 25 ml 0.005 M sodium iodoacetate for the pH determinations recorded every half hour for the first four hours post-mortem. Deionized water was used for the ultimate pH reading. Duplicate pH measurements were made with a Beckman, Model G pH meter or a Corning Model 12 pH meter. Statistical Analysis Analysis of variance was determined on the data from parts I, II and III. If a significant variance ratio was calculated between lots for a Specific characteristic, Duncans Multiple Range Test was calculated and applied to the lot means. Simple correlation coefficients were determined on some data in part 11 (Steel and Torrie, 1960). RESULTS AND DISCUSSION There were no Significant sex or breed differences between treatments or the chemical and physical characteristics studied in any of the six experiments. Thus, breeds and sexes within each treatment were combined for statistical analyses for the subsequent results and discussion. Part I. The Influence of Hypothyroid Function Upon Pale, Soft, Exudative Porcine Muscle Effect of goitrogenic drugs uponyporcine muscle properties. Two goitrogens, thiouracil and Tapazole, were fed to pigs to produce hypothy- roidism in order to study its affects upon porcine muscle characteristics. In experiment 1, Hampshire pigs were fed 35 gm of Tapazole per 100 lbs of feed from 160 lbs to slaughter weight. Thirty—five gm of Tapazole were selected for experiment 1 Since Romach ggng1. (1963) reported 33.75 gm of Tapazole per 100 lbs ration completely blocked 1131 uptake by the thyroid gland of pigs. The results of this study are shown in Table l. The ham and 1,,ggggg muscles from the five pigs fed Tapazole were firm and possessed normal color. They were similar to the controls for these characteristics. Extractability of sarc0plasmic and myofibrillar protein fractions of the l. dorsi muscle was not significantly different for Tapazole treated and control pigs. Several workers (Bendall and Wismer-Pedersen, 1952; McLaughlin, 1963; McLaughlin and Goldspink, 1963b; Sayre and Briskey, 1963) have shown that porcine muscle which exhibits the PSE appearance 24 hours post-mortem possesses poor protein extractability in either high or low -39- -40- ionic strength buffers. Extractability of the myofibrillar protein frac- tion was slightly higher for Tapazole treated pigs than controls. In addition, ultimate pH values between control and treated pigs were simi- lar. Table 1. Means1 for sarcoplasmic and myofibrillar protein fractions, pH and muscle color and firmness scores of pigs fed Tapazole l. dorsi muscle characteristics Soluble Soluble sarcOplasmic myofibrillar Color Firmness Lot pgotein <_protein2 ng score3 score I Control 48.8 75.3 5.47 2.7 2.7 II Tapazole 45.2 84.2 5.54 3.0 3.0 GIAll'means are statistically non-significant. g per gm of fresh muscle. 3A five point scale was used with 1 indicating the lightest appearing muscle. 4A three point scale was used with 3 indicating the highest degree of firmness. It is interesting to note that three of the five pigs fed Tapazole showed secondary effects such as loss of appetite, interruption of growth and edema. Similar secondary effects were observed by Ludvigsen (1953) among pigs fed thiouracil prior to slaughter. Briskey (1963) reported that Poland China pigs fed thiouracil produced soft, watery ham muscles which is in contrast to the effects of the goitrogen, Tapazole, fed in this study. After observing the results of experiment 1, it was decided to com- pare the influence of Tapazole and thiouracil upon some physical and chemical properties of porcine muscle. In addition, the relationship -41- between hypofunction of the thyroid and adrenal cortex was studied since pork muscle quality as reported by Forrest ggng1. (1963) and Judge g; 31. (1959) is affected by seasonal temperature variation. These Studies in- dicate that PSE muscle structure occurs most frequently among pigs slaughtered during seasons when temperature fluctuations are pronounced or those reared when environmental temperatures are high. These condi- tions no doubt provide a stress upon the pig. The thiouracil level selected for the second eXperiment was the same as that used by Ludvigsen (1953) to produce PSE porcine muscle. Since Tapazole is considerably more potent than thiouracil in its goitrogenic activity (Premachandra ggug1., 1960), 0.5 gm of Tapazole per day was com- pared with 1 gm thiouracil per day in this study. Protein extractability. There were no significant differences in extractability of the sarcoplasmic or myofibrillar protein fractions and non-protein nitrogen fraction of the 1, d2£§1 muscle from treated or con- trol pigs, Table 2. Results were Similar to those reported for exPeriment l. The 1, d2£§1_muscle from pigs in Lot V (1 gm of thiouracil for 10 da) and Lot IV (0.5 gm of Tapazole for 10 da) yielded slightly higher quanti- ties of extractable sarcoplasmic and myofibrillar protein fractions than controls. Values for extractable sarcoplasmic and myofibrillar protein fractions of the 1, dg£§1 muscle from pigs in Lot I (controls), II (0.5 gm of Tapazole for 21 da) and III (1 gm thiouracil for 21 da) were nearly identical. No significant differences for pH or NPN values of the.1. .dg£§1_muscle were obtained between any of the five lots. From these re- sults, it appears that neither thiouracil nor Tapazole affects the muscle prOperties characteristic of the PSE condition in the 1, dorsi muscle of the pig. -42- .0woq500m mo summon ummcw0n onu wc0umo0pa0 m 5003 com: 003 S0000 uc00d mounu 0m «N .oocmo0w0am0masoc o00000c0 mua0uomummbm on :u03 memo: .uonuo some 8600 economm0m m0uamo000aw0m no: ohm um0uomnom50 mean use 5003 00000000000050 some pom mamozH 0.0 00.0 0.0 00.0 0.0 00.0 0.0 00.0 0.0 00.0 00 0.03 0mGoHpm .>< 0.0 00:00.5 0.0 nan-0.0 o.N £06 0.0 Am.m 0.0 mw.m am 0.03 p0ouhsa 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 000000 mmoaeu0m G000 0.0 0.00.0 0.0 0.00.0 0.0 00.0 0.0 00.0 0.0 0.00.0 00000 mmoam00m emu 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 00000 60omsa S000 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 000000 @00058 as; .9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 000000 o0omae Em: .0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 00000 .0 .00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 00\0e 202 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 00\0s .uoua 0060m0dooumm 0.0 0.000 0.0 0.000 0.00 0.00 0.0 0.00 0.00 0.00 ew\0e .0000 000000000002 .>op amoz .>op cmoz .>op cmoz .>op new: .>op and: mo0um0uou000050 .000 .000 .000 . .000 .000 00000000 .00 00 .00 00 .00 00 .00 00 0000000 00 00000000 00000900nu o0oumema 0000090050 o0oumdma H 000 > 000 >0 000 000 000 00 000 o0onmemH mam 0000030050 pom mw0a mo munw0oa pam0w 00so0wm new 0000050 new ammuoom 00000 was mmmaen0m o0omoa :000 new Em: «me 0zmz 00:0000000 S0muoum m0omae mo 0a00000>op pumpamum new 0mnmo2 .N o0an -43- The influence of these two goitrogens upon the ham muscles differed from those of the 1, ggggi. As expected from the results of protein extractability data, visual color and firmness scores (panel of 5 judges) of the 1, d2£§1_muscle from pigs in the five lots were not significantly different from each other. However, the light ham muscles from pigs in Lot III and V which were fed thiouracil were lighter in color than con- trols (Lot 1) or the Tapazole fed pigs (Lots 11 and IV). Furthermore, ham muscle firmness scores for pigs fed 1 gm of thiouracil for 21 days were lower than controls and significantly (P‘< .05) lower than those fed 0.5 gm of Tapazole for 21 days. These differences in muscle firmness were not observed when the two goitrogens were fed for 10 days. The dark ham muscle scores were not Significantly different between treatments. No significant difference in thyroid weight was obtained between control and treated pigs when these two goitrogens were fed for 10 days; however, a highly Significant (P'< .01) increase in thyroid weight was obtained when they were fed for 21 days. The average weight of the thy- roid gland from pigs receiving Tapazole and thiouracil for 21 days was 9.3 and 9.1 gm, reSpectively, as compared to 5.8 gm for controls. Acevedo __£.g1. (1948) reported that continued administration of thiouracil caused hypertrophy of the thyroid gland. weights of the thyroid gland indicate that both Tapazole and thioura- cil produced approximately the same degree of hypertrOphy of the thyroid; however, slight evidence of PSE musculature was observed in some ham muscles, eSpecially the gluteus medius, from thiouracil fed pigs but not from those fed Tapazole. -44- The evidence of PSE appearing musculature in hams from pigs fed thiouracil is in agreement with the findings of Briskey (1963). However, it should be pointed out that only some ham muscles seemed to be affected and no PSE characteristics were observed in the 1, §g£§1_muscle by feed- ing thiouracil. Terrill 25,31, (1948, 1950) reported that feeding thioura- cil to pigs did not Significantly alter the physical or chemical composi- tion of the carcass. Thyroid-Adrenal Relationship Feeding thiouracil for either 10 or 21 days decreased adrenal gland weight. Essentially no differences were observed when Tapazole was fed. The average adrenal gland weight from pigs in the control lot was 1.93 gm. Adrenal weights from pigs fed 1 gm of thiouracil daily for 21 days averaged 1.60 gm and those receiving the drug for 10 days averaged 1.68 gm. These values were not Significantly different from controls; however, they approached Significance (P‘< .05). Adrenal weights from pigs fed 0.5 gm of Tapazole for 10 and 21 days averaged 1.84 and 1.93 gm, reSpect- ively, which were similar to the controls. Considerable variation in adrenal gland weight was observed among pigs fed thiouracil. It appears this drug had a greater affect upon some individuals than others within the same lot. The influence of thiouracil upon pig adrenal weights is in agreement with the adrenal cortex atrOphy noted by Leblond and Hoff (1944), Baumann and Marine (1945) and Lazo-Wasem (1960) for thiouracil fed rats. Lazo- wasem (1960) also reported that 0.3% thiouracil for three weeks brought about thyroid and pituitary gland hypertrOphy as compared to non-thiouracil -45- treated rats. He reported the pituitary ACTH content of thiouracil fed rats was less than one third that of controls. These data support the hypothesis that adrenal atrophy following thiouracil administration is caused by lowered ACTH titers. IMcCarthy‘ggng1, (1959) studied the influence of several goitrogens upon the adrenal cortex of the rat. These authors reported that both thiouracil and Tapazole when fed for 12 weeks produced atrophy of the adrenal gland. Adrenal atrOphy resulting from Tapazole administration to rats as reported by McCarthy 21H31. (1959) was not observed among the pigs which received Tapazole for 3 weeks in this study. Since Forrest ggflg1. (1963) and Judge ggug1. (1959) reported seasonal influences upon PSE porcine muscle, it is interesting to note the work of Maqsood (1950). He reported that high environmental temperatures alone caused a significant decrease in rat adrenal gland weights which he indicated was probably due to the decrease in thyroid secretion rate occurring at these temperatures. Addis g£_g1, (1965) found no signifi- cant differences in adrenal weights of the pig when subjected to temper- atures ranging from 1.1 to 10.4°C. It appears from these data that high environmental temperature results in decreased adrenal gland weight but variation at lower environmental temperature (1.1 to 10.4°C) has little influence upon adrenal gland weight. These data indicate that thiouracil and Tapazole have similar goitro- genic effects upon the thyroid gland of the pig but thiouracil appears to have a greater or more rapid appearing influence upon the adrenal gland than Tapazole. This difference may eXplain why some ham muscles from pigs -46- fed thiouracil were indicative of PSE musculature and these same ham muscles from Tapazole treated pigs were firm, dry and normal in color. Carcass characteristics (Table 3) such as average fatback thickness, lean cuts, and loin eye area were quite similar between pigs in the five lots. Therefore, the degree of muscling or finish probably had little influence upon physical or chemical muscle characteristics determined in this Study. Sodium and potassium levels in the 1, dg£§1,muscle were similar among all pigs in this study. These data indicate that if thiouracil has an effect upon the adrenal gland function, the mineralocorticoids are pro- bably not the major hormones affected. Because a rapid drop in pH occurs in muscle post~mortem which ulti- mately possesses PSE musculature (Wismer-Pedersen and Briskey, 1961a; Briskey and Wismer-Pedersen, 1961a; Goldspink and McLaughlin, 1964), the adrenal glucocorticoids might be involved via their gluconeogenic effects. Therefore, the free, l7-hydroxycorticosteroids (17 OHCS) in plasma were determined. Levels of these hormones from pigs fed the two goitrogens were lower than controls; however, the values were not significantly different. It should be pointed out that it is difficult to collect blood samples from pigs without excitation. The degree and length of excitation was not completely controlled in this study. The procedure for collecting blood samples was quite uniform, but some pigs struggled more than others. The effect of excitation and struggling upon the secretion of 17 OHCS prior to collection of blood samples, therefore, may have affected these results in this study. .00500 0055 50 05008 5003505 00050000000 05000005w00 02H 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 00 000\0 .0000 00 - 0.00 0.0000 0.000 0.0000 0.00 0.0000 0.000 0.0000 0.00 0.0000 000 .000000 00 0 7 .0 0.00 0.000 0.00 0.000 0.00 0.000 0.00 0.000 0.00 0.000 000 .0000.-0 00 00 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 .00.00 .0000 000 0000 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0 0.00 0 .0000 0000 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 .00 .0000000 .>ov 5mm: .>op 5mm: .>ov 5mm: .>op 5mm: .>op 5mm: mUHumHHouomHm5o .000 .000 .000 .000 .000 00000000 00 00000000 .00 00 .00 00 .00 00 .00 00 0000000 0000050050 0000050H 0000050050 o0onmmma 0 000 0 000 >0 000 000 000 00 000 00000509 050 0000050050 000 0w05 mo momo 00 080005 550 850000005 550 850500 000058 00000 000 5000 00050 5000 x 0000550050 5005500 mo 0500500>on pump5mum 550 005002 .m 0050H -48- Part II. The Relationship of Plasma 17 OHCS Levels to Some Chemical and Physical PrOperties of Porcine Muscle There is considerable evidence indicating that a rapid post-mortem pH drOp concurrent with high temperature of the muscle (37°C or above) alters porcine sarcoplasmic and myofibrillar proteins to such a degree that diminished water binding and PSE musculature results (Ludvigsen, 1953; Wismer-Pedersen and Briskey, 1961b; Bendall and Wismer-Pedersen, 1962; Sayre and Briskey, 1963; Sayre gg‘gl., 1964; Goldspink and Mc Loughlin, 1964). The effect of sudden ante-mortem environmental temperature change has shown that cold treatment (Sayre gt 31., 1961), heat treatment (Sayre £31., 1963) and combinations of the cold and heat treatments (Kasten- schmidt ggugl., 1964) alter color and structure of porcine muscle. Since these treatments and environmental temperatures (Ludvigsen, 1953; Forrest .§£.2lr: 1963; and Judge g£“§1,, 1959) provide stress conditions upon the pig, adrenal gland weights and plasma levels of free 17 OHCS were deter- mined and their relationship to some physical and chemical pr0perties of porcine muscle was studied. Porcine muscles were subjectively divided into three groups based upon visual appearance of color, firmness and degree of exudation. Ex- amples of the ham musculature representing each group are shown in plates I, II and III. Ham.muscles, particularly the piriformis, gluteus medius and tensor fagggg'lgggg, shown in plate I are typical of the severe PSE group. Plates II and III show examples of slight PSE and normal muscu- lature, respectively. —49- o mmm 0H U>wm .0 00000 _50- Slight PSE. Plate II. .Hmeuoz .HHH mumam _ 1L 5 _ -52- Protein extractability. Significantly (P < .05) lower quantities of sarcoplasmic and myofibrillar protein were extracted from A, g2£§i_muscles of the severe PSE group than the normal group (Table 4). These data are in agreement with the results of Bendall and Wismer-Pedersen (1962); Sayre and Briskey (1963) and Goldspink and McLoughlin (1964) who reported that PSE muscle possesses poor protein extractability in both high and low ionic strength buffers. However, the above authors did not report or discuss extractability characteristics of slight PSE muscle as studied in this experiment. Slight PSE muscle extractability did not differ signi- ficantly from the other two muscle groups for myofibrillar protein, but it had a significantly (P‘< .05) higher quantity of sarcoplasmic protein than the severe PSE muscle. Although the quantity of sarcoplasmic protein extracted from the slight PSE muscle was lower than that found in the nor- mal muscle group, the difference was not significant. Table 4. Means1 and standard deviations of some chemical and physical characteristics obtained from normal pigs and those exhibiting slight or severe PSE musculature. Severe PSE Slight PSE Normal 811L111; grOUP group Chemical and physical Std. Std. Std. characteristics Mean dev. Mean dev. Mean dev. Sarc0plasmic prot.,‘ . mg/gm 39.061 6.0 50.013 10.0 54.0b 10.0 Myofibrillar prot., mg/gm 48.03 11.0 70 oa,b 20.0 95.0b 15.0 pH, _1_. dorsi 5.13a 0.10 5.35b 0.10 5.463 0.11 NPN, mg/gm 3.2a 0.5 3.78 0.7 4.3b 0.6 Plasma, 17 OHCS 7/100 m1 17.1 5.8 17.8 5.7 20.4 6.4 Adrenal weight, gm 1.98 0.32 2.04 0.45 2.07 0.32 IMeans for each characteristic with the same superscript are not signifi- cantly different from each other. Means with no superscripts are not statistically significant. -53- Considerable variability in extractability of sarcoplasmic and myo- fibrillar protein fractions was found .mmong pigs in the slight PSE group. Some l, gg£§3.muscles in this group had protein extractabilities as high as muscles in the normal group while others extracted similar to muscles classed severe PSE. This is indicated by the large standard deviation obtained for the sarcoplasmic and myofibrillar protein values in this group. These results indicate that sarcoplasmic and myofibrillar protein extractability is not entirely consistent with the three visual classifi- cations of porcine muscle as normal, slight or severe PSE. Bodwell (1964) reported that high temperature and low pH combination ‘pg£.§g_did not result in PSE muscle as eXpected from the results of Bendall and Wismer-Pedersen (1962), Sayre gt 3;. (1963), and Goldspink and Mc Loughlin (1964). However, Bodwell (1964) did find that a high temperature- low pH combination resulted in decreased water holding capacity. Thus, the proteins responsible for hydration in porcine muscle were apparently altered by this treatment. Hamm (1959) reported myofibrillar proteins account for the major water binding sites of muscle and sarcoplasmic pro- teins have a much lower capacity for water binding. It is interesting to note that several muscles which appeared firm and normal or dark in color possessed protein extractability values which were much lower than other muscles in this group. A few of these muscles had extractabilities of sarcoplasmic and myofibrillar protein fractions similar to those in the severe PSE group. While the severe PSE muscles possessed low protein extractability with both high and low ionic strength buffers the variability in protein extraction between muscles in this group was not as extreme as those of the other two groups. -54- McLoughlin and Goldspink (1963a) reported the pale color character- istic of PSE muscle may result from the masking of muscle pigment by precipitated protein (poor protein extractability). This conclusion is not in agreement with the finding of this study since low extractability values were observed even among the normal or dark colored muscles. These data indicate the difficulty encountered in objective and subjective classi- fication of porcine muscle to facilitate study of the causative factors associated with PSE muscle. McLoughlin and GoldSpink (1963) reported the change in extractability of porcine muscle proteins occurred between 45 minutes and 24 hours post- mortem. All carcasses appeared relatively normal 45 minutes after slaughter but changes in texture, color and water holding were manifest during sub- sequent cooling of the carcass. From these observations, it may be con- cluded that the alterations in PSE muscle proteins were not due to degeneration of the muscle ante-mortem, but were produced by the post- mortem changes. The processes responsible for these protein alterations are still unexplained. Bendall and Wismer-Pedersen (1962) reported that loss of protein solubility in high ionic strength buffers is due to adsorption of denatured sarcoplasmic proteins onto the myofibrils. Reduced extractabi- lity of the sarcoplasmic protein fraction in the present study was accom- panied by reduced myofibrillar protein extractability. The denatured sarc0plasmic protein (not extractable) remaining in the centrifugate may possibly have adsorbed on the myofibrillar proteins reducing their ex- tractability. It might also be postulated that reduced solubility of -55- myofibrillar proteins in PSE muscle is due to denaturation of these pro- teins by the same processes involved in denaturation of the sarcoplasmic proteins. Ultimateng. Low pH values of the l: dorsi muscle were obtained in severe PSE muscles 24 hours post-mortem. The pH reading was significantly different between all three groups. These pH values were 5.18, 5.35 and 5.46 for the severe PSE, slight PSE and normal muscle, respectively. In one instance, an ultimate pH value of 4.87 was found in a severe PSE l, .ggrgi muscle. Low ultimate pH values were also recorded in the l, ggggi muscle by Lawrie (1958) and McLoughlin and Goldspink (1963b). These workers found that low pH was associated with marked exudation. However, in the present study, considerable exudation was observed in some of the post-rigor muscle samples without the occurrence of abnormally low ulti- mate pH values. This supports the work of Wismer-Pedersen (1959), Wismer- Pedersen and Briskey (1961a) and Briskey and Wismer-Pedersen (1961a). These authors reported PSE muscle resulted when post-mortem pH fall is rapid with or without the deveIOpment of abnormally low ultimate pH. Therefore, rate of postwmortem pH fall appears to be more important than ultimate pH in the development of PSE muscle. Non-protein nitrogen. The NPN content of the l, ggggiymuscle was significantly (P'< .05) lower in the severe and slight PSE group than normal pigs. No significant difference was found between the mean NPN values for severe or slight PSE muscle. It should be pointed out that the buffer volume recovered after extraction varied between and within groups, -56- but was usually lowest for muscles from the severe PSE group. Thus the buffer solution is apparently bound in some manner to the non-extractable proteins of the homogenized muscle. These volume differences are used in calculating extractability values of the NPN fraction as well as the sarcoplasmic and myofibrillar protein fractions and may, therefore, account for some of the differences in NPN or protein fractions obtained for the three muscle groups. McLoughlin (1963) reported higher NPN values for porcine muscle with an ultimate pH of 5.5 than those with an ultimate pH of 5.05. In the same study with a different group of pigs, the NPN-pH relationship was less well defined. The physiological role of muscle NPN components is not completely understood. However, two dipeptides included in muscle NPN, carnosine and anserine, have been studied by Davey (1960). The distribution of carnosine and anserine varies.from muscle to muscle. Red muscle (rich in myoglobin) containslittle or no carnosine and anserine; whereas, white muscles such as the l, ggggi_contain a higher concentration of these di- peptides. The concentration of carnosine and anserine appears to be more closely related (inversely) to the respiratory activity of the muscle than the myoglobin concentration. The importance of the buffering capacity of these dipeptides in the physiological pH range of 6.5 to 7.5 has been assessed by Davey (1960). The dipeptides can contribute as much as 40% to the buffering capacity in this pH range in resting, living muscle. Therefore, the dipeptides are important in stabilizing pH which would fall rapidly during muscle excitation due to lactic acid accumulation. -57- From Davey's observations (1960) the lower NPN values found in the severe PSE muscle group may play a role in the rapid pH drop of the PSE muscle reported by Briskey and Wismer-Pedersen (1961a). Further work on the buffering capacity of muscle NPN components must be conducted before definite conclusions can be made. Plasma 17 OHCS levels. Plasma levels of 17 OHCS of the severe PSE group were 3.3 7/100 ml lower than the normal muscle group, but only 0.7 7/100 m1 lower than the slight PSE group (Table 4). These differences were not statistically significant, but the levels between the severe PSE and normal group were approaching significance (P'< .05). Consider- able variation (rather large standard deviation) was observed in these hormone levels, especially in the severe PSE group. Five of the twenty pigs in the severe PSE group had plasma l7 OHCS levels below 10.6 7/100 m1. No such low values were observed in the normal group. However, some plasma 17 OHCS levels in the severe PSE group were as high or higher than the average value found in the normal group. The high 17 OHCS values of some pigs in the severe PSE group and the large variation of these hormones between pigs within the other groups may be eXplained by the method used to collect the blood samples. As was pointed out in Part I, it is extremely difficult to collect a blood sample from pigs without stressing them. Adrenal gland weight, plasma 17 OHCS levels and protein extractability data from carcasses with nonmal appearing muscle are presented in appen- dix IX. Most carcasses in this group which possessed lower than average myofibrillar and sarcoplasmic protein extractability also had lower than -53- average plasma 17 OHCS levels and some had smaller adrenal gland weights. This indicates a relationship exists between the adrenal glucocorticoids and protein extractability properties of post-mortem muscle. From these data it might be observed that adrenal weight is some- what indicative of plasma l7 OHCS levels.) However, when the average adrenal weights for the three muscle groups were compared, the values appear quite similar. The average adrenal weights for pigs in the normal, slight and severe PSE groups were 2.07, 2.04 and 1.98 gm, reSpectively. It may be pointed out, however, that in all three groups, pigs with very small adrenal weights usually had lower than average levels of plasma l7 OHCS. It appears from these data that lower levels of plasma glucocorti— coids are probably associated with the ultimate physical and chemical properties of post-mortem.skeletal muscle. This concurs with the work reported by Ludvigsen (1957) and Henry'ggngl. (1958). They found that pigs with PSE muscle possessed lower levels of ACTH in the pituitary gland and concluded that a lower adrenal cortex output of glucocorticoids was involved in the PSE condition. The mode of action of the glucocorticoids in the deve10pment of PSE muscle has not been elucidated. Since lactic acid accunulates rapidly in postdmortem PSE muscle, it seems feasible that the lower plasma 17 OHCS levels might influence this phenomenon by their effect on carbohy- drate metabolism. Engle (1953) reported the ll-oxycorticosteroids inhi- bited the hexokinase catalyzed reaction. If 17 OHCS levels are low, the reaction converting glucose to glucose-6-P04 is accelerated. If this -59- occurred in PSE muscle, more pyruvate available for lactic acid produc- tion may result. Ludvigsen (1957) suggested that adrenal glucocorticoids influence PSE muscle by their vasodilatory action. Furthermore, one of the major symptoms he observed in MD pigs was skeletal muscle vasoconstriction and decreased lactic acid in ear vein blood after exercise as compared to exercised controls. Forrest (1965) reported a significant increase in PC02 and decreased pH of blood collected anaerobically from pigs with PSE musculature indicating the circulatory system is altered. Hydrocortisate has been shown to have striking influence on regula- tion of the vasomotor mechanism (Schayer, 1964). The results of these investigations indicate the adrenal cortex apparently plays a role in removing lactic acid from muscle after exercise or excitation. The lower glucocorticoid levels in the plasma of PSE pigs may play a role in the vasomotor reSponse to accumulation of lactic acid in porcine muscle after excitation. The role of adrenal glucocorticoids in amino acid metabolism and protein synthesis is of interest in discussing PSE porcine muscle. Wool and weinsheldaum (1959) and Wool (1960) found that cortisone and cortisol participate in the mobilization of endogenous protein. Kaplan and Shimizu (1963) and Kostyo (1965) reported that cortisol and other glucocorticoids increased concentrations of virtually all amino acids and urea in skeletal muscle. These authors suggested the adrenal glucocorticoids cause an appreciable delay in the response of the muscle amino acid transport pro- CESS. -60- Turner (1960) indicated that adrenalectomized animals not only have an increased protein synthesis but also have a diminished rate of pro- tein catabolism. This accounts for the lowered nitrogen excretion in fasted adrenalectomized animals. The lower levels of plasma l7 OHCS and muscle NPN observed in the severe PSE group than in the normal group of pigs indicate the glucocorticoid involvement upon pig muscle NPN is similar to that found in the rat by W001 and Wéinshelbaum (1959), W001 (1960), Kaplan and Shimizu (1963) and Kostyo (1965). Sodium and potassium levels. Results for the l, gg£§i_muscle and plasma sodium and potassium levels are presented in Table 5. The severe PSE muscle group had a mean sodium and potassium content of 397 and 4217 ppm, respectively, as compared to 387 ppm sodium and 4360 ppm potassium for normal muscle. These differences were not significant and are in agreement with the work of Briskey 93 31. (1959) but disagree with the work of Henry £3 a1, (1958). The latter author reported a large increase in sodium and a decrease in potassium in PSE muscle. It should be pointed out that they only used 12 pigs in their study. Table 5. Means1 and standard deviations of muscle and plasma sodiun and potassium levels and l, dorsi area of pigs possessing normal or slight and severe PSE muscle. Severe PSE Slight PSE Normal Chemical or group group group physical Std. Std. Std. characteristics Mean dev. 'Mean dev. Mean dev. Muscle Na, ppm 397.0 59.0 395.0 40.0 387.0 50.0 Muscle K, ppm 4217.0 361.0 4208.0 351.0 4360.0 258.0 Plasma Na, mg % 325.0 32.0 363.0 32.0 347.0 32.0 Plasma K, mg Z 25.0 3.0 27.0 4.0 27.0 4.0 .l. dorsi area, sq. in. 4.6b 0.6 4.5b 0.5 3.9a 0.5 IMeans for each characteristic with the same superscript are not signifi- cantly different from each other. Means with no superscripts are not ~61- The average plasma potassium values for the three groups ranged from 25 i 3 to 27 i 4 mg % and from 325 i 32 to 363 i 32 for sodium. These values were not significantly different and are in the normal range reported by Widdowson and McCance (1956) and Bohstedt and Grummer (1954). Carcass muscling. Area of the l, dgzsi muscle, which is indicative of total muscling in the pork carcass, was significantly (P'< .01) larger for the severe PSE group (4.64 sq. in.) than normal pigs (3.94 sq. in.). A highly significant correlation coefficient (-.43) was ob- tained between 1. dorsi area and muscle firmness score for the pigs in this study. This indicates that the more muscular pigs have a greater prediSposition to soft, exudative muscle than pigs with a lesser degree of muscling. Vitlo (1965) reported a similar relationship (r = -.44) between loin eye area and muscle firmness score. It may be that selection for the "meat-type" pig has resulted in unintentional selection for characteristics associated with development of PSE muscle. .umw msoocmunocsm 0:0 scum woumummom wacfimw> mm; was uMOm ao>guuv=xo .uoaou CH mama ma unwws may no ammoumo 93 no 309:: a 0:9 .uwm msowamunonam 0:0 5:3 .Audfixoua omoHo CH can :53 «.03 £0.30 5 .2259“ mun ”Gm.“ web so $00qu 0:... mo 309:: a one .mmmonwo on”. GM 0.5”. 10.3098 mmm mo monomonn onu mouaoflpaw .31.st AmnmmumOuonm on”. Ca m wouwcwwmopv mwglowwnlwd. ecu zfiumanoflunmm «cowuosnn HopommonEDH use up meomse vomoaxw can we nowum>uomno .mmmosmo uHHmm one CH wouoouuv on coo upsuwanumne mmm can so: ouwuumsaaw ou wowsaoaw was woman mane 0.39:: mwm fiuws mmwuuwo ”MOMS?— HNEOC fi—HH3 mmMUHMU 3.. -62- .>H wumam -63- Part III. The Influence of Exogenous Adrenocortical -like Steroids Upon the Adrenal Gland and Various Chemical and Physical PrOperties of Porcine Muscle Three separate eXperiments are included in Part III; however, the objective of each was to block adrenal secretion of glucocorticoids by administering large doses of prednisolone or methyl prednisolone and study their influence upon chemical and physical pr0perties of porcine muscle. Every attempt was made to prevent excitation of the pigs prior to slaughter. Results of the three experiments are shown in Tables 6, 7 and 8, and will be discussed together. Adrenal gland atrophy. AtrOphy was produced by administering either prednisolone (delta-l-hydrocortisone) or methyl prednisolone (6-methyl- delta-l-hydrocortisone). Both drugs significantly decreased weight (25 to 29%) of the adrenal gland, to approximately the same degree, even though levels ranged from 100 mg/day for seven days to 450 mg/day for 25 days. Ingle $5.31. (1937, 1938), Boland and Headly (1949) and several other researchers working with laboratory animals showed that prolonged treatment with corticosteroids caused atrOphy and hypofunction of the adrenal cortex. Christy gghgl. (1956) administered prednisolone and cor- tisone to humans for one to two weeks and found prednisolone to be four or more times as effective as cortisone in suppressing plasma l7 OHCS levels. The mechanism.reSponsib1e for this reaction of corticosteroids is not completely understood. The view has generally been accepted that -64- corticosteroids exhibit pituitary ACTH production (Boland and Headly, 1949) and reduced adrenal function is due to absence of the ACTH stimu- lus. However, Peion g; 3;. (1960) and Fekete and Ggrgg (1963) suggested that corticosteroids have a direct adrenal inhibitory mechanism in addition to the regulatory mechanism mediated by the pituitary gland. Plasma 17 OHCS levels. Plasma 17 OHCS were markedly reduced when adrenal atrophy was produced. In experiment 1, the plasma l7 OHCS levels from pigs injected with 100 or 200 mg prednisolone for 7 days were sig- nificantly decreased by 8.0 and 10.1 7/100 ml, respectively. The blood sample was collected 24 hours after the last injection which indicates a rapid metabolic turnover of these hormones. In experiments 2 and 3, prednisolone and methyl prednisolone were administered in various concen- trations and for longer periods of time than in the previous experiment. Injection of 100 mg of prednisolone for 10 days blocked the secretion 036 7/100 ml lower than controls) of plasma l7 OHCS to approximately the same degree as the seven day injection. Blood samples were collected 24 hours after the last prednisolone injection in these two experiments. However, when 225 mg of methyl prednisolone were fed for 21 days and the plasma sample collected 5 days after the last hormone feeding, the plasma l7 OHCS were 2.7 7/100 ml lower than controls. This indicates that the pig adrenal cortex apparently slowly regresses after prednisolone with- drawal. This is further supported by experiment 3 when 450 mg of methyl prednisolone was fed for 10 and 25 days and the blood sample collected S three days after the last prednisolone feeding. As shown in Table 8, ~65- plasma 17 OHCS levels were 5.3 and 5.0 7/100 ml lower than controls for the 10 and 25 day treated pigs, respectively. The normal level of free plasma l7 OHCS in the pig appears to be similar to those reported for humans (16 to 18 7/100 ml) by Kruger gt 31, (1965). These values are considerably higher than those found in cattle (3 to 4 7/100 ml) by Brush (1960) and Shaw -e_t_§_1_. (1960) or in sheep (0.5 to 1.0 7/100 ml) by Lindner (1959). Bush (1953) suggested that Specie differences observed in adrenocortical secretion are geneti- cally determined and at present cannot be related to any known differ- ences in adrenocortical function. Further work is needed to more fully illucidate the so-called normal levels of 17 OHCS and other adrenal hormones in porcine blood. Musclegproteins. The influence of prednisolone administration upon the extractability of muscle proteins was determined in all three experi- ments. Many physiological reactions of adrenocortical steroids have been thoroughly investigated but little is known about their effect upon muscle tissues BSELEE: Widespread degeneration of skeletal muscle following administration of massive doses of cortisone to rabbits has been noted repeatedly (Ellis, 1956; Germuth 25‘313, 1951). The quantity of sarcoplasmic and myofibrillar proteins extracted from the prednisolone treated pigs in all three experiments was not sig- nificantly different from controls. This indicates that if degeneration occurred in porcine l, g23§i_muscle from large doses of prednisolone, the degeneration is not due to decreased quantities of extractable protein. -66- The quantity of sarcoplasmic and myofibrillar proteins from pigs injected with 100 and 200 mg of prednisolone for 7 or 10 days in experiments 1 and 2 was slightly higher than controls. However, when either 225 mg or 450 mg of methyl prednisolone was fed daily for 10, 21 and 25 days, gen- erally extractability of sarcoplasmic and myofibrillar proteins was slightly lower than controls. It should be pointed out that some pigs fed 225 to 450 mg of methyl prednisolone possessed l, gg£§i_muscles which were very exudative but darker in color than normal. In addition, some 1, §2£§i_muscles from the treated pigs showed slight exudation while others appeared firm and dry 24 hours post-mortem. Table 6. Means1 and standard deviations of chemical and physical charac- teristics from control pigs and those injected with prednisolone for 7 days Lot I Lot II Lot III Chemical or control 100 mngredgldayr 200 mggPred./day physical Std. Std. Std. characteristic Mean dev. Mean dev. Mean dev. Av. adrenal wt., gm 1.963 0.2 1.45b 0.3 1.49b 0.3 Plasma 17 OHCS 7/100 m1 21.8a 6.4 13.8b 6.3 11.7b 4.2 SarCOplasmic prot., mg/gm 53.0 6.0 56.0 6.0 53.0 8.0 Myofibrillar prot., mg/gm 92.0 14.0 96.0 4.0 93.0 12.0 NPN, mg/gm - 3.7a 0.2 4.3a,b 0.6 4.4b 0.4 pH,.l. dorsi » 5.62a 0.16 5.52b 0.07 5.47b 0.04 Na in plasma, mg Z 358.0 22.0 342.0 34.0 337.0 34.0 K in plasma, mg Z 27.7 0.8 30.4 4.0 32.4 4.0 AIMeans for each characteristic with the same superscript are not signifi~ cantly different. Means with no superscript are not significant. -57- Means1 and standard deviations of chemical and physical characteristics from control pigs and those fed or injected with methyl Prednisolone Table 7. Lot I Lot II Lot III control 100 mg Pred./day 225 mg Methyl for 10 days Pred./day for 21 Chemical and days3 physical Std. Std. Std. characteristic Mean dev. ‘Mean dev. Mean dev. Av. adrenal wt., gm 2.0661 0.2 1.52b 0.2 1.530 0.3 Plasma 17 OHCS, 7/100 m1 13.461 2.9 5.8b 0.86 10.7a 3.3 Sarc0plasmic prot. mg/gm 49.0 5.0 48.0 6.0 45.0 5.0 Myofibrillar prot. mg/gm 78.0 9.0 86.0 17.0 72.0 17.0 NPN, mg/gm 4.6 0.2 4.9 0.3 4.7 0.1 pH, 1. dorsi 5.47 0.12 5.49 0.09 5.37 0.16 Na in plasma, mg Z 365.0 19.0 360.0 26.0 351.0 15.0 K in plasma, mg 7. 31.2a 1.7 33.791 5.0 24.0b 1.4 Na in l. dorsi, ppm 332.0a 16.0 369.0b 8.0 319.0c 10.0 K in.l. dorsi, ppm 4326.0 184.0 4070.0 68.0 4103.0 61.0 IMeans for each characteristic with the same superscript are not signifi- cantly different. Means with no superscript are not significant. 2Intramuscular injection. 3Incorporated with the feed. -68- Table 8. Meansl and standard deviations of chemical and physical charac- teristics from control pigs and those fed methyl prednisolone for 10 and 25 days. Control 450 mg Methyl 450 mg Methyl Pred./day for 10 Pred./day for 25 Chemical and days days physical Std. Std. Std. characteristic Mean dev. Mean dev. Mean dev. Av. adrenal wt., gm 2.21{:1 0.2 1.59b 0.16 1.53b 0.2 Plasma 17 OHCS, 7/100 m1 12.2a 1.3 6.9b 3.1 7.2b 2.9 Sarc0plasmic prot., mg/gm 48.0 5.0 45.0 5.0 49.0 8.0 Myofibrillar prot., mg/gm 89.0 14.0 78.0 9.0 87.0 7.0 NPN, mg/gm 4.5 0.4 4.5 0.5 4.4 0.1 pH, L, dorsi 5.41 0.09 5.41 0.08 5.46 0.08 Na in plasma, mg Z 346.0 16.0 344.0 8.0 344.0 9.0 K in plasma, mg Z 27.4 4.2 30.1 2.0 28.3 4.6 Na in.l. dorsi, ppm 323.03 25.0 313.0651) 40.0 288.09 27.0 K in l. dorsi, ppm 4263.0 146.0 4121.0 241 4142.0 276.0 IMeans for each characteristic with the same superscript are not signifi- cantly different. Means with no superscript are not significant. -69- Nonfiprotein nitrogen. NPN values determined on the l, dgggi muscles from pigs injected with 100 and 200 mg of prednisolone for 7 days were higher than controls. The increase was significant (P'< .05) for the 200 mg injected group. This is in agreement with the work of Kaplan and Shimizu (1963) who report that virtually all amino acids and urea in muscle of fasted and non-fasted rats were increased by administration of cortisol. When high doses of methyl prednisolone were fed to pfigs for 10 to 25 days and the muscle sample obtained 3 to 5 days after the last hormone feeding, the NPN values were similar to controls. Ryan (1963) also found a variable effect upon muscle amino acids in rats, depending on the length of administration of hydrocortisone. Twenty-four hours after injection of hydrocortisone, an increase in the free amino acids of plasma and muscle was found. These acids were slightly decreased after 10 days of treatment with hydrocortisone. Because the pigs in this phase of the study were slaughtered 3 to 5 days after the last hormone administration, it was not possible to determine if the changes in NPN values resulted from the high doses of methyl prednisolone or adrenal atrophy follows pro- longed prednisolone feeding. Muscle pH. Ultimate pH values of the 1. dorsi muscle of treated pigs were similar to those for controls. Only pH values of muscles from pigs injected with either 100 or 200 mg of prednisolone daily were significantly different from controls. Since rates of pH fall has been reported (Briskey and Wismer-Pedersen 1961a) to be associated with PSE muscle as discussed -70- in Part II and plasma glucocorticoid levels might be associated with PSE muscle, the effect of prednisolone administration upon post-mortem rate of pH fall was studied. The l, dorsi muscle from pigs injected with 100 mg of prednisolone for 10 days was compared with controls. The results are shown in Figure 1. Initial pH was taken 40 minutes post-mortem and it is interesting to note that similar initial pH readings were found between control and treated pigs, but pH of the controls was slightly higher. One-half hour later, pH of the control group dropped considerably faster than predniso- lone treated pigs, but after one hour both groups had nearly identical muscle pH (approx. 6.0) values. The major changes occurred between 1 and 2 1/2 hours after the initial pH reading. During this time, the pH of the control group drOpped considerably faster than the prednisolone treated pigs. Between 2 1/2 and 4 hours, pH values of the control group remained relatively constant; whereas, pH of the prednisolone treated pigs continued to decrease, but very slightly. The ultimate pH values of the two groups were very similar (control 5.53 and prednisolone treated pigs 5.50). The _l..dg£§i muscle appeared firm and normal to dark in color in both groups. It appears from these data that the affects of prednisolone upon skeletal muscle results in a retardation of the rate of post~mortem pH fall. It may be possible that prednisolone has an influence on the buffer systems of the muscle since higher levels of muscle NPN were obtained when prednisolone was administered. Davey (1960) demonstrated that the NPN components, carnosine and anserine, influence buffering capacity in muscle. 6.6 d» -71- X injected with 100 mg prednisolone daily for 10 days . Controls Initial pH reading was 40 min. post-mortem 5.9 4. .. l 1 ‘1. 5.8 -;> \\ .7 9+- 5.6 db ' f 5.5 «I» ‘A X - / lyr/ 5-4-‘1- ° / g . O O U l 5.3 ' T i 5.2 4- _r i 5.1 f- a I z ‘ , 5.0 O O O O 0 .0.“ O O t L -~.~‘4 . 0 Hours post-mortem from initifl pH Figure l. Post-mortem pH curve. -72- However, the buffering capacity of the NPN fraction in muscle needs further study before any definite conclusions can be made about its influence upon rate of post-mortem pH fall. Plasma and muscle sodium and potassium content. Sodium and potassium levels were determined in the l, dgggiymuscle and plasma in experiments 2 and 3 and on the plasma in eXperiment 1. When working with glucocorti- coids, it is important to keep in mind their effect upon electrolyte balance is variable. The results are affected by dosage, time of admin- istration, specie and possibly by other variables not yet elucidated (Ingle, 1950). Results with pigs in this study indicate that prednisolone affects muscle electrolytes differently than methyl prednisolone. When 100 mg of prednisolone were injected for 10 days, muscle sodium content increased significantly, but feeding 225 and 450 mg of methyl predniso- lone daily for 21 and 25 days, respectively, significantly decreased the sodium content of muscle. When 450 mg of methyl prednisolone were fed for 10 days, muscle sodium decreased but not significantly. It should be mentioned, however, that the muscle samples for sodium analyses in the 'methyl prednisolone experiment were obtained 3 to 5 days after the last administration of the drug. Adrenal atrophy definitely resulted from this treatment and was manifest when the animals were slaughtered. There- fore, it is difficult to evaluate these findings since adrenal atrophy may also have influenced the electrolyte changes. Lyster gtwgl. (1957) found a slight sodium retention with predniso- lone administration and sodium and water diuresis activity during methyl prednisolone treatment with rats. -73.. Potassium content of the 1, d2£§i_muscle was not significantly altered by prednisolone treatment. Plasma levels of sodium and potassium were within the normal range for all prednisolone and and methyl predni- solone treated pigs except the potassium level from those fed 225 mg of methyl prednisolone. Plasma potassium levels in these pigs were signifi- cantly lower than controls. No other significant differences were ob- tained. Faludi 25:31, (1964) also found that plasma electrolytes were within the normal range for dogs treaed with prednisolone and methyl pred- nisolone. Tne marked changes in muscle sodium and the normal plasma sodium values in pigs injected with these hormones indicate that these hormones function to some degree at least at the muscle level. Swingle .ggngl. (1958) indicated potent glucorticoids function in a homeostatic mechanism in salt and water balance of the body by enabling the animal to freely transfer fluid and electrolytes from one body compartment to another. It appears from the data in the present study that prednisolone and methyl prednisolone influence the sodium homeostatic mechanism in the pig by keeping plasma electrolytes near normal levels by mobilizing muscle sodium. SUMMARY The results of this study were obtained from three separate investi- gations with 148 pigs. In Part I, the influence of thiouracil and Tapa- zole feeding upon adrenal and thyroid weight, plasma l7-hydroxycorticos- teroid (17 OHCS) levels, and some chemical and physical pr0perties of the _l..dg£§i_muscle was studied. Part II included the determination of plasma sodium, potassium, and 17 OHCS levels, adrenal weights and the extracta- bility of muscle proteins, pH, and sodium and potassium content of the-l. ‘d2£§2.muscle from normal pigs and those exhibiting slight and severe PSE musculature. In Part III, the influence of exogenous prednisolone and methyl prednisolone treatment upon plasma l7 OHCS, sodium and potassium levels and several porcine muscle characteristics was observed. Tapazole and thiouracil (Part I) both produced hypertrOphy of the thyroid gland to approximately the same degree, but thiouracil had a more pronounced effect upon adrenal atrOphy than Tapazole. L, dgg§i_mus:1e pH, myofibrillar and sarcoplasmic protein extractability, non-protein nitrogen and sodium and potassium levels were quite similar between goitro- gen treated and control pigs. Thiouracil treatment, however, produced a pale, soft, exudative (PSE) condition in the ham.musc1es of some pigs; whereas, Tapazole treatment resulted in normal colored, firm appearing hams in all pigs. Plasma 17 OHCS levels were lower than controls for both Tapazole and thiouracil treated pigs, but these differences were not sig- nificant. Significantly lower quantities of sarcoplasmic and myofibrillar pro- tein were extracted from PSE musclature than normal muscle, Part II. Also, -74- -75- normal muscle possessed significantly higher NPN values than the severe PSE group. Considerable variation in the quantity of extractable protein was found between muscle samples in the slight PSE group. Plasma 17 OHCS levels from animals possessing severe PSE muscle were 3.3 7/100 ml lower than the normal group. This difference was approaching significance (P < .05). Muscle pH in the severe and slight PSE muscle was significantly lower than normal muscle. Also, a highly significant correlation coeffi- cient (-.43) was obtained between 1, goggiymuscle area and muscle firmness scores for the pigs included in this phase of the study. Pigs fed or injected with either prednisolone or methyl prednisolone (Part III), showed adrenal atrophy and lower levels of plasma 17 OHCS at the levels of administration and durations studied in these experiments. Quantities of sarcoplasmic and myofibrillar proteins extracted from gluco- corticoid treated pigs in all three eXperiments were not significantly different from controls. Daily prednisolone injection (200 mg/day) for seven days resulted in significantly higheeruscle NPN values. Marked differences in rate of post-mortem muscle pH fall were obtained between prednisolone treated and control pigs. Also, prednisolone was found to have a sodiun retaining effect upon muscle while methyl prednisolone re- sulted in muscle sodium dimunition. Neither drug significantly altered potassium level of the l, dorsi muscle. BIBLIOGRAPHY Addis, P. B., M. D. Judge, R. A. Pickett and H. W. Jones. 1965. Envir- onmental factors associated with porcine adrenal size and muscle characteristics. J. Animal Sci. 24, 127. Acevedo, R., B. S. Schweigert, P. B. Pearson and F. I. Dahlberg. 1948. Effect of feeding thiouracil to swine on the rate of gain and weight of the thyroid gland. J. Animal Sci. 1, 214. Assoc. Offic. Agr. Chemists. 1960. Official Methods of Analysis. Assoc. Offic. Agr. Chemists, Washington 4, D. 0. 9th ed. p. 643. Bailey, K. 1937. Composition of the myosins and myogens of skeletal muscle. Biochem. J. 21, 1406. Bailey, K. 1954. Structure of proteins 11. Muscle. 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Corticosteroids and accumulation of C14-1abeled amino acids and histamine by isolated rat diaphragm. Am. J. Physiol. 199, 715. APPENDIX ~86- m m an.m Nm.¢ ~.on w.wm an.a mwo m m ow.n m¢.¢ m.nm m.w¢ om.a mmo m m m¢.n mo.¢ m.~m ¢.o¢ nm.a use m m am.m Hm.¢ ¢.ow o.m¢ m¢.H mmo m m mm.m mn.m ¢.nw q.c¢ oo.N me m m Nq.n om.m n.0m m.¢¢ mn.a MNH mameqcm woumouu oaonmemH 1 HH uou m m mm.m qm.m m.mm o.wm oo.a < H spasm seepage .H xaeaoaaa Appendix II. Thyroid Study 11 -37- Wt. Wt. Breed Fibrillar Sarc0plasmic right left 1=York protein protein Tattoo Date No. Lot adr. adr. 2=Hamp mg/g mg/g Controls 07 8-12-64 301 1 1.91 2.18 1 105.62 63.99 09 8-17-64 302 l 2.18 2.14 1 109.12 56.56 07 9- 1-64 303 l 1.61 1.49 1 101.81 48.43 05 9- 1-64 304 l 1.69 1.70 1 69.56 48.81 09 9- 1-64 305 l 1.41 1.47 2 95.81 47.98 06 9- 1-64 306 1 2.41 2.43 2 92.50 50.62 05E 8-12-64 307 1 2.20 2.29 2 112.69 56.75 Tapazole-3 weeks 15 8-17-64 308 2 1.39 1.58 1 98.62 52.70 06E 8-17-64 309 2 1.85 1.97 1 109.12 62.07 15E 8-28-64 310 2 2.01 1.89 1 106.62 49.50 17E 9- 1-64 311 2 1.98 2.20 2 98.37 48.88 X28 8-29-64 312 2 2.21 2.17 2 100.25 48.81 05X 8-29-64 313 2 1.71 1.92 l 82.50 42.37 06X 8-29-64 314 2 1.96 2.23 l 96.75 45.25 Thiouracil-3 weeks 07E 8-17-64 315 3 1.65 1.74 1 117.25 62.00 08E 8-17-64 316 3 1.52 1.31 1 96.81 53.69 05E 8-17-64 317 3 1.23 1.17 1 112.00 59.88 29E 8-25-64 318 3 1.57 1.59 1 96.69 48.19 08E 9- 1-64 319 3 1.71 2.17 2 85.50 47.89 15E 9- 1-64 320 3 1.91 1.85 2 80.06 44.69 19E 9- 1-64 321 3 1.46 1.64 2 96.81 59.25 Tapazole-10 days 08E 8-28-64 322 4 1.42 1.52 1 99.62 50.56 07E 8-28-64 323 4 1.96 2.16 1 101.87 56.56 05E 8-28-64 324 4 1.88 1.66 2 114.43 58.07 18E 8-28-64 325 4 1.96 2.04 1 109.31 52.88 06E 8-28-64 326 4 1.72 1.84 2 105.50 51.50 17E 8-28-64 327 4 1.76 1.63 1 112.31 59.75 09E 8-28-64 328 4 2.01 2.24 1 108.18 52.37 Thiouracil-10 days 26 8-25-64 329 5 1.45 1.52 1 111.12 54.63 16 8-25-64 330 5 1.46 1.63 1 112.25 53.75 17 8-25-64 331 5 1.72 1.86 1 114.18 55.56 19 8-25-64 332 5 1.60 1.66 1 122.25 65.06 18 8-25-64 333 5 1.72 1.92 1 116.06 56.37 27 8-25-64 334 5 1.86 1.87 2 95.43 58.44 25 8-25-64 335 5 1.69 1.70 2 114.00 59.00 Appendix II. Thyroid Study II (continued) -88- NPN Na in K in Sex Wt. of mg/g muscle muscle l=M 17-OHCS thyroid Tattoo Date 11 4ppm. ppm 2=F pH (1/100 m1 4gland Controls 07 8-12-64 4.83 345 4416 1 5.45 16.92 5.98 09 8-17-64 3.92 363 4336 1 5.60 24.86 4.95 07 9- 1-64 5.00 356 4407 2 5.67 18.00 5.42 05 9- 1-64 4.01 379 4502 2 5.68 16.90 9.33 09 9- 1-64 4.60 376 4342 1 5.77 26.86 4.92 06 9- 1-64 4.80 424 4270 l 5.92 13.76 5.30 05E 8-12-64 3.94 380 4356 2 5.60 17.57 4.98 Tapazole-3 weeks 15 8-17-64 4.70 362 4301 1 5.60 15.92 11.94 06E 8-17-64 4.52 359 4312 1 5.55 17.90 8.92 15E 8-28-64 5.60 404 4402 2 5.80 10.90 8.05 17E 9- 1-64 4.70 392 4475 l 6.12 18.60 9.64 X28 8-29-64 4.36 354 4493 2 5.50 11.63 9.17 05X 8-29-64 4.64 345 4407 2 5.50 26.10 8.89 06X 8-29-64 4.70 336 4090 2 5.50 17.26 8.56 Thiouracil-3 weeks 07E 8-17-64 4.45 330 4406 l 5.85 14.54 11.90 08E 8-17-64 4.23 346 4468 1 5.59 17.90 10.40 05E 8-17-64 4.74 327 4363 2 5.58 17.90 12.86 29E 8-25-64 4.74 394 4372 2 5.91 18.16 6.95 08E 9- 1-64 4.70 380 4306 2 5.68 16.00 5.78 15E 9- 1-64 4.90 406 4340 1 5.67 16.85 5.81 19E 9- 1-64 4.66 402 4435 1 5.93 17.62 10.14 Tapazole-10 days 08E 8-28-64 4.72 349 4375 2 5.80 8.00 6.24 07E 8-28-64 3.62 397 4385 2 5.80 16.80 5.60 05E 8-28-64 4.91 346 4248 2 6.04 14.40 5.25 18E 8-28-64 4.82 401 4445 1 6.00 9.60 7.25 06E 8-28-64 4.79 359 4486 l 6.00 14.40 4.65 17E 8-28-64 5.63 358 4166 1 6.00 16.00 8.47 09E 8-28-64 4.60 388 4356 l 5.80 17.60 7.68 Thiouracil-10 days 26 8-25-64 4.74 356 4449 2 6.19 16.90 6.38 16 8-25-64 4.74 318 4273 2 5.92 21.98 6.85 17 8-25-64 4.74 403 4458 l 4.74 12.36 4.22 19 8-25-64 4.65 293 4290 1 6.05 13.00 7.06 18 8-25-64 4.65 338 4368 1 5.62 10.40 7.29 27 8-25-64 4.70 338 4300 1 5.90 16.80 9.22 25 8-25-64 4.74 354 4276 1 6.08 12.36 8.26 ‘ -89- Thyroid Study 11 (continued) Appendix II. Ham Ham Loin Ham firm- HESS firm- Loin dark light Loin Fat- color score muscle muscle ness eye (sq.in) Lean back (in.) score score score CUtS score Date Tattoo Controls nvnwfiwfiwfiuQunu Quqa9a9m1aququ IOA5I0,0AJAJ.1 EJ9a1anv9_q:2 O O O O C O O 1.1.1.1.1.1L1. A.A.A.A.A.A.A. rblb,b,b.b,b-b . . - . - . . 9.7a1.1.1.1.7. 1.1. .1 . . . . _ _ - RVRVQIQJQ’Q’QU Wu 7.0/7.§JQJGvRJ flvnvnvnvnvnvnv Tapazole-3 weeks nvnvnvnvanwa Raquququh.9m9. nvnvnvnvanvRv o o o o O o o QJQanQJQu959. rOnJIO,b.U.DnJ 4.A.R.9.1.9.nv 0.00... 1111111.. A.A.A.A.A.A.A. rOrOrO,b,b,b,b .._.... 7.7.Ru1.010’01 1.1.9. 9.9.9. ....... RVRVRVQIRVRVRV wuwuwunuvava .3,0.D_/MM_D,O 1.00111. nvnv Rvnvnv9-nvnv9v o o o o o o o 7—qu9a1a1.959- 43,0.3,o.3,b.3 4.1-9-olo.9.h. O O O O O O O 3.1-1.1.0.1.1-1 .% e.4.4.4.4.4.4.4 w,6,b,6,6,0,o,o . - . - . . . 1.7.7.7.RJ111-1- .111-1-9_ 1.. - . - . . . MuRvR-R-Rvoloso, a r unfififlnbnnnnnbnfi Caloocan/Russo, “MAUnunuoznv1i1- T. .4.4,b.5.4nurb o o o o o o o Quqa1u1u24&.qu Raqanunun314,b 111LnLIOAU,b.D 000.... 111.1111 8 V14.4.4.4“M.4.4 8,0,0IOIO [Orb d....... RVRVRVRVRVRVRV nv959u9.9.9.9.9. 1....... &.8.5.5.5.5n5n5 1.- O unmannnnnnnnn.n Rv7.§JRvfiv7.0’ WLU.U.U.1.U.1.U T- 9_A.5vth.fivnv o o o o o o o qqqu9-9hq49—L. A.A.flw9.nwnv9. QanQJQ.A.A.L. nvnvnvnvflvh.7. nvnvfiunvqcfivnv “5.3.1,b.4,b-i 0.00... 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qu No.m Hm.Hm mm.HHH H om.H an.H mouNHnHH mmN «and mmm o¢.q oo.no om.ooH N do.N aw.H noun nHH on mqu wad H¢.¢ nN.oo oo.HHH H o¢.N HN.N moan uHH mmo mqu mud om.m hw.nn mm.NOH H 0H.N mw.H moumNuoH MNH mama qu mn.m oo.No mm.MNH H mo.H oo.H mouNNnoH who and and mHomoE mHomse w\wE w\we w\we wwmmum Hmcmuvm Handgun 00mm oouumH aH M GH mz zmz cHououd cHououa umoH uawHu oHEmdeooumm umHHHHHHm .uz .uz macho spam .HH Henna .HHH prepaaa -91- 0HmEmm n N .mHma n H "xwmn momupamH as «pamHom I m «menu a N axuow u H "madame NH.m m N.NN Hmm qN.@N Nm.m N douHNud moo 0N.¢ m w.om mam NH.mH HN.m N dons nq Mum No.¢ m o.wN mam wo.oH o¢.m N donoNuN mmH No.¢ m o.mN 5mm Nm.mH om.m H qouoNuN mmH Nm.m m ¢.qN ONm do.ON on.m H coumNsN moo om.m m m.¢N mom wN.mH Nm.n N docmNuN mwo Nm.m m N.HN ohm oo.MH m¢.m H doumNuN mmo wm.m m m.oN Hmm we.0N Nn.m H doumHnN MNH qu.m m m.NN mam wo.NN om.n H «ouNHnN mmo oH.¢ m m.NN omm Nn.mH nN.m N doINHaN mwo wo.m m 5.0N mmm mw.NH mN.m H doumHuN moo ww.¢ m n.oN omm w¢.0N m¢.m N «onHNuH me mN.m m m.mN mHm om.wN m¢.m H «ouqHuH Na mm.¢ m o.NN on wo.NN mm.m H douanH H* m m.mm Ham «m.mN w¢.m N mouNHnHH MoN m N.wN ooq Nm.HN ¢¢.m H mouNHnHH mmN m o.HN HmN Nm.nm Hm.m H noun nHH moH No.m m o.oq cHd oo.~H mm.m H moum uHH mwo m w.oN mom aw.mH oo.m N mouaNIOH HRH mw.m m m.Hm mam ow.oN m¢.m N mouNNuoH use moum mmmcEHHm wae N we memmHm.HE 00H\A mm exam oumn oouumH who mammHm mamde momOINH cHOH cH M GH 02 moum 1- AwmscHucoov esouu auHm «HH upsum .HHH vacmqm< -92- mHmaom n N 4onE n H uxwmn oomupamH n q «psmHom n m «memm u N «xnow u H "pomumm mwmq Ham om.m oo.q¢ mo.Hn N mm.H mo.H mouHNnm wN Home ¢H¢ an.¢ wH.~m mm.Nn m n¢.N mH.N monH 1N Nmmm meq mam o¢.m 0N.n¢ mm.om q do.N N¢.N douHNuq MmH mqu mom m¢.m mm.¢q no.¢q a om.H mm.H «onHNad mwo oHoq 0mm wN.N mo.om m~.Nw a mo.N mm.N «onHNuq umH omnm mwm Nm.m Hm.nm wo.mm q mm.N Hm.N «oaHNna Moo Hnmm mom No.N om.o¢ om.mn m Hm.H NN.H «mun as mwm omom on mm.N om.oq mm.mn m mn.H mm.H dean nq mmm Hme mum HN.m oo.m¢ om.m¢ m mm.N HN.N «caoHum MNH mmnm oqm oo.¢ oo.Nm nm.¢m m oo.N mm.N «onOHum mmH mmmd Nam mm.¢ om.o¢ mn.Nm a mo.N om.N donmNuN Mme mqu owm mm.¢ Hw.m¢ wH.¢n m Nm.H ow.H «oINHuN who «qu mom MH.m om.m¢ Ho.w¢ m oo.H «m.H donHNuH MmN meme anm mm.m mn.mo mm.Hn N mm.N no.N monHmuNH who mode mmq no.¢ Hm.wo mm.ooH N m¢.N mH.N mouHmuNH mmo HHoq mam mm.m nw.om No.am N o¢.H oH.H moaNHuHH who does Non mm.m om.mm om.mw N q¢.H ¢¢.H mouNHuHH mmo ooqq qu Nm.m mm.mo oo.moH N oo.N mo.N moum uHH me mqu mod oa.N m¢.oo mm.Nw H Ho.H mm.H moumNuoH Mme News wmm nm.¢ wH.mm wH.on H 0H.N NH.N mouNNIOH Moo and Eng. lw\wE .w\wE kw\we madman Hmcmuwm Honoupm puma oouuma oHomoe 0HomDE zmz nHououd :Hououd ummH uanu a. e a. «z oaempHaouumm upaaapnnm .03 .02 agape umm nemHHm .HH spasm .>H xapepaaa -93_ onaow u N «one u H "womb momuwamH I a «pamHom u m «@803 u N «xuow u H "vooumm H¢.m N m.oN dam Nm.HH om.m H mouHNum wN wN.¢ N m.mN HHd Nm.a c¢.n H monH 1N Mmmm H¢.q N o.HN on om.MH m¢.m N douHqu mmH No.¢ N m.NN mHm dw.NN om.m N «onHNuq mmo wq.¢ N N.oN ooq dN.mH mm.m N douHqu mmH mm.m N m.oN com ON.mN N¢.m N «onHNnd moo do.¢ N w.mN Hmm do.qH NN.m N cons uq mmm HN.N N N.NN mom Nm.oH om.m H dons sq mom N n.0N own Nm.NH mN.m N douoHum mNH N m.oN 0mm Nm.0N MN.n N douoHum umH mm.m N m.mN mos om.NH mq.m H «onmNnN mNo mm.¢ N o.¢N mom mo.Hm mN.m H donNHuN who mo.q N o.wN 0mm om.NH 0m.m H donHNuH mnN Nm.¢ N o.Nm mum do.wN mN.m H monHmnNH mNo om.¢ N o.¢m mom wN.HN Hm.m N monHmuNH mmo N N.Hm mam wo.mH mH.m N monNHuHH mNo oq.q N w.mm NNm Nm.HH om.m H mouNHnHH mmo NN.¢ N N.mN MHm Nm.HN mm.m H monm aHH me oo.q N m.Hm moq oo.NH Nm.n H moumNnOH woo ww.¢ N ¢.wN mNm ww.nH mq.m H mouNNuOH moo «mum wm0:EhHm xxwe N NE memmHm HE OOH\N mal exam ouma oouumH who mammHe memmHe moMOINH aHOH aH M aH 02 00am newscauaoov macho mmm newHHm .HH spasm .>H wacomq< -94- 0HmEom n N «one n H "some momupcmH I q «pcmHom n m «new: I N «Mnow u H Atacama Mqu Nam Nw.m oo.om Hm.w¢ m mm.H mH.H mcuHNum mmm mmoq qu mo.m mN.Hm NH.q¢ N mN.H dw.H mouHNnm mH moNq 0mm oo.m Hw.Hm mm.mn m co.H mN.H mouHNum mom mend HHm m¢.m mm.~m wH.mo N dN.N HN.N monHNum om «HH Mvsum .> ancmmm< -95- 0Hmemm n N «onE I H "wows oomuwcmH u a ”wamHom I m .eEmm n N «xuow I H "madame mm.¢ H o.HN Nmm oH.oH mH.m H mouHNum mmm om.m H m.0N mwm om.w mo.m H mouHNum mH wH.m H m.Hm ooq wo.mH wN.m H monHNum mom mm.m H N.mN mmm qw.mH No.m H mouHNum 0m «HH hpoum .> prcodm< -96- momuwcmH I a «pamHom u m «MEmew m Nxhwwwm M Hupwwwmm NHH¢ Nnm «o.¢ om.oo oo.NoH N mm.H donOMum Mom mqmm mom Hm.q NN.¢¢ co.mm N oH.H «ouomnm mwm oqoq mom mH.¢ m¢.mn NN.Nm N oo.N «ouomum mNm owH¢ mmm oo.¢ mH.m¢ Hw.Hw N mm.H donomum mom mqu mmm mo.m H¢.mm No.on N ¢N.H douOMam mmm oONq can Nm.m Ho.Ho wH.ooH N Nq.H douomum MmN mmoq qqm mo.m mo.oq Nw.n¢ N qq.H demon-m mwN mqu Nwm mn.¢ NH.Ho nn.om N oo.N «muonsm mNN mmoq dam «N.q 0N.mm mm.mw N mo.H douomum woN mooq mam cq.m mw.om No.wm N mm.H dcuOMnm mmN wmo¢ mum o¢.m wN.om Nm.Hm N Ho.N «anon-m MmH onm on on.m wm.om HN.Nm N Ho.H donom:m mwo oHoq moq Nw.m mo.Q¢ mm.oN N mo.N aosomum moo oqu mwm mN.m Hm.mm Hw.om N mm.H douomum Moo ommq dun «m.m mN.om mo.NHH N HN.N «ouomum mmo Ema and w\mE w\wE w\wE mpooum Hmcouwm ouwo oouumH mHomDE oHomse zmz cHououe GHououd .uB .>¢ cH M SH oz UHEmmHeooumm HmHHHHnHm H usoeHuoaxm10COHOchpwum «HHH Mpsum .H> wacmde< -97- « mHmaom u N «onE n H uxmmc momHoGMH n a ocmHom I m «new: a N «Muow n H Homoumm m o.mm mom om.¢ m¢.m H «onomnm mam m ¢.Hm on oN.HH om.m H .douomum mom m o.mN mum oH.oH om.m H donomum mNm m m.Nm NmN do.NH o¢.n H donomum Men M m.mm Nmm Nm.mH om.m H «ouomnm mnm N o.wm NHm dw.mN o¢.m H douomum MmN N m.mN omm m¢.N mm.m H conomum mmN N w.oN Hem w¢.NH on.m H douomum mNN N m.wN on o¢.nH nm.m H douomum moN N N.mN Nom oo.oH oo.m H donomnm mmN H m.oN mNm o¢.mH om.m H douomam mmH H w.0N mom NN.NH om.m H douomum mwo H H.mN omm ¢¢.mN om.m H donomum woo H m.wN omm oo.wN oo.m H «ouomnm moo H o.wN omm ¢¢.NH oo.n H douomnm Mmo uOH N we N we memmmeHE ooH\N mm Axum puma oouume mammHa mEmde mUMOuNH mosh a. M 5.. pz AoooaHuaoov H unmeHumexm:0dOHochooum «HHH hvoum .H> xHocona< -93- 0Hm50m n N «onE u H "xomn oomuoamm n a «pamHom n m «damn I N «Maow n H "oedema NNH¢ ooN mm.¢ oo.om wH.ow N mm.H douNHuHH moH dec mom mw.¢ mn.wm mm.Nm H oN.H douNHuHH mmH oNoq mom ow.¢ mN.Hm No.om H oN.H donoHuHH moo wood «om mm.¢ mN.mq HN.NN N mm.H ¢ouoHuHH mNo moHd omm NN.¢ om.m¢ mm.oo H wH.H #ouoHuHH mno oqod me ow.¢ NH.Hm Hm.Nw N Nw.H «cum uHH mmd moms mmm mo.¢ Nw.mq NH.Nm N NN.N «cum nHH moo momq Hmm mo.¢ om.Hm mH.HN N om.H «cum nHH mnq mmmq mmm om.¢ oo.H¢ oo.No H oN.N douNNnoH mmN ooqq wmm mw.¢ mN.on Nm.Hm H Ho.N douNNuoH mNo quq com mo.¢ mm.mq HN.Nm N mm.H «oumHuoH mNH omoa Hum mw.¢ oo.Hq NH.mw N N¢.H doanuoH moH Nmmm Hmm o¢.m mn.mm No.HoH N mN.H douoNcoH moo Hood «mm NH.m wH.mm No.om N wN.H donoNuoH moo mqu dam o¢.¢ om.¢¢ o¢.wm N dn.H «ouNNuoH mNN and Bad. w\me w\wE w\w5 moooum Hmaouom mumo oouumH mHomse. 0Homse. zmz cHououd aHououm .uB .>< GH M GH mz oHEmmHeooumm HmHHHHnHm N uddEHuoaxmumaoHOmHapoum «HHH hosum .HH> xapamaaa -99- "K0 weapon-mm n a «pamHonH n m a arm-meow Nu fawowH-wne Hn H3003 we m ¢.MN mom do.MH o¢.n N douNHuHH moH m o.mN com qN.NH om.m N douNHIHH mmH m N.¢N oqm Nm.mH oo.m H «ouoHnHH moo m m.mN mNm qo.o o¢.m N donoHnHH mNo m n.oN Nmm qw.m nH.m H «osoHnHH mmo H o.mm onm NN.NH o¢.m N «cum nHH mud H o.mm mom NN.NH oo.m N «mum nHH moo H m.mN wmm om.m mm.m H qoum uHH mmq H o.mN omm om.oH om.m H douNNuoH mwN H N.Hm ohm mw.mH om.m N douNNnoH mNo N o.Nm omm ww.q om.m H doumHuoH mNH N m.mm NNm No.q om.m N doannoH moH N o.¢m owm om.o Hm.m H donoNuoH moo N m.o¢ wmm «N.o No.n N douoNuoH moo N N.oN mmm om.o mm.m N douNNnoH mNN 00H NkwE N we memmmeHE ooH\N. mm. Axum puma oouuwa memmHm mEmmHe momOnNH ovum CH M cH mz AposaHuaooV N unweHuooxmnocOHochpoum «HHH hvsum .HH> xwwcomq< -100- on80m u N «mHmE u H "moon oomuocom u d «pawHom n m «dEmm u N «Mao» n H "powwow oomd omN Nm.d oo.No oN.ooH N oo.N mouHmnm mom moNd on md.d No.md om.oN N dm.N monHoum moo mood on od.o No.od oo.No N oo.N mouoHuo mNH mmmd mNm do.d Ho.dd mN.oo H No.H mouoHum moN ommm NNm mo.d NH.md md.oN N Hd.N monN no moo ond Hum Hm.d mN.Nm No.NoH H oH.N monN no moH oomd HNN Hd.m od.md oo.HoH H No.H mouHono moo Hood HNN oN.m oN.od No.do N mm.H mouHouo mom oood NoN oH.N oo.md mN.Ho H Nm.H mouomum moo ooom NNN oo.d oo.No Ho.oo H oo.H monomum mNo NNom Ndm mm.d md.om mN.mo H oo.H moqunm moo doHd NmN Nd.m No.md md.Ho N NN.H monoouo moo MNoo ddm mo.d No.md om.om H oo.H monNHum mNo moom Nom mN.d Hm.od No.oo H No.H mouNHam moo omod Hmm mN.d Ho.Hm mo.mo H oo.H nouNHam mNH mNNd doN NN.d om.om NH.Ho H NN.H monNHnm moo mood mdN NN.d oo.Md oo.HN N oo.H monoHum mom odmd ddm om.o No.oo oo.oN N NN.H mouoHnm moo End and w\wE w\mE wxme moooum Honouom mama oouumH oHomDE mHomse zmz aHouono cHououm .uB .>< CH M :H mz oHEmdeouumo umHHHHon m nameaumaxm-paoHomHapmum .HHH Npsum .HHH> waamaad -101- onEom I N «onE.I H uxoon oomuoamm n d «ocmHom I o «oEmm I N «Muow I H "coupon H o.MN mom oN.oH od.m N mouHmuo mom H m.oN odm oN.mH om.m N monHonm moo H m.Hm Hmo No.mH Ho.m N mouoHuo mNH H N.Hm don oo.HH md.m H mouoHum moN H o.NN de oo.oH mm.m H mouN no moo H N.om mom oo.HH od.m H mouN um moH m o.oN omm do.d od.m N monHmao moo m o.NN Ndm dN.o mm.m H mouHmum mom m o.Nm dmm Nm.HH od.m H mouoonm moo o m.do ndm oo.d oo.m H monoouo mNo m m.mN oom om.o md.n N monomam moo m o.NN odm om.o om.o N monomum moo N o.mm mom NH.d om.m H mouNHum mNo N H.oN mom oo.m od.m H mouNHum moo N H.Hm Ndm oo.oH md.m H mouNHnm mNH N d.om Ndm oo.m od.m N monNHuo moo N N.oN omm NN.oH mm.n N monoHno moo N m.oN mom oo.d mN.m N oouoHum moo com lea N we memo dxHE ooH\N. mal nxoo 00mm oouumH memmHm memmHe oomOuNH moum GH M GH 1..2 AomsaHusoov m ucoEHuooxmuocoHochooum «HHH moouo .HHH> premaad -102- Appendix IX. Adrenal weights, myofibrillar and sarcoplasmic protein values and plasma l7-OHCS levels from animals possessing normal muscle Wt. right Wt. left Myofibrillar Sarc0plasmic Plasma adrenal adrenal protein protein 17-OHCS Number gms Agms mglg mg/g .7/100 ml 1 1.60 1.63 123.4 62.1 26.8 2 1.89 2.16 107.8 57.8 15.8 3 2.21 2.46 111.1 60.3 17.6 4 1.84 2.04 100.5 67.0 37.9 5 1.79 1.90 111.4 51.3 21.9 6 1.68 1.65 103.2 63.2 23.8 7 2.32 2.22 103.0 72.3 22.1 8 1.74 1.85 108.9 73.2 28.9 9 1.74 1.83 97.7 51.1 20.5 10 2.53 2.35 79.2 46.9 12.8 11 2.57 2.90 78.1 51.4 18.7 12 1.80 2.04 89.9 41.9 22.1 13 1.59 1.49 68.8 36.7 14.1 14 1.70 1.72 67.1 50.0 17.7 15 2.53 2.24 70.8 35.0 20.7 16 1.89 1.87 68.4 37.7 11.5 17 1.69 1.99 115.8 40.8 13.6 18 2.17 2.41 80.9 43.3 17.3 19 2.47 2.43 108.2 53.4 20.6 20 1.80 2.07 78.6 67.1 13.9 21 1.92 1.94 81.7 48.9 10.8 22 1.57 1.78 76.7 39.3 14.5 23 1.78 1.97 77.4 45.6 17.1 24 1.44 1.71 43.6 37.8 17.9 25 2.37 2.73 83.7 51.3 17.4 I-