ABSTRACT PHYSICAL AND BIOCHEMICAL PROPERTIES OF PORCINE MUSCLE AS AFFECTED BY SOME ADRENAL HORMDNES AND POST-MORTEM GLYCOLYTIC PARAMETERS by Elton David Aberle This study consisted of two separate investigations and included 36 market-weight pigs. The adenylic acid deaminase activity and AMP and IMP levels of porcine longissimus dorsi, gluteus medius, and rectus femoris muscles were determined in Part I. The relationship of these parameters to rate of post-mortem glycolysis and pH fall and to development of pale, soft, and exudative muscle was evaluated. In Part II, the effects of ante-mortem epinephrine injection or of prednisolone plus epinephrine injection upon some physical and biochemical pr0perties of porcine muscle were studied. Rate of pH decline, adenylic acid deaminase and phosphory- lase activities, glycogen, AMP, and IMP concentrations, Munsell value, and transmission value were determined in the longissimus dorsi muscle. Munsell value, transmission value, phosphorylase activity, and glycogen, AMP, IMP, inorganic phosphate and ATP levels were contrasted in porcine muscles with fast or slow rates of post-mortem glycolysis among the pigs in the control group. Specific activity of adenylic acid deaminase was .031, .031 and .027 in the longissfmus dorsi, gluteus medius, and rectus femoris muscles, respectively. Adenylic acid deaminase, AMP, and IMP were not signifi- cantly different between muscles. Correlation analysis indicated a low positive relationship between rate of post-mortem pH decline and adenylic acid deaminase activity of all three muscles. Elton David Aberle Rate of post-mortem pH fall, ultimate muscle pH, Munsell value and transmission value were not significantly altered by either epinephrine or prednisolone plus epinephrine injection. Epinephrine injection re- sulted in significantly higher (P < .05) total phosphorylase activity and slightly elevated phOSphorylase 3 activity and Z phosphorylase a. How- ever, neither total phosphorylase nor phosphorylase a activity was related to the rate of post-mortem pH fall or frequency of PSE muscle development. Adenylic acid deaminase specific activity was lower (P < .05) in muscle from pigs injected with either epinephrine or prednisolone plus epine- phrine. AMP levels were 1.82, 1.72, and 2.10 u.mole/g fresh muscle in control, epinephrine, and prednisolone plus epinephrine treated pigs, respectively, and IMP levels were 2.53, 2.74, and 2.54 u.moles/g, respectively. Concen- trations of the two mononucleotides were not significantly different be- tween treatments. IMP concentration was highly related to rate of pH fall. Correlation coefficients between IMP and longissimus dorsi muscle pH were -.93 and -.94 at 15 and 45 min post-mortem, respectively. Muscle from prednisolone injected pigs contained more glycogen (P < .05) at both 15 min and at 24 hr post~mortem. ‘Muscle with slow glycolysis (pH < 5.65 at 45 min) had lower Munsell values and lower transmission values than fast glycolysis muscle (pH > 5.85 at 45 min). Total phosphorylase and phosphorylase 2 activities were not different between slowly and rapidly glycolysing muscle. IMP, ATP inorganic phosphate and glycogen levels were 3.51 u moles, 1.24 u moles, Elton David Aberle 25.6 u moles, and 6.27 mg/g fresh muscle, respectively, in muscles with rapid glycolysis. In slowly glycolysing muscles, the IMP, ATP, inorganic phOSphate and glycogen levels were 1.54, 2.49, 17.4 and 10.28, respectively. IMP, ATP, and inorganic phosphate differed significantly (P < .05) between the two types of muscle. PHYSICAL AND BIOCHEMICAL PROPERTIES OF PORCINE MUSCLE AS AFFECTED BY SOME ADRENAL HORMONES AND POST-MORTEM GLYCOLYTIC PARAMETERS By Elton David Aberle A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1967 f, f, I .v , fix / 1 ’ :../ // e2 “- (gr; ‘ I ACKNOWLEDGMENT The author wishes to express his appreciation to his major professor, Dr. R. A. Merkel, for his guidance and encouragement throughout this research and for his assistance in the preparation of this manuscript. The advice of Dr. C. H. Suelter of the Department of Biochemistry in the adenylic acid deaminase assay portion of this research is grate- fully acknowledged. Appreciation is expressed to Dr. B. S. Schweigert, Chairman of the Department of Food Science, to Dr. A. M. Pearson, and to Professor L. J. Bratzler for their interest and stimulation. Special thanks are expressed to Mr. Ken Kemp for his advice and assistance with the statistical analyses. The author is indebted to the National Institutes of Health training Grant No. ES 016 for financial support during his graduate study and to the American Meat Institute Foundation for partial support of this research. The Upjohn Company, Kalamazoo, Michigan is acknowledged for their generous contribution of the prednisolone used in this study. Lastly, the author expresses his appreciation to his wife, Carrie, and to his parents and family for their support and encouragement through- out his graduate study. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . . . 4 Relationship of Post-Mertem Changes to Deve10pment of PSE Musc1e O I O O O O I O I O O I O O O O O I I O O O O O 4 Normal muscle . . . . . . . . . . . . . . . . . . . . 4 Pale, soft and exudative muscle . . . . . . . . . . . 5 Protein Extractability of PSE Muscle . . . . . . . . . . . 7 Factors Which Influence Meat Hydration . . . . . . . . . . 9 Muscle proteins and water . . . . . . . . . . . . . . 9 Divalent cations and electrolytes . . . . . . . . . ll Temperature-pH relationship to meat hydration . . . . 12 Muscle location and hydration . . . . . . . . . . . . 13 Glycolytic Enzymes and Metabolites in Relation to the Deve10pment of PSE Muscle . . . . . . . . . . . . . . l4 Phosphorylase . . . . . . . . . . . . . . . . . . . . l4 PhosPhofructokinase . . . . . . . . . . . . . . . . . 15 Lactic dehydrogenase . . . . . . . . . . . . . . . . . l6 Glycogen . . . . . . . . . . . . . . . . . . . . . . . l7 Hexose phosphates . . . . . . . . . . . . . . . . . . l9 Myoglobin . . . . . . . . . . . . . . . . . . . . . . 20 Vitamins . . . . . . . . . . . . . . . . . . . . . . . 21 ZMinerals . . . . . . . . . . . . . . . . . . . . . . . 22 Histochemical observations . . . . . . . . . . . . . . 22 Hormone Relationship to PSE Muscle . . . . . . . . . . . . 23 Physiological Observations . . . . . . . . . . . . . . . . 27 Preslaughter Factors and Prevention of PSE Pork . . . . . . 28 EHERMNTAII mn-IODS O O O O O O O O O O O O O O O O O O O O O O 31 Slaughter, Cutting and Sampling Procedure . . . . . . . . . 32 iii MUSCIE pH 0 o o o o o o o o o 0 AMP and IMP Determinations Extraction . . . . . Chromatographic separation Quantitative evaluation 5'-Adenylic Acid Deaminase Phosphorylase Assay . . . . . . Percent Reflectance and Munsell Transmission Value . . Glycogen Determination Inorganic PhOSphate . . . Adenosine Triphosphate . Statistical Analyses . . RESULTS AND DISCUSSION . . . . Part I. Part II. Prednisolone Injected Pigs . Musc1e pH 0 o o o o Munsell value and transmission value PhOSphorylase . . . Adenylic acid deaminase, AMP and IMP Glycogen . . . . . . Adenylic Acid Deaminase in Porcine Muscle Observations on Control, Epinephrine and Observations on Porcine Muscle With Fast and Slow Post-Mbrtem.Glycolysis . . . . . . . . . . . Munsell value and transmission value . Phosphorylase and adenylic acid deaminase Glycogen I O O O O O O I O C C O O O O 0 AMP, IMP, ATP, and inorganic phosphate . SUMMARY . . . . . . . . . . . iv Page 33 33 33 34 35 36 38 39 40 41 42 43 43 44 44 50 51 52 56 58 61 62 65 66 67 69 75 Page LITEme CITED 0 O O O O O O O O O O O O O O O O O O O O O 0 O 77 APPENDIX C O O O I O I O C O O O O O O O O O O I O O O C O O O O 86 Table 10 11 LIST OF TABLES Page Means and standard error of the means of adenylic acid deaminase activity, AMP, and IMP in three porcine muscles 47 Simple correlation coefficients between pH, adenylic acid deaminase activity, and AMP and IMP levels in three porcine mUSCIeS O O O O O O O O O O O O I O O O O O O O I O O O O 49 Means and standard error of the means for pH, Munsell value, and transmission value in control, epinephrine, and predni- solone plus epinephrine treated pigs . . . . . . . . . . 54 Simple correlation coefficients between Munsell value, transmission value and pH of porcine muscle . . . . . . . 54 Means and standard error of the means for total phos- phorylase, phosphorylase a and % phOSphorylase a in control, epinephrine, and prednisolone plus epinephrine treated pigs . . . . . . . . . . . . . . . . . . . . . . 57 Simple correlation coefficients between phosphorylase activity and pH, transmission value and Munsell value of porcine muscle . . . . . . . . . . . . . . . . . . . . . 57 Means and standard error of the means for adenylic acid deaminase, AMP, IMP, and glycogen in control, epinephrine, and prednisolone plus epinephrine treated pigs . . . . . 59 Simple correlation coefficients between pH, adenylic acid deaminase activity, and AMP and IMP concentrations of porcine muscle . . . . . . . . . . . . . . . . . . . . . 59 Means and standard error of the means for pH, Munsell value and transmission value in porcine muscle with fast and slow rates of post-mortem glycolysis . . . . . . . . 63 Simple correlation coefficients between pH, MUnsell value, and transmission value in porcine muscle with slow and fast rates of post-mortem glycolysis . . . . . . . . . . 66 Means and standard error of the means for total phosphory- lase, phosphorylase a, and adenylic acid deaminase in porcine muscle with fast and slow rates of postdmortem glycolysis . . . . . . . . . . . . . . . . . . . . . . . .67 vi Table 12 13 Page Means and standard error of the means for glycogen, AMP, IMP, ATP, and inorganic phOSphate in porcine muscles with fast and slow rates of post-mortem.glycolysis . . . . . . 72 Simple correlation coefficients between pH and AMP, IMP, ATP, and inorganic phosphate in porcine muscles with fast and slow rates of post~mortem glycolysis . . . . . . . . 73 vii Figure LIST OF FIGURES Page 5'-Adenosine monophosPhate standard curve . . . . . . . . 37 Chromatogram of nucleotides extracted from muscles and separated by anion-exchange chromatography . . . . . . . 46 Total phosphorylase, adenylic acid deaminase and 15 min and 24 hr glycogen in control, epinephrine, and prednisolone plus epinephrine injected pigs . . . . . . . . . . . . . 55 Rate of pH fall in muscles with slow and fast glycolysis 64 Munsell value, transmission value, total phosphorylase and phOSphorylase a in porcine muscle with slow and fast rates of post-mortem glycolysis . . . . . . . . . . . . . 68 Glycogen at 15 min and 24 hr post-mortem in porcine muscles with fast and slow glycolysis . . . . . . . . . . . . . . 70 5'-Adenylic acid deaminase, AMP, IMP, ATP, and inorganic phosphate in porcine muscles with fast and slow glycolysis 71 viii LIST OF APPENDIX TABLES Appendix Page I Adenylic acid deaminase study . . . . . . . . . . . . 86 II Control, epinephrine and prednisolone plus epinephrine injected pigs 0 O O C O O O O O O O O O I O O O O O O 87 III Simple correlations for control, epinephrine, and prednisolone plus epinephrine injected pigs . . . . . 89 IV Simple correlation coefficients for control pigs . . . 91 V Simple correlation coefficients for epinephrine injected pigs 0 I O O O O O O O O O O O I I O O O O O 93 VI Simple correlation coefficients for prednisolone plus epinephrine injected pigs . . . . . . . . . . . . . . 95 VII Sources of chemicals . . . . . . . . . . . . . . . . . 97 ix INTRODUCTION The subject of porcine muscle quality has received considerably more attention during recent years, especially among American and EurOpean workers. ‘Muscle quality can be defined as those chemical, physical and structural properties which contribute to palatability of the meat. In the past, much emphasis has been placed upon improvement of the quantita- tive aspects of porcine production such as muscle to fat ratio, longissimus .dgrgi muscle area, and wholesale cut percentages while qualitative factors were usually ignored. This may have been due to the fact that pigs are usually slaughtered at a young age and thus are expected to have acceptable eating qualities. Also, the type of processing procedures utilized influ- ence palatability attributes. With the production of leaner, heavier muscled, earlier maturing porcine animals, problems have arisen in the qualitative aspects of pork production. The meat industry has recently become aware of the pale, soft, and exudative (PSE) condition in porcine muscle. This abnormal condition is characterized by excessive loss of fluids and shrinkage during processing and the muscle is less tender and juicy. Incidence of the PSE condition in porcine muscle has been reported to be as high as 25% during some seasons of the year. Recent research has also indicated that such factors as color, firmness, and texture are related to palatability of meat. Reports by Ludvigsen (1953), Briskey and Wismer-Pedersen (1961a,b), Bendall and'Wismer-Pedersen (1962) and'MeLoughlin and Goldspink (1963b) indicate that the development of pale, soft and exudative porcine muscle -1- -2- is related to post-mortem muscle temperature and pH. The PSE condition resulted when a rapid rate of post-mortem glycolysis and pH fall occurred while muscle temperature remained high. Numerous investigations have been conducted into the chemical and biochemical processes which occur in muscle post-mortem; however, the cause of the rapid glycolytic rate and pH drop has not been elucidated. The rate of anaerobic glycolysis in skeletal muscle can apparently be regulated at several discrete metabolic steps. It has been established that epinephrine affects glycolysis by increasing the activity of the phosphorylase reaction (Cori and Illingworth, 1956; Danforth 'gtugl., 1962). It has also been suggested that alterations in the activity of phosphofructokinase may be important in regulating the rate at which hexose mon0ph03phate is converted to lactic acid (Passonneau and Lowry, 1962; Karpatkin ggngl., 1964). The activity of phOSphofructokinase is strongly influenced in giggg by changes in concentration of a variety of cellular constituents such as 5'-adenosine tri-, di- and mODOPhOSphate, inorganic phosphate, ammonium ion, potassium ion, and fructose-6-phosphate. Whether these factors are Operative in the development of PSE muscle has not been established. After consideration of these facts, this study was undertaken with the following experimental objectives: 1. To determine if there is a possible relationship between muscle adenylic acid deaminase activity, mononucleotide (AMP and IMP) levels of muscle, rate of post-mortem glycolysis and procine muscle quality. -3- To study the effects of ante-mortem epinephrine and prednisolone injection upon post-mortem chemical events, muscle properties, and enzymatic activities. To determine the muscle concentration of some cellular consti- tuents which are believed to affect glycolytic enzyme activity and thus alter glycolytic rate. REVIEW OF LITERATURE Relationship of Post-Mortem Changes to Deve10pment of PSE Muscle The muscles of living porcine animals are moderately dark in color, firm in texture, and dry in appearance (Briskey, 1964). Upon death of the animal, anaerobic conditions develop and the biochemical and phy- siological reactions which then occur largely govern the ultimate quality characteristics of the muscle. The magnitude of change is a result of rate of post-mortem.anaerobic glycolysis and pH decline and temperature of the muscle at the onset and completion of rigor mortis. Normal muscle. Wismer-Pedersen and Briskey (1961a) stated that when post-mortem anaerobic glycolysis occurred at an intermediate rate, requiring 6 to 12 hr for complete lactic acid production and 4 to 6 hr for completion of rigor mortis, the muscles were normal in color, moder- ately dry, and moderately firm. These muscles had desirable characteris- tics for fresh or processed meats. .More recently, Sayre and Briskey (1963) found that muscle color and juice retaining properties were not greatly affected if pH was above 5.7 and temperature below 35°C at rigor mortis onset. In normal muscle, approximately 28% of the total nitrogen can be extracted as sarc0plasmic protein (soluble in 0.03M potassium phOSphate, pH 7.4) and 40% as myofibrillar protein (soluble in 1.1M potassium iodide in 0.1M potassium phosphate) as reported by Sayre and Briskey (1963). Bendall ‘g£,§l. (1963) found that creatine phosphate had fallen to 50% of initial concentration at 1 hr post-mortem in normal muscle while adenosine triphosphate (ATP) level reached 50% of initial concentra- tion at 2 1/2 hr post-mortem. Normal muscle also shows a slight but gradual decrease in water holding capacity (Briskey, 1964) which quite likely is associated with the onset of rigor mortis and disappearance of ATP. This is supported by a postulation of Hamm (1960) that the minimum in hydration corresponds to the maximum in muscle rigidity. The normal drOp in pH resulting from anaerobic glycolysis also is responsible for some decrease in water retention. Hamm (1960) suggested that two-thirds of the decrease in hydration in bovine muscle is due to the loss of ATP and one-third to pH fall. Pale. soft and exudative muscle. The early work of Ludvigsen (1953) showed that extremely low pH values (5.3 to 5.5) were achieved at 45 min post-mortem in the muscles of some Danish Landrace pigs while other pigs had pH values of 6.8 to 7.0. Those muscles with low pH were abnormally light colored and quite exudative. He termed the condition "muscular degeneration". Wismer-Pedersen (1959) reported that porcine muscle, in which a very rapid fall in pH post-mortem.was recorded, possessed lower water holding capacity. Significantly lower pH values at 40 min post~mortem.were also noted by Briskey gt al. (1959a) in muscles which ultimately became PSE, even though the values were normal at death. A rapid decrease in pH to values of 5.5, and in some cases to 5.1, at 1 1/2 hr post-mortem while Inuscle temperature remained at 36 to 41°C has been observed (Briskey and ‘Wismer-Pedersen, 1961a). Concurrently with the accelerated fall in pH, Briskey and Wismer- Pedersen (1961a) found a rapid decrease in labile phOSphate compounds. Lactic acid production and liberation of ammonia were especially rapid and approached completion in the first 2 hr post-mortem. Liberation of ammonia was accompanied by a sharp reduction in ATP (Bendall and Wismer- Pedersen, 1962). The rapid liberation of ammonia and reduction of ATP can be taken as indicative of the onset of rigor mortis during the initial 2 hr period as described by Bendall and Davey (1957). Recently, Kasten- schmidt g£_§13 (1966) found that ATP level was already lower at 2 min post-mortem in pork muscle with more rapid rates of glycolysis when com- pared to muscle with slower glycolytic rates. Briskey st 31. (1962) and Bendall g£_§l, (1963) next demonstrated by means of extensibility measurements of muscle strips that muscles which became PSE exhibited a very rapid development of rigor mortis. Rigor mortis onset was reported to occur when ATP concentration reached 30% of the initial level (Bendall $531., 1963). Bendall and Wismer-Pedersen (1962) stated that all the characteris- tics of pale, soft and exudative porcine muscle could be artificially induced by allowing normal muscle to pass into rigor mortis at 37°C. This was not consistently observed by Briskey (1964). The latter author found that when one side of the carcass was chilled normally while the other side was allowed to pass into rigor at 37°C, a wide range of muscle structures and appearances was noted, regardless of the holding tempera- ture. Bodwell g; a1. (1966) reported that subjecting pork carcasses to a 37°C treatment during rigor onset did not consistently produce PSE 'muscle. Thus, it would seem.that considerable variation exists among animals in their susceptability to deve10pment of PSE muscle in response to high temperature and low pH at the onset of rigor mortis. Protein Extractability of PSE Muscle Wismer-Pedersen (1959) demonstrated a direct relationship between rate of pH fall and water holding capacity of the muscle. He also found that mmscle with low water holding capacity had reduced protein solubility in 0.6M KCl. The postulation was that denaturation of actomyosin during accelerated pH dr0p could be responsible for lower protein solubility and that the denaturation would be reflected in lower ATPase activity of PSE tissue. However measurement of ATPase activity showed no close relation- ship to water retention. Bendall and Wismer-Pedersen (1962) reported that washed myofibrils obtained from watery muscle had lower water retention at low ionic strength and lower extractability at high ionic strength than the myofibrils from normal muscle. ‘Moreover, the washed myofibrils from watery muscle had a higher protein content than similar normal myofibrils. It was the authors' conclusion that the additional protein of the watery myofibrils was de- natured sarcoplasmic protein which had been deposited upon and firmly bound to the myofibrillar protein. They indicated that the layer of de- natured sarc0plasmic protein was bound sufficiently strongly so as to make the myofibrillar proteins resistant to high ionic strength extraction and -3- low ionic strength hydration. Bendall and Wismer-Pedersen (1962) also presented histological evidence for the presence of denatured sarc0plasmic proteins precipitated upon the myofibrils. The results of Bendall and Wismer-Pedersen (1962) were corroborated by McLaughlin (1963) and MeLoughlin and Goldspink (1963b). The extracta- bility of sarcoplasmic and myofibrillar proteins from exudative post- rigor muscle was reduced at low and high ionic strengths, respectively, and the solubility change was related to attainment of low pH values soon after death. McLaughlin and Goldspink (1963a) also reported that the pale color of exudative muscle was an effect of rapid glycolysis and low pH while muscle temperature remained high. Acceptable color of post-rigor ‘muscle could be maintained if temperature was reduced to about 30°C before the pH approached 6.0 (Goldspink and McLaughlin, 1964). The solubilities of sarc0plasmic and myofibrillar proteins were de- termined by Sayre and Briskey (1963) at the time of slaughter, onset of rigor mortis, completion of rigor mortis and 24 hr after death in porcine muscles exhibiting a wide range of physiological conditions during the initial postdmortem period. Those proteins soluble in 0.03M potassium phosphate, pH 7.4 were designated sarcoplasmic. Subsequent extraction with 1.1M potassium iodide in 0.1M potassium phosphate, pH 7.4, yielded the myofibrillar proteins. ‘Muscle protein solubility was greatly altered by both the temperature and pH condition which existed at the onset of rigor mortis or during the first few hr after death. Sarc0p1asmic protein solubility at 24 hr was decreased to 55% of the value found at 0 hr in muscle groups which exhibited high temperature, > 35°C, and low pH, 5.3 to 5.5, at the onset of rigor mortis. In contrast, only a 17% reduction in sarcoplasmic solubility was observed in groups with high pH, > 6.0. It should be noted that when rigor mortis onset occurred between pH 5.7 to 5.9, and < 35°C, sarcoplasmic protein solubility was reduced only 12%. Myofibrillar protein solubility ranged from no reduction during the first 24 hr after death when pH remained high at onset to a 75% reduction in muscle with low pH and high temperature at onset of rigor mortis (Sayre and Briskey, 1963). The 24 hr pH seemed to have only a minor influence on protein solubility. ZMuscle protein solubility appeared to be one of the major factors affecting the juice-retaining prOperties of muscle. Scapes (1964) studied the solubility characteristics of myofibrillar and sarcoplasmic proteins in various induced and simulated post~mortem conditions. Sarc0plasmic proteins were readily denatured at 37°C and pH values below 6.0 and at higher temperatures more independently of pH. Denaturation of sarcoplasmic protein in gitugwas associated with decreased myofibrillar solubility in 1.0MIKC1. Starch gel electr0phoresis indicated one major and several minor proteins which were specifically denatured by conditions of low pH and high temperature; the major protein was identi- fied as ATP-creatine phosphotransferase. Factors Which Influence Meat Hydration Muscle proteins and water. Hamm (1960) compiled a very comprehensive review of the basic concepts of muscle hydration. He stated that the "true hydrationdwater" of muscle is the amount of water that attaches to -10- protein by monomolecular and multimolecular adsorption. This water amounts to about 4 to 5% of the total water in the muscle tissue and it is bound directly to certain hydrOphilic groups, which include polar groups such as carboxyl, amino, hydroxyl and sulfhydryl groups. The physical proper- ties of fixed, bound water are different from those of "free" water. The bound water has a lower freezing point, a lower vapor pressure, and a lower dissolving power than normal water. {Most of the water in muscle is free water, chemically Speaking (Hamm, 1960). It freezes at the same temperature as normal water, has the same solvent power, and does not seem tightly bound to the protein molecules. ‘MOst of the free water can be considered as mechanically immobilized by the network of cellular protein membranes and protein filaments. Hamm concluded that changes in the water holding capacity of meat are not due to any changes in the true hydration water fixed to the polar groups of the protein. Hamm (1960) further stated that in contrast to the tightly bound water, the amount of free water immobilized within the tissue is strongly in- fluenced by the spatial structure of the muscle tissue. Tightening this spatial structure or network of proteins decreases immobilized water and increases easily expressible water and that loosening the protein structure has the Opposite effect. This so-called stereo effect (Hamm, 1959) is extensively influenced by changes in protein charges, by attraction or repulsion of charged groups. Adjustment of pH to certain values on the presence of certain ions greatly affects the spatial arrangement and there- fore affects water holding capacity. Hamm (1959) defines water holding -11- capacity as the ability of meat to hold fast its own or added water during the application of any force (pressing, heating, chewing, grinding, etc.). The influence of pH on meat hydration is a typical example of the importance of protein net charge (Hamm, 1959). The isoelectric point of muscle is about pH 5.0. At this point, the net charge of muscle proteins is at a minimum. Therefore, at this pH, meat hydration also has a sharp ‘minimum. The pH of normal meat is about 5.5, which is close to the iso- electric point, and consequently water holding capacity is low. Divalent cations and electrolytes. Hamm (1959) reported that in addition to hydrogen ions, divalent cations, which are naturally present in muscle, are also important in meat hydration in spite of their relatively low concentration. Magnesium (25 mg/100 g) and calcium (5 mg/100 g) were found to decrease water holding capacity of muscle. Removal of part of the calcium from the muscle by treatment with a cation exchanger resulted in a strong increase in water holding capacity at pH values above the isoelectric point. The increase in hydration by removal of divalent cations was thought to be a typical case of the stereo effect, whereby removal of metallic crosslinkages freed more charged groups for water binding. Swift and Berman (1959) found an inverse relationship between water retention and either magnesium or calcium content in a study of the factors influencing water retention in 8 bovine muscles. Adenosine triphOSphate, an organic polyphosphate, has the ability to produce a higher water holding capacity. The post-mortem dr0p in muscle hydration is proportional to the decomposition of ATP to ADP,.NMP and -12- finally to IMP (Hamm, 1959). Hamm (1959) proposed that ATP has an effect similar to the cation exchanger. Because of its ability to form strong complexes with alkaline earth metals, ATP ties up part of the divalent ions, probably the magnesium. Breakdown of ATP releases the cations since AMP and IMP are much less able to form the necessary complexes. Hamm (1959) reported that at pH 7, the structural proteins of bovine muscle immediately post-slaughter contained less bound magnesium than those of rigor or post- rigor muscle. Sherman (1961) indicated that polyphosphates and citrate influenced water retention due to their relatively high ionic strength and to their influence on muscle pH. These salts function primarily to form strong complexes with alkaline earth metals. They eliminate divalent cations in meat, especially magnesium ions. The addition of sodium chloride increased the water holding capacity of meat quite strongly (Hamm, 1959). This effect was due to the influence of chloride ions rather than to sodium ions. Salt crosslinkages may have been split off by binding of chloride ions and there was an increase of meat hydration of both net charge effect and stereo effect. Temperature1pH relationship £2_meat hydration. Briskey and Wismer- Pedersen (1961a) found that muscles which underwent a sharp decrease to pH 5.1 at 1 1/2 hr post-mortem, and a subsequent elevation to 5.3 to 5.6 as the carcasses cooled, were exudative. Bendall and Wismer-Pedersen (1962) stated that the immediate cause of wateriness in muscle was the combined effects of high temperature (37°C) and low pH upon the muscle proteins in agreement with Wismer-Pedersen and Briskey (1961b). Bendall and Wismer- -13.. Pedersen (1962) also concluded that the depression and elevation of pH values reported by Briskey and'Wismer-Pedersen (1961a) was due to the effect of temperature on the pK's of ionizable groups of the proteins and buffering substances. Therefore, they refuted the suggestion that the depression and elevation of pH was a factor in the deve10pment of exudative porcine muscle. Bendall-ggugl. (1963) reported further on the rates of ATP and creatine phOSphate disappearance in relation to pH and onset of rigorwmortis in muscle samples removed from the carcass at slaughter and subsequently held at a constant temperature of 37°C. iMuscle which eventually became exudative showed a more rapid ATP turnover and a shorter lag period to the onset of rigor mortis. The authors could not eXplain the reason for the differences in rates and were unable to provoke more rapid changes by varying ante-mortem.treatment. It was noted that muscle which was allowed to go into rigor at a constant temperature of 37°C always became watery and pallid in appearance. The authors concluded that the reason for pale, watery muscle was a combination of high temperature and low pH and that this could be prevented by rapid cooling to below 30°C. ‘Muscle location and hydration. Swift and Berman (1959) studied water retention and associated factors in 8 bovine muscles. The results indicated that the relative order of the muscles followed a pattern with regard to water retention and pH. The serratus ventralis from the rib and chuck and the rectus abdominus from the plate and flank had the highest pH and water retention values while the semitendinosus and semimembranosus from the round had the lowest values. -14- Using a modification of the Grau-Hamm procedure for determination of free moisture, Urbin.g£.§l. (1962) reported significant variation in free moisture due to location in the cross-section of the longissimus dorsi muscle. The medial portion of the 1,.dg£§i_had significantly lower free moisture values than did the lateral portion. Topel gtflal, (1966) reported on variation in juice retaining prOper- ties between and within certain selected porcine muscles. Among the ham muscles studied, the biceps femoris showed the lowest juice retaining prOperties while the rectus femoris had the greatest variation in juice- retaining properties between proximal and distal positions within the muscle. Glycolytic Enzymes and Metabolites in Relation to the Development of PSE Muscle Phosphorylase. Because it had been suggested that phosphorylase activity could influence the rate of glycogen breakdown (Cori, 1956), Wismer-Pedersen (1959) studied phosphorylase activity in 6 psoas major 'muscles which had different rates of post~mortem.pH fall in an attempt to ascertain the reason for the accelerated lactic acid production. The fresh tissue was macerated in 0.04M phosphate buffer, pH 7.4, containing 0.001M versene and 0.02M sodium fluoride. The macerate was incubated at 37°C for 30 min with an amount of glycogen corre3ponding to 1% of the weight of the meat. An indirect relationship was found between the 45 min pH and the amount of glycogen decomposed, which suggested that phos- phorylase activity was increased in muscles with lower pH. The author -15- also postulated that increased phosphorylase activity may have been a consequence of adrenalin release, since adrenalin is known to increase phosphorylase activity (Cori and Illingworth, 1956). In a later report, Sayre 23 al. (1963b) found that total phosphory- lase activity was not associated with rate of pH decline post-mortem, glycogen level of fresh muscle or with other ultimate physical prOperties of the muscle. In addition, phosphorylase activity was not affected by excitement, fasting, or sucrose feeding of the animals before slaughter. In general, most of the phosphorylase activity was in the b_form at 10 min post-mortem (Sayre 25 $1., 1963a). However, 3 animals possessed sig- nificant quantities of phosphorylase a, No values for the ratio of phos- phorylase a to phosphorylase b were given. It has been recognized that phosphorylase b_is active in living tissue only in the presence of adenosine monophOSphate (AMP) (Stetten and Stetten, 1960). Since AMP is normally present in very low levels in living muscle, it was suggested that phOSphorylase exists in two forms, as the active and inactive functional states. However, it is noteworthy that Morgan and Parmeggiani (1963) have suggested that post-mortem levels of AMP are suffi- cient for phOSphorylase‘b_activity and that activation of the b_form appears to be an important factor in the rapid rate of glycogenolysis under anaerobic conditions. Phosphofructokinase. The implication that phosphorylation of fructose- 6-phosphate (F-6-. P) was a rate controlling step in glycolysis (Passonneau and Lowry, 1962) and the observation that phOSphofructokinase limits the rate of glycolysis in liver flukes (Mansour and Mansour, 1962) led Sayre -15- 35:31, (1963c) to investigate phosphofructokinase activity of porcine muscle extracts in relation to PSE deve10pment of the intact muscle. Activity of this enzyme was not associated with rate of post-mortem gly- colysis or with physical prOperties of the muscle. Phosphofructokinase activity was not affected by ante-mortem sucrose feeding or heat treatment. While the previous observations would seem to preclude phOSphofructokinase as a contributing factor to the development of PSE muscle, the authors recognized that the conditions of enzyme assay may not have been the same as 3.21.112 conditions. Indeed, phosphofructokinase activity _i_r_1_ 11.13 has been shown to be sensitive to a variety of cellular constituents (Mansour, 1963), among them its substrate, fructose-6-phosphate, and AMP, inorganic phOSphate, ADP, ATP and ammonium ion (Ozand and Narahara, 1964; Regen SE 3;, 1964). Lactic dehydrogenase. The data from biOpsy samples reported by Briskey and Wismer-Pedersen (1961b) indicated that there were larger pyruvic acid pools in the living tissue of normal porcine muscle than in those which had a rapid postdmortem pH decline. Only minor differences were observed in pyruvic oxidase activities between the two types of muscle. However, muscle that elicited a rapid pH fall had a slower rate of methylene blue reduction (used as a measure of lactic dehydrogenase activity). If the samples showed rapid reduction of methylene blue, they were ultimately uniform in color, dry in appearance and firm in structure (Briskey and Wismer-Pedersen, 1961b). Zinc content was significantly correlated with methylene blue reduction, lactic dehydrogenase being a zinc containing enzyme. -17- Cassens 25 El, (1963) confirmed the above results in a study of por- cine muscles of varying function. The trapezius muscle, which is considered to be more active and has a slower rate of pH fall post-mortem than does the longissimus dorsi muscle, was reported to have a higher zinc content and higher lactic dehydrogenase (LDH) activity than the longissimus dorsi muscle. Charpentier and Goutefongea (1964) observed a marked decrease in the activity of lactic dehydrogenase in exudative muscles. Comparison of LDH from normal and exudative pig muscles by starch gel electrOphoresis re- vealed 3 anodic bands much reduced in staining intensity and a fourth anodic band much increased in patterns from exudative muscles. The authors suggested that the LDH pattern might be used as a diagnostic tool on the blood p1asma of the live animal. Glycogen. The amount of glycogen in the muscle at the time of death has been generally recognized to be important in determining post-mortem physical and chemical prOperties of muscle only if: a) the glycogen is available for degradation (Lawrie, 1955) and b) the enzymes are not inhi- bited by decreasing pH (Bate-Smith and Bendall, 1949). Briskey and Wismer- Pedersen (1961a) found that muscle which exhibited rapid post-mortem glycolysis had slightly higher glycogen levels than muscle with a normal rate of pH fall. It is recognized, however, that the rate at which glycogen is degraded post-mortem has a greater influence upon ultimate muscle pro- perties than the amount present at the time of death (Sayre gt 31., 19633,b; Briskey and Wismer-Pedersen, 1961b). -13- Lawrie 25 21. (1959) reported that glycogen isolated from bovine sternocephalicus muscle after onset of rigor mortis had significantly shorter external chain length than that isolated from pre-rigor muscle. This significant shortening of outer chains was suggested to indicate g3 lyi!2_heterogeneity with respect to branching characteristics. Glycogen isolated from post-rigor horse muscle did not differ from pre-rigor glycogen. This may have physiological importance since glycogen of high 'molecular weight appears to be preferentially degraded in gigg and HE yi££2_by muscle phosphorylase (Stetten et al., 1958) and to exchange its glucose units more vigorously with the environment than glycogen of low 'molecular weight. The average chain length of glycogen from porcine muscles decreased by approximately 1 glucose residue during post-mortem glycolysis as ob- served by Sayre 25 31. (l963d). There was, however, a more severe decrease in both external and internal chain lengths of glycogen from muscles of the Chester White breed which had a slow rate of anaerobic glycolysis while the muscles of Poland Chinas (rapid rate of glycolysis) had only slightly shortened chain lengths during the post-mortem.period (24 hr). Ante-mortem sucrose feeding tended to lengthen both the internal and external chain lengths of the muscle glycogen and accelerated the rate of anaerobic glycolysis. These findings suggest that glycogen structure may be altered by nutritional as well as genetic influences and in both in- stances may influence the rate at which the molecule is degraded (Sayre 31; 11; , 1963d). -19- Kjolberg 3; 31. (1963) determined the average chain length of porcine longissimus dorsi muscle glycogen to be 15 glucose residues. ‘Muscles from Hampshire, Poland China and Chester White pigs having PSE musculature had a lower glycogen content than non-affected animals but the molecular structurasof the polysaccharides were identical. Hexose phosphates. Kastenschmidt gt El. (1966) studied the levels of various glycolytic intermediates in porcine skeletal muscles possessing fast and slow glycolytic rates. The accumulation of lactate in muscles with fast glycolysis was nearly complete within 30 min post-mortem, but in muscles with slow glycolysis, lactate continued to rise for 180 min after death. The most striking differences in glycolytic intermediates were in glucose-6-phosphate (G-6-P) and fructose-1,6-diphosphate (F-l,6- diP). ‘Muscles with rapidmanaerobic glycolysis contained higher levels of G-6-P especially during the first hr post-mortem, which was interpreted to indicate increased phosphorylation of glycogen. Higher glucose-6- phOSphate to fructose-6-phosphate ratios in rapidly glycolyzing muscle than in muscle with slow glycolysis indicated that phosPhorylation of glucose was more rapid than its isomerization under these conditions (Kastenschmidt g£_§1,, 1966). A large decrease in F-1,6-diP was observed at 15 min postdmortem.in'musc1e with fast glycolysis and this intermediate was virtually absent at 30 min after death. The explanation for this observation was a) an inactivation of phosphofructokinase by decreasing pH and b) an activation of aldolase and subsequent steps in glycolysis. -20- Myoglobin. Since myoglobin plays an important role in the color of meat, Briskey gt al. (1959a) determined the myoglobin content of various classes of ham muscles. Myoglobin values appeared somewhat lower in pale, two-toned muscles than in normal muscles, but the differences were not statistically significant (P < .05). As reviewed by Briskey (1964), Hart (1962) reported a slight but significantly lower pigment content in PSE muscles from a large group of animals in which animal age and activity werecarefully controlled. Lawrie (1960) found a virtual absence of pig- ment in the muscles of English breeds of pigs affected by'"white muscle" disease. In this investigation, pigment was determined as alkaline hematin. Meyer ‘gtual. (1963) noticed a marked difference in myoglobin concentration between PSE and normal longissimus dorsi muscles in loins obtained from a commercial packer. Some of the pigment differences may have been due, however, to drip loss during the period before analyses were conducted. Sayre £5 31. (1964) found that exudative muscle lost 10% of its weight as drip during an 8-day holding period compared to a l to 3% loss for dry, firm.musc1e. The accumulated drip contained 11 to 14% protein, the protein being sarcoplasmic in origin and thus containing sOme myoglobin. Observations by MeLoughlin and GoldsPink (1963a) implied that the pale color of exudative pork muscle was due to rapid fall of pH after death so that low pH values (5.0 to 5.6) were attained while muscle temper- ature was still high. A cold water extract of freshly killed muscle was incubated at various temperatures and pH values and the deve10pment of turbidity observed. Turbidity increased and acid hematin remaining in -21.. solution decreased under conditions of increasing temperature and de- creasing pH. Thus, the combined effects of low pH and high temperature were interpreted to have caused precipitation of previously soluble sar- caplasmic proteins. The color of the muscle myoglobin was apparently masked by the precipitated protein and some myoglobin was also apparently adsorbed onto the precipitated material and removed from free solution. Vitamins. Niacin, thiamin and riboflavin were determined in fresh and cooked gluteus medius pork muscles with extremely different physical characteristics by Meyer gt 31, (1963a). Pale, soft and exudative tissue contained about twice as much niacin (P < .005) as dark, firm and dry muscle, both fresh and cooked. Fresh, normal muscle had slightly higher riboflavin and thiamin and in the cooked form had higher thiamin. The PSE muscles lost a higher percentage of their'vitamdn.content during cooking, either by heat destruction or drip loss. The authors postulated that the niacin differences reflect a difference in the pyridine nucleotide coenzyme content of the two types of muscle and this may in turn affect post-mortem metabolism of the muscles. In a later study (Meyer 35 2i: .1963b), only 20 to 25% lower niacin levels were again found in dark colored muscle as compared to lighter colored muscle. There was a tendency for pigs which were physically more active to have lighter colored muscles. The ratio of oxidized to reduced niacin-containing coenzymes was positively correlated (P < .05) with level of antedmortem activity and appeared to be related to muscle color. Additionally, the ratio of oxidized to reduced coenzymes appeared to be related to rate of glycolysis because of its negative correlation 'with pH values during the initial 2 hr post-mortem. -22- Minerals. Ludvigsen (1953), in his reports concerning ”muscular degeneration" in hogs, found that potassium levels in the blood serum were elevated to 50 to 60 mg/100 ml in animals which had died of the disease. Normal pigs had serum potassium levels of 16 to 20 mg/100 ml. Along with the rise in serum potassium, there was a decrease in sodium. Normally, sodium content ranges between 400 to 550 mg/100 ml, however, values from pigs affected with muscular degeneration were 300 to 360 mg/100 ml. Briskey SE al, (1959a), in a study of muscles of various classes, noted no difference between PSE and normal muscles in potassium content. However, PSE muscles contained significantly greater amounts of sodium (P < .05) than normal muscles. In a study of 60 market weight pigs, Tapel (1965) observed no differences in either muscle or blood plasma levels of sodium or potassium which were related to muscle quality. Severe PSE muscle 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 (T0pel, 1965). Cassens gt 31. (1963) observed marked differences in zinc content between active (dark) and inactive (light) muscles of individual animals; however, there was no variation in zinc content among muscles that exhi- bited fast or slow glycolysis and consequently became light or dark. The trapezius (dark) muscle contained 182 ppm zinc and the longissimus dorsi (light) muscle 69 ppm. Histochemical observations. Bodwell st 31. (1965) studied the effects of post-mortem heat or cold treatments by histochemical methods. UDPG- glycogen transferase (UDPG = uridine diphosphate glucose), phOSphorylase, -23- and branching enzyme activity were evaluated. No relationship was apparent between enzyme activity and ultimate muscle condition in muscles chilled rapidly immediately after slaughter. There was no detectable UDPG-glycogen transferase activity present at 3 hr post-mortem in muscles held at 37°C. The authors concluded that this enzyme was completely inhibited by condi- tions of low pH value and high muscle temperature. Heat treatment had a highly variable effect upon phosphorylase and branching enzyme activity. Hormone Relationship to PSE Muscle Ludvigsen (1953) cited factors which indicated the significance of hypothyroid function in the pathogenesis of "muscular degeneration" in pigs in Denmark. First, a pronounced seasonal variation in the frequency of the disease was demonstrated with a higher number of cases during the summer, when thyroid function is normally lower, and a lower frequency during the winter, when the activity of the gland is higher. Secondly, the pathological condition was produced experimentally in carcasses by blocking the thyroid gland with methyl-thiouracil. Feeding l g of this substance daily for 10 days prior to slaughter resulted in the development of PSE musculature. Experiments also showed that the muscle changes characteristic of "muscular degeneration" could be prevented by feeding iodinated casein containing approximately 3% free Thyroxin at a daily dose level of 2 g for 10 days starting 3 weeks prior to slaughter (Ludvig- sen, 1953). The author also found an increased incidence of PSE in Danish pigs subjected to forced exercise and excitement immediately preslaughter and a 75% reduction in the incidence if a 24 hr rest period was allowed before slaughter. -24- In a later report (Ludvigsen, 1957), a lowered function of the adrenal cortex in the deve10pment of PSE muscle was postulated since the cortico- trOphin content of the anterior lobe of the pituitary was found to be lower than that of normal pigs. Moreover, Ludvigsen (1957) found that in contrast to normal pigs, pigs with "muscle degeneration" showed no increase in lactic acid level in the blood during moderate exercise. He postulated this was due to vasoconstriction in the musculature. Pretreatment with hydrocortisate increased the lactic acid output in the blood to normal levels and no symptomatic reactions occurred during exercise. The author concluded that adrenal cortical hormones play a key role in the complex of reactions governing vasodilation in skeletal muscles and suggested a possible antagonistic reaction between adrenalin and cortisone. More recently, Briskey (1964) reported higher thyroid to adrenal gland ratios in pigs that ultimately deve10ped PSE muscle. The larger thyroid glands also showed histological evidence of hypo-activity. Tepel and Merkel (1966) studied the effects of exogenous goitrogens upon some pro- perties of porcine muscle. Both methyl-thiouracil and Tapazole caused hypertrOphy of the thyroid gland to approximately the same degree. Longissimus dorsi muscle pH, protein solubilities and non-protein nitrogen, sodium and potassium levels of the muscle were not affected by the goitro- gens. However, some methyl-thiouracil treated hogs deve10ped slightly pale, soft and watery muscles. While goitrogen treatment lowered the adrenal gland weights and plasma l7-hydroxycorticosteroid levels, the differences were not statistically significant. The authors concluded -25- that goitrogenically-induced hypothyroidism_pg£;§g.apparently had little effect upon the ultimate physical and chemical prOperties of porcine ‘muscle, in disagreement with Ludvigsen (1953). However, development of PSE musculature may be the indirect result of secondary effects of methyl- thiouracil upon the adrenal gland (Tapel and Merkel, 1966). Following injection of iodine-131, Judge ggugl. (1966) found a signi- ficantly higher percentage of the plasma iodine-131 present as protein- bound iodine-131 in pigs which ultimately became pale, soft and exudative compared to pigs with normal muscle. Thyroid iodine-131 was slightly lower in the PSE group. The authors could find no differences in urinary excretion of catecholamines, l7-ketosteroids, or 17-hydroxycorticosteroids when animals were grouped according to muscle color and structure and rate of pH fall. However, among the Poland China barrows in the study, in which the muscles varied from PSE to normal, positive correlations were found between 17-ketosteroids and gluteus medius quality score (r = 1.74; P < .05) and between l7-hydroxycorticosteroids and the length of delay phase to onset of rigor mortis (r = ~.76; P < .01) and the total time to completion of rigor mortis (r = .64; P < .05). Judge SE El: (1966) im- plied that pigs which rapidly develop the changes associated with rigor mortis in PSE muscle also have elevated blood levels of protein-bound iodine, depressed thyroid iodine uptake, and deficiencies in adrenocortical hormone production. Histological examination of porcine adrenal glands revealed two types of material which were stained by Sudan black B: 1) fine granules and 2) large masses (Cassens gtugl., 1965). Of special interest was the frequent appearance of large masses of sudanOphilic material localized in the zona -26- reticularis of adrenal cortices from Poland China animals (rapid glycoly- sis). The large masses of lipid were seldom seen in the glands from Chester White pigs (slow glycolysis). Although the border between the zona reticularis and zona fasciculata was not clearly defined, the large lipid masses, when present, always extended outward from the clearly de- fined border of the medulla. Cassens 2E 31. (1965) did not believe this phenomenon was due to pigment accumulation since staining with hematoxylin and eosin revealed no apparent accumulation of granules or pigment. It was suggested that the presence of large lipid masses might represent a degenerative change. Tepel 25 El- (1967) found that plasma levels of free l7-hydroxycorti- costeroids were 3.3 ug/100 m1 lower in pigs which were ultimately classified as severe PSE than in pigs classified as normal. This difference approached significance (P‘< .08). A considerable range in plasma l7-hydroxycorti- costeroid levels was observed among animals within each group and was attributed to the method of collection of the blood sample and the stress induced during collection. The authors also recognized that plasma levels of 17-hydroxycorticosteroids are not an accurate measure of adrenal secre- tory activity over a period of time but only reflect plasma levels at the time of sampling. Topel gtugl, (1967) stated that plasma glucocorticoid levels were associated with the ultimate physical and biochemical prOperties of post-mortem skeletal muscle, in agreement with Ludvigsen (1957). Tapel and Merkel (1967) also reported that feeding or injection of either prednisolone or methylprednisolone in pigs produced adrenal gland atrOphy and significantly lowered plasma levels of l7-hydroxycorticosteroids. -27- However, injection of prednisolone or methylprednisolone did not result in the deve10pment of PSE musculature in any of the animals or in fast rates of pH fall post-mortem. Prednisolone treatment caused a slight sodium retention in the muscle while methylprednisolone resulted in sodium dimuni- tion. Neither steroid affected longissimus dorsi muscle potassium content. Bendall and Lawrie (1962) reported on the effect of ante-mortem in- jection of various drugs on post-mortem changes in muscle. Injection of adrenalin 4 hours before slaughter significantly depleted muscle glycogen in rabbits and raised ultimate muscle pH. The accepted exPlanation for this effect is that adrenalin causes activation of phosphorylase so that glycolysis proceeds more rapidly along the anaerobic pathway to lactic acid (Cori and Illingworth, 1956). However, cortisone injection 20 hours before adrenalin administration completely protected the animal against the adrenalin effect without itself altering the normal ultimate pH or rate of chemical changes in the muscles. Physiological Observations Forrest (1965) reported on the effect of antedmortem heat treatment upon heart rate, respiration rate, partial pressures of oxygen (P02) and carbon dioxide (P002) in the blood, and blood pH of pigs and also the relationship of these factors to post-mortem muscle quality. Heat treat- ment caused a sharp rise in heart rate in pigs of the Poland China breed, a significant increase in P002, a sharp decrease in P02 and a fall in blood pH from 7.2 to 6.8. A sharp rise followed by a fast decline similar to respiratory arrest was noted in respiratory rate. In Chester White pigs, .Sfo. -28- heat treatment resulted in a slight elevation in heart rate, a continuous increase in respiration rate, decreased PCO2 and a slight increase in P02. The longissimus dorsi muscles of all treated Poland Chinas were extremely PSE at 24 hr post~mortem.while all Chester Whites had normal muscles. The author concluded that circulatory and respiratory difficulties leading to increased blood P002 and decreased P02 could be major contributors to the production of PSE muscle, particularly if the animals were subjected to warm temperatures and stress immediately pre~slaughter. Preslaughter Factors and Methods of Prevention of PSE Pork Briskey ggnal. (1959b,c) reported that exhaustive exercise immediately before slaughter significantly decreased initial muscle glycogen and in- creased 24 hr pH values. The muscles from exercised pigs were dark in color, dry and firm in appearance and possessed relatively low eXpressible water ratios. In contrast to this response, hogs which were placed on high sucrose rations and given no exercise had muscles that were pale, soft in structure and exudative in appearance. Sayre 25 El. (1961) placed marketdweight swine in cold water (0.5°C) for 30 to 40 min to simulate an environmental change. The extreme change from a warm to cold environment decreased the initial muscle glycogen level with a resultant decrease in lactic acid concentration and increase in color intensity of the chilled muscle. However, water binding capacity of the chilled muscle was not consistently affected. The authors concluded that factors other than fresh muscle glycogen content, chilled pH and rate of glycolysis were important in determining the water binding capacity of muscle. -29- Increased muscle temperature and a rapid rate of post-mortem glycoly- sis resulted upon subjecting pigs to elevated environmental temperatures immediately before slaughter (Sayre 35 31,, 1963c). Heat treatment produced decreased color intensity of the muscle and a marked acceleration of rigor mortis onset. Short-term excitement and exercise immediately prior to slaughter resulted in muscles with low color and texture scores and in- ferior water binding pr0perties (Sayre g5 al., 1963b). Long- and short- term sucrose feeding elevated the muscle glycogen level at slaughter and ultimately resulted in muscle that was slightly soft and pale. Fasting 70 hours prior to slaughter lowered the initial glycogen content of muscle and also slowed pH decline and color change during post-mortem glycolysis. Kastenschmidt_25 El. (1964) studied the effects of regulation of ante- mortem.temperature upon porcine muscle quality. Treatments were 1) warm (42-45°C for 30-60 min), 2) cold (1-3°C water for 30 min), 3) warm to cold, and 4) cold followed by warm. The warm treatment was reported to induce extremely pale, soft, exudative tissue with a marked loss of protein solu- bility. The most desirable muscles were produced by the warm to cold treatment, the muscles being dark, dry and firm. The other two treatments had less marked effects. In a later, more comprehensive study, Kasten- schmidt 25 31, (1965) found that mild warm air followed by cold air treat- ments, which were not severe enough to influence muscle temperature at slaughter, slowed the glycolytic rate and were most effective in improving muscle quality parameters. Borchert and Briskey (1964) removed the entire loin or the ham from the carcass immediately after slaughter and immersed them in liquid nitrogen -30- for varying lengths of time. Liquid nitrogen treatment was quite effective in preventing the development of PSE musculature and in improving water holding capacity and subjective color and structure scores. Equilibration temperatures, either 4 or -18°C, had no effect on the results. Borchert and Briskey (1965) also reported that liquid nitrogen treatment resulted in higher sarcoplasmic and myofibrillar protein extractability and also improved the emulsifying prOperties of the muscle proteins. EXPERIMENTAL METHODS This study was divided into two separate investigations. Part I consisted of a study of the adenylic deaminase activity of porcine muscle and its relationship to rate of post-mortem pH decline and visual prOper- ties of the muscle. Fourteen market weight (200 to 220 1b) Poland China pigs, purchased from a local breeder, were included in this study. Enzymatic activity and pH fall were determined in the longissimus dorsi, (gluteus medius and rectus femoris muscles since these muscles often vary considerably in quality within the same carcass. In addition, AMP and IMP levels were determined on samples obtained from the longissimus dorsi and gluteus medius muscles at slaughter. AMP and IMP were determined only on the first 8 animals slaughtered. In addition to the determination of adenylic acid deaminase activity and AMP and IMP levels, part II of the investigation also included deter- mination of phosphorylase activities, glycogen, inorganic phOSphate, and ATP levels as related to glycolytic rate and some objective indicators of muscle quality. Twenty-two market weight Poland China pigs were again obtained from a local breeder. The animals, most of which were gilts, were divided randomly among three treatment groups. The first group, including 8 pigs, served as controls. Each of the eight pigs included in group 2 was injected intramuscularly (biceps femoris) with 5 mg of ep- inephrine (1:1000 solution) per 100 lb body weight 10 min prior to slaughter. The third group of 6 pigs each received a daily intramuscular (biceps femoris) injection of 100 mg of prednisolone (delta-l-hydrocortisone) -31- -32- for 10 days prior to slaughter. The pigs were slaughtered 24 hr after the last prednisolone injection. Ten min prior to slaughter, the pigs in group 3 also received an intramuscular injection of epinephrine as previously described for group 2. Slaughter, Cutting and Sampling Procedure Animals were transported to the MSU Meat Laboratory and allowed to rest 4 to 5 hr prior to slaughter. water was provided ad libitum but feed was withheld during this period. The pigs were electrically stunned, bled and dressed according to conventional procedures. In part I of the study, 'muscle samples were obtained at 10 min post-mortem from the lumbar region of the longissimus dorsi and from the gluteus medius and rectus femoris muscles of the ham. Samples were frozen in liquid nitrogen immediately after excision from the carcasses. iMuscle pH was determined on samples of the longissimus dorsi at 15 min, 45 min and 1 hr post-mortem and at 30 min intervals thereafter until 3 hr post-mortem. Only the longissimus .QQEEE muscle was sampled in part II of the study. 'Muscle samples for analysis and pH determination were obtained and prepared as described in part I. After a 24 hr chill period, all carcasses were separated into wholesale cuts according to procedures outlined by the Pork Carcass Eval- uation Committee of the Reciprocal Meat Conference (1952). In part II, a l in. thick chap was removed from the 10th rib region of the loin (longissimus dorsi muscle) for determination of color and transmission value and then was frozen for subsequent 24 hr glycogen determination. -33- All frozen muscle samples in part II were powdered before analysis. Powdering was performed with pre-chilled equipment in a -20°C cold room. The frozen tissue block was chipped into smaller pieces and placed in a waring blender with an approximately equal weight of dry ice. The blender was operated at full speed until the sample was finely powdered, usually 30 to 60 sec. The dry ice was then allowed to sublime from the powdered tissue after which the powder was stored at -20°C until used for analysis. Muscle pH Approximately a 5 g muscle sample was homogenized for 1 min in a small Wering blender containing 25 m1 of 0.005M sodium iodoacetate for the pH determinations which were made during the first 3 hr post-mortem. Twenty-five ml of deionized water was blended with a 5 g muscle sample for ultimate pH determinations at 24 hr post-mortem. All pH estimates were performed in duplicate with a Corning Model 12 exPanded scale pH meter. AMP and IMP Determinations Extraction. Samples were not thawed prior to this extraction. In part I, 10 g of frozen tissue was weighed into a VirTis homogenizer flask (Dannert, 1966). Approximately 25 ml of distilled water was added and the sample was homogenized at maximum speed for 2 min. Following quanti- tative transfer to a 100 m1 volumetric flask, the homogenate was boiled for 5 min. It was then cooled before bringing the volume of the extract to 100 ml with distilled water. The extract was centrifuged for 20 min -34- at 2000 x G after which it was filtered through S & S No. 588 filter paper. In part II, 10 g frozen powdered muscle was placed in 50 to 60 m1 of boiling deionized water. The slurry was boiled for 10 min, quanti- tatively transferred to a 100 m1 volumetric flask and allowed to cool to room temperature before being brought to volume. This extract was centri- fuged for 10 min at 14,000 x G and subsequently filtered through S & S No. 588 filter paper. The clear filtrate resulting from the extractions in both parts I and II was adjusted to pH 8.0 with 1N sodium hydroxide. A 10 ml aliquot of the extract was then applied to an anion exchange column for separation of mononucleotides. Chromatographic separation. The method of Lento gt g1, (1964) as modified by Macy and Bailey (1966) was used for nucleotide separation. Bio-Rad Laboratories analytical grade anion exchange resin AG 1 x 8, 200- 400 mesh, was washed with distilled water to ranove fines. It was then packed in a glass column, 12 mm inside diameter, to a height of 160 mm. The resin was converted to the hydroxyl fonm by washing the column with 3 bed volumes of 1N sodium hydroxide. Excess sodium hydroxide was washed from the column with distilled water and the resin was next converted to the formats form by washing with 3 bed volumes of 6N formic acid. Excess reagent was again washed from the column and the aliquot of muscle extract was placed on the column. The resin was washed free of ultraviolet ab- sorbing materials with distilled water until a transmission of nearly 100% was obtained. The mononucleotides were now ready for elution. The columns were recharged after the nucleotides had been eluted according to the above procedure. Each column was recharged a maximum of 15 times. -35- The nucleotides were eluted by the three phase gradient system described by Macy and Bailey (1966). Three asPirator bottles were aligned one above another. The lower bottle leading to the column contained 125 ‘ml of deionized water, the middle bottle 250 m1 of 0.5N formic acid, and the upper bottle 500 ml of 0.2M sodium formate. Magnetic stirrers assured prOper mixing in the lower 2 bottles during elution. Flow rate was maintained constant at 2.5 ml per min by means of a chromatographic pump. The absorbance of the column effluent was monitored with an Isco, Model UA, ultraviolet flow densitometer at 254 mu and 10 m1 fractions were collected with a Rinco fraction collector. Ultraviolet absorbing peaks were combined, their volume measured and the nucleotide concentration determined as described below. anntitative evaluation. The method of Lento 25 g}, (1964), which is specific for 5'-ribonuc1eotides, was employed for this determination. One ml of 0.01N periodic acid was added to a 5 ml aliquot of sample and the mixture heated for exactly 3 min at 70°C in order to oxidize the ribose moiety between the 2 and 3 carbon position. Oxidation was stOpped by addition of 1 m1 of 0.05N sodium arsenite (NazHAs03) followed immediately by 1 m1 of concentrated hydrochloric acid, and then by 1 m1 of a saturated solution of 2,4-dinitr0phenylhydrazine (2,4-DNPH) in 2N hydrochloric acid. After mixing, the tubes were placed in boiling water for 10 min to hydro- lyze oxidation products and facilitate precipitation of the bis-hydrazone. The precipitate was collected on medium porosity fritted glass filters and washed free of unreacted 2,4-DNPH. The hydrazone was then dissolved -36- in acetone, dilutedixl volume and the absorbance of the yellow solution determined at 435 mu in a Beckman DU spectrOphotometer. According to Lento ggugl. (1964), the nucleotide content of each fraction can be deter- mined from a single standard, since the extinction coefficients of each 5'-nucleotide derivative formed are essentially the same. However, a standard curve using AMP was run and used for actual determinations (Figure 1). 5'-Adenylic Acid Deaminase The enzyme was extracted from frozen muscle tissue as described by Lee (1963). Frozen tissue, which had been stored a maximum of 7 days, was cut into small pieces and homogenized for l min in a Wering blender with 3.5 volumes of a cold buffer solution containing 0.3M potassium chloride, 0.09M potassium dihydrogen phosphate, and 0.06M potassium mondhydrogen phosphate, pH 6.5. The deaminase was extracted by efficient stirring at 3°C for 1 hr. Assay of enzymatic 'activity was conducted by the spectro- photometric method of Lee (1963) with modifications as suggested by Smiley 25 El: (1967). Reagent solutions needed for the assay were 0.15M succinate buffer, pH 6.5, and 3 x 10'3M 5km in distilled water. For the deter- mination of enzymatic activity, 0.1 ml of prOperly diluted (1:6 or 1:8 depending upon protein concentration and enzyme activity) muscle extract was added to 2.9 m1 of prewarmed (30°C) reaction mixture containing 0.1 ml of AMP solution, 2.0 m1 of succinate buffer and 0.8 ml of distilled water. The reaction is thus carried out at a total volume of 3 ml in the presence of 0.1M succinate and 1 x 10'4'M AMP. The rate of decrease (initial velocity) in absorbance at 265 mu was measured in a Beckman DU Spectro- -37- ABSOIBANCE (435 an) 2» 1 2 3 ug AMP/m1 acetone Figure l. 5'-Adenosine monOphOSphate standard curve. -33- photometer equipped with a Gilford, Model 220, absorbance indicator. Protein concentration was determined by 280/260 mu absorbance ratio. Specific activity of the enzyme (umoles AMP deaminated/min/mg protein) was calculated according to the following formula (Smiley ggfl§1., 1967): Absorbance units decrease/min S ecific activit = P y (8.86)(protein content in mg per assay) PhOSphorylase Assay Frozen muscle powder was extracted for phosphorylase within 12 hr post-mortem by the method of Posner ggugl. (1965). Powdered muscle (approximately 500 mg) was extracted at -20°C with 2‘m1 of a 60% glycerol solution containing 0.02M sodium fluoride, 0.005M ethylenediaminetetra- acetic acid (EDTA), 0.03M cysteine hydrochloride and 0.04M sodiumr/s- glycerOphosphate adjusted to pH 6.8 with sodium hydroxide. The material was allowed to extract for 20 min and was then diluted to 10 ml with a cold aqueous solution of the above salts. The suspension was stirred for 10 min (2 to 4°C), centrifuged at 2000 x G for 30 min and decanted. The supernatant was immediately assayed for glycogen phosphorylase. Phosphorylase was assayed as described by Illingworth and Cori (1953). Solutions required included: 4% glycogen, 0.064M glucose-l-phosphate (dipotassium salt), pH 6.8, and 0.064M glucose-l-phosphate containing 0.004M.AMP, also pH 6.8. Two-tenths ml of 4% glycogen was added to 0.4 ‘ml of enzyme extract (in duplicate) and the solution incubated at 30°C for 20 min. The reaction was started by adding 0.2 m1 of either the glucose-l-phosphate (for phosphorylase 3 activity) or glucose-l-phosphate -39- plus AMP (for total phOSphorylase activity) solution to the two samples. Final concentrations in the reaction mixture were: 0.01M sodium fluoride, 0.0025M EDTA, 0.015M cysteine, 0.02M sodium-fl-glycerophosphate, 1% glycogen, 0.016M glucose-l-phosphate and 0.001M AMP (when present). At appropriate times after starting the reaction (1 min intervals with AMP, 4 min intervals without AMT), 0.2 m1 aliquots were pipetted from the reaction mixture into 7 m1 of dilute (approximately 0.07N) sulfuric acid. The sulfuric acid stapped the reaction but was too dilute to allow much additional hydrolysis of glucose-l-phosphate at room temperature in 60 min (Illingworth and Cori, 1953). The amount of inorganic phosPhate released was determined by a modified Fiske-Subbarow procedure (Clark, 1964) and the amount of inorganic phOSphate present in the enzyme was corrected for by running blanks on the muscle extract. Protein concentration of the muscle extract was detenmined by 280/260 mu absorbance ratio. Results were expressed as u moles of inorganic phosphate released/min/mg protein either with AMP (total phosphorylase) or without AMP (phosphorylase-g). PhOSphorylase 3 activity was also eXpressed as a percentage of total phosphorylase activity. Percent Reflectance and Munsell Value Lightness or darkness of the longissimus dorsi muscle at 24 hr post- mortem was objectively determined using a Photovolt Photoelectric Re- flection Meter, Model 610. The instrument was standardized against a magnesium oxide block using the green filter. A chop removed from the longissimus dorsi muscle was allowed to oxygenate for 30 min prior to -40- measuring percentage reflectance at 3 positions in the cross-section of the muscle: in the medial one-third of the muscle, in the center one-third, and in the lateral one-third. The reflectance readings from the 3 posi- tions were averaged and converted to the equivalent Munsell value 'accord— ing to the conversion factors of Nickerson (1946). A Munsell value of 0 indicates pure white and a value of 10 pure black; intermediate values indicate various shades of gray. Transmission Value The transmission value method described by Hart (1962b) was used in this investigation as an objective measurement of muscle quality. The method is based upon the fact that PSE muscle has lower sarcoplasmic protein solubility than normal muscle. Transmission value is defined as the percentage of light transmitted through a water extract of muscle in which the proteins have been precipitated by a citric acid-phosphate buffer, pH 4.6. Since fewer water-soluble proteins are extracted from PSE muscle, a smaller quantity of proteins are in turn precipitated, the turbidity of the resulting solution is lower and the transmission value higher. Reagents for the test included 0.2M dibasic sodium phOSphate and 0.1M citric acid. The reagents were combined in the ratio of 9.35 ml sodium phOSphate and 10.65 m1 of citric acid solution to form a pH 4.6 buffer. Ten g of finely ground meat was placed in a centrifuge tube previously calibrated at 40 m1. Cold distilled water was added until the total volume was 40 ml and the mixture was then thoroughly stirred. -41- The mixture was held at 2 to 4°C for 20 hr after which it was stirred again and then centrifuged at 2000 x G for 20 min. The supernatant was filtered through S & S No. 588 filter paper. One ml of the clear filtrate was mixed with 5 m1 of pH 4.6 buffer solution, temperature 20°C, in a colorimeter tube. This mixture was held at exactly 20°C for 30 min. After this incubation, the solution was mixed again by inversion of the tube and read at 600 mu in aBausch and Lamb Spectronic 20 colorimeter against a blank containing 1 ml of muscle extract and 5 m1 of distilled water. Percent transmission was recorded as the transmission value. Glycogen Determination Glycogen was extracted from the frozen muscle powder as described by Hansen 2; 31, (1952). A 200 to 500 mg sample, accurately weighed, was hydrolyzed for 20 to 30 min in 3‘m1 of 30% potassium hydroxide in a boiling water bath. After cooling, 4 m1 of 95% ethanol was added and gently mixed with the contents of the tube. The tubes were heated in boiling water until bubbles appeared in the mixture and then allowed to stand at 2 to 4°C for 3 hr to allow complete precipitation of glycogen. The tubes were next centrifuged for 15 mdn at 2000 x G, the supernatant decanted and the precipitated glycogen dissolved in 3 ml of distilled water. Glycogen was reprecipitated with 95% ethanol, centrifuged and drained as before. The resulting glycogen precipitate was dissolved in 3 ml distilled water in the case of determinations on 24 hr muscle or 10 to 20 ml of distilled water for determinations at 15 min post-mortem. -42- Glycogen was assayed by the method of Dubois ggual. (1956). Two m1 of glycogen solution (containing 10 to 60 ug of glycogen) was placed in a test tube and 0.5 ml of 8% phenol solution added. Five ml of concen- trated sulfuric acid was then rapidly added and the contents of the tube were well mixed. After standing for 10 min, the mixture was incubated at 25 to 30°C for 20 min and then read at 490 mu in aBausch and Lomb Spec- tronic 20 calorimeter. The amount of glycogen was determined from a linear standard curve constructed for pure rabbit liver glycogen (Sigma Chemical Co.) between 10 and 80 ug of glycogen. Inorganic Phosphate The method of Lowry and Lopez (1946) was used for inorganic phosphate estimation. The powdered muscle sample was deproteinized with cold 5% trichloroacetic acid, brought to pH 4.0 to 4.2 by addition of 4 volumes of 0.1N sodium acetate, filtered, and the filtrate diluted with pH 4 acetate buffer. To each volume of diluted extract was added 0.1 volume of 1% ascorbic acid and 0.1 volume of 1% ammonium molybdate in 0.05N sulfuric acid. Readings of color development were taken at 5 min and again at 10 min after molybdate addition at a wavelength of 700 mu. Simultaneous readings were made on a standard and a blank. If a differ- ence was observed in the readings of the unknown at 5 and 10 min as compared to the standard, the values were extrapolated to zero time. -43- Adenosine Triphosphate IMuscle ATP was extracted from frozen powdered muscle with boiling distilled water. A 0.4 to 0.5 g sample was accurately weighed into the hot water and boiled for about 1 min then cooled and filtered. The fil- trate was assayed for ATP by the bioluminescence method described by Strehler and Trotter (1952) and Strehler (1953). In order to prepare the enzyme system needed for the assay, 100 mg of vacuum dried firefly lan- terns (Sigma Chemical Co.) were extracted with 0°C 0.1M sodium arsenate. The filtered solution from this extraction was diluted with 15 m1 of deionized water and then 48 mg of magnesium sulfate were dissolved in the solution. This enzyme solution was maintained constantly at 0°C. Before assaying for ATP, 0.5 ml of enzyme solution was placed in 2 m1 of distilled water and warmed to room temperature. The reaction was started by addition of 0.5 ml of unknown sample or standard to the diluted enzyme solution. Fluorescence was measured at 550 mu 30 sec after starting the reaction using a Aminco-Bowman SpectrOphotofluorometer. Statistical Analyses The statistical methods employed in this study were outlined by Snedecor (1956). Analyses of variance, standard deviations, and standard errors were calculated for the various treatment groups. Duncan's Multiple Range test as given by Steel and Torrie (1960) was used to test for differences among means. Simple correlation coefficients were determined between various factors both within and between treatments. RESULTS AND DISCUSSION Part I. Adenylic Acid Deaminase in Porcine Muscle Typical PSE porcine muscle is very pale, almost white, in color, soft in texture and extremely exudative in visual appearance. Many pro- cessing difficulties occur with PSE muscle, especially excessive loss of fluid and lower yield of processed product. PSE muscle yields a lower quality processed meat product. The deve10pment of PSE muscle is largely a direct result of extremely rapid post-mortem glycolysis and pH fall, with pH values of 5.5 and below being attained while muscle temperature is high (Briskey, 1964). This low pH is usually attained during the first hr postdmortem (Briskey and Wismer-Pedersen, 1961a). Thus, pH measurements must be taken during the first 60 min post-mortem in order tx>assess rate of pH fall. The combination of high temperature and low pH causes precipitation and insolubilization of the muscle proteins. The lower water holding capacity and pale color of PSE muscle are believed to be a direct result of the low pH - high temperature conditions. The objective in part I of this study was to determine the relation- ship of S-adenylic acid (AMP) deaminase activity of porcine muscle to the development of PSE musculature. AMP deaminase is responsible for the degradation of AMP to IMP. One mole of ammonia is released for each mole of AMP degraded. It is recognized that AMP influences the activity of certain glycolytic enzymes; notably phosphorylase b_is able to degrade glycogen to glucose-l-phosphate in the presence of sufficient quantities -44- -45- of AMP (Cori and Green, 1943). However, it has generally been accepted that AMP concentration is much too low in resting muscle to activate phosphorylase 2 (White _e_t__a_1_l_., 1964). Secondly phosphofructokinase ac- tivity isincreased by AMP as reported by Passonneau and Lowry (1962). It would be possible, therefore, that adenylic acid deaminase activity of porcine muscle might indirectly affect the rate of glycolysis and thus have some bearing upon ultimate muscle prOperties. In this study, deaminase activity was determined in three muscles, specifically chosen because of their varying susceptibility to the PSE condition. The gluteus medius muscle of the ham is readily subject to development.of PSE muscle morphology, the longissimus dorsi is somewhat intermediate in suscepti- bility, while the rectus femoris of the ham is quite resistant. The levels of AMP and IMP present in the longissimus dorsi and gluteus medius *muscles immediately after slaughter were also determined in addition to deaminase activity. An example of the elution pattern obtained upon separation of nucleo- tides on the anion-exchange column is shown in Figure 2. Although the method of separation is well documented in the literature, the eluted peaks were identified by applying known compounds to the column. Cytidine monOphOSphate was the first peak eluted, followed by AMP and IMP. Uridine monophosphate, when present, was eluted from the column between AMP and IMP. Recovery of AMP and IMP from the column was checked by chromato- graphing known amounts of the two nucleotides. Recovery values ranged between 95 and 105%. ~46- .zneoquuoeouno owootoxoucoHoo mo woumsoeom use oaomss scum concouuxo mopwuoofiooc mo Eonqueouto suntan 29-hotcu em av an as a— &--1 ml. 580 .N ouswwh SONVIIOSUV (“W OSZ) -47- As shown in Table 1, there were no significant differences in adenylic acid deaminase activity among the three porcine muscles studied. It is interesting to note, however, that the mean activity of the rectus femoris muscle is slightly lower than that observed in the longissimus dorsi or Agluteus medius. Table 1. Means and standard error of the means of adenylic acid deamin- ase activity, AMP, and IMP in three porcine muscles. Adenylic acid deaminase1 AMP2 IMP2 Muscle Mean Std. error Mean Std. error Mean Std. error lopgissimus dorsi .031 .001 .60 .11 6.15 .42 gluteus medius .031 .002 .50 .06 6.03 .14 rectus femoris .027 .002 - - -- - IAdenylic acid deaminase activity is expressed as umoles AMP deaminated] min/mg protein. 2umoles/g fresh muscle. The levels of AMP and IMP at 15 min postdmortem (Table l) were not statistically different between longissimus dorsi and gluteus medius muscles. Few data concerning porcine muscle nucleotide levels are avail- able in the literature and those values which have been reported were for muscle which had been held for varying lengths of time post-mortem. The nucleotide levels of such muscle would undoUbtedly vary greatly due to enzyme degradation of nucleotides. For example, at 48 hr post-mortem, Dannert (1966) found IMP concentrations of 3.12, 3.23, and 2.98 umoles/g in the biceps femoris, longissimus dorsi and semimembranosus porcine muscles, respectively, and AMP levels of 0.90, 0.33, and 0.33 umoles/g. -43- The IMP levels found by Dannert (1966) are substantially lower than those found at 15 min post~mortem.in the present investigation. It has been shown by several researchers (Briskey and Wismer-Pedersen, 1961a; Bendall and Wismer-Pedersen, 1962; Briskey g£_§l., 1962; McLoughlin, 1963) that an abnormally rapid post-mortem glycolytic rate is among the causative factors in deve10pment of PSE musculature. As a result of raphi glycolysis, pH drOp is also very rapid during the initial post-mortem period (1/2 to 1 hr). In this study, glycolytic rate was assessed by following rate of pH decrease in the musculature. In order to evaluate the relationship between PSE muscle development and adenylic acid deaminase activity, simple correlation coefficients were calculated between enzyme activity and muscle pH at 15 and 45 min post-mortem (Table 2). Positive correlations were obtained between adenylic.acid deaminase activity and muscle pH in the three muscles studied. However, only the correlation coefficients between deaminase activity and pH at 45 min (r = .71) in the ggluteus medius and enzyme activity and pH at 15 min (r = .56) in the rectus femoris muscle were statistically significant (P < .05). Simple correlation coefficients between muscle pH and AMP levels revealed no apparent relationship since all of the correlation coefficients were low. This would seem to be in conflict with the relationship observed between deaminase activity and pH decline. The reason for this difference is not evident from these data. IMP concentration was negatively correlated with pH in all but one case in which a low positive correlation was obtained (IMP and 15 min pH in the longissimus dorsi muscle). This negative relation- ship was most pronounced in the gluteus medius muscle with the correlation between IMP and 45 min pH being significant (r = -.84, P < .05). -49- Table 2. Simple correlation coefficients between pH, adenylic acid deaminase activity, and AMP and IMP levels in three porcine muscles. pH 15 min pH 45 min Lopgissimus dorsi muscle Adenylic acid deaminase .29 .41 AMP .23 .00 IMP .ll -.44 Gluteus medius muscle Adenylic acid deaminase .48 .71* AMP .17 .12 IMP -.65 -.84* Rectus femoris muscle Adenylic acid deaminase .56* .43 *Significant (P < .05) Deficiencies in adenylic acid deaminase activity in skeletal muscle have been reported in certain pathological conditions. Pennington (1961) reported that 5'-adenylic acid deaminase activity was nearly 3 times lower in skeletal muscles of dystrOphic mice than in those from normal mice. Engel ggugl. (1964) found a markedly lower activity of this enzyme in skeletal muscles of human patients suffering from periodic hypokalemic paralysis. Activity differences of this magnitude were not observed in -50- the present investigation. None of the animals used in this part of the study yielded carcasses with severe PSE musculature. However, a wide range of muscles qualities and rates of pH fall was present among the pigs in this study. Much greater animal variation in adenylic acid deamin- ase activity should have been noted if the enzyme were involved in deve1- Opment of PSE muscle. The absence of high positive relationships between rate of pH fall and both deaminase activity and AMP concentration in the muscle supports the conclusion that adenylic acid deaminase activity is not directly involved in the deve10pment of PSE porcine musculature. Part II. Observations on Control, Epinephrine and Prednisolone Injected Pigs The objective in the second part of this study was to determine the effects of epinephrine injection immediately pre-slaughter upon some phy— sical and biochemical prOperties of porcine muscle. Ante-mortem stress and excitement have been cited as causative factors in the deve10pment of PSE muscle (Briskey, 1964) and it was hoped to simulate stress conditions by epinephrine injection. Epinephrine was also shown to activate phos- phorylase by initiation of the p|to‘§_conversion (Cori and Illingworth, 1956; Danforth 55 al., 1962). It was thought that epinephrine injection might thus accelerate the rate of postdmortem glycolysis in porcine muscle and possibly increase the incidence of PSE development. The effects of epinephrine injection were also studied in pigs which had been injected with large doses of prednisolone in an attempt to suppress adrenal cortical secretion. It was of interest to study the -51- effects of epinephrine upon porcine muscle properties by supressing adreno- cortical secretion.since the latter has been implicated in the etiology of PSE musculature as well as stress conditions (Ludvigsen, 1957; Judge .EEuél-y 1966; Cassens ‘ggug1,, 1965; Tapel 55 Elf: 1967). The effect of prednisolone injection upon adrenal gland weight and plasma glucocorticoid level was not evaluated in this study; however, Tapel (1965) reported that prednisolone injected pigs, at the dosage used in the present study, had a 25 to 29% decrease in adrenal gland weight and an 8.0 ug/lOO m1 decrease in plasma l7-hydroxycorticosteroids. Normal porcine plasma con- centration of 17-hydroxycorticosteroids was reported to be 21.8 i 6.4 ug/lOO m1. Tapel (1965) has shown that muscle from.prednisolone injected pigs has a different postdmortem pH pattern than muscle from non-injected pigs. The results for control, epinephrine and prednisolone plus epine- phrine injected pigs are shown in Tables 3 through 8 and in Figure 3. Muscle pH, Ultimate muscle pH (24 hr post-mortem) was not signifi- cantly affected by either epinephrine or prednisolone plus epinephrine treatment (Table 3). Hedrick 25 a1. (1957) reported that injection of epinephrine at 24 hr and 12 hr prior to slaughter significantly elevated the ultimate pH of bovine muscle. Epinephrine injection 4 hr pre-slaughter significantly raised the ultimate pH of rabbit muscle (Bendall and Lawrie, 1962). The explanation for the elevated ultimate pH was that muscle glycogen was depleted ante-mortem by epinephrine injection thus decreasing the quantity of lactic acid produced during anaerobic, post-mortem gly- colysis. Since ultimate pH was not affected by epinephrine treatment in the present study, it indicates that muscle glycogen was not significantly depleted by the 10 min anteémortem injection. -52- Rate of postdmortem pH decline was not affected by epinephrine in- jection. ‘Muscle pH values did not differ significantly between control and treated pigs either at 15 min or at 45 min after exsanguination. However, Bendall and Lawrie (1962) found faster rates of pH fall in rabbits injected with epinephrine 4 hr ante-mortem, even though ultimate pH was higher in the muscles of treated rabbits. The rate of pH fall was unaffected by exogenous epinephrine in the pigs injected with 100 mg prednisolone for 10 days prior to slaughter. Hypoadrenocorticoidism is characteristic of an animal after prolonged prednisolone administration as shown by Tapel (1965), with resultant adrenal atrOphy and lower plasma 17-hydroxycorticosteroids. Glucocorti- coids play an important role in the ability of an animal to withstand stress (Guyton, 1961). Bendall and Lawrie (1962) showed that pre-injection of cortisone in rabbits (24 hr antedmortem) protected against the glycogen wasting effect of exogenous epinephrine (4 hr ante-mortem) and restored a normal rate of post-mortem pH fall and normal ultimate pH. It was ex- pected that simulated stress in the present eXperiment (epinephrine injection) might accelerate the post-mortem glycolytic rate in prednisolone injected animals. It was noted that after several days of prednisolone injection, the pigs.were less active physically and were easily exhausted by a small amount of exercise. Munsell value and transmission value. Munsell value did not differ significantly between control, epinephrine, or prednisolone plus epine- phrine treated pigs (Table 3). Higher values indicate that the muscle -53- was paler in color. However, it is of interest that the mean value for epinephrine injected pigs was slightly higher than controls while the value for prednisolone treated pigs was lower than controls. It is not surprising that significant differences were not observed for Munsell value since wide variation existed within groups. Hedrick ggnal. (1957) reported that darker colored bovine muscles resulted from 12 or 24 hr ante-mortem epinephrine injection and significantly darker colored porcine 'muscles were also observed CHedrick'ggngl,, 1964) following 24 hr ante- mortem.administration of epinephrine. Simple correlation coefficients were calculated between Munsell value and pH at various times post-mortem (Table 4). ‘Munsell value was nega- tively correlated with pH at 15 min post-mortem (r = -.69, P < .01) and at 45 min post-mortem (r = -.70, P < .01). This indicates that color intensity of porcine muscle was related to the rate of postwmortem pH decline in agreement with other workers (Wismer-Pedersen, 1959; Briskey and Wismer-Pedersen, 1961a; Sayre g£H§1., 1963c). iMuscle color was posi- tively correlated with muscle protein extractability as shown by the correlation (r = .78, P < .01) between Munsell value and transmission value. As shown in Table 3, transmission value was not significantly affected by epinephrine or prednisolone plus epinephrine injection (high trans- mission value indicates muscle with low protein extractability). Although transmission value for prednisolone plus epinephrine treated pigs was substantially lower than for the other treatments, the difference was not statistically significant. As expected, transmission value was highly -54- negatively related to rate of pH decline. Correlation coefficients be- tween transmission value and pH at 15 and 45 min post-mortem were -.76 and -.88, respectively (Table 4). This indicates the important relation- ship between rate of pH fall and porcine muscle quality. Hart (1962b) demonstrated that transmission value was very high in PSE (low quality) muscle and quite low in normal muscle. Table 3. Means and standard error of the means for pH, Munsell value, and transmission value in control, epinephrine, and prednisolone plus epinephrine treated pigs. Prednisolone Standard Control Epinephrine + epinephrine error pH - 15 min 6.35 6.25 6.29 .09 pH - 45 min 5.93 5.85 5.98 .17 pH - 24 hr 5.45 5.48 5.42 .02 Munsell value 5.58 5.70 5.43 .14 Transmission value 50.8 53.5 41.4 8.7 Table 4. Simple correlation coefficients between Munsell value, trans- mission value and pH of porcine muscle. pH pH 15 min 45 min Munsell value Munsell value -.69** -.70** Transmission value -. 76* * - . 88* * . 78* * *Significant (P < .05) **Significant (P < .01) -55- ccho‘..2‘hr(m'/‘) f\ ~O ‘0 V C") N '— O _ I G ’°°""15nlu(""/‘) , .3 2‘ .9 on o V N o ------------------- h : ------------------ cg " Eflfl33flfi§$fi§$§g33§§3§§3§§33§§33 . 0.... .C...D.........C.........‘.......0.0.0..'0'..0.......0.0.0.............0.0... . o‘o‘o‘vo-c 0 O OAO‘O‘OAO‘O.I_O‘OAO o o o o o o o o o o o o e o o o o o o o o o o e U ‘- «p 3‘ ‘I - . E“ = - - - - - - - - .2 - - - - - - - - «o C! II 2 : a. .3 '9 'i I. '8 2 < a 4: ll ‘ .2 o 7:. I .. at a g 0 ~ : fi -.------------ ° - .- ‘ 0 ------------ D E . . I°I°I°I°I°I°I°Z°I°Z°Z°I°I°I°I°I°Z° 09 <2 V. o: o unnoquou "301 O O O O O ‘Q C) CH " «sumac may auluapy ADRENAL"! PIEDNISOLONE CONTROI. I. ADRENAL"! Total phosphorylase, adenylic acid deaminase and 15 min and 24 hr glycogen in control, = umoles AMP deaminated/min/mg protein = umoles Pi released/min/mg protein and prednisolone plus epinephrine injected pigs. Adenylic acid deaminase activity Phosphorylase activity epinephrine, Figure 3. -56- Phosphorylase. Total phosphorylase activity was significantly in- creased by epinephrine injection (Table 5 and Figure 3). The activity of this enzyme in the muscle of control pigs was .40 umoles Pi released /min/mg protein compared with .53 for epinephrine injected pigs and .57 for prednisolone plus epinephrine treated pigs. However, the increased total phosphorylase activity in epinephrine treated pigs could not be entirely accounted for by higher phosphorylase g_activity. This may have been due to the fact that phOSphorylase 3.13 more active in the pre- sence of AMP (used for total phosphorylase assay) than in the absence of AMP as explained by Cori (1956), thus causing total phosphorylase to appear greater. Also, since approximately 10 min elapsed between bleeding of the animal and excision of the muscle sample, considerable breakdown of phosphorylase g;by phOSphorylase phosphatase could have occurred. Posner g; 31. (1965) reported 2 to 4 fold increases in the ratio of phos- phorylase activity without AMP to activity with AMP in frog and rat muscles 12 min after epinephrine injection.' In the present study, phOSphorylase 2 activity was slightly higher in epinephrine treated pigs. The differ- ence between controls and prednisolone plus epinephrine treated pigs approached significance (P < .10). PhOSphorylase a_activity, expressed as a percentage of total phOSphorylase activity did not differ signifi- cantly between treatments (Table 5). Simple correlation coefficients between phOSphorylase activity and pH values, Munsell value and transmission value are presented in Table 6. Low, nonsignificant correlations were obtained between total phosPhorylase activity and pH at either 15 (r = -.05) or 45 min (r = .05) post-mortem -57- Table 5. Means and standard error of the means for total phosphorylase, phosphorylase_ a and % phosphorylase_ a in muscle of control, epinephrine, and prednisolone plus epinephrine treated pigs. 1 Prednisolone Standard Control Epinephrine + epinephrine error Total phosphorylase activity .40a .53b .57b .03 Phosphorylase-a acitivityz .03 .05 .07 .01 % phOSphorylase g; 7.7 10.2 12.0 2.4 IMeans with different superscripts are significant (P«< .05). 2Phosphorylase activity = umoles Pi released/min/mg protein. Table 6. Simple correlation coefficients between phosphorylase activity and pH, transmission value and Munsell value of porcine muscle. Total % phosphorylase Phosphorylase g phOSphorylase g pH - 15 min -.05 .20 .22 pH - 45 min .05 .28 .30 Munsell value -.05 -.22 -.22 Transmission value -.05 -.31 -.33 (rate of pH fall). This is in disagreement with Wismer-Pedersen (1959) but agrees with the results of Sayre 25 e1, (l963b) who found no relation- ship between total phOSphorylase and rate of pH decline. Even though phosphorylase §_activity and % phosphorylase §;were slightly associated pH at 15 and 45 min post-mortem, the correlations did not approach signi- ficance (P < .05). In addition, low relationships were afiserved between Munsell value and transmission value and the various measures of phos- -53- phorylase activity. These results tend to negate any postulation that some abnormality in the phosphorylase system is responsible for deve10p~ ment of PSE muscle. Adenylic acid deaminase, AMP and IMP. Specific activity of 5'- adenylic acid deaminase (expressed as umoles AMP deaminated/min/mg protein) was significantly lower (P < .05) in the muscle of epinephrine (.025) and prednisolone plus epinephrine (.026) treated pigs than in control animals (.032) as shown in Table 7. However, differences between treatment groups in AMP and IMP concentrations were not statistically significant. ‘More- over, correlation analysis revealed that neither AMP nor IMP level was highly related to 5'-adenylic acid deaminase activity. The reason for lower adenylic acid deaminase activity in muscle from epinephrine treated pigs is not known. Low positive correlation coefficients were found between adenylic acid deaminase activity and the rate of post-mortem.pH fall in agreement with results of part I of this study. The values were r = .26 and r = .09 between enzyme activity and pH at 15 and 45 min post- mortem, respectively (Table 8). As in part I of this study, AMP level was not highly related to muscle pH at either 15 min or 45 min post-mortem, r = .24 and r = .13, respectively. In contrast, IMP level was very highly but negatively correlated with rate of pH fall, the values being r = -.94 at 15 min post- mortem and r = -.93 at 45 min. This almost linear, inverse relationship indicates that in porcine muscle with very rapid post-mortem glycolysis, there would also be a very rapid liberation of ammonia, 1 mole of ammonia -59- being released for each mole of IMP formed. Briskey and Wismer-Pedersen (1961a) reported that liberation of ammonia from PSE muscle was complete at 2 hr postdmortem.while normal muscle required 8 to 10 hr. Table 7. Means and standard error of the means for adenylic acid deaminase, AMP, IMP, and glycogen in control, epinephrine, and prednisolone plus epinephrine treated pigs.1 Prednisolone Standard Control Epinephrine + epinephrine error Adenylic acid deaminase activityz .032a .025b .026b .002 AMP3 1.82 1.72 2.10 .16 IMP3 2.53 2.74 2.54 .46 Glycogen - 15 min4 8.27a 5.85a 11.66b .87 Glycogen - 24 hr4 .34a .21a 3.14b .18 1Means with different superscripts differ significantly (P < .05). 2Adenylic acid deaminase activity = umole AMP deaminated/min/mg protein. Zumole/g fresh muscle. mg/g fresh muscle. Table 8. Simple correlation coefficients between pH, adenylic acid deaminase activity, and AMP and IMP concentrations of porcine muscle. pH pH 15 min 45 min Adenylic acid deaminase .26 .09 AMP .24 .13 IMP -.94** -.93** **Significant (P < .01) -60- The AMP and IMP levels found in this part of the study differ sub- stantially from those found in part I. In part II, AMP levels were 2 to 3 times higher and IMP concentrations approximately one-half of that reported in part I. These differences were due to the slightly different extraction procedure used in part II, in which approximately 5 volumes of boiling water was added directly to the frogen muscle powder. This resulted in almost instantaneous coagulation of the muscle proteins and much more rapid inactivation of the enzyme systems responsible for degra- dation of AMP and IMP. The extraction method of Dannert (1966) used in part 1 involved homogenization of frozen tissue with water and allowed 2 to 3 min before heat coagulation of protein, during which time consider- able change in nucleotide levels could conceivably take place due to enzyme degradation. Therefore, the AM? and IMP concentrations in part II more nearly reflect actual muscle concentrations. The mean AMP concentrations observed in this study were 1.82 umole/ g of muscle in controls, 1.72 umole/g in epinephrine treated, and 2.10 umole/g in prednisolone plus epinephrine treated pigs. Cori egflgl. (1943) and Cori (1956) have reported that rabbit muscle phOSphorylase h_is acti- vated 1/2 maximally in the presence of'l x 10'5M AMP. The concentrations reported above for porcine muscle are substantially greater than the amount of AMP needed for 1/2 maximal activation of phosphorylase 2, Morgan and Parmeggiani (1963) observed a 20 fold increase in rate of glycogen breakdown by rat heart muscle phosphorylase b_when the reaction “was carried out in .6 mM.AMP, 4 mM ATP and 10 mM inorganic phosphate (anaerobic levels) as opposed to conducting the reaction in .15 mM AMP, -6l- 8 mM ATP, and l-mM inorganic phOSphate (aerobic levels). They stated that phosphorylase §_activation would appear to be an Emportant factor in the rapid rate of glycogenolysis under anaerobic conditions. Based upon the AMP levels found in porcine muscle, it would there- fore be possible for phosphorylase §_to be active during post-mortem glycolysis. However, the AMP requirements of porcine muscle phosphorylase 2 may possibly be quite different from those reported for rabbit or rat muscle phosphorylase. Glycogen. Prednisolone plus epinephrine treated pigs had signifi- cantly (P < .05) greater amounts of muscle glycogen at 15 min post-mortem and at 24 hr post-mortem (Table 7 and Figure 3), than either control or epinephrine treated pigs. The average 15 min muscle glycogen level in epinephrine treated pigs was lower than that for controls, however, the difference was not statistically significant (P < .05). Lower muscle glycogen at 15 min post-mortem in the epinephrine treated group was no doubt a reflection of the higher total phosphorylase activity found in this treatment as compared to the control pigs. Yet, rate of pH fall was not different between control and epinephrine treated pigs. The fact that 15 min and 24 hr glycogen was higher in prednisolone treated animals and that initial rate of pH decline was not altered by this treatment indicates that the muscle glycogen reserves were higher in prednisolone injected pigs. The reason for this action of predniso- lone is not known. As stated previously, prednisolone injected pigs were less active and more easily exhausted after the 5th or 6th injection. -62- Limited information is available concerning the effects of adrenal steroids upon postdmortem muscle prOperties. Faludi 33 gl. (1964) re- ported loss of weight and muscle atr0phy in dogs injected with large doses of prednisolone and methyl prednisolone for prolonged periods. The dogs were also lethargic and quite weak after several days of in- jection. Other authors have reported similar effects in rabbits following large dosages of cortisone (Ellis, 1956; Germuth 23 al., 1951). One important function of the glucocorticoids is to enhance gluconez- genesis (Guyton, 1961; Zarrow 55 gl., 1964). Along with increased gluconeogenesis, there is greater storage of glycogen in the liver but not in muscle. This illustrates that there is a basic difference in the effects of glucocorticoids upon liver glycogen and cellular glycogen elsewhere in the body. As stated by Zarrow 2; a1. (1964), glucocorti- coids are believed to interfere with glucose utilization at the cellular level by inhibition of the hexokinase reaction. These data indicate that the incidence of PSE muscle was no greater in either epinephrine injected or prednisolone plus epinephrine injected pigs than in controls. Although total phosphorylase activity was increased by epinephrine administration, rate of post-mortem pH fall and frequency of PSE muscle were essentially unaffected. Observations on Porcine Muscle With Fast and Slow Rates of Post-Mortem Glycolysis Since variation in postdmortem pH fall existed among the control pigs in this study, they were divided into two groups on the basis of rate of pH decline. -63- Those with 45 min pH < 5.65 were designated as fast glycolysis while those with 45 min pH > 5.85 were designated slow glycoly- sis. Only 1 of the pigs in the slow group had 45 min pH below 6.0, the others being greater than 6.1. The data obtained on the slow and fast groups were subjected to analysis of variance, in order to observe any differences between the two groups. The striking difference in rate of pH fall between the two muscle types is shown in Figure 4. At 3 hr post-mortem, pH of the slow group had fallen to 5.80 while the fast group had reached pH 5.46 at the end of 1 hr. The difference in pH between the two groups was already evident at 15 min post-mortem, when the first determination was made and became more pronounced at 45 min post-mortem (Table 9). However, the ultimate pH (24 hr) did not differ significantly between muscles with rapid or slow pH fall. Table 9. Means and standard error of the means for pH, Munsell value and transmission value in porcine muscle with fast and slow rates of post-mortemglycolysis.1 Fast Slow Standard error pH - 15 min 6.188 6.521) .09 pH - 45 min 5.5281 6.33b .14 pH - 24 hr 5.46 5.44 .02 Munsell value 5.7881 5.37b .10 Transmission value 68.98 32.8b 6.8 lMeans with different superscripts differ significantly (P < .05). 6.8 6.7 6-6 .| 6.5 6.4 6.3 6.2 6.] two 5.9 5.8 5.7 5.6 5.5 504 5.3 Figure 4. -64- d— 5 LOW GLYCOLYSIS -*- FAST GLYCOLYSIS I 2 3 I 24 Time Post-Modem (In) Rate of pH fall in muscles with slow and fast glycolysis. -65- Briskey (1963) described 6 different types of post-mortem pH pattern in porcine muscle. Two very gradual types of pH fall were found, one of which resulted in ultimate pH of 6.0 to 6.5 and the other an ultimate pH of 5.7 to 6.0. At the other extreme, a rapid decrease to a pH of 5.1 to 5.4 at 1/2 to 1 1/2 hr and the retention of this low pH or a slight sub- sequent elevation to an ultimate pH 5.3 to 5.4 was also reported (Briskey, 1963). This latter pattern corresponds to the fast group observed in the present study; however, the very gradual types of pH decline were not observed. Munsell value and Transmission value. Average Munsell value and transmission value for muscles with slow and fast rates of pH fall are shown in Figure 5. 'Muscles with fast glycolysis ultimately became signi- ficantly lighter colored as indicated by the higher Munsell value (P < .03). This group of muscles also possessed a significantly higher transmission value indicative of some PSE muscle development. Transmission value of the slow group was 32.8 versus 68.9 for the fast pH decline group (Table 9). Hart (1962b) stated that normal high quality muscle has a transmission value of 0 to 20 while severe PSE muscle averages 80 to 100. In agreement with results reported earlier, Munsell value and trans- mission value were negatively related to rate of pH fall (Table 10). Although only one of the correlation coefficients was statistically signi- ficant, but they were all approaching significance at the 5% level. ‘Munsell value and transmission value were again positively related but not quite as highly as observed previously. Muscle color and morphology -66- Wenenot subjectively scored as described by Topel (1965), however a close relationship was apparent between the transmission value and the visual appearance of the muscle. 'Muscle which was visually classified as PSE always had a high transmission value. Table 10. Simple correlation coefficients between pH, Munsell value, and transmission value in porcine muscle with slow and fast rates of post-mortem glycolysis.1 pH pH ‘Munsell Transmission 15 min 45 min value value pH - 15 min 1.0 pH - 45 min .93 1.0 Munsell value -.69 -.63 1.0 Transmission value -.65 -.83 .58 1.0 IE1 > .71 are significant (P < .05) These results further substantiate the postulation that rapid rates of glycolysis and the concomitant accelerated pH fall are closely related to the ultimate quality of porcine muscle (Wismer-Pedersen, 1959; Briskey and Wismer-Pedersen, 1961a,b; Bendall and Wismer-Pedersen, 1962). PhOSphorylase and adenylic acid deaminase. Total phosphorylase and ph0$phorylase g activities of muscle with slow and fast rates of pH decline are shown in Table 11 and Figure 5. Muscles in the slow group had a slightly lower average value for total phosphorylase, however, there was rather large variation between animals and thus the differences were not statistically significant. Phosphorylase §_activity was higher in the slow group but a large standard error was also present and the differences -67- were not significant. It is concluded from these results that phosphory- lase activity, as assayed in this study, is not related to the rapid glycolytic rate often observed in porcine muscle or to the development of PSE muscle. There was no significant difference in adenylic acid deaminase activity between the muscle groups possessing fast or slow pH decline. Table 11. Means and standard error of the means for total phosphorylase, phOSphorylase g, % phosphorylase g, and adenylic acid deaminase in porcine muscle with fast and slow rates of post-mortem glycolysis.1 Fast Slow Standard error Total phosphorylas e activity2 .41 .38 .04 Phosphorylase‘gactivity2 .02 .04 .02 % phosphorylase g 5.85 9.52 2.65 Adenylic acid deaminase activity3 .033 .031 .004 1Means with different superscripts are different significantly (P < .05). 2Phosphorylase activity = u mole Pi released/min/mg protein. Adenylic acid deaminase activity = u mole AMP deaminated/min/mg protein. Glycogen. Glycogen content of the longissimus dorsi muscle at 15 min and 24 hr is shown in Table 12 and Figure 6. Fifteen min glycogen was significantly lower (P < .05) in muscles with rapid rates of glycolysis (6.27 mg/g) compared to muscles with slow glycolysis (10.28 mg/g). Muscle pH at 15 min post~mortem.was 6.18 in the slow group versus 6.52 in the fast group. Thus, the difference in glycogen is probably largely due to more rapid degradation as reflected in the lower initial pH rather than to differences in glycogen reserves at the time of death. There was no -68- Phosphorylase ; ‘v CH 0 o. .06 'fil Phosphorylase g m Total Phosphorylase asalnoudsou luoj Transmission Value 0 0 O l\ O ‘0 V 6") 9 8 2 Munsell value, transmission value, total phosphorylase and phosphorylase 3 in porcine muscle m Iunsoll VaIuo m Transmission Value T\ O in V m N '— amen ”nun" O O O O O O SLOW FAST SLOW FAST Figure 5. (phosphorylase activity - u mole P1 ‘with slow and fast rates of postwmortem glycolysis. released/min/mg protein). -69- difference between groups in 24 hr glycogen concentration. It should also be noted that all pigs used in this study were of one breed (Poland China). Sayre 5E eh. (1963a) reported that muscles from the Hampshire breed had 2 to 3 times higher initial glycogen than Poland China muscles. Chester White longissimus dorsi muscle also contained significantly more initial glycogen than Poland Chinas. Poland China muscle consistently had lower initial pH than muscle from the other two breeds. However, the authors did not specify the time lapse from exsanguination of the animal to sampling of the muscle. Kastenschmidt £5 31. (1966) found slightly lower initial glycogen levels (2 min post-mortem) in porcine muscle which exhibited rapid glycolysis as compared to slowly glycolysing muscle. AMP, IMP, ATP, and inorganic phosphate. The results of AMP, IMP, ATP and inorganic phosPhate determinations are presented in table 12 and illustrated graphically in Figure 7. AMP concentration at 15 min post- mortem.was slightly higher in the muscles possessing fast pH decline, however, this difference was not significant between the two groups as greater variation occurred between individuals than between groups. There was a highly significant difference (P < .01) in 15 min IMP level between muscle with rapid and slow glycolysis (pH decline). The fast group contained 3.51 u mole IMP/g while the slow group had 1.54 u mole/ g of muscle. Inorganic phosphate was also significantly higher (P < .02) in the rapidly glycolysing muscle, 25.6 u mole/g fresh muscle versus 17.4 u mole/g for slow glycolysis. However, ATP concentration was higher in the slow group of muscles (2.49 u moles/g versus 1.24 u moles/g of ‘muscle). This difference was statistically significant (P < .07). -70- I: Glycogems min G"°°g°"24 hr 12 IO 1 II 73 uafioai l9 Glycogenwmin O~ FAST SLOW Figure 6. Glycogen at 15 min and 24 hr post-mortem in porcine muscles with fast and slow glycolysis. (glycogen - mg/g fresh muscle) -71_ SLOW Inorganic PO4 AT P FAST 5'-Adenylic acid deaminase, AMP, IMP, ATP, and inorganic phOSphate in porcine muscles with fast and slow glycolysis. [:1 m O O O O 6") CV .— Foa ogualaoug A"? and I"? V C") N F- O 3 o d r m r—L—trrr.'.'.'.‘.'°.°'.iii°'-‘°.‘E:'° t-H E In? [a] Adenylic Acid Deaminase & ‘V"“"""""'""""""' F I ° ° ° °';I;I;.;I;I;I;I;3:133;2:2;I;I;I;I;I;I;I;I;I;I;I;I «n g 3....;.;.:.;.;.;.;.;.;.;.;.;.;.;.;.:.:.;.;.;.;.;.;.;.;.;.: g u. I ---r----------------- F Pu-J-nnnn-nnnuu-uunnn . l\ U c) C) C) c: (3 g v C”) N '- 00 E aseugmeaa may ogMuapv (Adenylic acid deaminase activity = u moles AMP deaminated/min/mg protein; AMP, IMP, ATP, and inorganic phosphate expressed as u mole/g fresh muscle). -72- Table 12. Means and standard error of the means for glycogen, AMP, IMP, ATP and inorganic phOSphate in porcine muscles with fast and slow rates of postdmortem glycolysis. Fast Slow Standard error Glycogen - 15 min2 6.273 10.28b 2.26 Glycogen - 24 hr2 .40 .27 .24 m3 1.88 1.74 .20 IMPB 3.5151 1.54b .35 ATP3 1.24 2.49 .40 Inorganic phosphate3 25.6a 17.4b 1.8 1Means with different superscripts differ significantly (P < .05). /g fresh muscle 3u moles/g fresh muscle Simple correlation coefficients between pH and AMP, IMP, ATP, and inorganic phosphate are presented in Table 13. It is evident from the low correlation coefficients between pH at 15 and 45 min and AMP levels that no relationship existed between rate of pH fall and AMP concentration. However, IMP, ATP, and inorganic phOSphate levels were closely associated with rate of pH decline. All of the correlation coefficients between these factors and pH at either 15 or 45 min post-mortem were highly significant (P < .01). From these results, it appears that a more rapid degradation of ATP occurred during the initial 15 min post-mortem.period in muscle with rapid pH fall, as indicated by the lower ATP level and higher inorganic phosphate concentrations. This is in agreement with Bendall 35 gl. -73- Table 13. Simple correlation coefficients between pH and AMP, IMP, ATP, and inorganic phosphate in porcine muscles with fast and slow rates of post~mortem glycolysis. pH pH 15 min 45 min AMP -.04 -.26 IMP -.95 -.95 ATP .93 .87 Inorganic phosphate -.88 -.95 r > .71 is significant (P < .05) r > .83 is significant (P < .01) (1963). The major pathways for resynthesis of ATP during the post-mortem period are either from creatine phOSphate or by anaerobic glycolysis as pointed out by Bendall (1963). The fact that ATP resynthesis is not keeping pace with ATP breakdown is also indicated by the higher IMP and inorganic phosphate levels in muscles with rapid pH fall. Therefore, the question which remains unanswered is what mechanisms are responsible for the rapid ATP degradation. Kastenschmidt e£_§1, (1966) reported that ATP levels were lower and IMP levels slightly higher at 2 min post-mortem in porcine muscle which elicited a very rapid pH decline (pH < 5.5 at 60 min post-mortem). Whether LEHYLEQ levels of ATP are lower in these muscles has not been determined. Phosphofructokinase, the glycolytic enzyme which catalyzes conver- sion of fructose—l-phosphate to fructose-l,6-diph03phate, has been shown to be a rate limiting step in glycolysis (Passonneau and Lowry, 1962; Mansour, 1963; Ozand and Narakara, 1964). ATP in high concentrations has been shown to be a potent inhibitor of phOSphofructokinase activity -74- in muscle (Lardy and Parks, 1956). Passonneau and Lowry (1962) demon- strated that ATP inhibition could be overcome by small increases in AMP, ADP and inorganic phOSphate. Ammonium ion (NHZ) was shown to be a potent stimulator of phosphofructokinase activity (Muntz and Hurwitz, 1951). It is possible, therefore, that increases in phOSphofructokinase activity could account for the rapid rate of pH fall observed in porcine muscle. Lower ATP levels and higher inorganic phosphate concentrations were observed in muscles with rapid pH decline in the present study. It must also be assumed that NHZ concentration would also be greater in this type of muscle, since greater amounts of IMP were found. One mole of ammonia (NH3) is released for every mole of IMP formed so NH: and IMP should be present in nearly equal molar quantities. SUMMARY The results of this study were obtained in two separate investiga- tions with 36 porcine animals. In part I, the adenylic acid deaminase activity and AMP and IMP levels of longissimus dorsi, gluteus medius, and rectus femoris muscles were determined. The relationship of these parameters to post-mortem glycolysis, rate of pH fall, and to development of PSE porcine muscle was evaluated. In part II, epinephrine injection immediately prior to slaughter and the effects of prednisolone adminis- tration prior to epinephrine treatment were studied for possible effects upon the development of PSE musculature. Of Special interest were the rate of post-mortem glycolysis, phOSphorylase activity and adenylic acid deaminase activity of the longissimus dorsi muscle as well as glycogen, AMP, IMP, ATP, and inorganic phosphate levels in the muscle. Phosphory- lase and adenylic acid deaminase activities and AMP, IMP, ATP, and inorganic phosphate levels were contrasted in porcine muscles with fast and slow rates of post-mortem glycolysis. There were no significant differences in adenylic acid deaminase activity, AMP, or IMP between the longissimus dorsi, gluteus medius and rectus femoris muscles. Simple correlation coefficients between muscle pH and adenylic acid deaminase activity indicated a low but positive relationship between enzyme activity and rate of post-mortem pH fall. Epinephrine or prednisolone plus epinephrine injection did not signi- ficantly alter the rate of post-mortem pH decline, ultimate muscle pH, -75- -76- ‘muscle color (Munsell value) or muscle quality (transmission value). Total phOSphorylase activity was significantly increased in muscle from epinephrine injected pigs and phosphorylase §_activity and % phosphorylase §_were slightly increased. However, neither total phosphorylase nor phOSphorylase.§_activity was related to rate of postdmortem.pH fall or incidence of PSE musculature development. Adenylic acid deaminase activity was significantly lower in the epinephrine treated muscles. AMP and IMP concentrations were not affected by either treatment, however, IMP level was highly related to the rate of pH fall. Epinephrine injection 10 min antedmortem did not significantly deplete muscle glycogen. 'Muscle from prednisolone injected pigs contained higher glycogen levels at both 15 min and 24 hr post-mortem. Pigs in the control group were divided into slow and fast glycolysis groups on the basis of postdmortem pH fall. ZMuscle with slow glycolysis had lower Munsell values and transmission values. Phosphorylase activity and adenylic acid deaminase activity were not significantly different between muscles with slow and fast glycolysis. IMuscle which elicited a rapid pH fall had higher IMP and inorganic phOSphate and lower ATP and glycogen levels than did muscle with slow pH fall. LITERATURE CITED Bate-Smith, E. C. and J. R. Bendall. 1949. Factors determining the time course of rigor mortis. J. Physiol..2§, 176. Bendall, J. R. and C. L. Davy. 1957. Ammonia liberation during rigor ‘mortis and its relation to changes in the adenine and inosine nucleotides in rabbit muscle. Biochim et BiOphys. Acta 2h, 93. Bendall, J. R. and R. A. Lawrie. 1962. The effect of pretreatment with various drugs on post-mortem glycolysis and the onset of rigor mortis in rabbit skeletal muscle. J. Comp. Path. Therap. 12, 118. Bendall, J. R. and J. Wismer-Pedersen. 1962. Some properties of the fibrillar proteins of normal and watery pork muscle. J. Food Sci. 21, 144. Bendall, J. R. 1963. Physiology and chemistry of muscle. Proc. Meat Tenderness Symposium. Campbell Soup Co. p. 33. Bendall, J. R., O. Hallund and J. Wismer-Pedersen. 1963. Post~mortem changes in muscles of Landrace pigs. J. Food Sci. 22, 156. Bodwell, C. E., A. M. Pearson and R. A. Fennell. 1965. Post-mortem changes in muscle. III. Histochemical observations in beef and pork. J. Food Sci. 29, 944. Bodwell, C. E., A. M. Pearson, J. Wismer-Pedersen and L. J. Bratzler. 1966. Post-mortem changes in muscle. II. Chemical changes in pork. J. Food Sci. 22, 1. Borchert, L. L. and E. J. Briskey. 1964. Prevention of pale, soft, exudative porcine muscle through partial freezing with liquid nitro- gen. J. Food Sci. 22, 203. Borchert, L. L. and E. J. Briskey. 1965. Protein solubility and asso- ciated properties of porcine muscle as influenced by partial freezing with liquid nitrogen. J. Food Sci. 22, 138. Briskey, E. J., R. W Bray, W. G. Hoekstra, P. H. Philips and R. H. Grummer. 1959a. The chemical and physical characteristics of various pork ham muscle classes. J. Animal Sci. 22, 146. Briskey, E. J., R. W. Bray, W. G. Hoekstra, R. H. Grummer and P. H. Phillips. 1959b. The effect of various levels of exercise in altering the chemical and physical characteristics of certain pork ham muscles. J. Animal Sci. 22, 153. -77- -73- Briskey, E. J., R. W. Bray, W. G. Hoekstra, P. H. Phillips and R. H. Grummer. 1959c. The effect of exhaustive exercise and high sucrose regimen on certain chemical and physical pork ham muscle character- istics. J. Animal Sci. 22, 173. Briskey, E. J. and J. Wismer-Pedersen. 1961a. Biochemistry of pork ‘muscle structure I. Rate of anaerobic glycolysis and temperature change versus the apparent structure of muscle tissue. J. Food Sci. 2_6_, 297. Briskey, E. J. and J. Wismer-Pedersen. 1961b. Biochemistry of pork muscle structure II. Preliminary observations of biopsy samples versus ultimate muscle structure. J. Food Sci. 22, 306. Briskey, E. J., R. N. Sayre and R. G. Cassens. 1962. Development and application of an apparatus for continuous measurement of muscle extensibility and elasticity before and during rigor mortis. J. Food Sci. 21, 560. Briskey, E. J. 1963. Influence of ante- and post-mortem handling practices on properties of muscle which are related to tenderness. Proc. Meat Tenderness Symposium. Campbell Soup Co. p. 195. Briskey, E. J. 1964. Etiological status and associated studies of pale, soft, exudative porcine musculature. In Advances 2h_Food Research (C. O. Chichester, E. M. Mrak and G. F. Stewart, eds.) Vol. 13, p. 89. Academic Press, New York. Cassens, R. G., W. G. Hoekstra and E. J. Briskey. 1963. Relation of pork muscle quality factors to zinc content and other properties. Food Technol. 21, 493. Cassens, R. G., M. D. Judge, J. D. Sink and E. J. Briskey. 1965. Porcine adrenocortical lipids in relation to striated muscle characteristics. Proc. Soc. Exptl. Biol. Med. 120, 854. Charpentier, J. and R. Goutefongea. 1963. Electrophoretic characteristics of lactic dehydrogenase in normal and exudative pig muscle. Nature 201, 1325. Clark, J. M. 1964. Experimental Biochemistgy p. 32. W. H. Freeman and Company, San Francisco. Cori, C. F., G. T. Cori and A. A. Green. 1943. Crystalline muscle phos- phorylase III. Kinetics. J. Biol. Chem. 151, 39. Cori, G. T. and A. A. Green. 1943. Crystalline muscle phosphorylase II. Prosthetic group. J. Biol. Chem. 151, 31. -79- Cori, C. F. 1956. Regulation of enzyme activity in muscle during work. In Enzyges: Units 22_Biologica1 Structure and Function. (0. H. Gaebler, ed.) p. 573. Academic Press, New York. Cori, G. T. and B. Illingworth. 1956. The effect of epinephrine and other glycogenolytic agents on the phosphorylase 2 content of muscle. Biochim. et Biophys. Acta. 22, 105. Danforth, W. H., E. Helmreich and C. F. Cori. 1962. The effect of con- traction and of epinephrine on the phOSphorylase activity of frog sartorius muscle. Proc. Natl. Acad. Sci., U.S. 22, 1191. Dannert, R. D. 1966. The determination of 5'-mononucleotides in meats. Ph.D. Thesis. Michigan State University. East Lansing. Dubois, Michel, K. A. Gibbes, J. K. Hamilton, P. A. Rebers and Fred Smith. 1956. Colorimetric method for determination of sugar and related substances. Anal. Chem. 22, 350. Ellis, J. T. 1956. Necrosis and regeneration of skeletal muscles in cortisone-treated rabbits. Am. J. Pathol. 22, 993. Engel, A. G., C. S. Potter and J. W. Rosevear. 1964. Nucleotides and adenosine monOphosphate deaminase activity of muscle in primary hypokalaemic periodic paralysis. Nature 202, 670. Faludi, G., L. C. Mills and Z. W. Chayes. 1964. Effect of steroids on ‘muscle. Acta endocrinologica.22, 68. Forrest, J. C. 1965. Porcine physiology as related to post-mortem muscle prOperties. Proc. 18th An. Reciprocal Meat Conf. p. 270. National Live Stock and Meat Board, Chicago. Germuth, F. G., Jr., C. A. Nedzel, B. Ottinger and J. Oyama. 1951. Anatomic and histological changes in rabbits with experimental hypersensitivity treated with compound E and ACTH. Proc. Soc. Exptl. Biol. Med. 22, 177. Goldspink, G. and J. V. MeLoughlin. 1964. Studies on pig muscle III. The effect of temperature on the solubility of the sarc0plasmic proteins in relation to colour changes in post-rigor muscle. Irish J. Agric. Res. 2, 9. Guyton, A. C. 1961. Textbook 92 Medical Physiology; p. 1002 W. B. Saunders Company, Philadelphia. Hamm, R. 1959. Biochemistry of meat hydration. Proc. 11th Research Conf. Circ. No. 50. p. 17. American Meat Institute Foundation, Chicago, Illinois. -30- Hamm, R. 1960. Biochemistry of meat hydration. In Advances 2h_Food Research. (C. O. Chichester, E. M. Mrak and G. F. Stewart, eds.) Vol. 10, p. 355. Academic Press, New York. Hansen, R. G., W. J. Rutter and E. M. Craine. 1952. A nephelometric method for the determination of glycogen. J. Biol. Chem. 195, 137. Hart, P. C. 1962a. Physico-chemical characteristics of degenerated meat in pigs. Tijdschr. Diergeneesk. 21, 3. Hart, P. C. 1962b. The transmission value, a method for meat quality evaluation. Research Institute for Animal Husbandry "Schoonoord". Zeist. The Netherlands. (mimeograph) Hedrick, H. B., D. E. Brady and C. W. Turner. 1957. Effect of ante- mortem stress on post-mortem beef carcass characteristics. Proc. 9th Research Conf. p. 9. American Meat Institute Foundation, Chicago, Ill. Hedrick, H. B., F. C. Parrish and M. E. Bailey. 1964. Effect of adrena- line stress on pork quality. J. Animal Sci. 22, 225. Illingworth, B. and G. T. Cori. 1953. Crystalline muscle phosphorylase. In Biochemical Preparations (E. E. Snell, ed.) Vol. 3, p. 1. John Wiley and Sons, Inc., New York. Judge, M. D., E. J. Briskey and R. K. Meyer. 1966. Endocrine related post-mortem changes in porcine muscle. Nature 212, 287. Karpatkin, S., E. Helmreich and C. F. Cori. 1964. Regulation of glycoly- sis in muscle 11. Effect of stimulation and epinephrine in isolated frog sartorius muscle. J. Biol. Chem. 239, 3139. Kastenschmidt, L. L., E. J. Briskey and W. G. Hoekstra. 1964. Prevention of pale, soft, exudative porcine muscle through regulation of ante- mortem environmental temperature. J. Food Sci. 22, 210. Kastenschmidt, L. L., G. R. Beecher, J. C. Forrest, W. G. Hoekstra and E. J. Briskey. 1965. Porcine muscle prOperties A. Alteration of glycolysis by artificially induced changes in ambient temperature. J. Foos Sci. 22, 565. Kastenschmidt, L. L., E. J. Briskey and W. G. Hoekstra. 1966. Metabolic intermediates in skeletal muscles with fast and slow rates of post- mortem.glycolysis. Nature 212, 288. Kjolberg, 0., D. J. Manners and R. A. Lawrie. 1963. a-(1,->4)-Glucosans 16. The molecular structure of some pig muscle glycogens. Biochem. J. 22, 351. -81- Lardy, H. A. and R. E. Parks, Jr. 1956. Influence of ATP concentration on rates Of some phOSphorylation reactions. In Enzymes: Units of Biological Structure and Function (0. H. Gaebler, ed.) p. 584. Academic Press, New York. Lawrie, R. A. 1955. Residual glycogen at high ultimate pH in horse muscle. Biochim. et Biophys. Acta.lz, 282. Lawrie, R. A., D. J. Manners and A. Wright. 1959. a-l,4-Glucosans 10. Glycogen structure and rigor mortis in mammalian muscles. Biochem. J. 13, 485. Lawrie, R. A. 1960. Post-mortem glycolysis in normal and exudative longissimus dorsi muscles of the pig in relation to so-called white muscle disease. J. Comp. Path. Therap. 19, 273. Lee, Y.IK. 1963. Crystalline adenylic acid deaminase from rabbit skeletal muscle. In Methods in_En§ymology. (S. P. Colowick and N. 0. Kaplan, eds.) Vol. VI, p. 102. Academic Press, New York. Lento, H. G., J. A. Ford and R. E. Denton. 1964. A method for determin- ation of 5'-mononuc1eotides. J. Food Sci. 22, 435. Lowry, 0. H. and J. A. Lopez. 1946. The determination of inorganic phOSphate in the presence of labile phosphate esters. J. Biol. Chem. 162, 421. Ludvigsen, J. 1953. "Muscular degeneration" in hogs (preliminary report). Proc. XV Intern. Veterin. Cong. Vol. I, part 1, p. 602. Boktryckeri, Stockholm. Ludvigsen, J. 1957. On the hormonal regulation of vasomotor reactions during exercise with special reference to the action of adrenal cortical steroids. Acta endocrinologica 26, 406. Macy, R. L. and M. E. Bailey. 1966. Modified method for rapid determina- tion of individual mononucleotides. Food Technol.‘2Q, 346. Mansour, T. E. and J. M. Mansour. 1962. Effects of serotonin (-5-hydroxy- tryptamine) and adenosine-3;S'-phosphate on phosphofructokinase of the liver fluke Fasciola hepatica. J. Biol. Chem. 237, 689. Mansour, T. E. 1963. Studies on heart phosphofructokinase: purification, inhibition, and activation. J. Biol. Chem. 238, 2285. NkLoughlin, J. V. 1963. Studies on pig muscle II. The effect of rapid post-mortem pH fall on the extraction of the sarc0plasmic and myo- fibrillar proteins of post-rigor muscle. Irish J. Agric. Res. 2, 115. -32- MeLoughlin, J. V. and G. Goldspink. 1963a. Post-mortem changes in the colour of pig longissimus dorsi muscle. Nature 128, 584. MeLoughlin, J. V. and G. Goldspink. 1963b. Studies on pig muscle I. Exudative pig muscle. Irish J. Agric. Res. g, 27. Meyer, J. A., E. J. Briskey, W. G. Hoekstra and K. G. weckel. 1963a. Niacin, thiamin and riboflavin in fresh and cooked pale, soft and watery versus dark, firm and dry pork muscle. Food Technol. 11, 485. Meyer, J. A., E. J. Briskey, W. G. Hoekstra and K. G. weckel. 1963b. The niacin and niacin-containing coenzyme contents of certain pork 'muscles and their relationship to certain postwmortem.muscle charac- teristics. Proc. 15th Research Conf., Circ. No. 74, p. 1. American Meat Institute Foundation, Chicago, Illinois Morgan, H. E. and A. Parmeggiani. 1963. Regulation of phOSphorylase activity in muscle. Federation Proc.-22, 298. 'Muntz, J. A. and J. Hurwitz. 1951. The effect of ammonium ion upon isolated reactions of the glycolytic scheme. Arch. Biochem. Biophys. 32, 137. Nickerson, Dorothy. 1946. Color measurement and its application to the grading of agricultural products. A handbook on the method of disk colorimetry. ‘Misc. Publication 580. United States Department of Agriculture. washington, D. C. Ozand, P. and H. T. Narahara. 1964. Regulation of glycolysis in muscle III. Influence of insulin, epinephrine, and contraction on phospho- fructokinase activity in frog skeletal muscle. J. Biol. Chem. 239, 3146. Passonneau, J. V. and 0. H. Lowry. 1962. Phosphofructokinase and the Pasteur effect. Biochem. BiOphys. Res. Comm. 1, 10. Pennington, R. J. 1961. 5'-Adenylic acid deaminase in dystrOphic mouse muscle. Nature 192, 884. Pork Carcass Evaluation Committee. 1952. Cutting procedure for pork carcasses. Proc. Fifth An. Reciprocal Meat Conf. p. 120. National Live Stock and Meat Board, Chicago. Posner, J. B., R. Stern and E. G. Krebs. 1965. Effects of electrical stimulation and epinephrine on muscle phosphorylase, phosphorylase 2_kinase and adenosine -3',5'-phosphate. J. Biol. Chem. 240, 982. -83.. Regan, D. M., D. A. B. Young, W. W. Davis, J. Jack and C. R. Park. 1964. Adjustment of glycolysis to energy utilization in the perfused rat heart. The effect of changes in the ionic composition of the medium on phosphofructokinase activity. J. Biol. Chem. 232, 381. Sayre, R. N., E. J. Briskey, W. G. Hoekstra and R. W. Bray. 1961. Effect of preslaughter change to a cold environment on characteristics of pork muscle. J. Animal Sci. 29, 487. Sayre, R. N. and E. J. Briskey. 1963. Protein solubility as influenced by physiological conditions in the muscle. J. Food Sci. 28, 675. Sayre, R. N., E. J. Briskey and W. G. Hoekstra. l963a. Comparison of muscle characteristics and post-mortem.glycolysis in three breeds of swine. J. Animal Sci. 22, 1012. Sayre, R. N., E. J. Briskey and W. G. Hoekstra. 1963b. Effect of excite- ment, fasting and sucrose feeding on porcine muscle phosphorylase and postdmortem glycolysis. J. Food Sci. 28, 472. Sayre, R. N., E. J. Briskey and W. G. Hoekstra. l963c. Alteration of post-mortem changes in porcine muscle by preslaughter heat treatmen: and diet modification. J. Food Sci. 28, 292. Sayre, R. N., E. J. Briskey and W. G. Hoekstra. l963d. Porcine muscle glycogen structure and its association with other muscle properties. Porc. Soc. Exptl. Biol. Med. 112, 223. Sayre, R. N., B. Kiernat and E. J. Briskey. 1964. Processing character- istics of porcine muscle related to pH and temperature during rigor mortis deve10pment and to gross morphology 24 hr post-mortem. J. Food Sci. 22, 175. Scopes, R. K. 1964. The influence of post-mortem conditions on the solubilities of muscle proteins. Biochem. J. 21, 201. Sherman, P. 1961. The water binding capacity of fresh pork I. The influence of sodium chloride, pyr0phosphate and polyphosphate on water absorption. Food Technol..l§, 79. Smiley, J. L., Jr., A. J. Berry and C. H. Suelter. 1967. An improved purification, crystallization and some properties of rabbit 5'- adenylic acid deaminase. J. Biol. Chem. (in press). Snedecor, G. W. 1956. Statistical Methods. 5th Edition. The Iowa State College Press, Ames. Steel, R. G. D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. MeGraw-Hill Book Company, Inc. New York. 784- Stetten, M. R., H. M. Katzen and D. Stetten. 1958. A comparison of the glycogens isolated by acid and alkaline precedures. J. Biol. Chem. 232, 475. Stetten, D. and M. R. Stetten. 1960. Glycogen metabolism. Physiol. Rev. 49, 505. Strehler, B. L. and J. R. Trotter. 1952. Firefly luminescence in the study of energy transfer mechanisms I. Substrate and enzyme deter- mination. Arch. Biochem. Biophys. 49, 28. Strehler, B.L. 1953. Firefly luminescence in the study of energy trans- fer mechanisms II. Adenosine triphosphate and photosynthesis. Arch. Biochem.Bi0phys. 43, 67. Swift, C. E. and M. D. Berman. 1959. Factors affecting the water reten- tion of beef 1. Variation in composition and properties among eight muscles. Food Technol. 13, 365. Tepel, D. G. 1965. Role of the adrenal cortex and thyroid gland function upon some physical and chemical properties of porcine muscle. Ph.D. Thesis. Michigan State University. East Lansing. TOpel, D. G. and R. A. Merkel. 1966. Effect of exogenous goitrogens upon some physical and biochemical prOperties of porcine muscle and adrenal gland. J. Animal Sci. 22, 1154. Topel, D. G., R. A. Merkel, D. L. Mackintosh and J. L. Hall. 1966. Variation in some physical and biochemical pr0perties within and among selected porcine muscles. J. Animal Sci. 25, 277. T0pe1, D. G. and R. A. Merkel. 1967. Effect of exogenous prednisolone and methylprednisolone upon plasma l7-hydroxycorticosteroid levels and some porcine muscle characteristics. J. Animal Sci. (submitted). Topel, D. G., R.A. Merkel and J. Wismer-Pedersen. 1967. The relationship of plasma 17-hydroxycorticosteroid levels to some physical and bio- chemical properties of porcine muscle. J. Animal Sci. (In press). Urbin, M. C., D. A. Zessin and G. D. Wilson. 1962. Observations on a method of determining the water binding prOperties of meat. J. Animal Sci. 21, 9. White, A., R. Handler and E. L. Smith. 1964. Principles of Biochemistry 3rd Edition, p. 416. MeGraw-Hill Book Company, New York. Wismer-Pedersen, J. 1959. Quality of pork in relation to rate of pH change post-mortem. Food Res. 24, 711. ~85- 1961a. Rate of anaerobic glycolysis Wismer-Pedersen, J. and E. J. Briskey. Nature 189, 318. versus structure in pork muscle. 1961b. Relationship of post- Wismer-Pedersen, J. and E. J. Briskey. Food Technol. 12, 232. mortem acididty and temperature. Zarrow, M. X., J. M. Yochim and J. L. MeCarthy. 1964. Experimental Endocrinology. Academic Press. New York. APPENDIX ~86- .nHououm w8\afla\woumawemov mz< moHoe : n kuw>wuom ommawamov wean caesameHuom ommnwewoo owom oaaeaoowuom ommahuoammoan o.HN ooH. on. m.N~ mH.m N¢.o mm.o so.o no w.oa moo. oo. o.mm do.o o¢.n oH.o ~¢.o mo m.w moo. no. o.w¢ om.m om.n Hm.o oo.o me o.- oaa. on. o.~¢ o¢.m mm.n ow.n N~.o Hq o.m wHo. we. o.HN a¢.n um.m N¢.o w¢.o oq o.n mmo. do. o.~w Ha.o N¢.m wm.m oo.o am oaaunmonwao + oGoHOmHaooum m.q muo. on. o.om oo.m ~m.m mm.o om.o mm H.m moo. no. o.mm om.m o¢.m m¢.m No.o mm m.¢ omo. ao. o.HN Hm.m w¢.n ma.o om.o om ~.¢ owe. me. o.mn oH.o ¢¢.n ¢¢.m om.o om o.oH omo. no. o.wm ow.m mn.m om.o o¢.o om ¢.¢N oHH. no. o.mm dm.n a¢.m mN.o n¢.o mm o.MH one. on. o.ww mm.o N¢.m om.m qm.m mm o.HH who. mo. o.qo nw.m mm.m m¢.m o~.o mm oawnsmoawam m.~ 500. mm. o.mm Hm.m mm.m wH.o m¢.o mm o.~H omo. No. m.m¢ mn.m ¢¢.n o¢.m No.o mm N.m mHo. Ho. o.mm «H.o m¢.m mo.m no.o om m.¢ omo. «q. o.mn oq.o m¢.m mo.m mm.o om w.HH ooo. 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No.n mn.m Hq.H mmo. oN o.HN oN.H mo. wo.o mo.N Ho.N wNo. nN o.ma mm.N NH. mm.NH mq.H mo.H mNo. «N o.mH ow.m ma. om.NH oo. Ho.H omo. MN o.oN mo.H oo. o¢.¢ oq.m ow.H «oo. NN N.HN ww.H ma. mm.o mo.N wo.N Nmo. HN Houunoo wwoaoe_= w\oaoa_= w\w8 w\we_ wxoaoa_s w\oaoe.= Nommaqamoo Hogans 3289... m3 2 a... an. 2 an an. 32. 35.3. ownmwuonH dowoohao nomoomaw owamaoo< .mwfim oouoonaw unaufimonfimo mafia oaoaomfiaooua oam oafiunmoaamo «Houunau .Aoosawunoov HH xwoaomm< ma. Nm. Nm.- om.- ma. Ho. u: eN - Gowoohaw em. 3. 3.- S: 3. mm. 33. 3 - amwoobo NH.- mo. mm. mm. mm.- oN.- mzH 0N. mo.- ma.- oN.: ma. «N. mz< mH.- Nm.- mo.- NN.- mo. oN. omoaNEmoo owom oHH>noo< so. mm. mm: 3.. om. NN. m mafia-8:82.. N 0., 8.. No. 3.- 2.- mm. 8. m omflhofimofi no oo.H oo.: oo.: oo. oo.: ommamuosnmonm Hmuoa oo.H on. oo.: oN.- osao> nofimmwamnmua oo.H om.- oo.- onHm> Naomnsz 84 mm. 52 ma - ma oo.H nae ma - mm n1“ cowamuosmmosm on~m> osHo> awe on see ma cowamuosmmonm Hmuoe nowmmHEmamuH HHomnsz mm mm onfiufimoafimo mDHm oaoaomficoonm one oawhsaonwmo «Houunoo mom .mem oouoonaw mnofiumHouHoo onEHm .HHH xwonoma< 84 E. 8? mm. 5.- mo. an «N - nowoofio 8; 3.- mm. .3.- mN. fie 2 - 589.8 co; 3.. ON.- AN... NEH 004 3. N. 8.2 so; 2.- mmmfiemue 3.9. 03.8%... oo.H m.omoamuo£mmosn N Mm M ommHm-aosamosm .. omoauuonmmona HouoH 09.1.5 nowmflamcmua osam> Haomnnx Se 3 .. ma 5.. 2 - a E 3.. Ea 2 EH a2 33.8% m nomoohaw Gowoohaw ofiom ommamuofimmoem 03.803 .\. .mem oouoonaa oawunmonwmo mafia oaoHOmHnooum one unannounwao «Honuaoo How mcoaumaouuoo oamaam Aoosnwuaoov HHH xwonomm< -91- N .mem Houuaoo mom muaofioflmmooo so. as. an. wm.- wo.- am. am. me< so.- we.- em.- an. on. ma.- mm.- mumeamoaa unamwuoaH NN. «a. eH.- m~.- no. am.. He.- He «N - amwousao on. om. me. ~a.- mm.- mm. «a. gas me - cowouazo He. m..- NH.- He. he. mm.- mm.- mzH oN.- w~.- o..- ms. em.- o~.- mo.- ~24 No.- mo.- Ns.- am. no. mN.- ao.- ommaaamoe ego. uaaaamua oo.. ma. oo. «0.- m~.- an. we. m.mmm.auoeamoea e oo.a He. mo.- s~.- mm. mm. m.ommasuoeamoem oo.. HH.- mm. am. am. mmmaauoeamoea Hmuoa oo.H mm. mm.- mo.- unem> scammHEmamua oo.H mo.- mo.- Q=Hm> Havana: oo.H mm. cae.mq - ma oo.H use.ma - ma mW- m. ommamuonamona o=Hm> osam> nae we owe ma ommamhonmmosm omoamuosmwonm HmuoH soammNEmamnH Haomnuz mm mm nowumaouuoo ngENm .>H xwonomm¢ -92- oo.H oo.- H¢.- oo. oo.: Ho. oH.u ma< oo.H ma. Hm.- Na. oo. no. oumnamone oaamwuonH oo.. «a. on. m~.- mH.- as «N - amwooaao oc.H on.- em.- mm.- qua ma - nmwoosao oo.H mo. 0H. mzH oo.H oa.n mz< oo.H ommafiemoo oaom ofiahaoo< m.ommamuosmmonn N m.ommamuo£mmo:m ommahuonawonm HmuoH osam> Cowmmwemnmua ooao> Havana: 5a 3 - a sea ma - ma mH< oumammonn- a: qN awe ma mzH mz< ommnHEmoo owamwuoaH cowooeau nowoo%aw oaom oaaaamca .mwwa Houuaoo How muaowofimmooo noHumHouuoo oHaENm .Aoosnwuaoov >H xwonoma< -97- Appendix VII. Sources of chemicals. Chemical Source Ammonium molybdate Baker AMP (sodium salt) Sigma Anion exchange resin AG 1 x 8 Bio-Rad Arsenic thioxide Baker Ascorbic acid Eastman ATP (disodium salt) Sigma Citric acid Baker Cysteine - HCl Eastman 2,4-Dinitr0phenylhydrazine Eastman Elon (p-methylaminOphenol) Sargent Ethylenediaminetetra acetic acid (disodium salt) Baker Firefly lanterns (vacuum dried) Sigma Glucose-l-phOSphate (dipotassium salt) Sigma Glycogen (rabbit liver) Sigma IMP (sodium salt) Sigma Periodic acid Smith Phenol National A Aniline Sodium-,CLglycerophOSPhate Sigma Sodium iodoacetate Eastman Succinic acid Baker Baker I J. T. Baker Chemical Co., Phillipsburg, N.J. Bio-Rad - Bio-Rad Laboratories, 32nd & Griffin, Richmond, Cal. Eastman - Eastman Organic Chemicals, Rochester 3, N.Y. National Aniline a National Aniline Division, Allied Chemical & Dye Corp., 40 Rector St., New York 6, N.Y. Sargent - E. H. Sargent & Co., 8560 W. Chicago, Detroit, Mich. Sigma = Sigma Chemical Co., 3500 DeKalb St., St. Louis, Mo. Smith - G. Frederick Smith Chemical Co., Columbus, Ohio III Ilt1ll‘ A I .lfl-l.‘ ‘1 IIJI [ I l