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1 DAVIS, David Ray, 19*+1- SOME EFFECTS OF COLD TEMPERATURE EXPOSURE OF RATS ON ASPARTATE AMINOTRANSFERASE AND ALANINE AMINOTRANSFERASE LEVELS IN PLASMA, LIVER AND MUSCLE TISSUES. The Ohio State University, Ph.D., 1970 Physiology University Microfilms, A XEROX Company, Ann Arbor, Michigan i THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED

2 SOME EFFECTS OF COLD TEMPERATURE EXPOSURE OF RATS ON ASPARTATE AMINOTRANSFERASE AND ALANINE AMINOTRANSFERASE LEVELS IN PLASMA, LIVER AND MUSCLE TISSUES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By David Ray Davis, B.Sc., M.Sc. * * * * * * The Ohio State University 1970 Approved by ffilmrtit'***1- AdviseJX. Department of Dairy Science

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4 ACKNOWLEDGMENT To my adviser, Dr. T. M. Ludwick, for his guidance throughout the course of this study and help in preparing this manuscript, my sincere thanks. To Dr. W. R. Gomes, 1 wish to express my thanks for permission to use his laboratory facilities. To Mr. William Crist, I wish to express my thanks for his assistance in one phase of this study. To my wife, Rita, for her encouragement throughout the course of this study and for typing this manuscript, my sincere thanks. ii

5 VITA August 10, Born-Patriot, Ohio 1959-I96I.... Rio Grande College, Rio Grande, Ohio The Ohio State University Columbus, Ohio B. Sc., The Ohio State University Columbus, Ohio Research Assistant, Department of Dairy Science, The Ohio State University, Columbus, Ohio * * M. Sc. The Ohio State University, Columbus, Ohio Research Associate, Department of Dairy Science, The Ohio State University, Columbus, Ohio Animal Husbandman, USDA-ARS, Dairy Cattle Research Branch, Columbus, Ohio Research Associate, Department Dairy Science, The Ohio State University, Columbus, Ohio

6 PUBLICATIONS Variations in Reproductive Performance of Dairy Cattle Managed Under two Systems of Housing. D. R. Davis, T. M. Ludwick, E. R. Rader, K. L. Barker, E. W. Brum and H. C. Hines* J. Dairy Sci. 47:709» 196^* (Abstract) Variations in S-GOT and S-GPT in Cycling Cows. D. R. Davis, T. M. Ludwick, H. C. Hines, and K. L. Barker. J. Dairy Sci. 48:807, (Abstract) Effects of Exogenous Estrogen and Progesterone on Serum Enzyme Levels in Dairy Cows. D. R. Davis, W. L. Crist and T. M. Ludwick. J. Dairy Sci. 49:731* (Abstract) FIELDS OF STUDY Major Field: Reproductive Physiology iv

7 TABLE OP CONTENTS Pago ACKNOWLEDGMENTS... ii V I T A... LIST OP TABLES... LIST OF FIGURES... viii iii vi INTRODUCTION Chapter I. LITERATURE REVIEW... 3 The Concept of Animal Response to Stress The Response of Animals to Low Environmental Temperatures Status of GOT and GPT Some of the Factors Associated With Elevated Levels of Serum and Tissue Transaminase II. MATERIALS AND METHODS General Procedure Experimental III. RESULTS AND DISCUSSION IV. SUMMARY AND CONCLUSIONS BIBLIOGRAPHY v

8 LIST OF TABLES Table Page 1. Repeatability of GOT and GPT Values for Rat Liver Homogenates Repeatability of GOT and GPT Values for Rat Plasma Effects of Liquid Nitrogen Freezing and Storage on GOT and GPT Activity in Liver and Muscle Tissues Diurnal Variation in GOT and GPT Levels in Rat Plasma Diurnal Variation in GOT and GPT Levels in Rat Liver Diurnal Variation in GOT and GPT Levels in Rat Muscle Effects of Caging on Tissue Levels of GOT and GPT in the Rat Effects of Feed Removal, on Rats Kept at 26 C, on GOT Activity in Plasma, Liver and Muscle Tissues * Effects of Feed Removal, on Rats Kept at 26 C, on GPT Activity in Plasma, Liver, and Muscle Tissues Effects of the Removal of Feed for 0 to 36 Hours on Adrenal Ascorbic Acid Levels in Rats Kept at 2 6 C Effects of Exposure of Rats for 0 to 36 hours at 3 C on the Level of GOT in Plasma, Liver and Muscle Tissues 63 vi

9 List of Tables cont. 12. Effects of Exposure of Rats for 0 to 36 Hours at 3 C on the Level of GPT in Plasma, Liver and Muscle Tissue Effects of Exposure of Rats for 0 to 36 Hours at 3 c on the Level of Adrenal Ascorbic Acid... * Variation in the Levels of GOT in Tissue When Rats are Exposed for Two Hours at -25 C Variations in the Levels of GPT in Tissue When Rats are Exposed for Two Hours at -25 C Effects of Two Hours Exposure at -25 C on the Level of Adrenal Ascorbic Acid in the Rat Effects of Peed on Tissue GOT and GPT Levels When Rats are Exposed for 24 Hours at 3 C Effects of Feed on Adrenal Ascorbic Acid Levels in Rats Exposed to 3 C for 24 Hours Effects of Peed on Tissue GOT and GPT Levels When Rats Are Exposed for 2 Hours at -25 C- (Rats Sacrificed 4 and 7 Hours After Exposure) Effects of Peed on Adrenal Ascorbic Acid Levels in Rats Exposed for 2 Hours at vii

10 LIST OS' FIGURES Figure Page 1. Comparison of Tissue (JOT Activity and Adrenal Ascorbic Acid Levels of Rats Exposed for 0 to 36 Hours at 3 C Comparison of Tissue GPT Activity and Adrenal Ascorbic Acid Levels of Rats Exposed for 0 to 36 Hours at 3 C Comparison of Tissue GOT Activity and Adrenal Ascorbic Acid Levels Following the Exposure of Rats for 2 Hours at -25 C Comparison of Tissue GPT Activity and Adrenal Ascorbic Acid Levels Following the Exposure of Rats for 2 Hours at -25 C viii

11 INTRODUCTION The future of animal agriculture promises a highly intensive and competitive system of operation. Under such a managerial regime, animals will he exposed to various environmental conditions. It seems likely that the efficiency of individual animal performance will depend on the ability of the animal to adapt to its environment. Therefore, any information concerning the response of animals to different environmental conditions is of utmost importance in planning for the future animal agriculture. Animal response to stress conditions has classically been measured by a decrease in blood eosinophils, a decrease in adrenal ascorbic acid, and an increase in plasma adrenocortical levels. It was not until recent years, however, that the relationship between the activity of certain enzymes and animal response to stress conditions was proposed. The broad class of transaminase enzymes has been studied most extensively with regard to the enzyme-stress relationship. Two of the more common transaminase enzymes studied are aspartate aminotransferase (GOT) and

12 alanine aminotransferase (GPT). The elevated level of these two enzymes in human plasma has become very important in the clinical diagnosis of certain cardiac and hepatic diseases. Following the establishment of the use of these enzymes as a diagnostic tool, a multitude of data has become available concerning the presence and activity of these enzymes in animal tissue. The relationships between plasma GOT and GPT, and certain physiological conditions of the dairy cow have been studied extensively in our laboratory, however, little information is available concerning the relationship of non-specific stress, without tissue breakdown, and enzyme levels. In view of this, the objective of this investigation was to study some of the early changes in tissue transaminase levels when animals are exposed to a non-specific stress. This investigation deals specifically with some of the early changes in plasma, muscle, and liver GOT and GPT levels, when rats are exposed to low environmental temperatures.

13 LITERATURE REVIEW The Concept of Animal Response to Stress All animals possess some ability to adapt to environmental changes. This ability to adapt is commonly called survival of the fittest in nature, but may similarly be called survival of the most efficient in our animal agriculture. The term "stress" is usually defined with respect to the parameters studied, but generally it refers to a situation which requires adaptation on the part of those exposed in order to survive. In terms of our modern livestock enterprises, where the efficient production of milk, meat, eggs and wool is of utmost concern, it does not seem unreasonable to define stress as any condition which will elicit a sufficient change in an animal which will reduce its economic performance. Animals often respond in a similar manner to a variety of stress conditions. This somewhat general response to systemic stress has been described by Selye (188) and later reviewed by Veilleux (209) as the general adaptive syndrome (GAS). According to the GAS there are

14 three phases of animal response to a prolonged nonspecific stress: (a) alarm reaction, in which the initial shock is followed by counter-shock reactions; (b) stage of resistance, in which adaptation is optional; and (c) stage of exhaustion, in which the stage of resistance could not be maintained. Our present concept of stress response is based on neuroendocrine relationships. The response of an animal's homeostatic mechanism elicited by stressful stimuli is for the most part mediated through the hypo- thalamo-hypophyseal-adrenocortical axis (121). The stress response is initiated by activation of the posterior hypothalamic centers (sympathetic system) by afferents from the rhinencephalic or mesencephalic limbic systems (195). This activation results in an immediate release of catecholamines from the adrenal medulla. It is the action of these hormones which allows the animal to overcome the initial shock of the stress condition. The second phase of the stress response is initiated by the activation of anterior hypothalamic centers (parasympathetic system), in the manner described above (195), to release corticotrophin releasing factor (CRF). CRF stimulates the anterior pituitary to release ACTH into the general circulation (6). The cortex of the adrenal gland is stimulated by ACTH to increase the synthesis and release of adrenal corticoids (179* 191* 89, 84-,

15 161, 72, 115)- It is the fast action of the second response coupled with the specific action of the adrenal corticoids which initiates the adaptive processes. The control of the hypothalamo-hypophyseal-adrenocortical axis seems to be mediated by a feedback of circulating corticoids on the anterior hypothalamus (195)* This control, however, is by no means absolute and can be overridden by continued stimulation of the hypothalamic centers (224). This model for the stress response, unites Cannon's (57) "emergency reaction" and Selyes's (188) "GAS" with the present concept that the hypothalamus is the central integrator of the stress response. The Response of Animals to Low Environmental Temperatures General Response. There are many different responses noted in animals when exposed to low environmental temperatures. Some of the more common responses are change in body flexure (96), increased pelage insulation (108), shivering (90, 61), increased food intake (50, 96), increased heat production (127, 50, 40, 182), increased cardiac output (62, 102), increased tissue enzyme and protein (40, 10, 157) increased size of body glands (107, 40), dehydration (81), and many other more

16 specific reactions, some of which are discussed later in this section. The mechanism of temperature regulation in homeothermic animals as reviewed by Dukes (67) sad Guyton (95) is pertinent to the current study. Body temperature is regulated by the stimulation and repression of hypothalamic centers. Centers located in the posterior hypothalamus protect the body from over-cooling. Similarly, centers of the anterior hypothalamus prevent overheating. The activation of one of the centers with a corresponding depression of the other center, is brought about by changes in blood temperature and or impulses from peripheral temperature receptors. It is the delicate balance between these two thermostatic centers which allows for a nearly constant body temperature. Other workers (193) have postulated that norepinephrine is a transmitter in the temperature regulating center of the hypothalamus. They found, by monitoring the turn-over of tritiated norepinephrine in the hypothalamus, that rats exposed to heat showed an increase in turn-over of norepinephrine whereas rats exposed to cold did not show any effect. Earlier work by Feldberg and Myers (73* 7*0 had also suggested the involvement of the amines in body temperature regulation. They reported that the injections of epinephrine or norepinephrine into

17 the hypothalamus caused a decrease in "body temperature while the injection of 5-hydroxytryptamine caused an increase. When animals are first exposed to low temperatures, the posterior hypothalamic center stimulates the heat conserving mechanism. (This system includes intense vasoconstriction throughout the hody, pilo-erection, and cessation of sweating. If the heat-conserving mechanism is unable to prevent a continued loss of body heat to the cool environment, then the sympathetic excitation of heat production is brought into play. The increased heat production is mediated by the action of epinephrine and norepinephrine to increase cellular metabolism. This process of chemical heat production is commonly referred to as non-shivering thermogenesis (NST). Non-shivering thermogenesis designates an increase in heat production, by an animal exposed to cold, without muscular activity (59)* In animals possessing inter- capalar brown adipose tissue, it is this thermosensitive tissue which generates much of the heat to maintain internal body temperature (29* Shivering is suppressed as long as sufficient heat is generated. Adipose tissue is not the only tissue involved in non-shivering thermogenesis. Jansky (125) and Davis (60) have reported that non-contracted muscle plays an important role in non-

18 shivering thermogenesis, however, Jansky (125) indicates that only about one half of the NST cquld be accounted for by muscle tissue and suggests that other tissues are similarly involved in NST. If NST is unable to prevent a negative heat balance, then the shivering mechanism is stimulated by the posterior hypothalamus (95)- The act of shivering is probably the most important means of protection against over-cooling in homeotherms. Animals generally adapt to cold temperatures by changes in NST. Bruck et al. (20) have reported that the rat can increase NST to 150% of basal metabolic rate in the adult stage. They indicated, however, that the rat should not be considered as a typical example for mammals. In the guinea pig, this high NST occurs only in the newborn stage while the miniature pig lacks NST in the neonatal form as well as adult (29). Cold adaptation in the rat is mediated by the action of norepinephrine, and to some extent epinephrine, to increase NST (130). In those animals, however, which do not exhibit NST, cold adaption is mediated by the ability of the animal to shiver more readily on exposure to acute cold (29). Thyroid response. The thyroid plays an important role in animal survival when exposed to acute cold stress.

19 Thyroidcctomized rats or rats given propyl thiouracil to block thyroid function, survive only a few days at low temperature (4-9)* Sellers and You (18?) likewise found that the increase in metabolic rate in animals after exposure to cold does not depend on a hyperthyroid state, but does depend on the presence of thyroid hormone and thus is associated with the ability of the animal to survive. The increased thyroid activity due to cold is thought to be due to an increased release of TSH by the adenohypophysis (4-9). Itoh et al. (122) reported a marked increase in rat blood TSH levels 30 minutes after exposure to 15, 8 or 0 C temperatures. Cottle (4*9) estimated that the amount of thyroid hormone produced is about twice as much at 5 C compared with 25 C. Similarly, Heroux and Brauer (106) reported that the thyroxine requirement of 6 C acclimated rats is twice as high as that of the 23 C acclimated controls. The period of maximal thyroid activity during exposure to cold varies with the experimental conditions. Cottle (4*9) reported that maximum thyroid activity in the rat occurs after 3 weeks exposure to 5 0, with a return to normal at 6 weeks. Leblond et al. (144) have indicated that rats exposed to 0-2 C showed little thyroid stimulation for the first 3 days; maximum

20 10 stimulation was after 26 days, and at 40 days the thyroid was again normal. Similar findings have been reported by Straw and Fregby (203), where the measurements of thyroid size, -^lj uptake, and release rates all indicated a hyperfunctional state of the thyroid which persisted for 4 weeks. Starr and Roskelley (202), however, reported a somewhat earlier thyroid response in rats. Their data showed hypertrophy of the thyroid epithelium after 3 days exposure to C. The work by Knigge (138) with the hamster, shows a maximum thyroid production occurring at 30 days when the adult animals were exposed to 5-7 C for 60 days. The action of thyroid hormone to increase cellular metabolism has been well established. Nevertheless, the hypothesis advanced by Weiss (214) suggests that the thyroid gland exerts its effects by way of a few selected tissues only (skeletal muscle, heart, and liver), in which it regulates the level of metabolism so as to provide adequate heat production in order that the animal may adapt to cold temperatures. Adrenal response. The adrenal gland responds to low environmental temperatures with a release of adrenocortical hormones. The adrenocortical response increases with the severity of the cold temperatures (24). In the normal rat the adrenal cortex secretes mainly cortico-

21 11 sterone when the animal is exposed to cold temperatures (24, 219). Schonbaum et al. (184) reported that rat adrenal ascorbic acid decreased and steroid production increased after 30 minutes exposure to -20 C. The exposure of rats to 4-5 C resulted in a significant increase in plasma corticosterone after 3 hours exposure (24). Sobel et al. (199) reported that the urinary corticoid excretion was 2.6 and 2.1 times control levels after six hours exposure to 2-4 C for male and female rats, respectively. In vitro studies (183) of adrenal steroid formation have indicated that when rats are exposed to acute cold, the adrenal responds during the first thirty minutes of exposure. Holzbauer and Newport (115) have indicated in their work that pregnenolone, a precursor of corticosterone, is increased in adrenal tissue of cold exposed rats. Another indication of adrenal response is an increase in adrenal weight. Heroux and Schonbaum (110) have reported adrenal hypertrophy in the rat within the first week of exposure to 6 C. They have suggested that the hypertrophy is due to an increase in the cell number of the zona fasciculata. Petrovic and Rajcic (168) have recently reported an increase in adrenal weight when rats were exposed to 96 hours of -2 C. A 35 percent increase

22 12 in adrenal weight over controls was reported by Woods (221), using rats exposed for 26 days to 0 C. Adrenal weight, protein, and RNA show an increase in the cold exposed rat, while DNA exhibits a decrease in these rats. The adrenal medullary hormones, epinephrine and norepinephrine, have been described previously with regards to their action in thermogenesis. Urinary levels of these hormones in the rat, during cold stress, have been reported by Leduce (145). He reports that norepinephrine increases after 12 hours exposure to 3 C and reaches a maximum level after 24 hours. Epinephrine increases more slowly and peaks after 1 week exposure. Leduce (145), however, reports that the increased level of epinephrine comes from the adrenal medulla while the increased level of norepinephrine comes from adrenergic nerve endings. Motelioa (156) has similarly reported a 3-4 fold increase in the rat urinary excretion of norepinephrine after 24 hours exposure to 3 C* Epinephrine only gradually increased upon exposure to cold. Electrolytes and water. The early response of electrolyte metabolism to cold temperature is characterized by a hemoconcentration of brief duration (8). In man a pronounced diuresis occurs. The concentration of plasma electrolytes remains relatively unaltered except for an increase in plasma potassium and probably magnesium

23 13 (8). Cohn et al. (4-5) have reported a decrease in nitrogen and calcium in the skeleton of cold-exposed rats. Baker (9» 8) has reported that the intracellular sodium of rat muscle was decreased by exposure to cold and that an increase in plasma sodium concentrations was observed. In a review by Hess and Baily (111) they reported an increase in plasma sodium and a decrease in haematocrit for rats exposed to cold temperatures. Generally, body water of animals increases when animals are exposed to cold temperatures (45, 103). Baker (8, 9) has reported that the total water of the skin of rats acclimatized to cold temperature increases due to an increased chloride space. However, he also reports that the total water of rat muscle is decreased while the chloride space is increased. Metabolic responses. One of the first responses of unacclimatized rats to a cold stress is an elevated metabolic rate, to compensate for the increased heat loss (194). Krog et al. (14-1) have reported that during exposure to cold stress, wild rats are able to increase body metabolism 4-6 fold, cold conditioned white rats 3 fold, and unacclimatized white rats even less. An increased oxygen uptake by tissues when es&osed to cold temperatures has been investigated (109$ 100, 213). Heroux et al. (109) have reported that 30 minutes

24 14 after exposure to 6 C, the cold acclimated rats showed a 2 fold increase in oxygen consumption, while warm acclimated rats demonstrated only a & increase. These data are substantiated by the in vitro work by Weiss (212) and Hannon (100) which indicated that tissues obtained from cold exposed rats exhibited an increased oxygen consumption. The metabolism of carbohydrates is generally increased during cold stress. Klain and Burlington (134) have demonstrated a decrease in glycogen levels and an increased expiration of COg from glucose-u- C when rats were exposed to 5 C. They have suggested that these findings indicate an increase in metabolic rate and a mobilization of body energy stores. The turnover of plasma glucose in cold stressed rats has 14 been reported by Depocas (6$). Using C glucose, he found a significant Increase in the rate of disappearance of the labelled glucose from plasma when warm acclimated rats were transferred to a 6 C environment. Cold acclimated rats, similarly treated, did not show this response. The report by Musacchia and Barr (137) indicated that glucose availability may also become a factor in the animals' ability to survive cold temperatures. Their work indicated that there is a decrease in glucose absorption from the intestinal tract when hamsters are

25 exposed to cold stress. They have postulated that 15 alterations in the active transport of glucose by the intestinal mucosa may be due to enzymatic changes which were elicited by the cold exposure. This decrease in absorption of glucose from the intestinal tract may help to explain the decrease in body weight seen in the hamster (157) and the rat (135) exposed to cold stress. Klain and Hannon (135) have reported that gluconeogenesis was more pronounced in both fed and fasted rats exposed to 5 C for 1,000 hours than in the 25 C controls. They suggested that changes in adrenocortical and thyroid activity seen during cold exposure lead to increased protein catabolism and an increased glucose production. Beaton (11) has indicated that with rats exposed to 2-3 C for 7 days, there is an increase in nitrogen retention for the first 24- hours, but a decrease in nitrogen retention occurs between hours of exposure. These data suggest that the catabolic effects of cold are not evident until after 24 hours of exposure. Cold acclimated rats have an increased capacity to oxidize long chain fatty acids, thus little deposition of fat in the liver occurs (152). Williams and Platner (218) found an increased level of unsaturation in white adipose tissue of the cold esqposed rat. Wilson et al. I

26 16 (220) have recently reported that plasma glycerol concentrations increased significantly in cold exposed men over the controls. An increased fat mobilization with an enhanced utilization of free fatty acids as a fuel substrate for heat production was suggested in the cold exposed men. The increase occurs immediately upon exposure and is still evident after 8 hours exposure. Other responses. There are many responses to cold temperature which could be discussed, however, only a sample of the multitude of data available is presented. Sundaresan ^et al. (204) have found that the depletion of vitamin A is increased in the cold exposed rat. Rats exposed to 0-2 C exhibited a decrease in the acetylation of p-aminobenzoic acid. Campbell 5t al. (35* 36) have reported an increase in coenzyme A in the liver of rats exposed to 0-2 C. The level of eosinophils has been known to decrease when animals are exposed to cold temperatures (64). In the rat, eaqposure to 4 C for 40 days resulted in a marked decrease in the albumin fraction (190). This is in contrast to the work published by Bopp and Platner (23) which indicated that in the prairie meadow mouse, the plasma albumin levels were significantly increased in the cold exposed mice. Huston and Sublas (120) have reported that in the chicken, total plasma protein

27 concentrations were significantly higher for birds kept at 8 C compared with those at 18 C or 50 C. For additional information concerning the response of animals to cold temperatures, the reviews by Hess (111), and Smith and Hajer (196) are most useful* Status of Aspartate Aminotransferase (GOT) and Alanine Aminotransferase (GPT). Transamination. The transamination reaction was first described in 1957 Braunstein and Kritzman (27) using pigeon breast muscle as the source of enzyme. Transamination is a chemical reaction involving the transfer of an amino group of one amino acid to a keto acid with the formation of a second amino acid and a new keto acid. It was postulated by these early workers confirmed later by Cohen and Hekhius (44) that enzymes, later called transaminases, were necessary catalysts in the transamination reactions. In 1940, Cohen and Hekhius (44) found that pigeon breast and pig heart muscle yield an enzyme or enzymes which catalyze the reversible reaction glutamic acid + oxaloacetic acid ketoglutaric acid + aspartic acid; and glutamic acid + pyruvic acid ketoglutaric acid + alanine. The enzymes which catalyze these reactions were given the name of glutamic

28 oxaloacetic transaminase (GOT) and glutamic pyruvic 18 transaminase (GPT), respectively. It was not until recently that the names of these enzymes were changed to aspartate aminotransferase and alanine aminotransferase, respectively. These two enzymes will be referred to throughout this paper as GOT and GPT. It was formerly thought that only alanine, aspartic and glutamic acids were involved in transamination. It was, however, reported by Cammarata and Cohen in 1950 (34*) that twenty-two amino acids in addition to alanine, aspartic and glutamic acids participated in transamination reactions. Snell, in (197)» reported that pyridoxal phosphate was a necessary coenzyme for the transaminase enzymes. Young rats fed a pyridoxine deficient ration for four weeks showed an 85 and 65 percent decrease in serum GPT and GOT, respectively (28). The incubation of the serum with pyridoxine restored part of the decreased enzyme activity. Prischer and Walter (75) have recently reported that the incubation of erythrocytes with pyridoxal phosphate produced an increase in GOT activity of these cells. The kinetic data for the transamination reaction catalyzed by GOT are consistent only with a "ping pong bi bi mechanism (104-). This indicates that during the reaction an alpha amino acid (aspartate) binds to

29 19 the enzyme (GOT) and the corresponding alpha keto acid (a-ketoglutarate) is released, followed "by the binding of the second keto acid (oxaloacetic) and the subsequent release of the second alpha amino acid (glutarate). There is little reason not to believe that a "ping pong bi bi" mechanism also operates in the transamination reaction catalyzed by GPT. The kinetic data presented by Bulos and Handler (31) indicate a high specificity of GPT for only the one transamination reaction. Presence and activity of GOT and GPT in animal tissue. Transaminases are intracellular enzymes which are widely distributed in animal tissues. GOT and GPT appear to have the highest enzymatic activity of the transaminases found in animal tissues and, therefore, have been more widely studied (130). With the use of chromotograpby and gel electrophoresis, it has been observed that GOT appears in multiple forms in animal tissues (128, 229)* Two isozymes of GOT have been reported (128, 76, 165)* GOT I, which has anionic properties and was observed in both tissue and serum, was postulated to be derived from the cytoplasm. GOT II, which has cationic properties and found only in serum was postulated and later confirmed (63, 18) to be derived from the mitochondria. In humans, however, there are reports (63, 18) which indicate that there exist

30 20 three anionic and two cationic isozymes with GOT activity in blood serum. Fewer reports are available concerning the isoenzymes with GPT activity. Ziegenbein (227) has reported two different forms of GPT. One which is found in the cytoplasm and the other which is located in the mitochondria. GOT and GPT have been found in high concentrations in cardiac and skeletal muscle, brain, liver, kidney and lung tissue (30, 7, 158). Generally, the skeletal muscle has the highest GOT activity while the liver has the highest GPT activity. The activity of GOT and GPT in many other tissues has been reported. Human blood platelets have been reported to possess high levels of GOT activity, but only traces of GPT activity (208, 150). GOT activity has been reported in the rumen liquor of sheep (170) and calves (200). The highest activity was found in the protozoan fraction. The presence of GOT (169, 71, 70, 126) and GPT (71) in human semen has been reported. Eliasson (70) and Pumpianski and Sharon (169) have reported a decrease in GOT activity in semen as sperm numbers decrease. Eliasson (71) observed that GOT activity is about tenfold higher than GPT activity in human semen. Other investigators have reported the presence and activity of GOT and GPT in the semen or

31 seminal plasma of the bull (77* 94)» rabbit (94) and 21 man (5)- There is general agreement that the activity of GOT in semen is about tenfold higher than blood serum GOT. GPT, however, is usually very low in the fluids and tissues of the male reproductive tract. With respect to the female, Hochman and Schenker (114) have reported that during the human menstrual cycle, there is a significant increase in GOT activity of the endometrium during mid-cycle at about the time of ovulation. It has been reported in the cow, that serum GOT activity is highest around the estrus period (176, 55, 59)- Davis et al. (59) have also indicated a secondary peak around the 15th day of the cycle. It is suggested that the variation in enzyme levels during the female cycle is probably under the influence of the ovarian hormones (114, 58). Other investigators have reported a relationship between serum GOT and GPT levels and certain physiological conditions in the bovine. Crist et al. (51* 52) have reported that plasma transaminase activity increases as milk production increases and that a highly significant increase in transaminase activity occurred with advancing stage of lactation. Stallcup et al. (201), however, have indicated a significant negative correlation between serum GOT activity and days of lactation. These workers have also observed no differences in transaminase

32 activities between open and pregnant cows in similar 22 stages of lactation. Grist et al. (52), however, found that plasma transaminase activity decreased with advancing stage of pregnancy. This work was later confirmed by Boots and co-workers (21, 22). Boots et al. (22) have reported a significant correlation between age and plasma enzyme levels in young bulls and open heifers. Roussel and Stallcup (177)> however, observed a decrease in plasma enzyme levels with increasing age of bulls. Season effects have been observed by Crist et al. (52) and Roussel and Stallcup (177)- Both groups of investigators have indicated an increase in plasma enzyme levels during the summer months. A discussion of the presence and activity of GOT and GPT in animal tissues of 23 different species is given in the reports of Zimmerman e al. (228, 230), Cornelius et al. (4-8) and Nagode et al. (158). The regulation of tissue enzyme levels. There are many pathological and environmental conditions which result in changes in tissue enzyme levels. It is the objective of this section to describe briefly some of the physiological factors involved in the control of tissue enzyme levels. GOT and GPT are intracellular enzymes. These enzymes diffuse into the surrounding fluid and are

33 taken into the general circulation. The movement of 23 these enzymes into the circulatory system occurs at a somewhat constant rate, thus allowing for the more or less constant level of GOT and GPT in the serum of a normal individual. The relationship of serum to tissue levels of GOT and GPT has been reported by Zimmerman et al. (228). They observed that generally, the content of GOT and GPT in tissues tended to be highest in those animals with the highest serum levels. however, proved to be an exception. The horse, Elevated levels of serum transaminases are the result of either cell necrosis or changes in cell permeability. It has been postulated (140, 113) that cell permeability changes are responsible for much of the increase in serum enzyme levels observed in those animals exposed to stress situations which do not involve muscular activity. Both the changes in cellular metabolism and certain hormone levels are capable of altering cell permeability. The level of tissue enzyme activity is the result of a balance between the rate of enzyme synthesis and the rate of enzyme degradation. The elevated levels of tissue transaminases observed in stressed animals are for the most part due to an increase in enzyme synthesis. This is substantiated by the reports (93* 217) which indicate that the induction of transaminase enzymes can

34 24- be prevented by protein inhibitors. There is little information to suggest that the elevated tissue levels are due to activation of already existing enzyme forms. There exists a number of situations which can result in changes in tissue transaminase levels. Some of these conditions are discussed in the next section. Hurwitz and Freedland (119) have observed in the rat that increasing increments of protein in the diet significantly elevated liver GOT and GPT activity. These findings have been reported also by Rosen et al. (173) and Freedland et al. (80). The reverse has been observed when rats are fed a protein deficient diet. Szepesi and Freedland (205) have observed a logarithmic decrease in liver GOT and GPT activity when rats were changed from 9096 protein to a 90% carbohydrate diet. A decrease in rat muscle GOT also has been reported with protein deficient diets (225). Nichol and Rosen (162) have reported that increased rate of gluconeogensis, glucocorticoid treatment, diabetes, starvation and high protein content would increase liver GPT activity, while growth of new tissue, tumor growth, fetal development and partial hepatectomy decreased GPT activity. The involvement of adrenal hormones with elevated transaminase levels has been well established (88, 101, 175» 174, 15). Critz and Withrow (56) observed that the

35 increase of GOT in the heart and muscle of the exercised rat, could be abolished by blocking or removing the 25 adrenal cortex. Freedland et al. (79) observed a decrease in liver GOT and GPT following adrenalectomy. Thus it is evident that adrenal hormones, especially adrenal corticoids, are important agents in regulating the tissue enzyme levels. The mechanism whereby adrenal corticoids exert their effect on enzyme synthesis is not completely understood. To date there is good agreement that the primary site of action of adrenal corticoids is at the transcriptional level of protein synthesis, to increase the synthesis of SNA (181, 211, 132). Lukacs and Sekeris (149) have reported that one of the first biochemical effects of cortisol was to activate SNA polymerase. These findings were later confirmed by Ohtsuka and Koide (165) The mechanism, of action of these hormones, resulting in an increase in enzyme synthesis is thought to be by derepression. Lukacs and Sekeris (149) suggests that cortisol increases template capacity of DNA by a derepression mechanism. Derepression of genetic material could result from binding of the hormone with, and inactivation of a repressor molecule localized on the chromatin itself. This theory is somewhat substantiated by the report of Hamilton (98) which found that estradiol binds to the

36 chromatin in the nucleus within a few minutes following 26 administration. Garren et al. (86) have reported that when enzyme levels reach a certain level, a repressor molecule is synthesized which slows down the protein synthesizing machine. It thus appears that a repressor- derepressor mechanism operates in regulating the level of enzyme synthesis. This type of mechanism coincides with the Jacob and Monod (123) theory of genetic control of protein synthesis. The rate of enzyme degradation is an important item when determining the level of tissue GOT and GPT. Segal et al. (186) reported the half-life of rat muscle GPT as 20 days whereas the same enzyme in the liver has a half-life of 3 days. Earlier Segal and Kim (183) reported the half-life of liver GPT to be 3.5 days in the normal animal but was 1.5 days in the prednisolone treated rat. Szepesi and Freedland (206) have recently reported a half-life of 40 hours for GPT and a halflife of 23 hours for GOT in cortisol treated rats. The degradation rate of other enzymes suggests that starvation (178), increased protein intake (206) and adrenal hormones (206), all effect the degradation of enzymes. The mechanism of enzyme degradation and the factors responsible are still to be elucidated.

37 Some of the Factors Associated With Elevated Levels of Serum 27 and Tissue Transaminases Since the discovery of the clinical importance of tissue transaminase levels in 1953* there have been numerous investigations which have described the relationship between certain pathological and environmental conditions and tissue transaminase levels. It is the purpose of this section to review some of the conditions which alter tissue transaminase levels. Disease. The american investigators La Due, Wroblewski and Karmen (143) were the first to draw attention to the clinical importance of the GOT-test for the early diagnosis of myocardial infarction in humans. They reported that patients with myocardial infarctions exhibited a 2-20 fold increase in serum GOT levels within 24 hours. It has been reported by other investigators (66, 148, 42, 154) that from 65 to 92 percent of the myocardial patients studied exhibited elevated serum GOT levels. The level of serum GOT seems to be dependent on the amount of tissue necrosis. Killen and Tinsley (133) found a correlation between the size of the infarct and peak serum GOT levels. The use of the serum GOT-test as a reliable tool in the diagnosis of cardiac diseases has been suggested (43, 91).

38 Not long after the early reports of the relationship of serum transaminase levels and cardiac diseases, Holander et al. (155) reported that serum GOT was a highly specific index of hepatocellular injury. He found that injecting rats with carbon tetrachloride resulted in hepatic cell necrosis and a subsequent rise in serum GOT. These findings were later confirmed by Cornelius and Kaneko (4-7) and Zimmerman et al. (229). Zelman et al. (226) observed an excellent correspondence between the extent of necrosis of liver cells and the rise in serum GOT and GPT levels. Hurtwell ejb al. (118) have reported elevated serum GOT and GPT levels in chimpanzees suffering from acute hepatitis. Abbruzzese and Jeffry (1) observed a fold increase in serum GOT in patients with chronic extrahepatic biliary disease. The use of elevated levels of serum GOT and GPT in the diagnosis of certain liver diseases has been established. Recently, however, Gabrieli and Orfanos (83) have indicated that the GOT isoenzyme pattern in the serum is a more important indicator of hepatic cell damage than total GOT. The relationship of muscle cell necrosis and elevated enzyme levels has also been established (17, 216, 105). Henson and Rao (105) observed that injecting rabbits with plasmocid dihydroiodide resulted in muscle

39 necrosis which was followed by an elevated level of 29 serum GOT. One of the more common diseases affecting muscle tissue is white muscle disease. Elevated levels of serum GOT and GPT, associated with this disease have been observed in calves (17)» lambs (17, 29* 142), chickens (210), and humans (26). The rise in serum enzymes associated with this disease is due to the leakage of the enzymes from the affected cells. It appears that the magnitude of the enzyme response is related to the extent of tissue damage. Other investigators have reported elevated serum transaminase levels with such diseases as renal infarction (87), canine distemper (198), riboflavin deficiency (41), and periodontasis (116). Physical stressors. Exercise, either by running or swimming has been a commonly used stressor in studying serum and tissue transaminase levels. Rats exposed to periods of exercise show an increase in serum transaminase levels (3» 192, 4, 85)* with a greater enzyme response in untrained versus trained rats. Critz (53)» however, observed a pattern of enzyme fluctuation in rat serum, while Critz and Merrick (95) earlier reported a decrease in serum GOT in rats subjected to swimming exercise. Cornelius (46) and Cardinet (38) both reported an increase in serum GOT in the horse following exercise.

40 30 Bedrak (14) observed an increase in serum GOT and GPT levels in the exercised dog. There is more controversy concerning the changes in serum transaminases when humans are exposed to exercise. There are reports which indicate that enzyme levels increase (171i 180, 212), do not change (159* 97i 78), and decrease (54, 160). In view of these findings, it appears that the level of serum enzymes is dependent on both the nature and duration of the exercise used. Other less frequently reported stressors which increase serum transaminase levels are, restraint (166), animal shipping (52), endotoxin shock (140, 19), hypoxia (112, 19), inflammation (19* and- burn shock (19)- Hormones. The inducible nature of tissue GPT by adrenal corticoids has been well established (101, 173* 174, 15). Gavosto et al. (88) have reported a significant increase in both hepatic GOT and GPT in cortisone treated rats. Earlier, Eischeid and Kochakian (69) reported a decrease in hepatic GOT in cortisone treated mice. Pearl et al. (166) observed an increase in serum GOT in the rat when injected with corticosterone. More recently, Ogawa et al. (164) reported, that adrenal steroids exert a differential affect on GOT liver isoenzymes. They observed that cortisone and corticosterone increased the activity of soluble GOT but decreased the

41 activity of mitochondrial GOT* The induction of 31 tyrosine transaminase by adrenal hormones has also been thoroughly reviewed (92, 131* 139* 207* 146)* Several other hormones have been reported to alter serum and tissue levels of GOT and GPT. Epinephrine (166, 113* 164) and norepinephrine (166, 113) have been found to increase serum GOT and GPT levels. This increase is thought to be mediated through tissue permeability changes* Litwack (147) has reported a decrease in transaminase response during thyroid administration. Mead (153)* however, observed no difference in serum transaminase levels between hypothyroid, hyperthyroid and normal patients. Curry and Beaton (57) observed an increase in tissue GOT and GPT in the glucagon treated rat. In vitro studies with thymocyte suspensions exhibited an increase in GOT with testosterone and progesterone administration. There was no detectable affect on GPT levels. Eckstein and Lavic (68) observed enhanced GOT and GPT activity in the ovaries and uteri of PMS and HOG treated immature rats. They have suggested that the enhanced GPT activity in the uterus of the treated animal is the result of sex hormones produced by the stimulated ovaries. Other investigators (114, 58) have also suggested the involvement of ovarian hormones in the alteration of transaminase levels.

42 Cold temperature. Articles concerning the relationship of cold temperature and serum and tissue transaminase enzyme levels are somewhat limited; nevertheless, Beaton (12) has reported a significant increase in tissue GPT in rats exposed for seven days to 2-3 C. He has also observed (10) a direct relationship between protein level and the activity of GPT. He demonstrated an increase in transaminase levels with an increase in protein of the diet, in the cold exposed rat. Klain and Vaughan (136) and Klain et al. (137) have observed an increase in liver GOT and GPT activity in the cold exposed rat. Their findings suggest that the increase in GOT and GPT is the result of an increased protein intake. This is in contrast to the enzymes tryptophan pyrrolase and tyrosine a-ketoglutarate transaminase which are increased by the cold. Beaton (13) has reported that liver GPT increases with cold exposure for a period of about 7 days in the rat. There was no indication of increased serum GPT level. Penney et al. (167) observed a decrease in muscle GOT level and an increase in serum GOT activity, when a limb of a rabbit was immersed in 2 C water for 24- hours. They suggested that changes in permeability of the muscle tissue were responsible for the elevated serum GOT. There are several enzymes which are useful in determining some of the factors which alter GOT and GPT

43 levels in the cold exposed animal, Harmon (99) has observed in rats which were exposed to 1-5 C for 3-4 weeks, that the levels of the pentose shunt enzymes are decreased, levels of glycolysis enzymes are increased and levels of glycogensis enzymes are unchanged, Mayer et al. (151) have found an increase in total lactic dehydrogenase in men exposed to 5 C. Blatt et al. (16), however, have reported a decrease in lactic dehydrogenase activity in the rat exposed to 5 C- 2?he activity of cytochrome oxidase has been reported to increase in the cold exposed rats. Jansky (124) reports that cold acclimatization makes full exploitation of the oxidase capacity possible in the cold acclimated rat. There exists a need for further information concerning the early response of enzyme systems in different tissue to changes in environmental temperature.

44 MATERIALS AND METHODS Two hundred eighty-two male albino rats of the Wistar strain were used to provide materials for 1460 enzyme determinations and 195 adrenal ascorbic acid assays. An additional 25 rats were used in preliminary standardization procedures, and to develop certain techniques. All rats were housed in a small animal room (26-2 C) in hanging wire cages with 2 or 4 rats per cage. The rats had free access to tap water and Purina Lab Chow at all times, unless otherwise stated. The lighting system was adjusted for 12 hours light and 12 hours darkness. This lighting regime was maintained throughout the study for both control and experimental animals. Rats were exposed to two different temperature regimes. A environment was provided by using a large walk-in cooler, which was maintained at 3-1 C. The other environment of -24 C was provided by using a walk-in deep freeze maintained at C. All rats, with the exception of those used in preliminary trials, were transferred to individual plastic cages (11 X 7 X 5 in.) with metal tops, just 34

45 prior to going on experiment. Both the control and 55 experimental animals remained in the plastic cages without bedding for the duration of a given experiment. Control animals, for the temperature experiments, were kept in the 26 C animal room. There was an attempt at all times to keep the amount of physical handling the same, for control and experimental animals. All rats were weighed prior to the time they were sacrificed. The mean weights of the groups within an experiment did not differ more than 10 percent. The activity of aspartate and alanine aminotransferase enzymes was determined in the blood, liver, and gastrocnemus muscle of the rats studied. The level of ascorbic acid in the adrenal glands of these rats was also determined. The procedure for enzyme analysis and ascorbic acid determination is outlined in the following sections. Tissue Collection and Processing Plasma samples were collected by cardiac puncture from ether anesthetized rats. The samples were taken with 10 ml glass syringes containing 1.0 ml of 5% sodium citrate. The blood samples were centrifuged at C g for one hour in a Servall table top centrifuge, which was maintained in a 3 C cold room. The plasma was

46 36 decanted, into clean test tubes for analysis. All samples which showed any evidence of lysis were discarded. The determination of GOT and GPT activity was made on the fresh plasma within 24 hours of collection. The large posterior lobe of the liver was excised, wrapped in aluminum foil, and quickly frozen in liquid nitrogen vapor. The tissues were frozen and stored in the top of a MVE Model A-8000 liquid nitrogen semen storage tank, until enzyme analysis could be determined. The right gastrocnemus muscle was excised and frozen in the same manner as described for liver tissue. At the time of enzyme analysis the liver and muscle tissues were allowed to thaw in the 3 C cold room. A center section of the liver lobe was removed, blotted with tissue, and weighed to the nearest milligram. Approximately 750 mg of liver tissue were used for enzyme analysis. An adjacent section of the liver lobe of about the same size, was used for dry weight determinations. Dry weight determinations were obtained by drying the tissue sample to constant weight in an oven maintained at 80 C. The liver section to be used in enzyme analysis was homogenized with a glass tube and a motor-driven teflon pestle in 3 or 6 volumes of 0.1 M potassium phosphate buffer (ph 7-4). The crude homogen&te was centrifuged