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UBC Theses and Dissertations

Estrogen inhibition of adrenocortical function in the lactating dairy cow Mason, Steve 1974

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ESTROGEN INHIBITION OF ADRENOCORTICAL FUNCTION IN THE LACTATING DAIRY COW by BRIAN DOUGLAS MASON B.Sc, University of British Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of ANIMAL SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1974 In presenting th is thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date A o c y O ^ . a o ; t TH i i ABSTRACT Acetonemia is a metabolic disorder common among dairy cattle in the period from 10 days to six weeks post partum. It is during this time that milk production is at its maximum and in addition, the cow is begin-ning to exhibit regular estrous cycles. Thus, the profound demands for the precursors of milk production are superimposed upon a relatively un-stable endocrine environment. The initial objective of this study was a clarification of the interactions between estrogens and endogenous substrates of glucose metabolism in the lactating cow. Since estrogen levels during the bovine estrous cycle had not been convincingly reported at the outset of this work, their measurement was the subject of the first experiment. Both estradiol and estrone exhib-ited peaks on the two days prior to the day of standing heat. These findings were later substantiated with the appearance of reports where plasma estrogen levels were measured by competitive protein binding and radioimmunoassay. A significant decline in plasma amino nitrogen concentration occurred after the estrogen peak, the decrease being reflected in marked reductions in the concentrations of threonine, methionine, proline and the branched chain amino acids. A notable hypoglycemia occurred somewhat later than the decline in amino acids. These findings were rationalized in terms of increased uterine and mammary uptake and/or decreased tissue mobilization of amino acids which may have given rise to the hypogly-cemia as a result of decreased availability of glucogenic substrate. A possible mechanism of decreased tissue mobilization of amino acids was considered to be the inhibition by estrogens of adrenal gluco-corticoid output, since glucocorticoids play a major role in regulating i i i amino acid balance in peripheral tissues. In addition, the efficacy of glucocorticoid therapy in the treatment of bovine acetonemia is well recognized. With these considerations in mind, the role of estrogens in the modification of ACTH-stimulated glucocorticoid secretion was assessed in two further experiments. Following suppression of endogenous ACTH secretion by dexametha-sone, both estrogen treated and non-estrogen treated cows were subjected to stimulation with incremental doses of exogenous ACTH. At all levels of ACTH stimulation, estrogen treatment resulted in reduced response when measured in terms of plasma glucocorticoid concentrations. However, estro-gen treatment had no effect on plasma binding of glucocorticoids. The concentrations measured were, therefore, a direct reflection of gluco-corticoid activity. In the second experiment to test adrenal inhibition by estrogens, plasma ACTH and glucocorticoid levels were measured during the estrous cycles of lactating cows. For this purpose, a radioimmunoassay for ACTH was developed, providing a sensitive, reproducible method for the measure-ment of this hormone in bovine plasma. A significant increase in circu-lating ACTH concentration was seen during the estrogenic phase of the es-trous cycle. No such increase was noted in the concentration of gluco-corticoids, levels fluctuating within the normal, low range. This result implied that during the estrogenic phase of the estrous cycle, inhibition of glucocorticoid secretion by estrogens resulted in a compensatory rise in circulating ACTH concentration, bringing the glucocorticoid level back to normal. iv The results of these experiments are discussed with reference to the possible role of alterations in endocrine balance in the etiology of bovine acetonemia. V TABLE OF CONTENTS Page Title Page 1 Abstract i i Table of Contents v List of Tables x List of Figures xi Acknowledgements xii Glossary of Terms xiii GENERAL INTRODUCTION 1. I. Glucose Metabolism in the Ruminant 2. A. Glucose Utilization in the Lactating Ruminant 2. 1. Oxidative substrate 2. 2. Synthetic substrate 3. 3. Lactogenesis 4. B. Glucose Supply in the Lactating Ruminant 5. 1. Dietary glucose 6. 2. Gluconeogenesis 6. a. From propionate 6. b. From glycerol 8. c. From lactate 9. d. From amino acids ' 9. C. Summary 13. II. Acetonemia 15. vi Pa£e III. Hormonal Control of Glucose and Amino Acid Metabolism 17. A. Glucose Metabolism 17. 1. Control by glucocorticoids 17. 2. Control by insulin 19. 3. Control by growth hormone 20. B. Amino Acid Metabolism 22. 1. Control by glucocorticoids 22. 2. Control by insulin 23. 3. Control by growth hormone 24. C. Hormones in the Etiology of Acetonemia 25. IV. Role of Estrogens in the Regulation of Substrate Metabolism . . . . 27. A. Direct Effects 27. B. Indirect Effects 29. 1. Mediated through alteration of glucocorticoid secretion 29. 2. Mediated through alteration of growth hormone secretion 31. EXPERIMENTS 33. I. The Influence of Estrous Cycle Hormones on Plasma Amino Acids, Glucose and Urea Nitrogen 33. A. Plasma Estradiol and Estrone Concentrations During the Estrous Cycle of the Cow 34. 1. Introduction 34. vii Pac[e 2. Materials and methods 35. 3. Results and discussion 36. 4. Conclusions 42. B. Plasma Amino Nitrogen, Amino Acids, Glucose and Urea  Nitrogen Concentrations During the Estrous Cycle of the Cow 42. 1. Introduction 42. 2. Materials and methods 43. 3. Results and discussion 45. 4. Conclusions 51. C. General Discussion 51. II. Estrogen Inhibition of Glucocorticoid Response to ACTH Stimulation in the Lactating Cow 53. A. Dexamethasone Suppression of Endogenous ACTH Production . . . 53. 1. Introduction 53. 2. Materials and methods 54. 3. Results and discussion 55. 4. Conclusions 57. B. Effect of Estrogen on Adrenal Response to Exogenous ACTH 57. 1. Introduction 57. 2. Materials and methods 58. 3. Results and discussion 59. 4. Conclusions 62. vi i i Page C. Estrogen Effect on Plasma Glucocorticoid Binding 64. 1. Introduction 64. 2. Materials and methods 65. 3. Results and discussion 66. 4. Conclusions 66. D. General Discussion 70. III. Plasma ACTH and Glucocortical Levels During the Estrous Cycle of the Cow 71. A. Radioimmunoassay of ACTH 71. 1. Introduction 71. 2. Materials and methods 71. •I oc a. Preparation and purification of I - ACTH 71. b. Assessment of purity and specific activity of labelled product 73. c. Blood sampling and extraction of ACTH from plasma . . . 75. d. a 1 _ 2 4ACTH antibody 77. e. Incubation of samples and standards 77. f. Separation of free from antibody-bound ACTH 77. g. Validation of assay 78. 3. Results and discussion 78. a. Radioiodination and purification of a ACTH 78. b. Radioimmunoassay 79. 4. Conclusions 82. ix Page B. Plasma ACTH and Glucocorticoid Concentrations During the Bovine Estrous Cycle 82. 1. Introduction 82. 2. Materials and methods 85. 3. Results and discussion 86. 4. Conclusions 89. GENERAL SUMMARY AND CONCLUSIONS 90. LITERATURE CITED 92. X LIST OF TABLES Page 1 Quantitative estimates of glucose utilization and supply in the lactating cow 14, 2 Plasma estrogen concentrations during four stages of the estrous cycle in the cow 40. 3 Mean concentrations of plasma metabolites during the four periods of the estrous cycle 47, 4 Comparison of amino acid concentrations in jugular plasma in cows before and after the proestrus plasma estrogen peak 49, 5 The effect of estrogen treatment on plasma cortico-steroid binding capacities in the dairy cow 69. 6 Schematic representation of ACTH radioimmunoassay procedure 76. 7 Effect of insulin and dexamethasone on plasma con-centrations of ACTH in two lactating non-pregnant cows 84. 8 Plasma ACTH and corticosteroid concentrations in 4 cows during the estrogenic and luteal phases of the estrous cycle 88. xi LIST OF FIGURES Figure Page 1 Plasma levels of estradiol and estrone during the bovine estrous cycle 39. 2 Alpha amino nitrogen concentrations in jugular plasma during the bovine estrous cycle 46. 3 Plasma corticosteroid concentrations on control day and on a day on which 10 mg dexamethasone were administered 56. 4 Pattern of corticosteroid response to hourly infusions of increasing doses of a'~^ACTH in a single animal 60. 5 Relationship between dose of ACTH and response of plasma corticosteroid concentration 61. 6 A comparison of the dose-response relationship for the dairy cow with that found in man 63. 7 Separation of bound from free Cortisol using small column gel filtration 67. 8 Regression of the inverse of bound upon the inverse of free Cortisol 68. 9 Radiochromatography scans of paper strips after chromatoelectrophoresis 74. 1 ?5 10 I-ACTH recovery from plasma as a function of the concentration of QUSO G32 in the extraction mixture 80. 11 Calibration curve for ACTH standards extracted from plasma 81. 1 -24 12 Recovery from plasma of a ACTH with reference to standards made up directly in diluent buffer before incubation 83. 13 ACTH and corticosteroid concentrations in plasma during the estrous cycle 87. xii ACKNOWLEDGEMENTS I am indebted to a number of individuals and institutions for their assistance in this project. Dr. W.D. Kitts, Chairman of the Department of Animal Science, made available facilities and equipment. Dr. J.A. Shelford, the late Mr. J.C. MacGregor, Mr. Don Cooper and Ms. Donna Mason provided valuable assistance in the preparation and handling of experimental animals. Dr. J.W. Kendall of the University of Oregon Medical School, through the National Pituitary Agency of the U.S.A. kindly donated the anti-ACTH serum without which the development of the radioimmunoassay for ACTH would have been much more difficult. QUS0-G32 was a gift of the Philadelphia Quartz Co., Philadelphia, Pa. Financial support for the research was provided by the National Research Council of Canada. I was fortunate to receive a Leonard S. Klinck Fellowship (1970-73) and a Kill am Predoctoral Scholarship (1973-74). Above a l l , I wish to thank my supervisor, Dr. C.R. Krishnamurti, for his guidance and continual inspiration throughout this work. xiii GLOSSARY OF TERMS Terms commonly employed for the description of parameters of glucose metabolism estimated by using isotopic tracers are defined here for sake of clarity. Entry rate - the rate at which glucose enters the sampled pool of gluco In this thesis, the term is synonymous with turnover rate. Pool size - the quantity of body glucose with which injected iso-topically labelled glucose mixes. Space - the volume of fluid through which the glucose pool is distri buted. Turnover rate - synonymous with entry rate at steady state. 1. GENERAL INTRODUCTION Under most practical feeding conditions, the contribution of alimentary glucose absorption to the total glucose requirement of the lactating bovine is, at best, relatively small. However, during early lactation, the stress upon the dairy cow for mammary lactose synthesis, and thus glucose supply, is profound and may result in hypoglycemia, giving rise to acetonemia. Since the major proportion of the glucose supply is met by way of hepatic gluconeogenesis, the demand for glucose precursors may limit the ability of the animal to meet her glucose re-quirements. Superimposed upon the lactational demand, other metabolic events may decrease the availability of substrates through direct competi-tion or inhibition of substrate supply. For example, estrogen-mediated protein anabolism may result in decreased availability of glucogenic amino acids by direct competition. Estrogens may also limit glucocorticoid-mediated amino acid mobilization from tissue stores through an inhibition of adrenal glucocorticoid synthesis. The role of estrogens in the control of substrate metabolism in the lactating cow is the subject of this inves-tigation. In particular, the inhibition of ACTH stimulated glucocorticoid secretion by estrogens is studied. 2. I. Glucose Metabolism in the Ruminant The metabolism of the ruminant is unique among mammals in several respects. To a major extent this is due to the fermentation of dietary carbohydrates in the rumen, giving rise to short-chain fatty acids which supply approximately 70% of the animals caloric requirements (Bergman ejt al_., 1965). In the monogastric animal -t carbohydrate digestion event-ually yields hexoses which are absorbed directly into the portal blood (Ballard ejt al_., 1969). The unique nature of ruminant glucose metabolism is reflected in a blood sugar level of 50-70:itig% compared with levels of 100-150 mg% in monogastrics. A. Glucose Utilization in the Lactating Ruminant 1. Oxidative substrate The contribution of glucose to respiratory carbon dioxide in the lactating ruminant is relatively minor, being 8-13% in the cow (Bartley and Black, 1966; Baxter et a]_., 1955; Kleiber et_ al_., 1958), 3-5% in the goat (Annison e_t al_., 1968) and 8-9% in the ewe (Bergman and Hogue, 1967). Although estimates for hon-1actating, non-pregnant cows (Bartley and Black, 1966) and sheep (Annison and White, 1961) may rise as high as 20-30%, these figures contrast sharply with estimates of 35-50% obtained in dogs under similar conditions (Ford, 1965). Baxter e_t al_., (1955) estimated that up to 40% of glucose turnover in the lactating ruminant was accounted for by oxidation to carbon dioxide. Ruminant nervous tissue, like that of other species, appears to utilize glucose as its principal fuel for oxidation (Bergman, 1973; Kronfeld et_ aj_., 1963; Setchell, 1961) except, perhaps, after prolonged negative nutrient balance when ketone bodies may be the primary substrate 3. (Cahill et al_., 1970). The role of glucose in the oxidative metabolism of skeletal and cardiac muscle, kidney cortex and leucocytes may be less important in the ruminant, being replaced largely by acetate and to some extent by ketone bodies (Ford, 1965). Long-chain fatty acids are used to approximately the same extent in the ruminant and monogastrde CFord, 1965). 2. Synthetic substrate Although liver glycogen turnover in monogastrics is a major glucose homeostatic mechanism, the ability of ruminant hepatic tissue to synthe-size glycogen from glucose is less than 1/20 that of the monogastric (Ballard et_ al_., 1969). This is largely due to a marked reduction in hepatic glucokinase (EC activity and is also reflected in de-creased activities of UDPG-glycogen synthetase (EC and UDPG-pyrophosphorylase (EC and a hepatic glycogen content in the adult ruminant amounting to less than 40% of that found in the adult monogastric (.Ballard ejt al_., 1969). Similar observations on muscle glycogen synthesis in ruminants are unavailable. However, muscle glycogen content would be expected to be similar in all species since glycogen is essential as an anaerobic energy source during exercise when oxygen is limiting (.Bergman, 1973). Resynthesis of this glycogen via the Con" cycle is relatively efficient and results in only a small requirement for new glucose carbon. Another striking discrepancy in glucose metabolism between the mono-gastric and ruminant lies in the synthesis of triglyceride. In monogastric mammals given a high carbohydrate diet, triglyceride is formed from glucose in both liver and adipose tissue, whereas in the ruminant little or no glucose is converted to fatty acid in these tissues* the major sub-4. strate being acetate (Ballard et al_., 1972). The enzymes which are absent in the ruminant liver are glucokinase, ATP citrate lyase (EC and NADP malate dehydrogenase (EC and in adipose tissue; ATP citrate lyase, NADP malate dehydrogenase and pyruvate car-boxylase (EC (Ballard e_t al_. , 1972). Some glucose catabolism, however, remains essential for triglyceride synthesis in order to supply NADPH via the pentose phosphate pathway and to produce et glycerol phos-phate for esterification (Bergman, 1973). 3. Lactogenesis During lactation, the glucose economy of the entire animal is dominated by the requirements of the mammary gland. In lactating goats, lactogenesis accounts for 60-85% of. the total glucose utilization (Hardwick et al_., 1963; Smith, 1971) whereas in cows it accounts for at least 50% (Kleiber et a]_., 1955) but perhaps 70-90% (Lindsay, 1970). Of prime importance, the synthesis of lactose accounts for 60-85% of the mammary glucose uptake in the cow (Kleiber e_t al_., 1955; Lindsay, 1971; Smith, 1971), 50-60% in the goat (Lindsay, 1971) and 60% in the lactating ewe (Bergman and Hogue, 1967). It is largely the availability of gljucose for lactose synthesis which determines the level of milk production (Hardwick et al_., 1963; Kronfeld et al_., 1963; Smith, 1971). Approximately 39% (Hardwick ejt al_., 1963) of mammary glucose util-ization in the goat is oxidized to carbon dioxide, largely via the pentose phosphate pathway which supplies NADPH for milk fat synthesis (Smith, 1971). This represents 50% of the carbon dioxide produced by this organ, which is significantly higher than the 8-13% contribution of glucose to carbon dioxide production by the animal as a whole (Bartley and Black, 1966). 5. Black et al_., (1955) estimated that 4% of piasma=derived glucose appeared in casein in the lactating cow. These authors also proposed that 25% of the alanine and serine, 10% of the aspartate and glutamate and 7% of the glycine in cow's milk protein were derived from this source. In the lactating goat, 25% of serine, 14% of alanine, 8% of glutamate plus glutamine and 8% of aspartate plus asparagine residues in milk are derived from plasma glucose (Linzell and Mepham, 1968). A relatively small proportion of milk fat is glucose-derived. Hardwick e_t al_., (1963) found that only 0.5% of milk triglyceride fatty acids and 23% of triglyceride glycerol were synthesized from plasma glucose. These findings may be accounted for by the observation that in ruminant mammary gland, the oxidation of glucose to acetyl CoA may be limited in favour of oxidation via the pentose shunt (Chesworth and Smith, 1971). In addition, like ruminant liver and adipose tissue (Ballard et al_. , 1972), ruminant mammary tissue may be deficient in ATP citrate lyase and NADP malate dehydrogenase (Smith, 1971). B. Glucose Supply in the Lactating Ruminant Kronfeld et al_., 0 971) have estimated the glucose entry rate for the lactating cow to be approximately 1 g/min for animals weighing 423-508 kg which amounts to 560-648 mg/kg^/hr. Earlier estimates (Kronfeld and Raggi, 1964) of approximately 590 mg/kg^/hr are also in this range. Entry rates for lactating goats of 636 mg/kg^/hr (Lindsay, 1970) and for lactating sheep of 495-676 mg/kg^4/hr (Bergman and Hogue, 1967) are comparable. 6. 1. Dietary glucose Alimentary glucose supply plays a relatively minor role in the glucose economy of the lactating ruminant since most of the dietary car-bohydrates are fermented by rumen micro-organisms to yield short chain fatty acids. The actual amounts of carbohydrates which escape fermenta-tion and are available for absorption depend largely upon the nature and physical form of the diet and the frequency of feeding (Leng, 1970). For sheep on roughage rations probably no more than 8 g/day of glucose is absorbed. Values as high as 30 g/day may, however, be obtained on all concentrate diets. Cattle may absorb as much as 50 g/day on high con-centrate rations but under practical feeding conditions 10 g/day would seem a more reasonable estimate (Lindsay, 1970). With these observations in mind, it is clear that the lactating ruminant must depend upon endog-enous glucose synthesis to satisfy its requirements and that the glucose entry rate is a good indication of net gluconeogenesis in the animal (Katz and Bergman, 1969). 2. Gluconeogenesis a. From propionate Of the major short chain fatty acids absorbed from the rumen, only propionate can contribute significantly to glucose synthesis (Annison et al_., 1963a; Bergman and Wolff, 1971; Black ejt al_. , 1961 , 1966 and 1972; Leng and Annison, 1963). The net contribution of propionate to glucose synthesis has been the subject of some controversy. Bergman et a l . , (1966), using a rummal vein infusion of C-propionate in sheep estimated-that approximately 50% of the propionate entering the portal bed was con-verted to glucose, accounting for 27% of the glucose entry rate. However, 7. infusing C-propionate directly into the rumen of the sheep, Leng et a l . , (1967) proposed that approximately 54% of the glucose entry rate was derived from propionate produced in the rumen, with only 32% of this propionate being converted to glucose. The observation that up to 70% of the propionate produced in the rumen was first converted to lactate in the rumen epithel ium apparently justified the differences (Leng. et al_., 1967). However, since jugular blood was used in this analysis, it seems likely that the major portion of propionate which was converted to lactate was first converted to glucose in the liver (Weigand et a]_., 1972). Much of the apparent gluconeogenesis may, therefore, have been due to recycling of Cori cycle intermediates. Using Holstein calves with blood sampling from thoracic aorta and portal vein, Weigand et al_., (1972) estimated that only 1.0 - 4.6% of rumen-derived propionate was converted to lactate by the rumen epithelium. Although no direct studies have appeared on the glucogenic contri-bution of propionate in the lactating cow, some approximations can be made. The production rate of rumen propionate in lactating Holsteins has been estimated at 10.5 (Knox et al,. , 1967) and 12.8 (Hungate e_t al_., 1961) moles per day. Approximately 20% of the rumen propionate production, how-ever, is recycled in the rumen (Knox e_t al_., 1967). Therefore the irre-versible loss of propionate from the rumen, which is roughly equivalent to thecrate of absorption, can be estimated at 8.4 - 10.2 moles/day. Assuming that 50% of this propionate is converted to glucose, as in the sheep (Bergman et al_., 1966; Judson and Leng, 1968), approximately 750-920 g of glucose/day .could be derived from this source. This is 52-64% of the net glucose entry rate of 1440 g/day estimated by Kronfeld et a l . , 8. (1971). Wiltrout and Satter (1972) calculated a 45-60% contribution of propionate to gluconeogenesis in lactating cows. The relative importance of propionate as a glucogenic substrate is clearly dependent upon feed intake and composition (Leng, 1970; Lindsay, 1970). In sheep receiving half of their normal ration, the percentage of glucose derived from propionate fell from 27% to 13% (Bergman et a l . , 1966). Intraruminal or intramesenteric vein infusions of propionate stimulated glucose synthesis from this substrate (Judson and Leng, 1973) suggesting that the rate of gluconeogenesis is substrate-limited. Vari-ation in the grain contents of isocaloric diets fed to sheep markedly affects the contribution of propionate to glucose synthesis (Judson et a l . , 1968). Increases in the caloric content of rations fed to lactating dairy cows may result in profound propionate production (Bauman et a]_. , 1968; Esdale e_t al_., 1968). b.. From glycerol In the non-1actating, non-pregnant fed sheep, the glucogenic con-tribution of glycerol carbon amounts to no more than 5% of the total glucose entry rate (Bergman e_t al_., 1968). During periods of lipid mobilization, however, triglyceride glycerol may account for as much as 40% of the animal's glucose production. Since the actual glucogenic con-tribution of glycerol is regulated by the rate of utilization of free fatty acids liberated simultaneously from adipose tissue, it is unlikely that glycerol ever contributes more than approximately 20% of glucose carbon (Leng, 1970). It must also be kept in mind that the source of triglyceride glycerol is glucose itself. However, during early lactation, the cycle is essentially unidirectional in favour of glycerol release from adipose tissue. 9. c. From lactate It is difficult to evaluate the net contribution of lactate to gluconeogenesis in the ruminant due to recycling of this substrate. Perhaps 15% of the glucose is synthesized from lactate in sheep (Annison et al_., 1963), but this glucose is reutilized in peripheral tissue for glycogen synthesis and anaerobic catabolism to lactate again, returning to the liver for resynthesis to glucose via the Cori cycle. Thus, no new glucose is derived. Variable amounts of lactate are absorbed from the ruminant digest-ive tract as a result of rumen fermentation (Bergman, 1973). In addition, propionate is to some extent converted to lactate during passage through the rumen epithelium (Weigand e_t al_., 1972). However, no estimates of glucose entry from these sources have been made. d. From amino acids Gluconeogenesis from amino acids can occur in both liver and kidney cortex with the liver accounting for approximately 85% of the net glucose production from this source in fed sheep (Bergman ejt al_., 1970; Kaufman and Bergman, 1971). Almost all amino acids are glucogenic to some extent with lysine, leucine and isoleucine being the notable exceptions. The estimation of the proportion of the total glucose production arising from amino acids has been fraught with difficulty. Based upon the passage of protein through the abomasum, Leng (1970) calculated that a maximum of 70% of glucose synthesis could be derived from dietary amino acids. The use of urinary nitrogen excretion and urea production rates are also of limited value since considerable amounts of ammonia are ab-sorbed from the rumen to be converted to urea in the liver and urea itself 10. is recycled through secretions into the digestive tract (Bergman, 1973). 14 Several investigators have infused C-amino acids in order to estimate their glucogenicity. Reilly and Ford (1971b) using a mixture 14 of U- C-ammo acids isolated from chlorella protein, found that 28% of the total glucose turnover in the sheep was derived from amino acids. These authors discussed the importance of arterial rather than jugular blood sampling. In earlier work using the latter method, Ford and Reilly (1969) estimated a contribution of 12.8 - 14.7% to gluconeogenesis by amino acids. Hoogenraad e_t al_., (1970) introduced 14C-labelled Bacillus subt.ilis and Escherichia coli into the abomasum of sheep and found, using jugular blood sampling, that 16% of the glucose entry rate arose from bacterial protein carbon. Black e_t al_., (1968), infusing single U-^C-amino acids in lactating cows, calculated that amino acid carbon could contribute 33-50% of the 14 new glucose synthesized. Infusion of a mixture of C-amino acids derived from algal protein yielded an estimate of 12% of milk lactose produced from protein in the cow (Hunter and Millson, 1964). On this basis it can be calculated that up to 40% of the glucose entry rate is protein-derived. 14 Studies such as these, using C-amino acids, probably underestimate the actual glucogenic contribution of amino acids due to the randomization of label between the tricarboxylic acid cycle and the gluconeogenic pathway 14 (Krebs et al_., 1966). In addition, C-amino acids should be adminis-tered separately since a substantial glucogenic contribution by one (e.g. alanine) can be confounded by an actual anti-glucogenic effect of another (e.g. leucine). 11. Recent studies (Wolff et al_.., 1972; Wolff and Bergman, 1972a and 1972b) in fed sheep have assessed the uptake of amino acids by the liver and the transfer ratios of several glucogenic ^4C-amino acids to glucose in the whole body. The first studies overestimate glucose production from amino acids by liver while the second yield underestimates for their glucogenicity in liver plus kidney. Hepatic uptake experiments estimate a glucogenic contribution of amino acids of about 30%. Transfer ratios calculated from infusions of alanine, glutamate, aspartate, serine and glycine yield an estimate of 11% of total glucose synthesis in liver and kidney from these amino acids. As might be expected, diet markedly affects the synthesis of glu-cose from amino acids. As protein intake in sheep was raised from 1 g/kg/day to 3 g/kg/day, glucose entry rate increased from 1 mg/kg/min to 2 mg/kg/min (Reilly and Ford, 1971b). Judson and Leng (.1973) noted similar results when casein hydrolysate was infused into the abomasa of fed sheep. Glucogenic amino acids arise both from the alimentary tract and from tissue mobilization. Assuming a steady state in tissue protein, all amino acids used in glucose synthesis must ultimately be derived from the diet or the products of digestion. However, even in the fed sheep, tissue pro-tein turnover may contribute significantly to the total amino acid entry. For example, the mean proportional contribution to amino acid entry rate made by portal absorption in fed sheep was only 33.5% (Reilly and Ford, 1973). It should be realized that much of this portal entry arose from the portal arterial supply. Calculations based upon the work of Wolff and Bergman (.1972b) for five glucogenic amino acids (alanine, aspartate, glu-tamate, glycine and serine) estimate that only about 10% of the total 12. amino acid presented to the liver arose from gut absorption. An estimate of the contribution of tissue mobilization to glucose entry in the lactating cow is of interest here. Although the fed, non-lactating ruminant represents a steady state with respect to tissue mobil-ization, the lactating cow can often not consume enough nutrients to main-tain this condition. In the first two months of lactation, a cow producing 25-50 kg of milk/day may utilize 1-2 kg/day of body fat and up to 400 g/day of body protein (Kronfeld and Emery, 1970). Paquay e_t al_., 0972) found that under extremely variable conditions of nutrient intake, the cow may be able to lose 20-25% of her body nitrogen reserves. Although no quan-titative estimates are available, it is apparent that the contribution of body protein reserves to gluconeogenesis in the lactating cow must be substantial. 13. C. Summary Applying the figures obtained by different workers for glucose supply and utilization to the turnover rate determined by Kronfeld (1971, experiment 5) for a 464 kg cow producing 20 kg milk/day, estimates have been made of the amount of glucose required or used for various metabolic purposes (Table 1). The figures can clearly be only rough approximations of the actual values because of the different conditions under which the data were obtained. 14. Table 1. Quantitative estimates of glucose utilization and supply in the lactating cow. Glucose entry rate g/24 h 1700 Reference Kronfeld et a l . , 1971 Glucose requirement Oxidation CO2 Total Mammary Extra mammary Lactogenesis Lactose Protein Fat 680 410 270 780 70 170 Baxter e_t al_., 1955 Hardwick et a l . , 1963 Kleiber et al_., 1955 Black et al_. , 1967 Hardwick et a l . , 1963 Glucose supply Alimentary Gluconeogenesis From propionate From glycerol From amino acids 10 850 340 500 Lindsay, 1970 Wiltrout and Satter, 1972 Leng, 1970 Black et a l . , 1968 15. II. Acetonemia An extensive review of bovine acetonemia with respect to glucose metabolism has recently appeared [Bergman, 1973). Kronfeld 0 971) has elaborated on glucose kinetics in acetonemic animals. In this disorder, hypoglycemia may result from either an increase in glucose space or a decrease in precursor availability at a time when the demand for glucose is especially high, namely during early lactation. The former possibility is supported by the observation (Kronfeld, 1971) that the glucose trans-fer rate was normal in cows early in the course of the disorder, whereas the glucose space was greatly enlarged. However, as the acetonemia pro-gresses, glucose entry rate does diminish and a supply of glucose or glu-cose precursors will ameliorate the condition (Schultz, 1971). The pathology of bovine acetonemia is reflected in the decreased availability of glucose to tissues as a result of hypoglycemia. Increased fatty acid mobilization from adipose tissue results in increased hepatic lipid oxidation (Bergman, 1971). Incomplete oxidation of fatty acids, perhaps due to a hepatic lesion arising from a lack of glucogenic pre-cursor (Krebs, 1966; Baird ejt al_., 1968), gives rise to increased ketone body formation and ketosis. Kronfeld (1971) argues that a subclinical ketosis may itself give rise to the characteristic hypoglycemia. Several notable changes occur in the livers of spontaneously ketotic cows. Among these, the concentrations of glucogenic amino acids are significantly reduced (Baird ejt al_., 1968) while the activities of phosphoenolpyruvate carboxykinase (EC and pyruvate carboxylase (EC remain unchanged (Baird et al_., 1968; Ballard et al_., 1968) and the activity of NAD malate dehydrogenase (EC decreases sig-nificantly (Ballard et al_., 1968). These observations suggest that gluco-16. neogenesis is reduced in the livers of acetonemic cows due to both the decreased substrate availability and the decline in activity of NAD malate dehydrogenase. 17. I l l . Hormonal Control of Glucose and Amino Acid Metabolism A. Glucose Metabolism 1. Control by glucocorticoids Glucocorticoid release from the adrenal cortex is stimulated by adrenocorticotropic hormone (ACTH) secreted by the adenohypophysis (Yates and Urquhart, 1962). The origin of the stimulus is, however, the hypothalamus (Guillemin, 1971) which produces corticotropin*n releasing factor (CRF) in response to a number of neural and metabolic threats to homeostasis (Selye, 1959). The major metabolic effector is probably hypoglycemia (Donald, 1971). Classically, glucocorticoids are thought to have two major effects on glucose metabolism. The first is a direct stimulation of hepatic gluconeogenesis [Landau e_t al_. , 1962). In addition, glucocorticoids inhibit peripheral substrate metabolism (Ariyoshi and Plager, 1970). In the rat, hepatic activities of glucose-6-phosphatase (EC, phosphoenolpyruvate carboxykinase (EC and pyruvate carboxylase (EC increase in response to glucocorticoid administration (Lardy, 1965). However, the effects seen in the domestic ruminant are quite different. Filsell ejt al_., (1969) demonstrated a significant reduction in hepatic pyruvate carboxylase activity while no change was seen in the activity of phosphoenolpyruvate carboxykinase in glucocorticoid-treated sheep. The administration of a therapeutic dose of a synthetic gluco-corticoid (dexamethasone 21-isonicotinate) to the dairy cow resulted in a slightly increased activity of glucose-6-phosphatase but a markedly de-creased activity of phosphoenolpyruvate carboxykinase and pyruvate kinase (EC (Baird and Heitzman, 1970). Furthermore, dexamethasone 18. administration to the rat results in decreased activity of citrate syn-thase (EC while the cow responds with an increase. Thus, the response to glucocorticoid in the rat is one of increased gluconeogenesis from oxaloacetate while that in the cow is one of increased tricarboxylic acid cycle activity (Heitzman ejt a]_., 1972). The latter observation accounts for the marked antiketogenic action of glucocorticoids in the bovine (Baird and Heitzman, 1971). Although marked changes in hepatic enzyme activities are seen with large doses of glucocorticoids, it has recently been suggested that under physiologic conditions, the role of these hormones is only a permissive one. Exton ejt al_., (1972) have suggested that glucocorticoids may be required for the normal activation of hepatic effects by other hormones such as glucagon and insulin but that the peripheral effects of the steroids constitute their primary function (Exton et al_., 1970). Likewise, Basset e_t al_., (1966) have demonstrated that the hyperglycemic effect of Cortisol in sheep cannot be accounted for by increased gluconeogenesis. The peripheral catabolic effects of glucocorticoids are a result of their inhibition of glucose uptake by skin, lymphoid and adipose tissue (Munck, 1971), muscle (Plageman and Renner, 1972) and mammary tissue (Hartmann and Kronfeld, 1973). These effects are likely biphasic (DeLoecker and Stas, 1973). Within two hours after Cortisol treatment of sheep, a moderate hyperglycemia is accompanied by a decrease in glucose entry rate and a decrease in glucose space (Reilly and Black, 1973). A maximum plasma glucose concentration is seen after 24 hr by which time the glucose entry rate may increase by 40% (Reilly and Ford, 1971a). Dexamethasone treatment of lactating dairy cows results in hyperglycemia and a decreased 19. mammary uptake of glucose after 12 hr (Hartmann and Kronfeld, 1973) accompanied by a normal glucose turnover rate and a redistribution of glucose space (Kronfeld and Hartmann, 1973). The observation that there was no overall reduction in glucose utilization rate may be due to the effect of the hyperglycemia compensating for reduced efficiency of util-ization in non-mammary tissue (Reid, 1968). Such compensation may be mediated to some extent by increased insulin secretion (Bassett and Wallace, 1968; Owen and Cahill, 1973). 2. Control by insulin Although glucose provides the major stimulus to the release of insulin in most species (Kipnis, 1972) and may have a permissive role in the stimulation by other secretogogues (Metzger e_t al_., 1973), release of the hormone may also be provoked by ketone bodies (Madison et_ al_., 1964), short-chain fatty acids (Horino et a1_., 1968), medium=chain fatty acids (Greenberger et al_., 1968), long-chain fatty acids (Crespin ejt a]_., 1969) and secondarily by several other hormones (Kipnis, 1972). In the ruminT ant, propionate and butyrate may also be insulinotropic (Manns and Boda, 1967). Consumption of diets containing carbohydrate or protein stimulates insulin secretion by way of a portal mechanism in the monogastric animal (Young, 1970), while protein digestion regulates a similar mechanism in sheep (Bassett e_t a]_., 1971). Insulin, like glucocorticoids, has two primary effects, one hepatic, the other peripheral. In the case of this hormone, however, its hepatic effect is more clear cut than that of.the glucocorticoids. Insulin exerts direct inhibitory effects on hepatic glycogenolysis and gluconeogenesis, the net effect being a reduction in glucose output "CLevine, 1972; Madison, 20. 1969). This process, is sensitive to very slight elevations in circulating glucose concentrations in man (Cahill, 1971). In the ruminant, however, these effects may be relatively less sensitive since the activity of hepatic glucokinase (EC is markedly lower than in other species (Kronfeld, 1970). Although the effects of insulin on hepatic metabolism are independent of glucose uptake by the hepatocyte (Cahill ejt al_., 1958), the peripheral effects of the hormone are primarily dependent upon its activity in influencing membrane permeability (Cuatrecasas, 1969). The actions of insulin on muscle and adipose tissue are less sensitive to alterations in the level of circulating glucose presented to the 3 cell. Only a fairly large glucose load stimulates insulin release to the extent that peripheral uptake is effected (Cahill, 1971). In this respect, insulin release may be less responsive in the ruminant since cir-culating glucose levels are normally low and fluctuations relatively slight. Insulin promotes the incorporation of glucose into glycogen in muscle (Viilar-Palasi and Larner, 1961) and triglyceride in adipose tissue (Ball and Jungas, 1964), the latter being the most sensitive peripheral effect of the hormone in both man (Cahill, 1970) and the ruminant (Kronfeld, 1971). 3. Control by growth hormone Of the stimuli which effect pituitary growth hormone (GH) release, hypoglycemia is the most potent (Roth et al_., 1963) and the most important in the context of this thesis. Conversely, glucose administration sup-presses GH release. It is of interest to note that GH secretion may persist after acute hypoglycemia and during prolonged hypoglycemia, in contrast with ACTH release, which may be increased with acute but probably 21. not with chronic depression of blood glucose CGIick e_t al_., 1965). It is likely that the hypoglycemic stimulus exerts its effect on a centre common to both GH and ACTH secretion (Nakagawa e_t al_., 1971). In add-ition, it is apparent that glucocorticoids may exert a negative effect on GH secretion as well as on ACTH release (Frantz and Rabkin, 1964; Wakabayashi et al_., 1971). Reports on the action of GH on glucose metabolism have been con-flicting. For example, after the administration of 4-8 mg of GH to human subjects (Zahnd et al_., 1960; Daughaday and Kipnis, 1966) or 8-10 mg to sheep (Bassett and Wallace, 1966) plasma glucose initially falls, indica-ting an insulin-like activity, then rises, demonstrating an anti-insulin effect. However, when GH is infused in low doses in man (Merimee and Rabin, 1973), no early insulin-like effects are observed. These early effects are likely a pharmacologic action of the hormone, the direct physiologic actions being decreased glucose utilization by both muscle and adipose tissue resulting in hyperglycemia and free fatty acid mobil-ization (Bassett and Wallace, 1966). An additional action of GH on glucose metabolism is its insulino-tropic effect (Merimee and Rabin, 1973). Prolonged elevations in blood GH potentiate the release of insulin in response to hyperglycemia result-ing in improved glucose tolerance. 22. B. Amino Acid Metabo1 ism 1. Control by glucocorticoids The primary gross effect of glucocorticoids on amino acid metabo-lism is the mediation of their release from cardiac and skeletal muscle. This effect is probably not a direct one but arises from the inhibition of glucose uptake by these tissues, discussed earlier. The mechanism of this action involves a reduction in protein synthesis [Kaplan and Nagareda Shimizu, 1963; Young, 1970) and accumulation of free amino acids in muscle (Betheil et aj_., 1965). Smith and Long (1967) demonstrated the mobilization of amino nitrogen which occurred 2-3 hr after the administra-tion of Cortisol to rats and their results were confirmed in the sheep by Bassett (1968). More recently, analyses of the individual amino acids released by muscle (Felig ejt al_., 1970b) have demonstrated the predominance of alanine in this role. The large proportion of this amino acid likely arises from the amination of pyruvate (Pozefsky and Tancredi, 1972), the carbon for which may be derived from the anaerobic oxidation of circula-ting glucose (Felig, 1973). However, under conditions of limited glucose uptake by muscle, mediated by glucocorticoids, the carbon is likely derived from the catabolism of other amino acids, notably leucine, iso-leucine and valine (Odessey and Goldberg, 1972; Goldberg and Odessey, 1972) which are released in relatively small proportions. The rate of release of amino acids from peripheral tissues can be . an important factor in the control of hepatic gluconeogenesis (Mallette et al_., 1969a). Alanine, in particular, is strongly glucogenic and its administration in fasting (Aikawa et al_., 1972; Felig ejt a]_., 1969) and human pregnancy (Felig et^  al_. , 1972) results in prompt increases in 23. hepatic glucose production. In studies on childhood ketotic hypoglycemia, Pagliara et al_., 0 972) noted that cortisone treatment produced a 3 to 4-fold increase in plasma alanine within 4 hr with complete remission of the hypoglycemia. Reilly and Black 0973) noted a significant increase 14 in the fractional rate of C-alanine incorporation into glucose when adrenalectomized sheep were treated with Cortisol. In addition to supplying glucogenic substrate, glucocorticoid mediated amino acid mobilization results in increased pancreatic a cell release of glucagon (Marco et_ al_., 1973; Wise et al_. , 1973). Amino acids which enter the gluconeogenic pathway via pyruvate and which probably pro-vide most of the amino acid derived glucose, have the greatest glucagon stimulating activity while only valine, leucine and isoleucine fail to stimulate glucagon release (Rocha et. al_., 1972). Glucagon, in turn, stimulates the hepatic gluconeogenic mechanism and the release of the branched-chain amino acids (Mallette et al_., 1969b). In summary, the actions of glucocorticoids which are rate limiting for gluconeogenesis take place in extra-hepatic tissues (Exton, 1972) causing increased substrate flow to the liver with the subsequent stimu-lation of glucagon-mediated glucose synthesis. 2. Control by insulin In common with glucocorticoids, the major site of the action of insulin on amino acid metabolism is muscle tissue (Young, 1970). The hormone enhances the transport of some amino acids into the tissue (Manchester, 1970), increasing the size of the free intracellular pools of many (Hider e_t al_., 1971). In post-absorptive human subjects, the close intra-arterial infusion of insulin resulted in a 74% decline in total amino 24. nitrogen output from forearm muscle (Pozefsky ejt al_., 1969). While most amino acids appeared to be involved, consistent decreases were seen only for threonine, isoleucine, leucine, tyrosine, phenylalanine, glycine and a amino butyrate. Insulin failed to affect alanine release. The effect of insulin on glucose and amino acid uptake is, however, insufficient explanation to account for a large increase in protein syn-thesis which is seen as a result of its action on the tissue (Manchester, 1972). Insulin mediates the assembly of polysomes and alters the ability of ribosomes to translate mRNA (Wool, 1972). Thus, its effect on protein synthesis is to increase protein chain initiation (Jefferson e_t al_., 1972). Of additional interest in relation to insulin activity is the metabolism of leucine. This amino acid very specifically provokes insulin release (Lernmark, 1972) and in diabetic keto-acidosis, the plasma levels of leucine, valine and isoleucine are notably elevated (Felig et a l . , 1970a). This work suggests that in the absence of insulin, mobilization of these amino acids, and the failure of liver to utilize them, results in their accumulation in plasma. 3. Control by growth hormone Although several amino acids provoke GH secretion (Knopf et a l . , 1965) only arginine and perhaps lysine and histidine are likely of phys-iologic importance (Merimee et al_., 1967). Leucine, isoleucine and valine initiate GH release through their insulinotropic activity.. The early effects of large doses of GH upon amino acid metabolism are analogous to those on glucose metabolism. These effects are insulin-like and can largely be accounted for by increased tissue permeability (Knobil, 1966). Again, these actions of GH are probably pharmacologic. 25. However, in this case the physiologic role of GH in amino acid metabolism by muscle is probably similar to that seen when large doses of the hormone are administered (Merimee and Rabin, 1973).. Physiologically, these effects are likely not mediated by GH itself, but are executed by somatomedin (sulfation factor: Salmon and DuVall , 1970). In either case, the net result is the same, conferring upon GH activities which are glucocorticoid-like with respect to glucose metabolism and insulin-like with respect to amino acid uptake. C. Hormones in the Etiology of Acetonemia The efficacy of glucocorticoids in the treatment of acetonemia is well established (Braun et_ al_., 1970). The pioneering work in this area was performed by Shaw (1956) who found several interesting histopathological conditions associated with the disorder (Shaw et al_., 1951). Enlargement and degeneration of the adrenal cortex were associated with regressive changes in the anterior pituitary and acute involution of the pancreas. These observations suggested a disorder of the pituitary-adrenal axis, a hypothesis which was tested by the administration of cortisone or ACTH to ketotic animals (Shaw e_t al_., 1953). Both treatments were successful, suggesting that the ultimate lesion was inadequate pituitary ACTH production. Since the histological changes seen in the adrenal cortex were character-istic of non-functional enlargement due to an inhibition of steroid secre-tion (Turner, 1966), it might be rational to propose thatsuchan inhibition leads to a compensatory increase in CRF stimulation to the pituitary via decreased negative feedback (Yates and Urquhart, 1962). Prolonged hyper-secretion of ACTH by the anterior pituitary could result in substrate de? pletion and the regressive changes observed with an ultimate inability to 26. compensate for the adrenal inhibition. The mechanism of the therapeutic effect of glucocorticoids is unclear. Administration of dexamethasone to ketotic cows (Baird and Heitzman, 1971) resulted in increased- hepatic levels of six glucogenic amino acids suggesting, perhaps, increased mobilization from the periphery (Young, 1970). The authors (Baird and Heitzman, 1971), however, attributed the effect of the steroid to its hepatic anti-ketogenic activity referred to earlier (p. 1:8). Kronfeld (1972) attributes the action of glucocorticoids in aceto-nemia to a diminished mammary glucose uptake rather than increased glucose production. The decreased milk production (Braun et al_., 1970) and glucose output (Hartmann and Kronfeld, 1973) ameliorate the characteristic hypoglycemia. These proposed mechanisms of steroid therapy are not mutu-ally exclusive but likely, complementary. Insulin has been implicated in bovine acetonemia since the changes in glucose kinetics found in ketotic animals are similar to those induced by this hormone (Kronfeld, 1971). A subclinical ketonemia may provoke the release of insulin resulting in hypoglycemia and hypophagia. Such changes are the reverse of those induced by glucocorticoids and Kronfeld (1971) considers that they are consistent with the relative adrenocortical insuffic-iency hypothesis proposed by Shaw (1956). There is little evidence for a role of growth hormone (GH) in aceto-nemia, although Kronfeld (1965) induced ketosis in lactating cows by admin-istering large doses of the hormone. In this respect the adipokinetic action of GH has been emphasized. Patterson and Cunningham (1969) have considered the possibility of GH hypersecretion in acetonemia. Growth hormone administration to rats (Bennett ejt al_., 1948) resulted in decreased 27. terminal fatty oxidation in liver, giving rise to ketosis. Assan and Tchobroutsky (1972), Chernick e_t al_., (1972) and Unger (1965) have noted elevated GH levels in diabetic ketoacidosis. IV. Role of Estrogens in the Regulation of Substrate Metabolism A. Direct Effects During the estrous cycle of the ewe, the lowest arterial amino nitrogen levels are found at estrus. Exogenous estrogen treatment yields a similar hypoaminoacidemia (Curet et_ al_., 1970). This effect is apparently due to estrogensmediated amino acid uptake into the uterus (Curet and Caton, 1971) and mammary gland (Frantz and Rabkin, 1965; Sinha and Tucker, 1969) as a result of increased blood flow across these tissues (Curet and Caton, 1971). Dunn et al_., (1972), studying glucose kinetics during the ewe's estrous cycle,.^ observed a significant hypoglycemia at estrus. In addition, the lowest glucose entry rate was found at this time. The observations of hypoaminoacidemia, hypoglycemia and decreased entry rate at estrus may be rationalized in terms of an estrogen mediated competition for glucogenic substrate. Calculations based on the data of Dunn ejt al_., (1972) and Curet and Caton (1971) allow a rough estimate of the importance of uterine amino acid uptake in the glucose economy of the ewe at estrus. Curet and Caton observed an increased uterine uptake of 1.8 g/hr upon estrogen treatment of the ewe (assuming a 60 kg ewe). Assuming that 100 g of mixed amino acids have the potential to contribute 55 g.of glucose via gluconeogenesis (Leng, 1970), this uterine uptake could have given rise to approximately 28. 1 g/hr of glucose. The total glucose entry in the ewe, however, decreases from 9.24 g/hr during proestrus to 5.16 g/hr during estrus-metestrus (Dunn ejt al_. , 1972). Thus, amino acid uptake by the ovine uterus can only account for approximately 25% of the observed decrease in glucose entry. An additional effect of estrogen, although inconsistently observed, is a depression of voluntary ration intake (Forbes, 1972). Curet and Caton (1970) have concluded, however, that this effect was insufficient to account for the hypoaminoacidemia which they observed at estrus since, in ewes fasted for 3 days, the plasma amino nitrogen concentration decreased only slightly, from 9.56 mg/100 ml to 9.44 mg/100 ml. 29. B. Indirect Effects 1. Mediated through alteration of glucocorticoid,secretion. Among their multiple effects upon the hypothalamo-pituitary-adrenal system, estrogens have been shown to inhibit corticosteroid pro-duction and secretion from the adrenal cortex. Pincus and Hirai (1954) noted a significant difference between the elevated production of cortico-sterone in rats during diestrus when compared with that at estrus. Women receiving contraceptive preparations containing estrogens demonstrate a significant decrease in Cortisol secretion rate (Beck et a l . , 1972; Turksoy, 1972). Hexosterol administration to rats results in a similar depression of corticosterone secretion, accompanied by a rapid loss of lipids, hypertrophy and an increase in vascularity of the adrenal cortex (Vogt, 1957). These effects are dependent upon the integrity of the pit-uitary and have been termed "non-functional enlargement" (Turner, 1966). Estrogens may reduce cortical steroid production by competing with NADP for binding to NADP-specific dehydrogenases. Such inhibition may be com-pensated for by an increase in corticotropin secretion (McKerns, 1963). Kitay (1963), in fact, observed increased adrenal weight accompanied by a 2.5-fold increase in pituitary ACTH content in estrogen-treated rats. The administration of ACTH to these animals resulted in significantly re-duced responses with respect to corticosterone production. Under similar conditions, decreased adrenal corticosterone output with increased plasma ACTH concentration was demonstrated (Fonzo et al. , 1967). Gemzell (1952) also measured increased ACTH secretion in estrogen treated rats. Although estrogens may inhibit corticosteroid synthesis resulting in a compensatory increase in ACTH secretion through decreased negative feedback they also act upon at least two other levels in this system. Intra-hypophyseal 30. or intra-hypothalamic estrogen implants enhance corticosteroid activity without adrenal enlargement. This effect occurs with no detectable periph-eral release of estrogen, indicating estrogens may have a direct effect on ACTH release (Richard, 1966). In humans, estrogen treatment results in increased plasma corticosteroid binding capacity (Doe et al_., 1964; Mills and Barter, 1959; Sandberg and Slaunwhite, 1959) indicating higher blood levels of corticosteroid binding globulin (transcortin), the a globulin which specifically binds these hormones. This increased binding has several effects since it is the plasma concentration of unbound steroid which determines the magnitude of its effect on target tissues (Sandberg and Slaunwhite, 1963), its rate of hepatic oxidation (Gillie et al_., 1973) and the degree of pituitary feedback activity (Acs and Stark, 1973). In the rat and the human, the net outcome of these multiple effects of estrogens on the pituitary-adrenal axis is an increase in the plasma concentrations of both free and bound corticosteroids (Colby and Kitay, 1972; Ghosh, 1971) largely due to increased plasma transcortin concentra-tion. However, the ruminant fails to respond to estrogen treatment with increased transcortin synthesis (Krulik and Svobodova, 1969; Lindner, 1964) and the normal response to estrogens would likely be a transient decrease in corticosteroid output, followed by compensatory ACTH secretion bringing the corticosteroid secretion rate back to normal. Of interest with respect to estrogen inhibition of glucocorticoid synthesis is the action of the pharmaceutical metyrapone. This drug is generally used in a test to assess the capacity of the pituitary to secrete ACTH. Metyrapone blocks 11 e-hydroxylation in the adrenal cortex (Sprunt et aJL, 1968) resulting in decreased blood levels of Cortisol and cortico-sterone with increased plasma concentrations of 11-deoxycortisol and 11-deoxycorticosterone (Clements and Newsome, 1973) and increased urinary 31. excretion of their metabolites. Reduced plasma glucocorticoid levels result in decreased negative feedback on the pituitary and stimulation of ACTH release. Hypopituitary patients fail to respond with an increment in plasma ACTH (Keenan et_ al_. , 1973). Concomitant with ACTH secretion, normal patients respond with increased growth hormone secretion (Lee et a l . , 1973; Sawano e_t al_., 1972). Thus, the effect of metyrapone on the pituitary-adrenal axis may be analogous to.that of estrogen, although the primary site of action is likely different. 2. Mediated through alteration of growth hormone secretion The major direct effect of estrogens on growth hormone (GH) secretion is a potentiation of the stimulatory effects of various.GH secretagogues. Frantz and Rabkin (1965) noted low basal GH levels throughout the menstrual cycle in women. When exercise provoked GH release, however,.much higher values were found during the estrogenic phase.of the.cycle. These higher values were not seen in ambulatory men unless concomitant estrogen treatment was given. A similar pattern is found when hypoglycemia-provoked GH ele-vations are examined. When plasma glucose concentration decreased by 20-30 mg/100 ml, GH increased in the pre-ovulatory phase to 18 ng/ml, in the post-ovulatory phase to 14 ng/ml and during menstruation to only 7 ng/ml (Merimee and Fineberg, 1971). Similar effects are observed in cattle. Trenkle (1970) noted sig-nificant increases in plasma GH concentration when beef animals were fed diethylstilbestrol. In lactating dairy cows, plasma GH concentrations during the estrogenic phase of the estrous cycle were significantly higher than those during the luteal phase (Koprowski and Tucker, 1973). 32. In the present context, it is of an inhibitory effect of progesterone on GH release (Bhatia et al_., 1972; Simon interest to note observations of hypoglycemia or arginine provoked et al_., 1967). 33. EXPERIMENTS I. The Influence of Estrous Cycle Hormones on Plasma Amino Acids, Glucose  and Urea Nitrogen. Acetonemia in dairy cattle typically appears 10 days to 6 weeks following parturition with the maximum appearance being about 3 weeks post-partum (Shultz, 1971). Milk production at this time is at its maximum and the animal is beginning to demonstrate regular estrous cycles, the first of which normally occurs about 14 days post-partum (Wagner and Hansel, 1969). Thus, this is a period of renewed cyclic appearance of the hormones of the estrous cycle superimposed upon a profound demand for precursors for milk production. With these considerations in mind,.the objective of this study was to clarify the interactions between estrous cycle hormones and substrate metabolism in the post-partum lactating dairy cow. The primary hormones controlling estrous cycle function and appear-ing in peripheral circulation in the bovine are luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone and the estrogens. Of these, the trophic hormones (LH and FSH) would not be expected to have significant extra-ovarian effects with respect to substrate metabolism (Turner, 1966). Peripheral progesterone concentrations during the bovine estrous cycle have been measured by several investigators (Donaldson et al_., 1970; Garverick et al_., 1971; Henricks et al_., 1970 and 1971; Plotka et al_., 1967; Pope et al_., 1969; Snook et al_., 1971; Stabenfeldt et al_., 1969; Wetteman e_t al_., 1972). The pattern which has emerged is one of increasing progesterone concentration beginning about day 3 or 4 of the cycle (day 0 being estrus), reaching a plateau near day 12 and perhaps a peak of 5-12 34. ng/ml plasma 4-6 days prior to the ensuing estrus. Prior to the present work, several authors had studied estrogenic activity in bovine blood during gestation (Bitman e_t al_., 1958; Pope et al_. , 1965; Robinson e_t aj_. , 1970; Saba, 1964; Soliman et al_., 1964; Szego and Roberts, 1946). In addition, estrogens had been demonstrated in urine during the estrous cycle of the cow (Erb et al_. , 1971; Garverick e_t al., 1971; Mellin and Erb, 1966). At the outset of this work, it was necessary to define the cyclic appearance of estrogenic activity in the estrous cycle of the lactating cow in order to assess its role in associ-ated metabolic events. A. Plasma Estradiol and Estrone Concentrations During the Estrous Cycle  of the Cow. 1. Introduction Previous studies of the concentrations of estrogens in the peripheral blood of the dairy cow during the estrous cycle had been few and the results, inconclusive. Soliman e_t a]_. , (1964) using a mouse uterine weight assay found 34.3 and 61.0 yg estradiol equivalent/1 during diestrus and estrus, respectively. Ayalon and Lewis (1961) using a similar procedure, detected 3 yg estradiol-17e equivalent/1 at estrus, 6 yg at 1, 2, 9 and 11 days, 9 yg at 14 days and 12.5 yg at 18 days after estrus. Pope ejt al_., (1965), using a colorimetric assay, measured 1-10 ng estrone equivalent/1 in the blood of cows at estrus. Fluctuating concentrations with a mean estrous peak of 36 yg total estrogen/1 were noted by Higaki et al_., (1959) using a bioassay. The present experiment was therefore designed to determine sys-tematically the peripheral plasma concentrations of estradiol and estrone throughout the bovine estrous cycle. 35. 2. Materials and methods Twenty-two Hoi stein, Ayrshire and Hoi stein-Ayrshire crossbred cows were sampled during a period in which they were showing regular estrous cycles. The animals were from two herds, maintained at the University of British Columbia campus or at the U.B.C. Oyster River Farm. Blood samples were taken 30-100 days post-partum. Estrous.samples were those from cows which first showed heat in the morning of the days on which blood was collected. The day of the estrous cycle was calculated for other samples as the number of days before or after heat. Since the estrous cycle length was variable among animals, results are based upon a 21-day cycle (Cupps e_t al_., 1969) standardized as described by Garverick ejt aj_., (1972). For the purpose of this discussion, the phases of the cycle are defined as follows. Proestrus starts on day 15, when progesterone levels begin to drop markedly (Plotka et a l . , 1967; Henricks et a l . , 1970; Garverick et a l . , 197o) and ends at the onset of standing heat. Estrus is synony-mous with the period of sexual receptivity. Metestrus extends from the cessation of standing heat until day 3, when progesterone again begins to dominate the cycle. Diestrus comprises the remainder of the cycle from days 4 to 14, inclusive. Approximately 100 ml of blood were collected by jugular venipuncture in a bottle containing heparin. Samples were divided into duplicate portions and.centrifuged at 12000 g for 10 min at 0 C. [6,7-3HJ Estradiol (6000-7000 cpm) and £6,7- 3 H] estrone (6000-7000 cpm), (both obtained from Amersham-Searle Corporation) in ethanol (0.2 ml) were added to each plasma sample (20 ml). Plasma was extracted and processed according to the method of Moore et a l . , (1969). Methanolic solutions were evaporated to dryness and the residues were taken up in 12 ml of 1 M NaOH. NaHCO- (1.6 g) was 36. added and toluene extracts of these solutions were assayed for unconjugated estrogens. Methylation and chromatography on alumina columns (2 g) were performed as described by Brown (1955). Estradiol and estrone were eluted as separate fractions, 10% of each fraction being taken for isotope count-ing. The remainder was subjected to fluorometry as described by Brown et al_., (1968). Sym-tetrachloroethane was used in the Ittrich reagent and fluorescence was measured on an Aminco-Bowman 4-8202 SPF spectrophoto-fluorometer using a mercury-xenon light source. The correction factor (Brown et al_., 1968) used was based upon the extinction of standards at 546/565 nm (excitation/emission) extrapolated to 0 ng and the common extinction of standards at 490/520 nm. In most cases, the correction factor (k) used was in the range 0.15 - 0.35. Concentration in plasma was calculated with correction for isotope recovery. Assay values were rejected if (1) the standard error of the mean exceeded 25% of the mean for duplicate samples, or (2) the recovery of isotope was less than 50%. A few samples,..collected from animals in the metestrous phase of the cycle (days 1-3) were eliminated due to poor re-producibility. The mean recovery for the assays reported was 64.0 ± 1.0% (SEM). 3. Results and discussion The plasma concentrations of estradiol ranged from 17.6 to 117.5 ng/100 ml, while those of estrone varied between 23.1 and 226.9 ng/100 ml (Fig. 1). These values are comparable with estradiol values measured in the ovarian vein blood of the cyclic ewe, using the present assay method (Moore e_t al_., 1969) and with levels obtained for the ewe using radio-immunoassay ( al_. , 1970). Previous reports on the levels of estrogens in the peripheral plasma of cycling dairy cows had been incon-37. sistent. For example, levels of 0.1 mg estrone equivalent per 100 ml (Ayalon and Lewis, 1961), 6100 ng estradiol equivalent per 100 ml (Soliman et a]_., 1964) and 660-3600 ng total estrogens per 100 ml (Higaki et a l . , 1959) have been reported for animals at estrus. Estrone concentrations found in bovine jugular blood during gestation, using the present method (Robinson et al_. , 1970) have been, as expected, considerably higher than those re-ported here. In the present study, the major peaks in plasma estradiol and estrone occurred on days 19 and 20 of the standardized 21-day cycle. Relatively high levels of estradiol were also found in some of the samples taken from animals on the day of estrus. It is possible that these samples represent animals in early estrus. Because of the difficulty inherent in the estimation of the exact time of onset and cessation of estrus, indiv-idual day zero samples may represent stages of estrus up to 24 h. apart (Cupps et al_., 1969). This variation becomes apparent in the large stan-dard errors of the mean estrogen concentrations during the ascending and descending portions.of the peaks (Table 2). The occurrence of peaks in the concentrations of estrogens in plasma 1 or 2 days before heat is consistent with the peaks observed in the urinary excretion of estrogens on the day of estrus by Garverick et al_., (1971) or on the day preceding estrus by Mel 1 in and Erb (1966). The urinary ex-cretion peak is likely to occur later than the plasma peak since Mel 1 in and Erb (1966) found that the maximum excretion of labelled urinary estrogen occurred 9 h after an intravenous injection of 4.5 uCi (150 yg) of [4- CJ estradiol-173 and 3-5 h after the i.v. administration of 13.8 uCi (5.1 mg) [16-14CJ estrone. 38. It would appear that the peak level of estrone in the present study (Fig. 1) occurred slightly before the peak of estradiol since, in a few proestrous samples, the estrone concentration was markedly increased while the estradiol concentration was maintained at a moderate level. In addition, relatively high levels of estradiol were found in some estrous samples while the corresponding estrone levels had fallen to lower values. Mean estrogen concentrations for the four periods of the estrous cycle are given in Table 2. Since the completion of this investigation (Mason et_ al_., 1972) several authors have reported estrogen concentrations during the bovine estrous cycle, determined by radioimmunoassay (RIA) or competitive pro-tein binding (CPB) assay. Shemesh et al_., (1972) using the latter method detected a major peak of plasma estradiol the day before estrus with minor peaks at 4 and 11 days post-estrus. The highest concentration measured by these authors was 17 ng/100 ml. Peaks of approximately 1.2 ng estradiol per 100 ml were observed one day prior to and on the day of estrus by Wetteman et al_., (1972) using both RIA and CPB assay. Other investigators, using RIA have noted estradiol peaks of 0.5 ng/100 ml (Glencross and Pope, 1972), 2.5 ng/100 ml (Henricks et aj_., 1971) and 80 ng/100 ml (Hoffman, 1972) on the day before estrus. Glencross and Pope (1972) observed a postovulatory estradiol peak on day 6 as well as a rise in estrone 1 and 2 days prior to estrus. Although the absolute values vary considerably, these studies support the present observations in terms of the timing of concentration peaks relative to estrus. In order to further substantiate the present findings, it is pert-inent to consider the relationships between estrogens and other circu-lating hormones during the estrous cycle. The timing of estrogen peaks 39. < O o 120 100-80 -6 0 -4 0 2 0 H 0 225 -2 0 0 -175-o 150" 125-100-7 5 -5 0 -25 -0 • o o E S T R A D I O L o o o J O g o o I I I I I—I I I I I I—I—I—I I I I I I o E S T R O N E o T 1 I I I 1 I 1 I 1—I—I 1 1 I I 15 17 19 E 2 4 6 8 10 12 14 DAY OF E S T R O U S C Y C L E Fig,. 1. Plasma levels of estradiol and estrone during the bovine estrous cycle. Day E is the day on which animals displayed signs of heat. Values for estradiol represent unconjugated a and B isomers. • single samples from individual cows, o samples from an individual cow through the course of one estrous cycle. Table 2. Plasma estrogen concentrations during four stages of the estrous cycle in the cow. Values given are mean ± standard error of the mean. Total estrogen is the sum of estradiol plus estrone. Concentrations (ng/100 ml) during periods of estrous cycle Proestrus Estrus Metestrus Diestrus Estradiol Estrone 72.3 ± 9.9 50.2 ± 8.6 41.0 ± 3.3 122.5 ± 23.5 69.8 ± 9.5 46.6 ± 10.4 47.9 ± 2.8 69.4 ± 9.8 Total Estrogen 194.7 ±31 .4 119.9 ± 1 2 . 9 87.6 ± 8.2 117.3 ± 9.8 No. Obs. 10 11 13 41. found here, is consistent with that of luteinizing hormone (LH) in both the dairy cow and the ewe. Several workers (Henricks et al_., 1970; Garverick et_ al_., 1971; Snook et al_., 1971; Swanson and Hafs, 1971; Swanson et a l , , 1972) have noted a preovulatory LH peak in the bovine which occurred before or slightly after the initiation of estrus (Hansel and Snook, 1970). Scaramuzzi ejt al_., (1970), assayed.both LH and estrogens in the ovarian vein blood of the ewe and found that the first rise in estrogen concentration occurred 1 day before the rise in LH. Furthermore, the administration of estradiol benzoate to the ewe is followed by a peak of LH secretion (Goding ejt al_., 1969; Radford et al_. , 1969) and estrous be-haviour (Scaramuzzi et al_., 1971). This evidence i s con si stent with the view that a positive feedback effect of estrogen on the pituitary is the cause of the LH surge which results in ovulation. The preovulatory sequence of endocrine events in the cow might be seen as follows. The rapid decline in plasma progesterone levels, be-ginning at about the 15th day of the cycle (Garverick ejt al_. , 1971; Henricks e_t al_., 1970; Plotka et al_., 1967) gives rise to the release of follicle stimulating hormone (FSH) by the anterior pituitary. Hackett and Hafs (1969) found that the preovulatory release of FSH precedes that of LH by about 2 days. It is possible that this FSH release results in the production of estrogens which reach a peak level in the systemic plasma on days 19 to 20. This estrogen peak may then be followed by an LH surge at about the time of initiation of estrus with subsequent ovula-tion, luteinization and progesterone synthesis (Hansel and Snook, 1970). We have noted marked increases in plasma estrone on days 6, 7 and 8 (Fig- !)• This is in agreement with the findings of Glencross and Pope (1972) and Shemesh e_t al_., (1972) who reported mid-cycle estradiol elevations 42. and with Varman ejt al_., (.1964) and Mellin and Erb (1966) who demonstrated peaks in estrogen excretion between days 6 and 8 post-estrus. The rising plasma level of estrone may be coincident with the climax of a first wave of follicle growth (Rajakoski, 1960; Cupps et al_., 1969) and is consist-ent with observations of mid-cycle elevation in LH (Schams and Karg, 1969; Henricks et a]_., 1970; Snook et^  al_-» 1971) and decreases in pro-gesterone (Plotka et a]_., 1967; Pope e_t al_., 1969; Henricks et a l . , 1970). 4. Conclusion The primary peaks in plasma estrogen during the estrous cycle of the lactating dairy cow occur 1 and 2 days prior to the day on which standing heat is exhibited. The elevation in plasma estrone apparently occurs slightly before that of estradiol. A secondary peak in estrone and/ or estradiol may occur near mid-cycle. These findings are substantiated by comparisons with the levels of LH, FSH and progesterone given in the literature. B. Plasma Amino Nitrogen, Amino Acid, Glucose and Urea Nitrogen Concentra- tions During the Estrous Cycle of the Cow. 1. Introduction During the estrous cycle of the lactating cow, the increased meta-bolic demands of uterine and mammary tissue may lead to a decreased availability of substrate for lactogenesis. Such competition might aggra-vate an already precarious balance between the supply of substrate and its utilization. 43. Using the concentration of amino nitrogen as a measure of total plasma amino acids, Curet et al_., (1970) have shown that the lowest levels during the ovine estrous cycle occur at estrus. Curet and Caton (1971) have demonstrated that estrogens enhance the uptake of amino nitrogen by the ovine uterus. Dunn e_t al_., (1972) observed a significant decrease in plasma glucose concentration from late diestrus to estrus in the ewe, accompanied by a marked, but non-significant decrease in glucose entry rate. Conceivably, these effects were a result of decreased amino acid availability for hepatic gluconeogenesis. In the present experiment, plasma concentrations of amino nitrogen and glucose are measured throughout the bovine estrous cycle in an attempt to establish relationships similar to those seen in the ewe. In addition, plasma urea nitrogen is assayed since decreased gluconeogenesis might be expected to result in decreased urea formation through reduced deamination of amino acid (Metzger e_t al_., 1970; Nolan and Leng, 1970; Oltjen and Lehman, 1968). Preston (1968) demonstrated a reduction of plasma urea nitrogen with estrogen treatment in lambs. 2. Materials and methods The animals sampled and the method of blood collection are described in the previous experiment. Alpha amino nitrogen concentrations in blood plasma were determined using the colorimetric method of Frame et a l . , (1943) and Russel (1944) with slight modification of the deproteinization procedure. Plasma (0.2 ml) was diluted with water (3.6 ml) and mixed. Proteins were pre-cipitated with the addition of 0.1 ml of 10% sodium tungstate and 0.1 ml of 0.67 N hydrochloric acid. 44. Plasma amino acids were assayed by ion exchange chromatography using an automated amino acid analyzer (Phoenix Precision Instrument Company, Model M-7800) after deproteinization with sulfosalicylic acid added to give a final concentration of 3%. Plasma for the urea nitrogen assay was deproteinized with tungstic acid using the procedure employed for alpha amino nitrogen. Urea nitrogen was determined using urease and mercuric iodide (Sigma Chemical Company; Technical Bulletin No. 14). Plasma glucose was measured using gliucostat enzymic assay (Worthington Biochemical Corp.). All determinations were performed on duplicate samples. Data were analyzed using least squares analysis with the following model: V = u + H i + pd + E i jk where Y... = observation of dependent variable (a-amino nitrogen, 1 J K plasma urea nitrogen, glucose) for the k individual in the j period of the estrous cycle and the i herd, u = overall mean with unequal subclass numbers H- = effect of the i t h herd as a deviation from the overall mean P^  = effect of the j^*1 period as a deviation from the overall mean ^ijk = r a n c ' o m e r r o r Period mean differences for each dependent variable were tested for significance using Kramer's modification of Duncan's new multiple range test. 45. 3. Results and discussion Least squares means for plasma alpha amino nitrogen, urea nitrogen and glucose concentrations during the four periods of the estrous cycle are given in Table 3. a. Alpha amino nitrogen Figure 2 gives alpha amino nitrogen concentrations through the estrous cycle. The lowest period mean was found during estrus. Increasing levels of alpha amino nitrogen were seen through metestrus and diestrus, with a peak mean level occurring during proestrus. The highest individual levels were found on days 16 and 17.following the expected decline in plasma progesterone concentration (p. 33) and prior to the peak peripheral levels of estrogenic activity (Mason e_t al_., 1972). All differences between period least squares means were significant (P<0.05) as was the herd mean difference (P<0.05). The coefficient of determination (r ) for the model was 0.35. The wide range in values apparent in day 0 samples reflects the difficulty inherent in the estimation of the exact time of onset and cess-ation of estrus and the rapid changes in endocrine events occurring during this time. Individual samples taken on this day may represent stages of estrus up to 24 hours apart. b. Amino acids The concentrations of acidic and neutral amino acids were determined on samples which gave high alpha amino nitrogen values on the two days prior to the peak of plasma estrogens and on samples which gave low alpha amino nitrogen values on the two days following the plasma estrogen peak. The object was to determine which of the amino acids measured might account for the significant decrease in plasma alpha amino nitrogen levels at estrus. 46. o o o Q O o -1 1 1 1 1 1 1 1 1 1 1 -15 17 19 E 2 4 6 8 10 12 14 DAY OF E S T R O U S C Y C L E Alpha amino nitrogen concentrations in jugular plasma during the bovine estrous cycle. Values are corrected for herd effect. • single samples from individual cows, o samples from an individual cow through the course of one estrous cycle. 47. Table 3. Mean concentrations of plasma metabolites during the four periods of the estrous cycle. All period mean differences for alpha amino nitrogen are significant (P<0.05). Concentrations (ng/100 ml) during periods of estrous cycle Proestrus Estrus Metestrus Diestrus Plasma Metabolite Alpha Amino Nitrogen 4.85 Urea Nitrogen 15.44 Glucose 65.18 3.86 4.06 4.53 15.61 15.73 15.57 71.57 50.66 61.08 48. The results are given in Table 4, where levels of each amino.acid before and after the estrogen peak were compared using a Student's t-test. The concentrations of threonine, valine, methionine, isoleucine and proline decreased significantly (P<0.05) from days 17-18 to days 0-1. A marked, though non-significant, decrease was also seen for leucine. c. Plasma urea nitrogen Although the mean plasma urea nitrogen level was significantly different between herds (P<0.05), period mean differences were all non-significant. The model accounted for 70% of the variation in assay values (r 2 = 0.70). d. Glucose Period mean differences for glucose were all non-significant (P<0.05). However, the mean metestrus value was 29.2% lower than the mean estrus value. The herd difference was significant (P<0.05) and the co-efficient of determination for the model was 0.28. The significant decline in plasma alpha amino nitrogen concentration at estrus is consistent with the results of Curet et aj_. , (1970) in the ewe. This observation may be rationalized in terms of estrogen mediated protein synthesis in the uterus (Curet and Caton, 1971) and mammary gland (Sinha and Tucker, 1969), decreased amino acid mobilization from protein depots and/or decreased amino acid absorption from the alimentary tract due to a decline in feed intake. The latter possibility seems unlikely since fasting for 3 days results in no significant change in alpha amino nitrogen concentration (Curet et_al_., 1970). In view of the previous discussion of tissue amino acid mobilization (p. 22), the predominance of valine, isoleucine and perhaps leucine in the 49. Table 4. Comparison of amino acid concentrations in jugular plasma in cows before and after proestrus plasma estrogen peak. Values are means of four samples each. *Difference significant at P<0.05. Days of Estrous Cycle % Difference Amino Acid 17-18 (A) 0-1 (B) A-B 100 mg/100 ml ~A~~ x Threonine 1.05 0.64 39.0* Valine 4.70 3.25 30.9* Methionine 0.48 0.28 41.7* Isoleucine 2.36 1.67 29.2* Leucine 2.79 1.92 46.1 Tyrosine 1.16 0.72 37.9 Serine 1.19 0.83 30.2 Asparagine-Glutamine 9.23 7.56 18.1 Proline 1.34 0.70 47.8* Glutamic Acid 0.90 0.91 - 1.1 Glycine 3.50 2.87 18.0 Phenylalanine 0.93 0.78 16.1 Alanine 2.45 1.67 31.8 50. decline in plasma amino nitrogen at estrus becomes interesting in the context of this thesis. Since the hepatic release of branched-chain amino acids accompanies gl uconeogenesi s from protein (Mallette et al_. , 1969b), the notable decreases in these amino acids seen here might reflect de-creased hepatic glucose production. Although these observations are far from conclusive, it might be reasonable to postulate a decreased availa-bility of amino acids for gluconeogenesis as a result of increased mammary and uterine uptake and decreased tissue protein mobilization, accounting for the hypoaminoacidemia observed during estrus. In addition, the apparent metestrus hypoglycemia may be a reflection of this decreased hepatic glucose production. This hypothesis gains support-from the obser-vations of Dunn et al_., (1972) who measured glucose kinetics during the estrous cycle of the ewe. Those authors noted a significant estrus-metestrus hypoglycemia coincident with a lowered glucose entry rate. Furthermore, calculations based upon the work of Dunn ejt al_., (1972) and Curet and Caton (1971) suggest that uterine uptake may be insuff-icient to account for such a decrease in glucose turnover (p. 27). A decreased gluconeogenesis from amino acids would be expected to result in decreased hepatic urea production (Metzger e_t al_., 1970). However, such a decrease was not demonstrable in this study in terms of plasma urea nitrogen concentration. It is possible that the effect of decreased gluconeogenesis upon plasma urea concentration is confounded by utilization of endogenous urea in the digestive tract through recycling (Nolan and Leng, 1970) and by the hepatic synthesis of urea from ammonia absorbed from the rumen (Bergman, 1973). 51. 4. Conclusions A .significant hypoaminoacidemia was associated vyith estrus in the lactating cow. Measurement of individual amino acids indicated a general decrease with significant reductions of valine, isoleucine, threonine, methionine and proline. 'A'46% decrease in plasma leucine was also observed. These findings were rationalized in terms of increased uptake of amino acids into the mammary gland and uterus with decreased tissue protein mobilization. Reduced availability of amino acid for gluconeo-genesis may have given rise to an apparent metestrus hypoglycemia. C. General Discussion The possibility that tissue protein mobilization might be influ-enced by the increased estrogenic activity seen during proestrus is of particular interest for several reasons. Of these, the importance of amino acids as glucogenic substrates has been repeatedly emphasized. In addition, modification by estrogens of growth hormone and cortico-steroid secretion (p. 29), the role of GH and glucocorticoids in tissue amino acid metabolism (p. 22), and the possible role of these hormones in the etiology of bovine acetonemia (p. 25) have been discussed. The possibility that estrogens reduce tissue amino acid mobiliza-tion by way of inhibition of glucocorticoid synthesis and potentiation of GH release thus arises. The increased estrogenic activity found during proestrus (p. 40) may provoke increased uptake of amino acids into uterine and possibly mammary tissue (Curet and Caton, 1971; Sinha and Tucker, 1969). Inhibition by estrogen of adrenal response to ambient ACTH activity (p. 29) could result in decreased negative feedback on the 52. pituitary and a compensatory increase in ACTH secretion, bringing the plasma glucocorticoid concentration back to normal (Garverick et a l . , 1971). In addition, the estrogens may sensitize the response of GH secretion to various stimuli, resulting in elevated plasma GH concen-tration (Koprowski and Tucker, 1973). Thus, a situation arises which is analogous to that which may exist in acetonemia (P. 25), that is; adrenal inhibition with pituitary overproduction of ACTH and GH, resulting in decreased amino acid mobil-ization and hypoaminoacidemia (p. 47). A reduced glucose entry rate due to lack of substrate may give rise to hypoglycemia and marked decreases in plasma levels of branched chain amino acids (p. 49). The critical point in the development of this hypothesis is whether estrogens do, in fact, inhibit glucocorticoid synthesis in the lactating cow, thereby resulting in increased ACTH secretion. It is with this point that the remainder of this thesis will be concerned. 53. II. Estrogen Inhibition of Glucocorticoid Response to ACTH in the  Lactating Cow. Large doses of ACTH have previously been administered to dairy cows, both in the treatment of acetonemia (Shaw, 1956; 600 IUVI.M.) and in the assessment of adrenal function (Venkataseshu a n c j Estergreen, 1970: 200 IU I.M.; Reigle and Nellor, 1967: 100 IU I.V.). However, the use of such pharmacologic doses reflects only the maximum ability of the adrenal to respond. In addition, glucocorticoid production is still influenced by endogenous ACTH. The assessment of adrenocortical sensitiv-ity (Landon et al_., 1967) requires the administration of incremental physiologic doses of ACTH in the absence of endogenous ACTH. The adrenal response is measured in terms of the plasma corticosteroid concentrations after stimulation. On the basis of previous discussion (p. 29), estrogens might be expected to inhibit glucocorticoid production under the condition described. A. Dexamethasone Suppression of Endogenous ACTH Production 1. Introduction The suppression of endogenous ACTH production necessary to the asse ment of adrenal sensitivity is accomplished through negative feedback on the pituitary (Morgner and Kruskemper, 1973). The synthetic fluorinated glucocorticoid, dexamethasone (Decadron, Merck, Sharpe and Dohme) is use-ful for this purpose since the doses required for suppression do not sig-nificantly impair adrenocortical function (Landon ejb al_., 1967). In addition, the steroid is not measured in the competitive protein binding assay used to assess adrenal response. 54. 2. Materials and methods Three mature, lactating, non-pregnant dairy cows (one Ayrshire, Topsy; two Jersey, Helen and Ella) were used in this study. The animals were milked twice daily (0400 and 1700 h) and since parturition, had been receiving a ration consisting of dairy pellets and hay. During the course of these experiments, the cows were housed in a stall which was separated by a plywood partition from an area from which blood collections and infusions were performed. Permanent jugular cannulae were established at least 20 h prior to experiments and were kept patent by twice daily flushing with 5 ml of 100 U heparin/ml saline. During an experiment, a polyethylene extension connected the cannula with the collection/infusion area in order that operations could be performed without disturbing: the animal. Blood samples (10 ml) were collected into heparinized, evacuated glass tubes and chilled in ice immediately. Plasma was removed after centrifugation and frozen within one hour of collection. All experiments were conducted between days 9 and 15 post-estrus in an attempt to mini-mize the effects of fluctuating hormone activities during the estrous cycle (ExperimentrIA).' In the first series of experiments (control day), blood was coll-ected at half-hour intervals from 0800 to 1530 h inclusive. On a second day dexamethasone phosphate (Decadron; Merck, Sharpe and Dohme, 10 mg) was administered intramuscularly at 0800 h. Blood was again collected at half-hour intervals. Corticosteroids (Cortisol, corticosterone) were extracted from plasma (0.2 ml) with dichloromethane (0.8 ml). Extracts (50-200 ul) were evaporated to dryness at 25 C under vacuum. Corticosteroids were determined by the competitive protein binding method of Bassett and 55. Hinks (.1969) with minor modification. Dexamethasone is not measurable with this method and the recovery of added C o r t i s o l was greater than 98%. Standard curves were linearized by the transformations: Y = 1/HC and; X = 1/(1 - B/BQ) where: HC = concentration of corticosteroid B = dpm corticosteroid bound BQ = dpm corticosteroid bound at zero concentration o Excellent fit (r = .98) was obtained by simple linear regression of the transformed variables. Regressions and calculations of plasma concentra-tions were performed using a Fortran IV computer program. Assays were rejected when the coefficient of variation (SD/mean x 100) for duplicates exceeded 20%. 3. Results and discussion Control day corticosteroid levels were variable both among and within animals being subject to fluctuation in response to endogenous ACTH stimulation (Fig. 3). The mean concentration for Topsy and the lower concentrations for Helen and Ella were in a range comparable to those found by Heitzman ejt al_., (1970), using the present method, and by several other workers using essentially similar methods (Macadam and Eberhart, 1972; Smith et al_., 1973; Wagner and Oxenreider, 1972; Whipp and Lyon, 1970; Willett and Erb, 1972). Since it was impossible during the whole course of an experiment to maintain the animals in an absolutely "stress-free" environment, the elevations in plasma cortico-steroids which were observed can clearly not be associated with a basal 8 9 10 II 12 13 14 15 16 T I M E Fig. 3. Plasma corticosteroid concentrations-in three cows on control day (no treatments) • and on a day on which 10 mg dexamethasone were administered at 0800 h o . 57. state or even with diurnal variation. Under these conditions, adrenal sensitivity trials would be pointless, since environmental responses would certainly mask the small responses to exogenous ACTH which were expected. The administration of 10 mg of dexamethasone baseline corticosteroid levels which, within two hours of injection, were always less than 2 ng/ml (Fig. 3). This effect is similar to that seen when direct measurements of corticosteroid secretion rates were made using dexamethasone suppressed sheep with adrenal autotransplants (Espiner et a l . , 1972). This procedure has also been utilized in adrenocortical sensitivity trials in humans (Landon e_t al_., 1967; Leclercq et a l . , 1972a; McDonald et al_., 1969). Landon et al_, (1967) found that dexa-methasone did not impair the response to exogenous ACTH but, as shown here, was of value in lowering basal corticosteroid levels such that further stimulation trials with exogenous ACTH were free from the influence of fluctuations in the activity of the endogenous hormone. 4. Conclusions During an 8 h experimental period, wide fluctuations in plasma corticosteroid concentrations were seen in untreated cows. Administration of 10 mg of dexamethasone (I.M.) effectively produced steady baseline con-centrations of 2 ng corticosteroid/ml plasma within 2 h after treatment. B. Effect of Estrogen on Adrenal Response to Exogenous ACTH 1. Introduction Adrenal insufficiency may arise as a result of limited ACTH stimu-lation of the adrenal cortex (secondary insufficiency) or inadequate 58. adrenocortical response to ACTH activity (primary insufficiency). Abso-lute primary insufficiency may result when the adrenal reaches its maxi-mum ability to secrete. Relative adrenal insufficiency might be used to describe a condition in which the adrenal remains responsive to ACTH stimulation ,!.but in which an increased stimulus is necessary to elicit a normal response. As discussed previously (p. 29), estrogens, by in-hibiting corticosteroid synthesis, may provoke relative adrenal insuffic-iency. It is the purpose of this experiment to examine such a possibility. Natural corticotrophin preparations are unsuitable for use in trials such as these since they are difficult to standardize and administer on a quantitative basis. For these reasons, the synthetic peptide which represents the biologically active 24 N-terminal amino acid sequence of 1 -24 all native ACTH preparations has been used. The quality of a ACTH (tetracosactrin, tetracosapeptide, Cortrosyn, Synacthen) is uniform, its dosage can be determined accurately by weight, and the possibility of adverse immunological reactions is minimal (Greig et al_. , 1968). 2. Materials and methods The animals used in this study and the methods of blood collection were as described in the previous experiment (p. 54). Stimulation experiments were begun at 0800 h with the administra-1 -24 tion of dexamethasone as above. At 1000 h, 6.25 yg of synthetic a ACTH (Cortrosyn; Organon) dissolved in 1 ml saline adjusted to pH 2 with HC1 to prevent ACTH adsorption to the syringe, was administered through the jugular cannula. The syringe and cannula were subsequently flushed with 10 ml of saline. Doubling doses of ACTH in 2 ml of acidified saline were given at hourly intervals until 1500 h when a final dose of 250 yg ACTH 59. was administered. Blood was collected at 5, 10, 15, 20, 30, 45 and 58 min after each infusion. In a second series of experiments, 5 mg of 173-estradiol (Sigma) in 2 ml of 70% ethanol was administered intramuscularly 18 and again 2 h prior to the beginning of a series of ACTH infusions identical to those described above. Plasma corticosteroid concentrations were determined as previously described (Experiment IIA). 3. Results and discussion A typical pattern of response to graded doses of Cortrosyn at hourly intervals is shown in Fig. 4. Response was measured in terms of peak plasma cortcicosteroid concentration after stimulation. At fixed blood flow through the adrenal, a linear relationship exists between the corticosteroid secretion rate and the logarithm of ACTH concentration in arterial blood (Yates et al_., 1969). Therefore, linear regressions of adrenal response on the logarithm of the ACTH dose were fitted (Fig. 5). Analysis of covariance (Snedecor and Cochran, 1968) revealed no significant differences between cows within estrogen or non-estrogen-treated groups with respect to variance, slopes or elevations. Data were thecefore pooled within treatments demonstrating significant (P<0.05) linear relationships in both cases. Comparison of treatments re-vealed no differences in regression coefficients although estrogen admin-istration resulted in a highly significant (P<JD.01) decrease in elevation. That is, at all levels of ACTH stimulation, estrogen treatment resulted in significantly decreased corticosteroid response. This finding is supported by those of other workers (p. 29). Assuming blood occupies 6% of body weight (Oser, 1965), the initial 60. e> UJ CO o CL CO UJ 80H 7 0 j 6 0 1 5 0 i 4 0 3 0 H O cr UJ h-CO o 2 20 Q : o ° 10 0 A C T H H E L E N NO E S T R O G E N IIIII i i m i l i i m i l i i m i l i i i n n i t i 12 13 TIME 14 15 16 Fig. 4. Pattern of corticosteroid response to hourly infusions of increasing doses of a^-^ACTH in a single animal. 61. CD 9 0 % 8 0 z o CL to 70 0 6 0 i LU § 5 0 o 1 4 0 -o NO ESTROGEN T 1 ESTROGEN 30^ 6-25 12-5 25 5 0 100 2 5 0 MICROGRAMS ACTH Fig. 5. Relationship between dose of ACTH and response of plasma corticosteroid concentration. The points are mean values for three animals. The vertical bars represent the magnitude of the standard error of the mean. 62. instantaneous ACTH concentration in blood can be calculated by dividing the dose given by the blood volume. Using this estimate as a correction for body size, the dose-response relationship found in human subjects (Landon e_t al_., 1967; Leclercq et al_., 1972b) has been compared with that found here in the dairy cow (Fig. 6). The refractory nature of adrenal response in the cow is obvious: For example, a response of 60 ng cortico-steroids/ml of blood is elicited by an initial ACTH concentration of approximately 20 pg/ml in the human. An equivalent response is reached in the cow only when the initial concentration reaches about 700 pg/ml. Comparison with responses to stimulation in the sheep yields similar results (Bassett and Hinks, 1969). Support of this observation is found in studies of adrenal size. Adrenals of adult female cattle are much smaller in relation to body weight (0.0056 - 0.008%) (Swett et al_., 1937) than adrenals found in other adult mammals (0.02 - 0.03%) (Christian, 1953). The significance of these data in support of a hypothesis of adrenal insufficiency in the dairy cow is clear. 4. Conclusions Estrogen treatment significantly (P<0.01) reduced the adrenal res-1 -24 ponse to all doses of a ACTH administered.;. 'Even with no estrogen treat-1 -24 ment it was found that the doses of a ACTH elicit responses were much higher than those required in similar human trials, although corrections were made for differences in metabolic size. o 0 — i 1 1— 1 1 — — I 10 100 1000 10000 INITIAL BLOOD ACTH CONC- P G / M L ig. 6. A comparison of the dose-response relationship for the dairy cows in the present study with that found in man (Leclercq . et al_., 1972b). Initial blood concentration is the dose of ACTH given divided by the estimated blood volume. 64. C. Estrogen Effect on Plasma Glucocorticoid Binding 1. Introduction The multiple effects of estrogens on the pituitary-adrenal axis have been discussed (p. 29). Of these, plasma binding is significant in the mediation of the biological effects of glucocorticoids in most mam-malian species. On the basis of previous work, however, ruminants do not appear to respond to estrogen treatment with increased transcortin (cor-ticosteroid binding globulin) synthesis (Krulik and Svobodova, 1969; Lindner, 1964; Shayanfar et a]_., 1973). The latter authors measured plasma binding capacity using the method of DeMoor et al_., (1962). In this method, plasma is incubated with labelled Cortisol, with free and bound label being separated at 4 C using gel filtration on columns 1.2 x 30 cm with flow rates of about 0.5 ml/min. Under these conditions, the protein bound fraction requires at least 30 min to be eluted from the column. This time approximates the half-time of dissociation of transcortin bound Cortisol in ovine plasma at 2 C (Patterson, 1973). It is apparent then, that the actual amount of steroid binding capacity found in plasma may be significantly greater than that estimated using the method of DeMoor et a l . , (1962). It is also apparent that any method of separating free from bound steroid will under-estimate the actual endogenous binding. With these considerations and limitations in mind, the present experiment was designed to verify, at least in qualitative terms, the plasma transcortin binding capacity in the dairy cow using a method which more closely estimates the actual binding capacities of the plasma in question. 65. 2. Materials and methods The animals used in this experiment and the method of blood coll-ection have been described (p. 54). Plasma from blood samples collected at 1000 h, before ACTH stimulation, were assayed for corticosteroid bind-ing capacity using small (10 mm x 35 mm) columns of Sephadex G-25 fine (Pharmacia) packed in disposable 3 ml plastic syringe barrels. A disc of 18XX serigrapher's silk supported the gel and a 17 cm extension of poly-ethylene tubing (ID 0.86 mm) was attached to the outflow end to attain a flow rate of 0.3 ml/min. Cortisol ([1, 2-3H] C o r t i s o l ; SA = 25 nCi/ng; New England Nuclear) in redistilled ethanol was added to small glass tubes in amounts of 2.5, 5, 10, 25 or 50 ng and evaporated to dryness. Plasma (0.2 ml) was added, the tubes were incubated at 45 C for 30 min and cooled to 4 C. Columns, maintained at 4 C, were loaded with 0.1 ml of sample and protein-bound corticosteroids were eluted with 1.5 ml of 0.04 M phosphate buffer (pH 7.2) directly into 20 ml scintillation vials. Free Cort i so l was then eluted with an additional of buffer. Cocktail (10 ml of PCS, Amer-sham Searle) was added and samples counted on a Nuclear-Chicago Isocap 300 liquid scintillation counter with a counting efficiency of 45 ± 2%. Recovery of isotope was 92 ± 5%. A simple linear regression of the inverse of bound upon the inverse of free Cortisol was calculated by least squares for the five concentrations of Cortisol added to plasma. The Cortisol binding capacity is the recip-rocal of the ordinate intercept (Tait and Burstein, 1964): — = — — x — " - - — CCT] K Am [C F] [T] 66. where: [Cj] = bound Cortisol concentration [Cp] = free Cortisol concentration [T] = concentration of transcortin binding sites = affinity constant for the association of transcortin and C o r t i s o l The gel filtration method used here measures the binding of Cortisol to transcortin exclusive of albumin binding (DeMoor et al_., 1962). 3. Results and discussion The elution pattern for the small column gel filtration of plasma is shown in Fig. 7. The elution of protein—bound Cor t i so l was essentially complete within 4 minutes, making the estimates of binding capacity ob-tained here closer to the level of endogenous binding than the estimates obtained by previous workers (Krulik and Svobodova, 1969; Lindner, 1964; Shayanfar et al_., 1973). A plot of the regression used to calculate C o r t i -sol binding capacity is shown in Figure 8. Estrogen administration failed to affect plasma Cortisol binding capacity (Table 5) although significant animal differences were demonstrated. The experiment thus verified qual-itatively the results of other investigators. The measured plasma cortico-steroid levels were, therefore, a direct reflection of the glucocorticoid activity. 4. Conclusions Plasma Cortisol binding capacity was not significantly affected by estrogen treatment. 67. I-4H I-2H O -8H § - 6 H o 4H 2H 0 DROPS 0 MIN 0 BOUND 100 2 0 0 3 0 0 4 8 12 E L U T I 0 N V O L U M E / T IME Fig. 7. Separation of bound from free Cortisol using small column gel filtration. Flow rate - 25 drops/min (72 drops/ml). 68. Fig. 8. Regression of the inverse of bound upon the inverse of free Cortisol. Plasma transcortin binding is determined from the intercept of the computed regression line. 69. Table 5. The effect of estrogen treatment on plasma corticosteroid binding capacities in the dairy cow. Non Estrogen Estrogen Treated Treated Binding Capacity (ng cortisol/ml plasma) Topsy 61.7 + 3.1a 49.5 + 2.9 Helen 70.9 + 2.9 71.9 + 2.9 Ella 38.0 + 3.2 35.7 + 3.1 a Mean ± standard deviation 70. D. General Discussion The inhibition of adrenal response to ACTH by estrogen has been demonstrated. Under conditions of normal pituitary-adrenal function, however, it is likely that the decreased negative feedback resulting from such inhibition would provoke a compensatory rise in ACTH secretion. Thus, the circulating plasma corticosteroid levels during estrus would be expected to be normal or near normal as has been demonstrated by Swanson e_t al_., (1972). Secondary adrenal insufficiency (hypopituitarism) would, of course, result in a failure of the plasma corticosteroid level to return to normal, but such a condition has never been dairy cattle. The present hypothesis (p. 51) requires a demonstration of compen-satory ACTH secretion induced by estrogen inhibition of corticosteroid synthesis. Such a demonstration is the aim of the ensuing experiments. 71. III. Plasma ACTH and Glucocorticoid Levels During the Estrous Cycle of  the Cow. Although it has been demonstrated (Experiment II B) that exogenous estrogens will inhibit adrenocortical response to exogenous ACTH, the possibility remains that the effect is pharmacologic rather than physio-logic. The following experiments were designed to obtain further evidence of adrenocortical inhibition by estrogens. Initially it was necessary to develop an assay method for plasma ACTH. Finally, the concentrations of both ACTH and glucocorticoids were measured during the estrous cycle with no exogenous treatment applied. A. Radioimmunoassay of ACTH 1. Introduction The method of radioimmunoassay developed by Yalow and Berson (1959) has been utilized in the assay of many peptide and steroid hormones. Its application to the measurement of ACTH has, however, been particularly troublesome due to the low concentrations found in the plasma of normal subjects and the relative lack of antigenicity of this hormone. Although measurements have been made of plasma ACTH in human subjects (Berson and Yalow, 1968; Demura et al_., 1966; Landon and Greenwood, 1968), rat (Rees et al_., 1971) and sheep (Alexander et^  al_., 1971), no previous measure-ments of ACTH in the plasma of the dairy cow have been reported. This experiment describes the development of a radioimmunoassay suitable for such measurements. 2. Materials and methods 125 a. Preparation and purification of I - ACTH The method used for the preparation of labelled ACTH was based upon 72. the method developed for human growth hormone by Hunter and Greenwood (1962). Throughout the procedure and subsequent assay, contact of neutral pH ACTH solutions with glass surfaces is rigorously avoided since sig-nificant losses of the peptide by adsorption to glass can occur (Landon et_ al_., 1967). Such adsorptive losses are minimized when solutions are acidified to pH 2 and when polystyrene and polyethylene equipment is employed. Three buffers were utilized in this procedure. Buffer A was 0.2 M phosphate, pH 7.5. Buffer B was 0.04 M phosphate, pH 7.5. Diluent buffer was buffer B containing 5 mg crystalline bovine albumin/ml and 0.5% mercaptoethanol. The iodination reaction was carried out in a small (12 x 75 mm) polystyrene tube. To 10 yl of buffer A were added sequentially: 2.5 yg of a 1 _ 2 4ACTH (Cortrosyn, Organon) in 10 yl of dilute HC1 (pH 2), 1 mCi of Na 1 2 5I (>14 mCi/yg, Amersham Searle) in 10 yl of dilute NaOH solution (pH 8.9),;.and 25 yg of chloramine-T in 10 yl of buffer A. The solution was agitated for 30-40 sec and the reaction was then stopped with the addition of 200 yg of sodium metabisulfite in 10 yl of buffer B. Diluent buffer (10 ml) was added immediately to prevent any further damage to the peptide, the albumin serving as a trap for free radicals produced by disintegration of the isotope. Purification of labelled ACTH, based on the method of Yalow and Berson (1966) for the purification of labelled parathyroid hormone, was carried out at any time up to a few weeks after iodination. To 0.5 ml of the di-luted reaction mixture was added 0.5 ml of diluent buffer containing 2 mg of microfine precipitated silica granules (QUS0 G32, a gift of Philadelphia Quartz Co., Philadelphia, Pa.). The suspension was agitated periodically 73. over a 30 minute period to allow adsorption of ACTH to the silica then centrifuged at 1500 xg for 10 min. The supernatant which contained dam-125 aged ACTH fragments and unreacted I (Fig. 9d) was discarded and the pellet washed by resuspending in 1 ml demineralized water and recentri-fuging. After washing three times, the QUSO pellet was resuspended in 0.5 ml aqueous 40% acetone/1% acetic acid. The suspension was allowed to stand, being agitated repeatedly for 30 min, in order to elute the ad-sorbed ACTH. Following centrifugation, the supernatant was aspirated and the pellet discarded. b. Assessment of purity and specific activity of labelled product Aliquots (usually 50 yl) of diluted reaction mixture and the purified product (diluted 10 times in diluent buffer) were assessed for purity using chromatoelectrophoresis (Berson e_t al_., 1956). Paper strips (Whatman 3MM, 2.5 x 65 cm) were soaked in pH 7.4 phosphate buffer (0.1 M ionic strength) and stretched across supports 45 cm apart. The ends were immersed in buffer tanks filled with the buffer described above. After a short period (10-20 min) of air drying, the samples were applied on a line 15 cm from centre towards the cathode. Since separation is partly a func-tion of chromatography, a canopy was placed over the apparatus such that a limited amount of evaporation would take place but complete drying of the strips would not occur. A potential of 300 V was applied for 150 minutes, the current reaching a final value of 3.5 mA per strip. Strips were air "I pc dried and scanned for I using an Actigraph III (Nuclear-Chicago) strip scanner. Typical radiochromatography scans are shown in Fig. 9. 74. 1261 - ORIGIN Fig. 9. Radiochromatography scans of paper strips after chromato-electrophoresis of: (a) product of iodination reaction, (b) iodination product after storage at 4 C for two weeks, (c) purified iodination product, (d) supernatant from puri-fication of iodination product, (e) paper strip stained with amido black showing position of albumin band. 75. c. Blood sampling and extraction of ACTH from plasma Blood samples were drawn into small (12 x 75 mm) evacuated poly-styrene tubes containing -200 U of sodium heparin in 50 yl of physiologic saline. Samples were immediately cooled to 0 C on ice and then centri-fuged (1500 xg; 0 C; 15 min). Plasma was aspirated with an Oxford sampler (Oxford Laboratories) using a polyethylene tip and frozen at -20 C within 30 min of sampling. ACTH was extracted from 0.1 ml of plasma in a small polystyrene tube with the addition of 0.1 ml of diluent buffer containing 5 mg QUS0 G32 adsorbent. The suspension was agitated frequently and after 30 min was centrifuged at 1500 xg for 5 min. The pellet was washed twice with 1 ml of deionized water and finally the adsorbed ACTH was eluted with two sequential additions of 0.5 ml aqueous 40% acetone/1% acetic acid followed by centrifugation. The supernatants were pooled and evaporated to dry-ness at 55 C under a stream of nitrogen. In order to obtain control plasma, free of endogenous ACTH, blood was collected from a cow which had been treated 3 hours previously with 10 mg of dexamethasone (IM) to suppress ACTH secretion. Residual ACTH was removed by extraction of the plasma with QUS0 G32. One set of standards 1 -24 (a ACTH) was made up in this control plasma and extracted in parallel with samples as described above. Recovery of standards from control plasma was calculated by comparison with a second set of standards made up in diluent buffer and incubated directly without extraction. In order to 1 -24 further quantitate the extraction procedure, labelled a ACTH was added to control plasma and recovery calculated. These steps are outlined in Table 6. 76. Table 6. Schematic representation of ACTH radioimmunoassay procedure Control plasma Samples Add 1 2 5 1 ACTH Add a 1 _ 2 4ACTH Extract with QUSO G32 -Elute from QUSO G32 • Evaporate to dryness - - Second set of I I standards in I I 100 yl diluent Dissolve in 100 yl diluent buffer buffer Add Add antibody in 100 yl diluent buffer 1 2 5 1 a 1 _ 2 4ACTH in 100 yl diluent bu Incubate 65 hours at 4 C fer Separate free from antibody-bound ACTH Count bound fraction 77. d. a'""ACTH antibody The antibody employed in this assay was a gift of the National Pituitary Agency of the National Institute of Arthritis and Metabolic Diseases, U.S.A.. It was prepared and characterized by J.W. Kendall as described by Rees e_t al_., (1971). e. Incubation of samples and standards Samples and standards which had been extracted from plasma and dried 1 -24 were dissolved in 100 yl of diluent buffer. Standards (a ACTH) made up directly in diluent buffer without extraction, were dispensed in 100 yl quantities into small polystyrene tubes. To all samples and standards were added anti-o ACTH serum diluted 1:25000 in 100 yl of diluent buffer and 10-15 pg of 1 2 5I-ACTH also in 100 yl of diluent buffer. The final incubation volume was 300 yl and the final antiserum dil-ution was 1:75000. Damage control samples contained 200 yl of diluent buffer and labelled ACTH in 100 yl diluent buffer. These samples allowed an estimate of the labelled hormone which did not bind to antibody be-cause of damage in addition to that which failed to adsorb to QUSO G32. All tubes were tightly capped and incubated at 4 C for 65 hours. f. Separation of free from antibody-bound ACTH This procedure was performed in an ice bath. QUSO G32 (2 mg in 0.5 ml diluent buffer) was added to each incubation tube in order to re-move free ACTH from solution by adsorption. The suspension was agitated briefly and centrifuged at 1500 xg for 15 min at 0 C. Aliquots (0.5 ml) of supernatant containing antibody-bound ACTH were transferred to scin-tillation vials, 10 ml of PCS scintillation cocktail (Amersham Searle) were added and samples counted on a Nuclear Chicago Isocap 300 liquid scintillation counter. The bound fraction was calculated as dpm for sample 78. minus dpm for damage controls. These procedures are demonstrated schem-atically in Table 6. g. Validation of assay Since the specificity of the antiserum used in this assay has been extensively defined by Rees et al_., (1971), no further characterization was considered necessary here. By way of validation of this assay procedure, blood samples were collected from two lactating, non-pregnant cows before and 45 minutes after intravenous injection of 4 mg (100 IU) of bovine insulin. Two hours later, 10 mg of dexamethasone (IM) were administered as described on p. 54 and after an additional three hours, blood was again collected. Insulin was expected to provoke hypoglycemia and a subsequent increase in ACTH secre-tion (Donald, 1971). Dexamethasone suppresses endogenous ACTH production as described previously (p. 53). These treatments were designed to demon-strate that the assay procedure described here was capable of measuring the changes in circulating ACTH levels anticipated. 3. Results and discussion 1 -24 a. Radioiodination and purification of a ACTH The quality of labelled ACTH, assessed by chromatoelectrophoresis, was consistently high. A radipchromatographic scan of the reaction prod-125 uct (Fig. 9a) demonstrates two distinct peaks of I activity. The major portion of the activity resides at the origin where intact ACTH adsorbs to the paper. A smaller peak appears on the anode side of centre and repre-125 sents unreacted Na I. Damage, when it occurs, is apparent as activity coincident with the tracking dye and albumin position on the strip. Specific activity of the labelled product was calculated as the ratio of the total peptide bound radioactivity obtained by integration of the peaks 79. and the quantity of ACTH used in the reaction. For example, in Figure 9a, 85% of the 1 2 5 I is peptide bound (15% free 1 2 5I). Thus, .85 mCi is 1 -24 associated with 2.5 yg a ACTH, giving a specific activity of 340 yCi/yg. Upon storage for even a few days, activity associated with intact ACTH decreases, with concomitant increases in activity appearing in the damaged peptide and free I peaks (Fig. 9b). Purification of the labelled product using QUSO G32 eliminated a 1 p5 major portion of damaged peptide fragments and free I (Figs. 9c and 9d) 125 yielding a product which was consistently better than 95% intact I-ACTH. This product was used immediately in the radioimmunoassay incubation, b. Radioimmunoassay The lability of ACTH in plasma makes its extraction of prime import-ance to any prolonged assay procedure. Besser et al_., (1971) have demon-strated rapid losses as well as the dissociation upon incubation in plasma of the biological and immunological ACTH activities present. Losses during incubation and damage to the labelled hormone are considerable when unextracted plasma is assayed (Ratcliffe and Edwards, 1971). 125 As demonstrated in Fig. 10, the efficiency of I-ACTH extraction from plasma increases with the concentration of QUSO G32 employed. However, when the quantity of QUSO in a single extraction tube exceeded 5 mg, wash-ing and elution of the adsorbed ACTH became very inefficient due to diffi-culty in resuspending the pellet. A standard curve for samples extracted from plasma as described (p. 75) is shown as Fig. 11. This is, in fact, the standard curve obtained for the assay reported in Experiment III B (p. 82). The sensitivity (the lowest level of ACTH which can be distinguished from zero) was found to be 9 pg. When the values (dpm or bound/free ratio) obtained from such a 80. mg Q U S O / 0-2 ml Fig. 10. I-ACTH recovery from plasma as a function of the con-centration of QUSO G32 in the extraction mixture. Absolute amounts of QUSO in excess of 5 mg per tube resulted in poor recoveries due to difficulty in resuspending the pellet after centrifugation. 81. • 7 H •6H •5 • B F • 4 -• 3 H • 2 H i 1 • — i — 1 " — 0 62-5 125 187-5 2 5 0 pg cc 1 " 2 4 ACTH Fig. 11. Calibration curve for ACTH standards extracted from plasma. B/F is the ratio of l 2 5I-ACTH bound to antibody to that remaining unbound after incubation for 65 hours at 4 C. Vertical bars represent the magnitude of the standard de-viation for means of triplicate measurements. 82. standard curve are compared with standards made up in diluent buffer and incubated directly, the linear relationship shown in Fig. 12 is obtained. The regression coefficient of such a relationship (b = 0.88), is a measure of the extraction efficiency and compares with the 90% efficiency of ex-traction of labelled ACTH (Fig. 10). As expected, insulin administration produced elevated plasma ACTH concentrations while dexamethasone treatment resulted in very low circu-lating ACTH levels after 3 hours (Table 7). Plasma levels have been corrected for the difference in molecular weight between native bovine ACTH 1 -24 and the a ACTH used here as a standard. 4. Conclusions A radioimmunoassay suitable for the measurement of ACTH in bovine plasma is described. Extraction of ACTH from plasma by adsorption to and subsequent elution from QUSO G32 microfine precipitated silica granules resulted in reproducible recoveries in the order of 90%. Radioimmunoassay of plasma extracts provided a sensitive (lower limit of detection = 9 pg 1 -24 a " ACTH) and, on the basis of previous work (Rees e_t. al_., 1971), a specific method for the measurement of ACTH in bovine plasma. B. Plasma ACTH and Glucocorticoid Concentrations During the Bovine  Estrous Cycle 1. Introduction It has been hypothesized (p. 51) that increased estrogenic activity during the proestrus phase of the estrous cycle may result in inhibition of the adrenocortical response to ACTH stimulation. At first sight, one might expect such an effect to produce a decrease in circulating gluco-corticoid concentrations. However, Garverick et a l . , (1971) found no 83. 2 5 0 1 o Ld > UJ CO CQ o X o < eg 187-5 H 125 H 62-5 i Q. 0 A 0 62-5 125 187-5 2 5 0 pg o c ' " 2 4 A C T H PREDICTED 1 -24 Fig. 12. Recovery from plasma of a ACTH with reference to standards made up directly in diluent buffer before incubation without extraction. Vertical bars repre-sent the standard deviation of triplicate estimates. Table 7. Effect of insulin (4 mg IV) and dexamethasone (10 mg IM) on plasma concentrations of ACTH in two lactating, non-pregnant cows. Plasma ACTH concentration (pg/ml) Pre- 45 min post- 3 h post-Cow Treatment Insulin Dexamethasone Nemesis 3 4 0 1 8 4 0 5 0 (Hoi stein) Helen (Jersey) 530 2020 40 85. significant differences in glucocorticoid levels between days of the estrous cycle in lactating cows. Likewise, Swanson et al_., (1972), sampling cycling heifers and Christensen et al_., (1974), sampling cycling beef cows, found no significant changes in plasma glucocorticoid levels throughout the estrous cycle. If estrogens do, in fact, inhibit the adrenocortical response to ACTH, then the pituitary may compensate with increased ACTH secretion, bringing a transient decrease in adrenal glucocorticoid secretion back to normal. The present experiment was designed in order to demonstrate such a compensatory increase in ACTH secretion in terms of plasma concentration of the hormone. 2. Materials and methods Four lactating, non-pregnant dairy cows (three Ayrshire, ArTis, Abigail, Chrystal; one Holstein, Nemesis) were used in this study. Blood samples were collected by jugular venipuncture shortly after the animals' arrival in the barn for afternoon milking. In addition, two of the cows were sampled at 0900 h on the day on which they exhibited standing heat. Sampling was complete within 1 minute of approaching the animal, the details of the blood handling being the same as those described in Experiment III A (p. 75). Plasma glucocorticoids were measured using the competitive protein binding assay described previously (p. 54). Radioimmunoassay (p. 71) was used to determine plasma ACTH. The estrogenic phase of the estrous cycle is defined as the period of increased circulating estrogen levels. This was found previously to occur on days 19 and 20 and on the day of estrus in a standardized 21-day 86. cycle as defined by Garverick et al_.,. (1971). The luteal phase of the cycle refers to the non-estrogenic period (days 1 through 18). Student's t-test was used to compare mean hormone levels between the two phases. 3. Results and discussion The results of this experiment are illustrated in Figure 13 and Table 8. Glucocorticoid concentrations did not vary significantly between the estrogenic and luteal phases of the estrous cycle. Plasma levels varied between 4.2 and 20.2 ng/ml, exhibiting low level fluctuations sim-ilar to those seen in Experiment II A (p. 56) and to those measured by Garverick et al_., (1971). Such fluctuations can be considered basal vari-ation. The mean plasma ACTH concentration during the luteal phase of the estrous cycle was 730 pg/ml. If luteal phase concentrations can be con-sidered basal, ACTH levels found during this period are considerably higher than those considered basal in man (Landon and Greenwood, 1968; Berson and Yalow, 1968), in the rat (Matsuyama et_ al_., 1971; Rees et al_., 1971 hand in the pig (Donald e_t al_., 1968). However, the validity of assuming luteal phase levels to represent a basal condition may be questionable since the cows used in this study were lactating heavily and were feeding at the time of sampling. In addition, these somewhat elevated levels may reflect upon other aspects of adrenocortical function unique to ruminants in general and to dairy cattle in particular as discussed previously (p. 62). Lindner, for example, (1964) found low plasma corticosteroid con-centrations in ruminants together with low levels of plasma binding of these steroids. Furthermore, the relatively small adrenal size in female cattle (Christian, 1953; Swett e_t al_., 1937) and the refractory nature of adrenal response to exogenous ACTH stimulation (p. 57) have been noted. to o o lei CO o o h-< z o o c C O R T I C O S T E R O I D S 2 0 H - r -14 18 i — r E 2 6 DAY 3 0 0 0 2 0 0 0 H 1 0 0 0 A C T H i 14 18 E i 2 1 — 6 DAY Fig. 13. ACTH and corticosteroid concentrations in plasma during the estrous cycle. Day E is the day on which animals exhibited standing heat. Animals are: • Arlis, o Nemesis, A Abigail, A Chrystal. 88. Table 8. Plasma ACTH and corticosteroid concentrations in 4 cows during the estrogenic and luteal phases of the estrous cycle. The mean ACTH concentrations are significantly different between phases (p<0.01). Plasma concentrations (mean ± sd) Luteal Estrogenic Hormone Phase Phase ACTH (pg/ml) 730 ±310 1630 ±490 Corticosteroids (ng/ml) 10.4 ± 3.9 13.3 ± 5.4 No. of Observations 17 13 89. Plasma ACTH concentrations rose significantly (p<0.Ol) to a mean of 1630 pg/ml during the estrogenic phase of the estrous cycle. The observa-tion that there was no increase in glucocorticoid concentration concomitant with the rise in ACTH supports the hypothesis of adrenal inhibition by estrogen (p. 51). Without such inhibition, an increase in ACTH should be reflected in a similar elevation of glucocorticoid concentration unless increased glucocorticoid excretion also occurred. There is no evidence however, which would support the latter possibility. 4. Conclusions A significant increase in plasma ACTH concentration was noted dur-ing the estrogenic phase of the estrous cycle. No such increase was seen with respect to the plasma glucocorticoid level implying inhibition of adrenal response to ACTH by estrogen. 90. GENERAL SUMMARY AND CONCLUSIONS Carbohydrate metabolism in the lactating dairy cow involves a pre-carious balance between precursor supply and glucose demand. During early lactation, when milk production is at its peak, the balance is strongly negative, the animal's feed intake being less than that required to main-tain its lactational output. At this time, mobilization of body reserves of protein and lipid must play a major role in satisfying the substrate requirements of the heavily lactating cow. In addition, during early lac-tation, the cow is beginning to demonstrate regular estrous cycles. This, then, is a period of profound demand for substrate for gluconeogenesis, together with renewed cyclic fluctuations in hormone levels. The initial objective of this study was to evaluate the influence of estrogens on plasma metabolites involved in glucose production from tissue reserves. Measurement of plasma estrogens during the estrous cycle of the cow indicated a major peak which appeared on the two days prior to and on the day of standing heat (Fig. 1, p. 39). Plasma amino nitrogen exhibited a significant fall during this period (Fig. 2, p. 46) after which a marked hypoglycemia was also seen (Table 3, p. 47). These results indi-cated that the estrogens had a negative influence on the availability of amino acids for glucose synthesis. Two possible mechanisms were consid-ered: the first, that estrogen-mediated^uptake of amino acids by the uterus and mammary gland could result in direct competition for substrate; the second, that estrogen could limit endogenous substrate mobilization by way of inhibition of adrenal glucocorticoid secretion. The latter possi-bility became the,subject of the remainder of this investigation. Three lactating dairy cows were subjected to stimulation with incre-mental doses of ACTH with or without prior treatment with estrogen. 91. Responses, measured in terms of plasma glucocorticoid concentrations, were significantly depressed following estrogen treatment (Fig. 5, p. 61). In a later experiment, a radioimmunoassay for ACTH was developed in order that both glucocorticoids and ACTH could be measured during the estrous cycle of the lactating cow. The results^of such measurements on four cows demonstrated a significant increase in plasma ACTH during the estrogenic phase as compared to the luteal phase of the cycle (Fig. 13, p. 87). Glucocorticoid concentrations were, however, not significantly altered, fluctuating in the normal basal range throughout the sampling period. High ACTH concentrations during the estrogenic phase of the estrous cycle likely compensated for inhibition of glucocorticoid secre-tion by the estrogens. It seems probable that the changes in plasma substrate concentrations noted earlier are the result of an interacting sequence of endocrine alter-ations. Estrogens inhibit glucocorticoid output by the adrenal cortex and the pituitary compensates with increased ACTH production, bringing the glucocorticoid level back to normal. Growth hormone secretion is also increased at this time (Koprowski and Tucker, 1973), provoked by a mechanism common to both ACTH and GH production by the pituitary. 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