EXGCRENE FANCREATEC ENZYME WNTHESES AND! SECREI’EON, AND ENTES‘FINAL FRGTEGLYTEC ACTNET‘Y AND STABILITY EN CALVES Thests ‘0? ”10 Degree of DB. D. MICHEGAN STATE UNWERSITY Albert D. L. Gerri“ 1966 THESIS LIBRAR" . Michigan Sta . University This is to certify that the thesis entitled Exocrine Pancreatic Enzyme Synthesis And Secretion, And Intestinal Proteolytic Activity And Stability In Calves presented by Albert D. L. Gorrill has been accepted towards fulfillment of the requirements for Ph.D. degree inDairy Nutrition // ' V‘Major professor ,V/ Date October 18, 1966 0-169 ,. u t..l|lllllul||. ABSTRACT EXOCRINE PANCREATIC ENZYME SYNTHESIS AND SECRETION, AND INTESTINAL PROTEOLYTIC ACTIVITY AND STABILITY IN CALVES by Albert Do L. Gorrill Tissue slices of bovine pancreases were incubated in Krebs media with and without metabolic inhibitors, acetylcholine, pancrea- zymin and supplementary amino acids. Release of amylase and protec- lytic enzymes into the incubation media was primarily due to passive discharge from the tissue, since this process was not inhibited by anaerobiosis or dinitrophenol, or stimulated by acetylcholine. Pancreozymin provided some stimulation of enzyme release. A small increase in total trypsin and chymotrypsin activity occurred during incubation of pancreatic tissue slices in Krebs media supplemented with amino acids. This increase in enzyme activity was inhibited by puromycin and potassium cyanide. Pancreatic duct reentrant cannulas were placed in six two-day- old calves. Volume, protein, trypsin, chymotrypsin and amylase secre- tion in pancreatic juice from.4 to 21 days of age were several fold lower from three calves fed a high-soy (60% of total protein supplied by a 50% crude protein soybean flour) than from three fed an all-milk protein diet. Specific activities of trypsin and chymotrypsin were also reduced in pancreatic juice from calves fed the highpsoy diet. Volume secretion of pancreatic juice collected at h and 21 days of age was 0.16 and 0.29 ml/hr/kg body weight, respectively. Total output of Albert D. L. Gorrill protein, trypsin and chymotrypsin tended to be greater in juice collected 1 hr after feeding than in that collected before or 12 hours after feed- ing. Trypsin and chymotrypsin concentrations were highly correlated with protein concentration of pancreatic juice, but were not related to volume of juice secreted. Calves were fed whole milk, the allmmilk, highpsoy or promosoy (86% of protein supplied by a 72% protein soybean flour) diets and sacri- ficed after one, three or five to six weeks of feeding (average ages of 10, 27, and 41 days). The high-soy diet did not support body growth in calves. Calves fed the promosoy diet and whole milk grew at comparable rates. Total trypsin and chymotrypsin activities in the pancreas and intestinal contents, stability of these enzymes during in vitro incuba- tion of intestinal contents, and in vitro protein digested per unit of intestinal trypsin or chymotrypsin activity were less from.calves fed the highpsoy diet than those from calves fed the other diets. In vitro protein digestion in small intestinal contents from calves fed the high- soy diet was 0.06 g/2 hr, compared with 2.0 to 3.3 g/2 hr in contents from calves fed other diets. The highmsoy diet, and abomasal and intes- tinal contents from calves fed this diet contained high levels of soy- bean trypsin inhibitor (6.2 mg/g of diet vs. 0.15 mg/g in the promosoy diet). The relationships between the trypsin inhibitor and the physio- logical effects of the highesoy diet on calves are discussed. Pancreases from calves at 10 and #1 days of age were 0.5 and 0.7 g/kg body weight, reapectively. Total chymotrypsin and trypsin activities in the pancreas and small intestinal contents and in vitro intestinal protein digestion also increased during this age period. Concentrations of these enzymes in the pancreas did not change. Albert D. L. Gorrill Digesta from the middle third of the small intestine contained higher concentrations of protein, trypsin and chymotrypsin, and more protein was digested in vitro, than in contents from the upper and lower sections. Protein digestion per unit of trypsin and chymotrypSin activity was greatest in upper and least in lower intestinal contents. Protein digestion was closely related to the level of protein in con- tents from the different sections before incubation. Greater distruc- tion of chymotrypsin than trypsin occurred during incubation of intestinal contents. Chymotrypsin-to-trypSin ratios before and after incubation of contents were 0.30 and 0.19, respectively. Intestinal contents from calves fed diets containing soybean protein contained interfering substance(s) which produced an apparent esterase activity. Certain substance(s) were precipitated by calcium chloride in the esterase assays for trypsin and chymotrypsin, and increased the absorbancy changes. Most of these substance(s) were removed by treatment of digesta with ammonium sulfate, calcium chloride and adjustment to pH 8. The esterase enzyme activities of the result- ing supernatant fluid were also augmented by this treatment. EXOCRINE PANCREATIC ENZYME SYNTHESIS AND SECRETION, AND INTESTINAL PROTEOLYTIC ACTIVITY AND STABILITY IN CALVES By 3 .o\{\ t" SOV Albert De L. Gorrill A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1966 To My wife, Eva, and my parents who sacrificed so much that I might obtain a higher education. 11 AUTOBIOGRAPHY I was born in my parents' farm home on January 28, 1935 at Bulyea, Saskatchewan. Canada. Hy education begain in a one-room country school (Yunghill), and was continued at Bulyea Public and High School. I COEh pleted Grade 12 at Strasbourg, Saskatchewan. I enrolled at the Univer- sity of Saskatchewan, Saskatoon, in September. 195“ and obtained a Bachelor of Science in Agriculture (Animal Husbandry Option) degree in May, 1958. Two years later, the requirements for a Master of Science degree were completed, with research conducted in swine nutrition under Dr. J. M. Bell. Employment to 1957 consisted of helping my father operate a mixed farm (wheat, oats, barley, cattle). Summer and part-time winter employ- ment during 1957-1958 was performed for the Animal Husbandry Dept., Univ. of Saskatchewan. Beginning September. 1960, I was employed.by the Research Branch, Canada Dept. of Agriculture, Experimental Farm, Charlotte- town, Prince Edward Island. I was in charge of research related to Dairy cattle management, nutrition and genetics, and Poultry genetics. The Canada Dept. of Agriculture granted me Educational Leave to attend Michigan State University, beginning September, 1963. Upon completion of the Ph. D. requirements I will be returning to the Canada Dept. of Agriculture, Research Station, Fredericton, New Brunswick. Scholarships and prizes which I have received include: (1) Sas- katchewan Mutual Insurance Co., Entrance Scholarship (1954), (2) Bowman Bros. Prize in Field Husbandry (1956), (3) Pioneer Grain Co. Scholarship (1956), (0) National Research Council of Canada Bursary (1958-59). and iii (5) National Research Council of Canada Special Scholarship (1960-66). At the present time I am a member of the following professional organi- zations: (1) Agricultural Institute of Canada, and Prince Edward Island Institute of Agrologists, (2) Canadian Society of Animal Production, (3) Nutrition Society of Canada, (A) American Society of Animal Science, (5) American Dairy Science Assoc. (student member), and (6) Sigma II. I married Eva Mae.Kerr frommStrasbourg, Saskatchewan, on July 19, 1957 and have two daughters, Glenda, age 8 and Gayle, age 6. iv ACKNOWLEDGMENTS The author wishes to express sincere appreciation to Dr. J. W. Thomas for guidance in conducting the research, and for the construc- tive criticism in the preparation of this thesis. Suggestions and advice from Dr. R. S. Emery, Dr. H. P. Hafs, Dr. E. P. Reineke (Physi- ology) and Dr. H. Lillevik (Biochemistry) regarding experimental techniques and reading this manuscript are gratefully acknowledged. Collection of data on rate of pancreatic secretion would not have been possible without the cooperation of Dr. W. E. Stewart, University of Maryland, in surgically placing pancreatic duct cannulas in the calves and collection of pancreatic juice. Amylase determinations of the pancreatic juice were carried out by Dr. J. L. Morrill, Kansas State University. Technical assistance was rendered by the following: Judy Olney, Dave Schingoethe, Mike HcGilliard, Bruce Benjamin and Ron Dubouy. Feeding and management of calves by the Dairy Barn staff and use of facilities of the Meats Laboratory and Animal Pathology Laboratory are appreciated. Excellent cOOperation was obtained from Van Alstine's abbatoir in supplying bovine pancreases. Appreciation is extended to the Research Branch, Canada Department of Agriculture, in granting Educational Leave to attend Michigan State University. The author is grateful to the following for financial assistance: Dr. C. A. Lassiter for an assistantship (Sept.. 1963 - April, 1964), the National Research Council of Canada for a Special Scholarship (April, 1961!, - December, 1966), and to the College of Agriculture for a Fellowship in 1964 and 1965. This thesis would not have been possible without the moral support, technical assistance, and preliminary typing by ny wife. V TABLE OF CONTENTS LIST OF Tm I O O O O C O O 0 LIST OF FIGUE O O O O O O C 0 APPENDIX TABLES . . . . . . . . ”PmDIx FIGURE 0 O O O O O O O I C IMWWCTION O O O O O O O 0 II. LITERATURE REVIEW 1. 2. 3. Physiological control of pancreatic enzyme and secretion A. B. C. D. E. Neural control . . . Hormonal control . . (i) Gastrin . . . (ii) Secretin . . . (iii) Pancreoeymin . (iv) Anterior pituitary, thym hoanSeeeoeeee (v) Insulin Bill-90000000000000 Dietary conStituents a e e e e Neural-hormonal relationships id and Pancreatic enayme synthesis and secretion A. B. C. In vivo results synthesis 0 O Q 0 0 OOOOOLOOOO ad mn O O O (i) Collection and flow of pancreatic juice . (ii) Incorporation of labelled amino acids into pancreatic juice proteins . . . . . . In vitro pancreatic tissue slices Relative rates and ratios of enzyme synthesis and secretion Age and dietary factors affecting pancreatic enzyme prOduC'tioneeeeeoeeoeeeeeeeeeoe A. B. Prenatal development c e o e e o a o e e e e e Postnatal development (11) Hormonal induCtion o e a o e a e e o e e (1) Relative activities in pancreas with age . (iii) Dietary control and enzyme adaptation . Pancreatic enzymes in intestinal contents A. B. Assay and maintenance of activity Relative activity and stability vi rE xii xiii xii h‘hlhi h*k* NNN Hoxooooo O‘nan’tUIUU h>hs 0CD N N 25 26 26 27 28 30 31 III. 50 Soybemsmdmimalgromheeeeeeeeeeeeeoa A. Raw soybeans and growth inhibitors . . . . . . . . . B. Heated soybean meal and isolated soy proteins . . . 6. Pancreatic proteolytic enzyme assays . . . . . . . . . . A. Release of zymogen granules from.pancreatic 12188110..................... Be Activation Of trypsinogen o o e e e e e e a (i) Enterepeptidase (enterokinase) activation (11) Autocatalytic activation with trypsin . . (iii) Miscellaneous activators . . . . . . . . . Ca ACtiVCtion Of chymotryPSinogen e e e e e e e e e D. Activation of pancreatic homogenates pmcm‘tic‘juiceaeeeeeeeoe EeEnzymeassayseeeooeeeeee (1) Total proteolytic activity . . (ii) Esterase activity . . . . . . F. Inhibitors of trypsin and chymotrypsin is. O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O C O O O O O O O 0 MATERIALS AND METHODS Experiment 1. Experiment 2. Experiment 3. Experiment h. RESULTS Experiment 1. Experiment 2. Engmnt 3 0 Factors affecting in vitro synthesis and secretion of digestive enzymes from pancreatic tissue slices . . . . . . . . . . (1) Tissue preparation and homogenization . (ii) In vitro synthesis and secretion from tiSSHOSIiceS..o........o. (iii)EnzymeaSSay8............. Exocrine pancreatic secretion by calves with reentrant pancreatic duct cannulas . . . . . Effect of soybean and milk protein on growth, pancreas size and enzyme activity, and proteolytic enzyme activity of intestinal contentseeeeeeeeeeeeeeeeeo Interfering substance(s) in intestinal contents . . . . . . . . . . . . . . . . . . Factors affecting in vitro synthesis and secretion of enzymes from.pancreatic tissue 311005 O O O O O O O O O O O O O O O O O O O Exocrine pancreatic secretion by calves with reentrant pancreatic duct cannulas . . . . . Effect of soybean and milk protein on growth, pancreas size and enryme activity, and proteolytic enzyme activity of intestinal contents.................. vii 53 62 67 72 VI. VII. VIII. Experiment 4. Interfering substance(s) in intestinal contentSeeeeeaeeeeeeeee DISCUSSION 1. In vitro synthesis and secretion of enzymes from pancreatic tissue slices . . . . . . . . . . . . . 2. Milk protein vs. soybean protein for calves . . . 3. Size and trypsin and chymotrypsin activity of calf pancreases, and intestinal proteolytic enzymes as influencedbyago................ 4. Physical. chemical and enzymic properties of calf intestinal contents, pancreas - intestinal inter- relationships, and apparent esterase activity . . INTEGRATED DISCUSSION AND CONCLUSIONS . . . . . . . . WEOOOOOOOOOOOOOOOOCOOOCO APPEN'DIX O O O O O O C O O O O O O O O O O O O O O O 0 viii 91 102 105 111 11h 121 126 1&8 LIST OF‘TABLES Table gang; 1. ”Normal" flow rates of pancreatic juice . . . . . . . . . 15 2. Protein secretion by the pancreas of different species . 17 3. Pancreatic secretion response to feeding of dogs, edvas md Sheep 0 O O O O O O O O O O O O O O O O O O O 19 4. Enzyme composition of pancreatic juice proteins . . . . . 23 5. Ratio of chymotrypsinogen to trypsinogen in pancreas mdpmchCticjuicoeeeeeeeeeeeeeeeeee 26 6. Hormonal induction of rat pancreatic enzymes . . . . . . 28 7e KNbSbic‘rbon‘tOMdiaeeaeeeeeeeeeeeeee “6 8. Composition of all-milk and high-soy milk replacer diOts e e e e e e e e e e a e e e e e e e e e e e e e e a 52 9 0 D08 ign or c .11. Oxpa rimnt 3 e e e e e e e e e e e e e e e 5“" 10. Composition of promosoy milk replacer . . . . . . . . . . 55 11. Amylase activity in bovine pancreatic tissue slices before incubation andixxtissue slices and incubation media after in vitro incubation in the presence or absence of oxygen, dinitrophenol, acetylcholine and atropine........................ 63 12. Comparison of trypsin and chymotrypsin activity from incubated and non-incubated calf pancreatic tissue slices in homogenates and supernatant fluids when homogenized in saline or saline plus Triton X-100 . . . . 65 13. Volume, protein and enzymes secreted by calf'pancreases “ban f“ two different diets O O O O O O O O O O O O O O 68 1#. Amylase concentration in pancreatic juice of calves as influenced by diet, age and time after feeding . . . . 69 15. Volume, protein and enzyme secretion by the calf pancreas as influencedby age and diet . . . . . . . . . 70 16. Feeding response on volume, protein and enzyme secre- tionbythOClepmchCSeeeeeeeeeeeeeeee 71 Igblg 17. 18. 19. 20. 21. 22. 23a 2“. 25. 26. Relationship between pancreatic protein and enzyme secretion....................... Growth, pH of abomasal contents, and size and enzyme activity of pancreases of calves fed four different diets and sacrificed after one, three and five to six weeks on the respective diet . . . . . . . . . . . . . Volume, pH, protein, and concentration and in vitro stability of trypsin and chymotrypsin of contents from the upper, middle and lower third of the small intestine of calves fed four diets and sacrificed after one, three and five to six weeks of feeding . . . Protein content and in vitro digestion, and trypsin and chymotrypsin activities before and after incuba- tion of digesta from the upper, middle and lower sections of the small intestine of calves as influenced by diet 0 O O O O O O O O O O O O O O O O O O O O C O O In vitro digestion of intestinal protein, and the relationship to the trypsin and cbymotrypsin activ- ities of intestinal contents, during a two hour incubation as influenced by contents from three sections of the small intestine from calves fed four different diets and sacrificed after one, three and five to six weeks on the respective diet . . . . . . . Trypsin inhibitor activity in three milk substitute diets, and in abomasal and intestinal contents from CdVOSdethOSOdietseeeeeeeeeeeeeeee Measurement of interfering substances in calf intes- tinal contents as affected by methanol concentration in the chymotrypsin asszy and treatment of contents with calcium chloride in the presence or absence of mnium sulf‘tQ md pH Idju3t0d to 8 e e e e e e e e e Trypsin and chymotrypsin activity of intestinal contents from a mature bovine as influenced by acidification to pH b and dialysis of the acid superb natant fluid against 0.001N HCl for 12 hours . . . . . Recovery of trypsin and chymotrypsin from the super- natant fluid of rat intestinal contents acidified to pH 1+ or adjusted to pH 8 and excess CaClz added . . . . Trypsin and cbymotrypsin activity in the supernatant fluid of calf intestinal contents from the upper, middle and lower third of the small intestine before and after the addition of ammonium.sulfate, and simultaneous addition of excess calcium chloride and .djuStmnttOPHBeeeeeeeeeeeeeeeeee 73 74 77 79 86 89 93 98 100 Table 27. 28. Proportion of dietary protein digested during incubation of intestinal contents from calves fed four different diets and killed after one or five tosnweeksoffeedingeeeeeeeeeeeeeeeee 107 Comparisons of dietary protein intake and total protein output in pancreatic juice collected from calves at different ages and fed two different diets . . 113 LIST OF FIGURES 1. Effect of pancreatic juice protein concentration on activation of trypsinogen by enteropeptidase at 37 C . . . “9 2. Effect of pancreatic juice protein concentration on activation of cbymotrypsinOgen by enterOpeptidase ‘t 37C 0 O O O O O O O O O O O O O O O O O I O O O O O O 50 3. Inhibition of crystalline trypsin with purified soybeantryPSininhibitor...............o g h. In vitro secretion of proteolytic enzymes from bovine pancreatic tissue slices in the presence of pmcreozymnaeeeeeeeeeeeeeeeeeeeeee 6a 5. Trypsin asszy of intestinal contents from.h1-dzyaold C‘lvoseeaeeeee.eeeeeeeeeeeeeeeeee 81 6. Interference of esterase enzyme asszys by intestinal contents mdpurified bile 3‘1t3 e e e e e e e e e e e e e 92 7. Sephadex.G-25 column chromatography of pancreatic micemdNflSIYCOChOl‘teeeaaeaeeeeeeeeee 95 W 1. Effect of age on apparent in vitro synthesis of proteolytic enzymes by bovine pancreas tissue slices . . . 159 2. Proteolytic enzyme activity of bovine pancreas as “feet“ by ‘8. Ind length or fasting e e e e e e e e e e 160 xii Table I. VI. VII. VIII. IX. LIST OF APPENDIX TABLES Volume and protein concentration of pancreatic juice frOMC‘lveseeeeeeeeeeeeeeeeeeeeeee Enzyme activity of pancreatic juice from calves . . . . . Pancreas size, dry matter and enzyme content, and pH of abomasal contents from calves fed whole milk, all- milk' high-Soy md promosoy diets e e e e e e e e e e e e Volume, pH and protein concentration of small intes- tinal contents from calves fed whole milk, all-milk, high-soyandpromsoydietS............... Trypsin and chymotrypsin activities of intestinal contents before and after in vitro incubation from calves fed four different diets and sacrificed at three ‘888 O O O O O O O O O O O O O O O O O C O O O O O Trypsin and cbymotrypsin activity in the supernatant fluid of intestinal contents from calves fed the promosoy diet before and after treatment of the contents with ammonium sulfate, calcium chloride and adjustment to pH 8. O O O O O O O O O O O O O I O O O O O O O O O O O 0 Release of proteolytic enzymes from bovine pancreas tissue slices during in vitro incubation and total enzyme activity in tissue plus media as influenced by pmcreozymn C O O C O O O O O O O O O O O O O O O O O 0 Total proteolytic activity in bovine pancreas tissue slices and incubation media before and after in vitro incubation as influenced by amino acid supplementation . Total trypsin and chymotrypsin activity in the incuba- tion media and incubated pancreas tissue slices from calves fed four diets at three ages and the effect of mo ‘Cid supplamntition Of the ”(11‘ e e e e e e e e e xiii 148 149 150 151 153 155 157 157 158 I. INTRODUCTION The new-born calf has the ability to efficiently utilize dietary nutrients from the dam's milk. Due to economic considerations milk sub- stitutes (replacers) have been formulated containing dry milk and plant byqproducts supplemented with minerals, vitamins and antibiotics. Infe- rior growth and feed utilization by calves fed diets containing plant nutrients led to the hypothesis that certain digestive enzymes were not synthesized in sufficient quantities. Supplementation of vegetable-type udlk replacers with exogenous digestive enzymes have given negative results. The exocrine secretion by the pancreas plzys a major role in the digestion of carbohydrates, fats and proteins in the small intestine. Dietary effects on the digestive enzyme content of the pancreas per se have been reported on a limited number of animals. Such data, however, mzy have limited application in determining the rate at which pancreatic enzymes are secreted into the duodenum in response to chyme entering from the abomasum, as well as their activity and stability. Measurements of pancreatic juice flow rate in calves have recently been obtained at the University of Maryland, but the effects of preaweaning diets were not determined. The purpose of the research reported herein was to study age and dietary factors affecting in vitro and in vive Synthesis and secretion of pancreatic amylase and proteolytic enzymes. In vitro studies were carried out with pancreatic tissue slices, and were based on earlier reports with the pigeon and mouse pancreas. Reentrant pancreatic duct cannulas were utilized to follow the secretion of protein, and digestive enzymes as a function of age, time after feeding and diet. The trypsin and chymo- trypsin content of the pancreas and intestinal contents provided addi- tional indices of pancreatic enzyme production. The stability of intes- tinal trypsin and chymotrypsin, and proteolysis were asszyed under in vitro conditions. Dietary comparisons were based primarily on milk or soybeans as protein sources. II. LITERATURE REVIEW 1. szgiologiggl gontrgl of pancreatic enzyme gypthgsis and eggggtion The most extensive recent review of the exocrine pancreatic secre- tion was published by the Ciba Foundation (224). Grossman (85), MbClure (166) and Magee (171) have also made short reviews of this broad topic. Three phases of pancreatic exocrine secretion are generally recog- nized as due to cephalic, gastric and intestinal stimulation. The cephalic phase is considered of minor importance, although an zpparent conditioned reflex to the sight of food or anticipation of eating was reported to increase the flow rate of pancreatic juice in pigs (204). External stim- uli or sham feeding of dogs augmented volume and protein output by the pancreas (35, 211). Vagotomy or atropine inhibited the pancreatic response to sham feeding (149). A gastropancreatic reflex, involving increased volume and protein output by the dog pancreas due to stomach inflation (282), contradicted earlier reports indicating the absence of a gastric phase of pancreatic secretion (149). The above reflex was under neural rather than hormonal control and its absence in anesthetized dogs might explain many negative results in the literature (282). Hormonal control of pancreatic secretion by the stomach.will be discussed later. The intestinal phase of pancreatic secretion is by far the most important and the many factors involved will be discussed in detail. A. Ne o trol Vagal stimulation has invariably resulted in enhanced secretion of pancreatic enzymes (92. 96. 101. 176). Little or no increase in volume flow was found in dogs or cats (92, 101), but a marked increase 4 was noted in pigs (101, 176). Nervous control of pancreatic secretion has been ascribed to two mechanisms: (1) constriction or relaxation of the pancreatic ducts, and (2) secretory fibers to the pancreatic exocrine cells (140). The latter mechanism is particularly prominent in rabbits (9). Species differences to vagal stimulation have been related to the cholinesterase activity of pancreas tissue (38). Pig, horse, ass, and human pancreases contained numerous structures which stained for acetyl- cholinesterase and resembled small, compact ganglia. These structures were related to the profuse flow of pancreatic juice on vagal stimula- tion. Similar structures were absent or few in pancreas tissue from cats, dogs and sheep. ReSponse to vagal stimulation can be mimicked by parasympa- thomimetic drugs (9, 40, 58, 101, 162). Injection of pilocarpine or carbamylcholine in rats and mice resulted in a prolonged decrease in the total protein and enzyme content of the pancreas (40, 58). Incor- poration of Ola-amino acids into pancreatic protein was also enhanced (58). There were no changes in the nucleic acids, amino acids, pep- tides or total non-protein nitrogen (40). This response may be inhib- ited or diminished by atropine (84, 101), although no change was noted in rats (165). The synthetic anticholinergics, probantheline bromide (Probanthine) and iSOpropamid iodide (Darbid) more effectively blocked nerve conduction at parasympathetic endings than atropine (266). Elmslie et a1. (55) reported that the atropine inhibition of basal flow of pancreatic juice in humans (84) did not occur when gastric contents were completely eliminated from the duodenum by gastric aSpiration. Atropine might act indirectly to reduce the flow of gastric chyme enter- ing the duodenum. Species differences and the abundance of parasympa- thetic secretory fibers in the pancreas (38) may partially explain the contradictory results. Vagotomy has generally resulted in decreased enzyme output by the pancreas (98), but the response has varied within and between species (9, 96) and may be related to flow of gastric con- tents (55). In rats, vagotomy decreased the secretin and increased the pancreozymin content of the intestinal mucosa (52). Although vagotomy had no marked effect on pancreatic secretion in rabbits, section of the splanchnic nerves produced a marked and lasting suppression (9). The latter technique has produced an increase, decrease, or no consistent effect on pancreatic secretion in dogs (39, 96, 98), and may be related to tone of pancreatic blood vessels (96) or tone of ducts per se (140). In this respect, epinephrine decreased both volume and enzyme output in humans (149). B. Hormonal control (1) Gastrin Release of gastrin from denervated gastric pouches or injection of purified gastrin increased both the bicarbonate and protein output by the intact or denervated dog pancreas (209, 210, 213, 214). Neither histamine nor gastric distention stimulated pancreatic secretion. Enhanced protein secretion from the dog pancreas during sham feeding was attributed in part to vagal release of gastrin (211). (ii) Secretin Secretin injections (intravenous or intra-duodenal) have resulted in a profuse flow of pancreatic juice of low enzyme content (101, 126, 170, 276). The discovery of secretin by Bayliss and Starling (10) placed major emphasis on hormonal control of pancreatic secretion. Entrance of acid chyme into the duodenum was believed to cause release of secretin from the epithelial cells, which was absorbed into the blood and carried to the pancreas. Farrell and Ivy (59) employed pancreas transplants to demonstrate the humoral nature of secretin. Secretin has been found to be widely distributed in the small intestine: eXtending to the large intestine in goats , the first ten feet in pigs, and the first few inches in cats (183). Jorpes and coworkers (127, 189) have purified secretin and carried out partial amino acid analysis. Pancreatic enzyme secretion was not stimulated by purified secretin, when nervous stimulation was abolished (126). In vitro enzyme secretion was not stimulated by secretin as shown by enzyme assays (49) and histology of zymogen granules (94, 120, 190). Contamination of secretin with pan- creozymin could explain earlier reports of enhanced enzyme secretion. A log-dose relationship was reported between secretin and output of pancreatic juice in sheep (170). Oxygen uptake by pancreatic tissue slices but not other body tissues was enhanced by secretin (49, 67). Numerous studies have indicated that secretin was rapidly inactivated in the body. Administration into a peripheral vein rather than the portal vein resulted in a greater stimulatory effect on the pancreas (192, 250), whereas subcutaneous injection was without effect (213). An enzyme has been purified from dog“S liver which rapidly destroyed secretin (28). Secretin was active for a much longer interval when the pancreas was perfused without the liver (190). Destruction of secretin activity by in vitro incubation in dogs' blood has been attrib- uted to a heat labile enzyme called secretinase (81), and a similar ther- molabile component was prepared from urine (82). Direct inhibition of pancreatic secretion was ascribed to a second urine compound, called uropancreatone. (iii) Pancreogmg Pancreozymin has been shown to exert a potent stimulatory effect on pancreatic enzyme secretion with no appreciable change in volume out- put (91, 93, 126, 276). Harper and Raper (95) first described hormonal control of pancreatic enzyme secretion due to pancreozymin and its phys- iological properties were reviewed by Harper et al. (93). Although preparations practically devoid of secretin activity have been prepared, cholecystokinin has not been separated from pancreozymin (129), and in fact, one hormone may possess both properties (128). Extracts of the entire small intestine of dogs have contained pancreozymin activity, whereas only the upper half of the small intestine of cats and pigs con- tained this hormone (95). No pancreozymin response was noted from extracts of the stomach mucosa. A linear log-dose response to pancreo- zymin has been illustrated in dogs, even after acinar cells were prac- tically devoid of zymogen granules (162). This response was shown by Harper and Raper (95) to be independent of vagal stimulation and not altered by atropine or vagotomy. Large doses of pancreozymin markedly depleted the acinar cells of zymogen granules (94, 120, 190). Under in vitro conditions, the response of pancreatic tissue slices to pan- creozymin required oxygen (49), but was not altered by atropine (94). Pancreozymin was reported to stimulate parallel secretion of oc-anylase (O(-1, 4-glucan 4oglucanohydrolase, EC 3.2.1.1)(2a) and trypsinogen (95), and non-parallel secretion of trypsinogen and chymotrypsinogen, since trypsinogen secretion was enhanced to a much greater degree than chymo- trypsinogen (226). The activity of pancreozymin extracts has been maintained.for extended periods in the dry state (95). These extracts were also stable in hydrochloric acid, trypsin (EC 3.4.4.4), or boiling water for 30 min- utes, but were destroyed by pancreatic juice activated with enteropep- tidase (EC 3.4.4.8, enterokinase). The relatively short response to pancreozymin in vivo may be due to deactivation or metabolism.by the liver or blood serum (29, 80, 93, 190), or destruction in the intestine by activated pancreatic juice. The physiological control of pancreo- zymin activity in the pancreas per so may be related to a trypsin-like enzyme, amidase (acylamide amidohydrolase, EC 3.5.1.4), produced in the pancreas (33). Nevertheless, pancreozymin activity has been detected in an active form in urine, and called uropancreozymin (262). (iv) Antgrior pituitaryI thyroid and adrenal hormones Hypophysectomy or thyroidectomy have resulted in both reduced pancreas weight and enzyme concentration (5, 21, 195, 245, 271). Nishikawara et al. (195) reported complete restoration of pancreas weight per unit body weight by administering a mixture of adrenocor- ticotrophic hormone (ACTH) and thyroid extract to hypophysectomized rats. Cortisone, corticosterone, thyroxine and somatotrophin have also been partially effective (4, 195). Sesso et al. (246) suggested differ- ent modes of action for thyroxine and cortisone. During short term trials (6 days) thyroxine caused a marked increase in the flow of panw creatic juice and enzyme secretion, and depletion of acinar cell zymogen granules. Cortisone exerted little effect on flow of pancreatic juice or enzyme secretion, but a marked accumulation of enzymes and particu- larly amylase in the zymogen granules was observed. (v) Insulin Subcutaneous injections of insulin have been reported to increase both the concentration and total amylase content of the pancreas (14, 149), although the opposite effect was produced by injecting two units protamine zinc insulin per day to rats (87). Alloxan injections to rats (180 mg/kg body weight/day) for 7 days markedly lowered the amylase con- tent of the pancreas, but ii;'was restored to normal by‘5 or 10 insulin units subcutaneously for 3 to 7 days (14). Blood glucose levels as well as pancreatic chymotrypsinogen varied in the opposite direction. Reten- tion of enzymes in the pancreatic zymogen granules by the action of insulin was also suggested by Sergeyeva (242). The effectiveness of hormone therapy in restoring the pancreatic enzymes of hypophysecto- mized animals was enhanced by insulin (4). These results in conjunc- tion with pancreatic adaptation due to diet indicate that amylase secretion may be controlled directly or indirectly by either the blood glucose levels or insulin output by the pancreas. A high blood glucose level would therefore decrease insulin output by the pancreas and in turn increase amylase secretion or reduce the amylase content of the pancreas. C. [Bilg Ox bile placed in the duodenum of cats was reported to markedly stimulate secretion of pancreatic juice low in enzymes (149, 183). The response was nearly equal to maximum doses of secretin. Purified bile salts gate a less prolonged pancreatic response, while bile pigments were ineffective (183). The longer lasting effect of ex bile was attributed to the mucin which delayed absorption of the bile salts. Bellanby (183) postulated that bile salts acted by releasing secretin and the duration of the bile salt effect was determined by the distri- bution of secretin in the intestine. A variable pancreatic response to bile was reported by Thomas and Crider (268). Gall bladder contraction has been recorded in response to inges- tion of dietary nutrients, resulting in a high concentration of bile acids in the upper small intestine (284). However, dietary nutrients or hydrochloric acid have provoked a pancreatic response following 1O ligation of the bile duct (96, 122, 123, 269). Ivy (122) concluded that bile was only an adjuvant to pancreatic secretion, and.not an essential stimulating agent. D. Dietgry constituents The ability of dietary nutrients to ellicit pancreatic exocrine secretion has been reviewed by McClure (166). A secretin response was simulated by the presence of acid in the duodenum (123, 149, 242, 266, 269, 275), and was directly related to the amount of acid introduced (212). A close correlation was observed between the buffering capacity of a solution and the pH at which it began to stimulate pancreatic secretion (pH threshold) (26?). The pH threshold of HCl was less than 3: lactate, acetate and glutamate were between 4.5 and 5, and gastric chyme was estimated to be between 4 and 5. Infusion of HCl into the duodenum of sheep increased both volume and amylase output by the pan- creas (170). ‘ Fats and soaps or soap extracts of intestinal mucosa placed in the duodenum of cats and dogs produced potent pancreatic stimulation of both volume and enzyme secretion (63, 166, 175). The slow’prolonged response (275) was attributed to a secondary flow of bile which in turn stimulated the pancreas (122). The failure of olive oil or oleic acid introduced into the sheep duodenum to motivate pancreatic secretion (170) may reflect adaptation to lower levels of dietary fat normally fed to ruminants vs. cats and dogs. A marked pancreozymin—type pancreatic response has been produced by products of protein digestion, including peptones and amino acids in the duodenum (219, 266, 269, 275). Single amino acids including leucine, tryptophan, and phenylalanine exerted an effect similar to pePtODOG (275)- 11 Isoleucine, threonine and lysine increased amylase but not protease secre- tion, and methionine produced the opposite response (175). Meat placed directly into the duodenum of cats or dogs gave no pancreatic response (149). indicating the need for a certain degree of gastric digestion. Injection of a casein hydrolysate solution into the duodenum of sheep increased both volume and amylase output, while a peptone solution gave no response (170). Dialyzed sheep abomasal contents were also a more effective stimulant of amylase secretion by the sheep pancreas than whole abomasal contents. Intraduodenal injections of carbohydrates have generally exhib- ited little or no excitatory influence on pancreatic secretion (59, 149, 270, 275). Carbohydrates and secretin may act synergistically to enhance pancreatic nitrogen output (270). Harper and'Vass (96) observed a paral- lel rise in amylase and trypsinogen secretion by either feeding a casein or starch meal. or placing them.directly into the duodenum. Since saline and water elicited a similar response, the carbohydrate effect may not have been a true response. With the exception of data reported by Harper and'Vass (96), saline or water injections into the duodenum have exerted little or no exocrine pancreatic response (123, 269, 275). 'Wang and Grossman (275) concluded that water was not a strong stinmlus for the release of secretin or pancreozymin. E. Ne -ho n l t one i s The relative importance of neural and hormonal control of pancre- atic secretion was reviewed by Grossman (84). The results of Hang and Grossman (275) suggested a dominant role of gastrointestinal hormones in the regulation of pancreatic activity in normal digestion. However, 12 several instances of a reduced response to these hormones following vagotony or atropine-like drugs (52, 85. 93. 173, 266) indicate that both routes of pancreatic stimulation are operative. Similarly, a synergistic action between the gastrointestinal hormones and parasym- pathetic stimulation have been reported (161, 176). Magee and‘Hhite (176) noted that vagal stimulation enhanced the sensitivity of the pig pancreas to secretin. Reports of non-synergistic action between neural and hormonal stimuli (91, 162), coupled.with Hickson's conclu— sion that vagal nerves played a greater role in pancreatic secretion in pigs than in dogs (101), imply important species differences. 2. Pingrggtig enzymg synthesis 32d secrgtion A. In viyo results (1) Co e tion d ow re ti McCormick (167) and Thomas (265) reviewed surgical techniques to collect pancreatic juice. Gastric fistulas as well as pancreatic cannulas have been employed in some cases (170), the former being used to prevent chyme from.entering the duodenum as well as for placing solutions into the duodenum. McCormick (167) made continuous record» ings of pancreatic juice flow rates in calves by allowing the juice to pass through an electronic dropcounter and thence back into the duode- num. Pekas (201) reported that reentrant cannulas in pigs necessi- tated a oneawzy valve to prevent peristaltic pressures in the duodenum from reversing the flow of secretions and ingesta into the pancreas. This was not a major problem.in calves (167). Magee and Hong (175) measured the total daily output of pancreatic juice from.dogs by collec- tion in balloons containing glycerol. Butler et al. (32) experienced 13 plugging of the cannulas in bovine species when the flow was slow and viscous. Intravenous infusions of Ringer's solution decreased the vis- cosity of juice with no apparent effect on the enzyme concentration. A disadvantage to non-reentrant pancreatic cannulas has been the loss of pancreatic juice from.the duodenum. Exclusion of the pan- creatic juice from the duodenum had only a moderate effect on the pH of the intestinal contents (3, 103, 206). Taylor (264) however, noted that the amylase content of sheep pancreatic juice always fell 30-60$ below the initial value 5 to 8 hours after starting a collection. This depression in amylase secretion was prevented by simultaneous infusion of pancreatic juice into the duodenum. Flow of pancreatic juice in calves was not adversely affected by exclusion of pancreatic juice from entering the duodenum (167). Under similar conditions, an increased rate of pancreatic secretion has been reported in dogs (3, see Table 1). The ability of the bovine to withstand a continuous loss of pancreatic juice (79), with no ill effect on the animal, has been attributed to a secondary duct system (278), which hypertrophied and became functional following cannulation of the major duct (277). No accessory pancreatic duct was found in swine and this animal succumbed to total collection of pancreatic juice, unless at least a portion of the juice was returned to the duodenum (279). The discovery of a secondary pancreatic duct in the bovine complicates attempts to correlate flow of pancreatic juice with age of calves, as reported by McCormick (167). Pekas (204) noted that no pancreatic secretions were obtained from.pigs for two or three days after surgery and some failed to flow at all. Due to the operating procedure, it was concluded that pancre- atic juice could not be procured from pigs before four weeks of age. 1!. Similar difficulties have not been encountered with dairy calves when operated on as early as three days of age (167). Herbivores and most common laboratory animals, other than cats and dogs, have been shown to secrete pancreatic juice continuously (102, 171). Spontaneous pancreatic secretion in rabbits, devoid of neural or hormonal stimulation, was revealed by Baxter (8). The exocrine pancreatic secretion rate under 'normal' and fasting condi- tions, as reported in numerous articles, has varied widely both within and between species and are summarized in Table 1. Pekas et al. (202) noted that the flow rate from the cannulated pig pancreas was highly irregular and unpredictable. A comparison of published data was made difficult by the wide diversity of units employed to express pancreatic flow rates. However in Table 1, the secretion rates were corrected for differences in body size where feasible. The resulting average flow rates of pancreatic juice from dogs, bovine and sheep were 0.63, 0.52, and 0.39 mllhr/kg, respectively. Body weights were not reported for Ithe swine data. McCormick (167) concluded that pancreatic secretion rate in man was about equal to the dog, but had a wider range. Further research would be necessary to prove or disprove the apparent disparity in flow rates per unit of body weight noted above between dags and rumi- nants. The average protein content of pancreatic juice from the litera- ture reviewed was 16.0 mg/ml (Table 2). However, the reported values ranged from 0.1 to 1&5 m/ml. No apparent species differences were evi- dent from.the limited data available. Protein content and enzyme activity of pancreatic juice are closely related. Digestive enzymes have comprised 80% of the total I 'F‘v I a... as F .O"»H°-n-ah La.» lel... 'u. and-e I.‘ e. Q‘I'Qeevl a eZea 15 Gm not... 30-8 s 8.“ I .2 3295.. 8 I pod ¢3_heon B so»? No ads. an. I 9:2 $3.. «.9 I cadences anenvneehloo: aw.hvon nod .858 3.. 3.3 N... I 9:3 in}... 3 I meanness aceupneeu as neon be 3. .3. 3 £25.. a on. I 3.5 3.2.. a I «2 I- 3:3. RINN .52.. RINN o8. «voted .3 o .8.“ on« who .ueevm mafianeeh In mm :Ha\da mm. at noauoeHHoo osoondunoo 2. s... m £8 tn N. I Rm .32: OS I onion em" I- I Sum” as. 33. m.~..~ I 36 huewusn henna an . cam 28.... .2... .3 .mé 0.8 8....8 aid. menus 33 33.3 S“ .5 mean .3 e33 ..I «a. It. I 9.3 $3.. file I N8 and .3 8 aw. a.“ no. 8:5 a; 8:3 «E 3 £9.88. BE: 2. I 8 as. 3:: c3: om I 9N and .3 8 I m 5a. m <1 N I an“ exepcd boom mapped N mw.«|n: m.ouuw.n M"e\as monies“ waned a o 00 o: o I . ll mm ps_mewmdww\cawuuuou mm mu m cam“ and mm mpg. «.52: .5 in R. 3 3 9.82:... TN 8-3 m 2.8%... 83. a I 0&4. .. 8.3. I monsoon boom. n e882 83. :1 I 3.0% .5 n 3.5 as 37% I lulu on s. .33 I! @b 3?: Saw a: saw. £333.: BE seam . ..eoaon.oaueehonea no eeaeu seam aHeaAoz: .« canes ! «Calla-a. ! .fialiene V 'Ib F Iihu a I. !~|-IIIIee‘a.‘ .\ a.‘ I'- I ‘1. ’aw i r‘ en h l'laeuh a,IZee l h I! !,.Q'.‘. 16 sow cannons queens» - «.3H can on\Hw Ha.m - :8 539a ads I in no. 83. mm... ooduenoeu ascenepsone «Hi\emohe «my son .eao nave msIns - n.an-o can ofi\uaoue omfi-o - now «was eao a: n-s - «H-o un\da Hfi-o -- .mmmm Nod grog. s «a up. -- c.5fi-5.HH as s~\da «ms-mam -- an an too new - codaoeHHoo oceanooo n.3n. II mx\ug\as 5.3m. :- ona aoaoaasaauu cabana.» cs. -- mx\:aa\aa coco. -- ona . case an maIsm «a. :.~H can nfl\da H.n co co” . an condo page. as. - mx\ng s~\He m.m - muumm 8“ 3. 8 e I 30 we: 3 I we“ was a: a - «.3 a«e\u H. -- .meeu one no when mm a no“ as com .uua an. .nda - eN-n.m un\as om-n.n - e no“ eHo nave owe-cod - ~.ss an sm\da owed - a no“ can «are nm-Hm - m.m an s~\da onm II x be“ was nave n-m - m e we s~\da mad -- nosaao ms «mflnmm Amie nudes .«wmu as eeamemm -3n2floammr eofiflam 8: goo-m- ..AeesnducooV sedan oaveeuenem,uo.eeaeu scan mHQBHoZa .« edges 17 protein in bovine pancreatic juice (13“). Volume of pancreatic secretion in dogs was correlated with total protease (r = 0.80) and amylase (r = 0.88) output (175). A gradual decline in the amlyase content of pancreatic juice from sheep and rats has been observed following cannulation of the pancreatic duct (#7, 170). In contrast, lipase (glycerol-ester hydrolase, EC 3.1.1.3) and chymotrypsinogen rose sharply in rat pancreatic juice. These changes might have been altered or prevented by simultaneous return of pancreatic juice to the duodenum (260). Table 2. Protein secretion by the pancreas of different species. Unitsgg Proficin conc. . Species Protgial .quporteg (calculated) Refgrenge mg ml Bovine: Yearling steer 1 g/hr -- 135 14.01 mg/ml 1fl-hi 135 500‘ mg/hr -- 136 “cl-10e3 138/1111 (bl-10.3 136 Mature cow 0.5-0.7 % protein 5—7 79 Shegp (adults) 3.0-4.5 % protein 30-05 102 223.8. 1~5 s/day 10 98 (150 ml juice) 25 /20 min. mil 7 ml juice) 10.7 282 126-206 mgN/3 hr (52-150 8.6.15.11 3 ml juice) 1Nitrogen x 6.25. The total exocrine volume and enzyme output by the pancreas in monogastric animals has been shown to increase soon after feeding (55, 18 166, 175, 203, 200, Table 3), in proportion to the amount of food ingested (175). Flow of pancreatic juice may cease completely in fasted dogs (206), although a low basal flow has generally been recorded under these condi- tions (213, 282, Table 1). No marked response in pancreatic juice flow in ruminants has been reported as a result of feeding, or fasting for 2h hr (102). A small transient increase in amylase output by sheep sometimes occurred after feeding (26“). In addition, volume and amylase content of pancreatic juice collected 20 hr after complete removal of contents and washing the rumen was reduced considerably (Table 3). Stimulation of pan- creatic secretion in calves has also been observed during feeding (Table 3), drinking and While ruminating (167, 168). Hay ellicited a greater pancre- atic response than concentrates. Decreased flow rate in calves approxi- mately two hours post-feeding (Table 3) was related to ingesta passing beyond the area of the cephalic stimuli. There was no apparent gastric phase of pancreatic secretion; the intestinal phase began 2 to 3 hr and reached a peak 6 to 9 hr post-feeding. The basis for differentiating between cephalic, gastric, and intestinal phases, however, was not described, and may in fact overlap. The results reported to date for both mature and immature ruminants attest to the need for further research to fully eluci- date the physiological control of pancreatic secretion. (ii) Inro tion 0 1 be mino a ds to The ability of the pancreas to rapidly synthesize large quantities of protein has been measured by the appearance of radioactive labelled amino acids in pancreatic juice protein. Intravenous injections of Gin-amino acids or SBS-methionine have resulted in secretion of labelled pancreatic juice proteins after 15 to 60 min (130, 136, 144). Maximum activity was attained in human pancreatic juice at 8 hr post-injection (10“), and in 19 Table 3. Pancreatic secretion response to feeding of dogs, calves and sheep. Volume ——'—fiiUnits Experimental S e es ut ut 0 ed c nditions Re e ce Q25; 0 --- fasting 206 25 ml/10 min (1 hr post-feeding 7 ml/10 min 3 hr post-feeding 3.1-1 m1/15 min fasting 212 20 ml/15 min 3 hr post-feeding 140-273 ml/day 450 gm feed/day 175 160-4032 m1/day 900 gm feed/day Cglvgs 9-11 ml/hr pro-feeding 167 mg. of 8) 1t- ml/hr 1 hr post-feeding 9 ml/hr 2 hr post-feeding 18 ml/hr 8 hr post-feeding 12 ml/hr 12 hr post-feeding 114 m1/4 hr pro-fasting 167 48 ml/4 hr 61 hr fast 142 m1/4 hr 24 hr post-feeding Sheep 0.3-0.7 ml/kg/hr normal feeding 264 0.05-0.153 ml/kg/hr 24 hr after removal of all rumen contents and washing 1Protein output increased from 90 to 500 mg/15 min. 2Protease and amylase also increased by 30-40%. 3Amylase reduced in same proportion. 20 bovine juice after 3 to 4 hr followed by an exponential decline (135). Incorporation of $35-methionine into human pancreatic juice protein was nine times greater than into serum.proteins (144). The calculated turn- over times and daily protein turnover fer pancreatic and serum.proteins were 4.7 and 11.2 days, and 5.1 and 14.1 g , respectively. Turnover times for trypsinogen, chymotrypsinogen, and ribonuclease (EC.2.7.7.16, EC 29292117) in the mature bovine have been estimated at 157, 153, and 127 min, respectively (135). High levels of S35-cystine have also been detected in the pancreas and intestinal contents of rats sacrificed after $35-methionine injection (7). Parasympathetic stimulation increased incorporation of Gib-amino acids into pancreatic proteins (58), suggesting a direct relationship between synthesis and secretion. B. In vitro pgpcregtig tissue sliggs Hokin (104) first reported in vitro synthesis of amylase using pigeon pancreatic tissue slices, and has subsequently reviewed the lit- erature (108, 109). Since in vitro synthesis was prevented by anaero- , biosis or metabolic inhibitors, the increase in amylase was considered de novo synthesis. However, the following alternatives to de novo syn- thesis have been suggested (18): activation of an inactive form of the enzyme, elimination of an inhibitor or formation of an activator, and the incorporation of labelled amino acids into total proteins or iso- lated enzymes may result from amino acid exchange in already formed molecules. Proof of de novo synthesis of cbymotrypsinogen by mouse pancreases in vitro was demonstrated by isotope dilution, and isolation of newly formed chymotrypsinogen (18). Chymotrypsinogen synthesis was equivalent to 3.3 mg/g dry tissue/hr, or 24,000 molecules/cell/sec. 21 Madman in vitro amylase synthesis by pigeon pancreatic tissue slices has been attained by supplementing a.Krebs media with either casein hydrolysate, ten essential amino acids or their ketonic acids (105, 109). Lipase and ribonuclease synthesis were not alwzys enhanced by amino acid supplementation (238). Haltose, soluble starch, triiodo- thyronine, testosterone, hydrocortisone, and several antibiotics had no stimulatory effect on amylase production by the pigeon pancreas (291). Several reports using mouse pancreases indicated a lower or negligible rate of in vitro amylase synthesis compared with pigeon pancreases, and with no stimulating effect by amino acid supplements (60, 111). Rychlik and coworkers (232, 233, 234) however, reported increased in vitro syn- thesis of protease and amylase in mouse pancreases by supplementing the media with casein hydrolysate, and a more marked stimulation by aSpara- gine, glutamine or their peptides. The latter reSponse was noted only after the tissue was preincubated to reduce the endogenous levels of these nutrients. Pancreatic homogenates or subcellular systems, except micro- somes, have resulted in little or no in vitro enzyme synthesis (200, 220, 263). The negative results have been ascribed to the high ribo- nuclease content of pancreatic tissue, but the real reason is not known (200). Secretion (active extrusion) of enzymes from.pancreatic tissue slices has been determined by the release of digestive enzymes into the incubation media (104). This process was stimulated by the presence of cholinergic drugs or pancreozymin in the media (43, 104, 107, 111, 112)- but not by secretin (107). Atropine blocked the effect of acetylcholine, but not that of pancreozymin (43, 107). In vitro secretion was inhibited 22 by lack of oxygen or metabolic poisons such as cyanide (104). Stimula- tion of in vitro secretion, or release of enzymes from tissue slices into the incubation media did not increase the rate of in vitro amylase synthesis by pigeon or mouse pancreases (108). Active extrusion of pan- creatic enzymes in vitro has been associated with an increased uptake of P32 into pancreatic phospholipids (110). However, since P32 incorpora- tion also occurred when secretion was blocked, this phenomenon may be involved in transmembrane transport of proteins across intracellular membranes to form zymogen granules, rather than the release of enzymes per se. Passive discharge of amylase from.pancreatic tissue slices has been observed under anaerobic conditions, and was attributed to loss of enzymes from cells damaged in the slicing technique (111, 112). Recently, however, enzyme leakage from.parotid gland slices was associated with repeated exposure to ice cold media during preparation and preincubation (237). Lipid components in the zymogen granules possibly "contract" in the cold to open gaps permitting leakage of amylase, and cast consider- able doubt on the usual practice of holding tissue in the cold prior to in vitro incubation. Fortunately, however, the addition of long-chain fatty acids, such as sodium oleate, prevented amylase leakage (237). C. Relgtive rates gag ratios of gnzymg synthesis 5nd secretign The proportions of exocrine enzymes produced by the pancreas tend to be species specific (Table 4), although age and dietary factors are also important (Section 3). Enzymes in pancreatic juice have been.quan- titated by ion-exchange chromatography and other characterization tech- niques (134, 178). Bovine pancreatic juice amylase and lipase represented less than 2% of the total juice protein, while proteolytic enzymes 23 .sowomdmnhnuoahzo masses menow .memosdmmhnp mHesuo was .Am.¢.«.m um .eueaoaemnoefipooaoomomaao eoeeaoesonaahxoeev eaeoHossonfiAmKoonm Amééa 8 32.369 ofiueH-q-Heeaeeofi m ogaeeoaenonho .3.~..~.n 8 .3352 Boa-ofifi-A-Heflsmoé < caedoeoafiofiaom .hmmeamopeaoago emcegoNeIsed An pageaomoa mmdeuoumfi and mum med 8N on and e newsman m.m n.m n.m seen» 23.3 on III III e.ma II n.md n.m« pom ma III III mm II n.ofi mm esdouom n.u III III II II ma 3N endouom III III III II s 0H II onwbom m.m III m mm m 3“ mm endpom NV e.a e.m om 0H ma 3H ocdbom Hw.. new so one mom: u ._ . "Hmnu. . e aunnnmmmmummw {an unlammmmmnmmummmunu 50.? one ewe P.3oseodwsfi as seasoned “Has. on... hp .3388» enthuse use 539a .0552? .3 0.33. 71 .033 :ofipoodmoe on 26 :35 heaven sesame ho. 93.53000 enema soot—poo. oesoaommflu a mo haaflfinonoum: .9 93:. .N 305.com comm .NH 93:. .e on. mm 3.238% oomN .3 0.3:. .H ovospoom coma 80 36 om.v S.v 30 i- 3.0 .2333 a 0. Mod newt om on «non; doomed flame. 3 m .an .m «was... mm o: How . a downs . m a .HS . o 55%.: owl-MN Nm 9: male; «#2.: 33. H ménfim an? a: 8 .nmoé nouns «H.143. o Afiiofisov Safiofiosv Anibal 2:35 “canine Awoaoooensooo 2: So a in a 532m one—Hos coaooofioo «.3085930 on» he oodaohooo (game one 538m 35—Hour so someones gosh .wu oases 72 The trypsin and chymotrypsin concentrations of pancreatic juice were highly correlated with protein content (r = 0.84 to 0.92, Table 17). The close relationship between protein and enzyme content, as shown previously by Keller et al. (134), has been used to estimate pancreatic enzyme secretion by assaying only for protein (98). Considerable error may arise, however, in estimating individual pancreatic enzymes, as illustrated in Table 17, due to dietary effects on the regression equations for trypsin and chymotrypsin. These equations reflect dietary effects on the specific activity of the enzymes presented in Table 13. Volume of pancreatic secretion was not correlated with protein, trypsin or chymotrypsin content of the pancreatic juice (r = -0.03 to -0.16).1 The ratios of chymotrypsin-to-trypsin activity in pancreatic juice from calves fed the milk and soy protein diets were 0.17 and 0.15, respec- tively (P3'.05), indicating a parallel reduction in secretion of both enzymes by soybean protein. Age of calves and collection time before and after feeding also exerted no change in chymotrypsin-to-trypsin ratios in the pancreatic juice. EIEEEEEEEEQl- JEffegt of soybegr 52d milk prgterr on growthI prrcregs si e d enz tivit and roteo ic z ' gctivitr of intestinal contents.1h The high—soy diet produced varying degrees of diarrhea in calves at all times, while the all-milk diet resulted in periodic diarrhea. Little or no scouring was evident in calves reared on whole milk or the promosoy diet. There was no curd formation in abomasal contents from calves fed the three milk replacer diets and sacrificed 1“Presented in part to the Canadian Society of Animal Production, Winnipeg, Manitoba, June, 1966 (72). 73 Table 17. Relationship between pancreatic protein and enzyme secretion. sin Allemilk protein 91-: 3.93 + 13.0 x2 (r = 0.84) High-soy protein 9 = .032 + 10.7 x (r = 0.91) C at sin All-milk protein 9: 0.59 + 2.20 x (r = 0.85) Highpsoy protein 9: -.025 + 1.55 x (r = 0.92) 19’: estimated enzyme level (units/ml) 2x = protein level (mg/ml) 1 to 1.5 hr post-feeding. Abomasal contents from calves fed whole milk were almost completely coagulated. The pH of abomasal contents from calves fed whole milk was lower (P< .0005) than that from calves fed the 3 milk substitutes (Table 18). Abomasal contents from calves fed the promosoy diet had the highest pH (6.17). The acidity of abomasal contents was the same in calves sacrificed after 1, 3, or 5-6 weeks on the respective diets. . The average ages of calves sacrificed after 1, 3 and 5-6 weeks on trial were 10, 27 and 41 days, respectively. Live body weights of calves after one week of feeding were generally less than the initial weight. The average daily gain of calves sacrificed after 3 and 5-6 weeks of feeding were 0.18 and 0.24 kg, reSpectively (Table 18). Growth rate tended to be negatively correlated with calf weights at 3-5 days of 7n .emwe ad mobfleo no owe omcaohdn .mo. VA on nodes ocean—eon» soosuob ecosystem ufioumgmde one .vsasueoup on one can» heaven eon-mo kn msaaaae oases scarves eocoaoumfie a no huaadoenoamw .Na edges .3 eposuoom oo .9. fi 232. seen no soothe .23... noes-a be oozes-o does no oofloflfis oofioa be 3822 nauseous... Shogun .NH ode-a .n evosuoou oomN 34v mood imam .Enmwwuww J 32% owe. new. mm.n can. Aaav as bun 33. .en. en.“ .2. :8 s. m to. new. 8% n8: n83 a: H afldololoflflfl e.n.z 86 no. v 86 mo.v 30.0 38. v 380 ones a a“. nmm n.emm. one: n.amm.: one. o.ama.w anm. hoeoaosm o.oom.m one.u nontnmem n.o$.n one. field one; doom. is. £25 43m a no so deflofl 3.3. 92 .936 seduced»: one. so eases fine 3 some Become» .oso 8%.,eooduauoeo one 3a.? esteemed much new ask—nae no oooeososen no enhance oflhwso use code one .oasopsoo Hones—one no we £9395 .3 Smash. 75 age (r = -0.20, P:>.1). Calves fed the highpsoy diet continued to lose body weight for the entire duration of the trial, and several died while on this regime. The all-milk replacer sustained relatively low weight gains (0.08 kg/day) compared with whole milk and promosoy. Pancreases from.ealves fed the promosoy diet represented a smaller fraction of the total body weight (0.h7 g/kg vs. 0.57 to 0.66 g/kg) than those from calves on the other 3 diets (P< .005, Table 18). The size of the pancreas glands per kg body weight were comparable when calves received whole milk. all-milk and high-soy diets. The concentration and total activity of trypsin and chymotrypsin in the pancreases of calves fed the highpsoy diet were less than those from the pancreases of calves fed whole milk (P‘<.05). Total pancreatic trypsin and chymotrypsin activity/kg body weight increased.with age for calves fed the milk protein diets, with an opposite trend for calves fed the highpsoy diet. Dietary treatment had no effect on the pancre- atic ratio of chymotrypsin-to-trypsin (Table 18). The value of 0.2 approximated that found in pancreatic juice (Experiment 2). Growth rate of calves fed for 3 or 5-6 weeks was not related to the total trypsin (r = 90.20) and chymotrypsin (r = 0.19) content of the pancreas. Pancreas size increased from 0.51 up to 0.66 g/kg body weight for calves 10 and 41 days of age, respectively (Table 18). Correspondp ing values in g/kg bodyweight'75 were 1.3 and 1.8. Promosoy-fed calves did not follow this age trend, and deletion of pancreas weights for these calves from the above calculations gave an average of 0.75 g/kg. or 109 8/k8.75 at #1 days of age. Concentration and total tryptic activity of the pancreas tended to increase with age relative to chymo- trypsin activity. This resulted in a reduction in the chymotrypsinpto- trypsin ratio from.0.24 at 10 days to 0.18 at #1 days of age (P.05). The influence of diet on the average pH of intestinal contents from all sections of the small intestine followed the same pattern as noted previously for abomasal contents. Intestinal contents from calves fed whole milk had the lowest pH (6.05) and those from calves fed the promosoy diet had the highest pH (6.87). Abomasal and upper intestinal contents from calves fed promosoy had the same pH (6.17 vs. 6.22). The pH of upper intestinal contents from calves fed whole milk, all-milk and high-soy diets were 1.1, 0.4, and 0.7 pH units higher, respectively than those for abomasal contents from calves fed these diets. The pH values of lower intestinal contents from calves fed all diets were above 7. Protein concentration of intestinal contents was not altered appreciably by diet or length of time calves were fed the diet. Values for calves fed whole milk, all-milk and high-soy replacers represent soluble protein remaining in the supernatant fluid following centri- fugation at 1300 g for 30 min, whereas data for promosoyhfed calves include the total soluble and insoluble protein. The centrifugal force applied to intestinal contents from calves fed whole milk sedimented only large feed particles, and almost the entire contents were retained as a semiliquid supernatant fluid. Contents from the upper and lower sections of the small intestine contained 20 and 50% less protein, respectively, than contents from the middle section. However. a signifi- cant interaction occurred between diets and intestinal sections (Table 20). 77 .m.z do.v .m.z nooo.v 0H.v. .m.z .m.z .mudv m .. ww. on. seem ms. «moo: 0.3“ on.o 0.3“ “flee xx cum -- mm. as. p..~na on. .emmw a.~. mm.e m... lame a. n a- we. on. .mes as. amass e.mfl ee.e ~.HH maofiv x: H . can» e se.. moo.v nooo.v mooo.v mmo.v mo.v .m.z nooo.v. w.m.z u.oeae a mm. mm. 0mm. n..NHe .ma. n..oeam 3.0H osm.e m.~e nauseous mo. em. new. seem new. news. a... .me.e o.m. a...sw.m mm. we. ems. e.em:w eww. n.eemm~ m.mfi ewo.o w.mfi xHHsaHH< ea. en. .03. ammo“ n..ms. .mmam H.0H ems.e m.o« gees oaon: . poem AH .32... an? 5.. .38. 8i» :H . 3.8. mm“. s an. s o .nnnnmmmmuummmummuunu _: _ saumumm .madeeeu,uo execs wde o» seen one eons» .eco hound eooamaaoeu one anode noon eon nebaeo mm endueepud Hanan on» no enanu aeseH on. canvas .uemm: env_aoam announce mo :«edhuucahne one ndedhfiv no haHHanepe ouvub ad one :ouveupneenoo use .cdepeum .mm .oisao>. .ma canes .ma canoe .u eponuoom oomw .wa canny .m oponpoom comm .ma edges .n opozuoom oemN .oddpoOpnd Hanan chance Beam avocados you hvfisavoe Heuop “NH canoe .3 eponpoom come .oudunouud HHQSu chance scum announce new Anabapoe Haven "NH canoe .m ovozpoom comm .ondvmeuqa Hausa chance onv.mo eacevcoo pom cadHo>d .oecsoamdqwan opened on sees no: one unsaved pmHueuuodsm .mcodpoon Hendpnopna one scavenge mcdooom .uodo one Avopensoca .n> mopenoocaonmv nanopcoo Hsnapmovca Mo pcoapaoup cesspon encapoehovca you madman .moadadn pepebsocdacoa mo mm mflwbduoe oshndo Henflmdno on» on 0 mm an a: N you movensocd mucopcoo Hecdamopca Ho hpfibdpoe eEhNCo no ofiaomm .mfl oan.a .H escapees come .m.z mmo.v .m.z moo.v mooo.v nooo.v .m.z .ooeo a nu mm. we. emmfi nu. same om.m eum.u she 9030A .. em. on. sewn on. omen“ om.mfi nwo.e mHN mavens 2. R. R. .3... a. .NR .3. .86 on tap eoo.neo so» nee oaaan a. pe>aoou naaaoas. possess. . seasons m ossao> -gH -seonm any.» cH Hesoa one.» aH fiasco can » can a wMIIImwmmHmmmmHMMIllll. mmnmumm Aeoanapooov .me oanuy 79 Table 20. Protein content and in vitro digestion, and trypsin and chymo- trypsin activities before and after incubation of digesta from the upper, middle and lower sections of the small intestine of calves as influenced by diet. Protein —lnvitro eniyme stability Pre-incubation In vitro Igtgzagtion centr ion i est 0 sin C sin (mg7ml) (mg7ml) Di t est nal section Whole milk - upper 22.7 7.80 .68 .36 - middle 19.6 7.75 .82 .uZ - lower 6.1 2.69 .70 .01 All-milk - upper 8.6 2.64 .83 .5“ - middle 15.5 5.65 .89 .53 - lower 12.7 1.69 .74 .28 High-soy - upper 11.9 0.35 .h8 .05 - middle 20.8 1.50 .67 0 - lower 11.7 1.33 .77 .66 Promosoy - upper 17.6 3.66 .91 .65 - middle 22.6 h.h2 .62 .65 - lower 9.0 2.h3 .81 .08 P aura <.025 <.05 <.025 (.0005 1See footnote 2, Table 19. 2See footnote 5. Table 18. 80 There was a smaller reduction in protein concentration in lower intes- tinal contents from calves fed the allomilk and highosoy diets, compared with those fed whole milk or promosoy (P‘<.025). The concentrations and in vitro stabilities of proteolytic enzymes in intestinal contents exhibited large individual animal varia- tions, tending to overshadow dietary and age effects. A number of calves were sacrificed during moderate or severe attacks of diarrhea. Intestinal contents from these animals contained very low trypsin and chymotrypsin activities and these data were eliminated from the statis- tical analyses. The total trypsin and chymotrypsin activities of intestinal contents from calves receiving the high-soy diet were lower than those from calves fed whole milk and the allomilk replacer (Table 19). Total enzyme activities of intestinal contents from calves fed the promosoy diet were not significantly greater (P3>.05) than those from calves fed the highusoy diet. Spectrophotometric assays for trypsin and chymotrypsin in intestinal contents from calves fed the high-soy diet were characterized by a marked curvilinear reaction rate (Fig. 5). The initial rapid increase in absorption was later shown to be due to a reaction with calcium ions in the Tris buffer to form a precipitate, which continued at a decreasing rate for the duration of the assay (5 to 6 min). Increases in absorbancy, due to precipitate formation. rather than due to hydrolysis of the synthetic substrate esters (TAME. BTEE) by trypsin and chymotrypsin were referred to as apparent esterase activity (73). The cause of the non-linear reaction rates for these enzyme assays of intestinal contents was not known when assays were performed on contents from calves receiving the high-soy diet. Although no correction for the interfering substance(s) of these data could be 81 .poae a...eman ecu se.. .uemaa noose xaaauaa. eon new. .neog .mopauo eaouaweuaa_aono usoooooo H.:ao..ooa no as... onuaaue .n .mfia Anamv mzHavonauqmm s m m a e . m m a a _ - q a q — Na. (db Luz) Louvsuossv 82 applied, the error was minimized by determining enzyme reaction rates (AA/min) after the first 3 to h min of the reactions. Intestinal con.- tents from calves fed the promosoy diet contained less interfering sub. stance(s) than contents from calves fed the highusoy diet. Corrections were made for trypsin and chymotrypsin assays of intestinal contents from calves fed the promosoy diet by subtracting the ASA/min of blank reactions (TAME and BTEE replaced by water and wateramethanol, respec- tively) from that obtained in the presence of these enzyme substrates. Intestinal contents from calves fed whole milk and the all-milk diet, except from calves sacrificed after 1 week of feeding, contained negligible substance(s) which interfered with esterase activity (Fig. 5). For these reasons, the actual enzyme activities of intestinal contents from calves fed the high-soy diet would be less than the values shown in Table 19. Differences between the activities of these enzymes in contents from.ealves fed the high-soy diet and those from intestinal contents arising from calves fed the other diets would also be greater than indicated. There was a 2- to 3-fold increase in the total trypsin and chymotrypsin activities of intestinal contents from calves sacrificed at #1 compared with those sacrificed at 10 days of age (Table 19). This age difference might be even greater if corrections had been made for interfering substance(s) in intestinal contents from calves fed whole milk and the all-milk diet for 1 week and that from all calves fed the high-soy diet. Correlation coefficients between growth rate of calves from 3-5 days of age to slaughter at 27 or #1 days of age and the total trypsin or chymotrypsin activities of intestinal contents were 0.25 and -0.18, respectively. Contents from the middle third of the small intestine 83 contained nearly twice the trypsin and 30% more chymotrypsin activity than contents from.the upper or lower segments. The latter two were nearly identical in total enzyme activity. Correlation coefficients between the total enzyme activity in the pancreases (enzyme concentra- tion x pancreas dry matter weight) and total enzyme activity of small intestinal contents were 0.11 and 0.03 for trypsin and chymotrypsin, respectively. Intestinal contents arising from the promosoy or all-milk diets retained about 80% of the original trypsin activity, following a two-hour incubation at 37 C, while contents arising from the high- soy diet retained only 60% (Table 19). Only Zhfi of the original chymotrypsin activity of intestinal contents from calves fed the high- soy diet was present after incubation whereas an average of #3 and 59% of it was retained in intestinal contents from calves fed the two milk protein and promosoy diets, respectively. The recovery of trypsin and chymotrypsin activity after incubation of intestinal contents was similar from.the three sections of the small intestine, when values for contents from calves fed all four diets were combined. An inter- action between diets and intestinal sections was present for the stability of these enzymes in incubated intestinal contents (Table 20). Contents from the upper or middle sections tended to retain more of the original trypsin and chymotrypsin activity following incubation of the contents than that from the lower section when the all-milk and promosoy diets were the source of nutrients. Recovery of these enzymes in incu- bated contents from calves fed the high-soy diet, in contrast, was greatest from the lower third of the small intestine. Little or no chymotrypsin.was present in upper and middle intestinal contents from 8h calves fed the high-soy diet after incubating the contents for two hours. Several instances of greater enzyme activity were noted after incubation of intestinal contents, particularly from the upper third of the small intestine. There was no apparent relation to diet or length of time calves were on the diets. Enzyme recovery after incubation of contents was not significantly related to the protein concentration of intestinal contents. Correlation coefficients between the final protein level of intestinal contents after incubation and the proportions of intestinal trypsin and chymotrypsin retained during the incubation were -0.11 and 60t12, reSpectively. Trypsin stability was not significantly ralated to pH of intestinal contents. The correlation coefficient between these two variables was 0.16. The length of time calves were fed had no effect on enzyme stability in incubated intestinal contents. The proportion of original trypsin retained during incubation of digesta was greater than that for chymotrypsin. This was illustrated by a reduction (P‘<.0005) in the ratio of chymotrypsin-to-trypsin from 0.3” to 0.19 for intestinal contents before and after incubation, respectively (not shown in Table 19). Proportionately more chymotrypsin was destroyed during incubation of intestinal contents from calves fed the high-soy diet than that destroyed in intestinal contents from.ealves fed the other diets (Table 19). The ratio of chymotrypsin-to-trypsin in non-incubated intestinal contents from calves fed the highpsoy diet was also lower than that for intestinal contents from calves fed the remaining three diets. Contents from the middle section of the small intestine prior to incubation tended to have a lower chymotrypsin-to-trypsin ratio than those for contents from the upper or lower sections (P>.05). The 85 ratio of chymotrypsin-to-trypsin in digesta from the small intestine of calves 10 days of age was 0.02 compared with 0.28 in digesta from.01- day-old calves (P > .0 5). Digestion of protein (mg/ml) during incubation of intestinal contents from calves fed whole milk was nearly two times greater than that from calves fed the all-milk or promosoy diets, and six times greater than that from calves fed the high-soy diet (Table 21). Total in vitro protein digestion in contents from the entire small intestine, however, ranged from 2.0 to 3.3 g/Z hr from calves fed the two milk protein and promosoy diets, and only 0.06 g/2 hr from calves receiving the highpsoy diet. Differences due to diets were not altered appreciably by correcting for the final body weights of the calves. Approximately 38, 30, 25 and 8% of the protein present in intestinal contents from calves fed whole milk, allamilk, promosoy, and high-soy diets, respectively, was digested during the two hour incubation. Protein digestion was positively correlated with the protein concentra- tion of intestinal contents before incubation (r = 0.03, P‘<.001). The amounts of protein digested were subsequently adjusted by covari- ance analysis for differences in protein concentrations. Differences in protein digested per milliliter of intestinal contents from calves fed the four diets, however, were not primarily due to differences in protein concentrations. Adjusted protein digestion was still greatest for intestinal contents from calves fed whole milk and least for con- tents from.calves fed the high-soy diet. Body weight changes from 3:5 days of age to slaughter at 27 or 01 days of age were related to total in vitro protein digestion in the entire small intestinal contents from these calves (r = 0.07, P< .05). 86 Table 21. In vitro digestion of intestinal protein, and the relation- ship to the trypsin and chymotrypsin activities of intes- tinal contents, during a two hour incubation as influenced by contents from three sections of the small intestine from calves fed four different diets and sacrificed after one, three and five to six weeks on the respective diet. Efficiency of Protein digestion protein digestion“ Adjusted 2 for protein Total content3 T sin C at 51 (mg7517 (272 hr) (mg7ml) (mg/unit) fima7unit5 Whole milk 6.08b 3.29“ 5.90b 1.42“ 3.96“-° All-milk 3.32“ 2.41“ 3.70“ 1.29“ 3.31""b High-soy 1.08° .46b 1.09c .48b 2.02b Promosoy 3.51a 2.46“ 3.29“ 1.75“ 5.61c P diff.5 <.0005 <.005 <.0005 <.005 <.o1 Duration of feeding 1 wk (10)6 3.68‘“b 1.04“ 3.31“ 1.66“ 4.26 3 wk (27) 2.56“ 2.47b 2.88‘ .71b 2.65 5.6 wk (41) 4.87b 3.88° 4.91b 1.34‘1 4.12 P diff. .<.o1 <.005 <.o1 <.005 N.s.7 Small intestinal section Upper 3.84""b -_ 3.75 1.82b 3.85 Middle 5.03“ -- 4.35 1.068 4.08 Lower 2.05b -- 2.81 .78a 2.97 P diff. (00005 -- (010 (9001 NOS. 1See footnote 1, Table 15. 2Total protein digested in combined contents from the three sections of the small intestine. 3Protein digestion adjusted by covariance analysis for differences in “the protein concentration of intestinal contents prior to incubation. Ratio of mg/ml of protein digested to units/ml of trypsin or chymo- trypsin activity present before incubation of intestinal contents. iSee footnote 5, Table 18. 6See footnote 7, Table 18. 7See footnote 6, Table 18. 87 A correlation coefficient of 0.21 was obtained when data from calves sacrificed at all three age periods were considered. Body weights of calves sacrificed at 10 days of age were generally less than the initial body weight (Table 18), which would lower the correlation between protein digestion and weight changes. The ratio of protein digested in vitro (mg/ml) to the trypsin or chymotrypsin activity (units/ml) of intestinal contents before incubation was calculated to estimate the efficiency of protein diges- tion by these enzymes. Protein digested per unit of tryptic activity was nearly equal for intestinal contents from calves fed the two milk protein and promosoy diets, but was markedly reduced (P <.005) in intestinal contents from calves fed the high-soy diet (Table 21). Intestinal contents from calves receiving the promosoy diet had the greatest protein digested per unit of chymotrypsin (5.6 mg) and con- tents from calves fed the highesoy diet had the least (2.0 mg). Protein digestion per unit of the combined trypsin and chymotrypsin activities of intestinal contents from calves fed whole milk, all- milk, promosoy and high-soy diets was 1.10, 0.80, 1.28 and 0.01 mg, reapectively (not shown in Table 21). Protein digestion in contents from the entire small intestine increased as the calves became older (P< .005), except for those from calves fed the high-osoy diet (Table 27). Total protein digestion per kilogram of body weight was 0.025, 0.054 and 0.074 g/Z hr for intes- tinll contents from calves at 10, 27 and 01 days of age, respectively (not, shown in Table 21). Protein digested per unit of trypsin or clmno- trypsin was nearly equal in intestinal contents from calves sacrificed 't 10 and 01 days of age, but for no apparent reason was reduced in cont Grits from 27-day-old calves . 88 The greatest in vitro protein digestion, irrespective of dietary source, occurred in contents from the middle portion of the small intes- tine, followed by that from.upper intestinal contents and the least protein digestion in contents from the lower section (Table 21). An interaction existed, however, between diets and intestinal sections (Table 20). Protein digestion in middle and lower intestinal contents from calves fed the highpsoy diet was nearly equal (1.50 vs. 1.33 mg protein digested/ml). These values were four times greater than those in upper intestinal contents (0.35 mg/ml) from calves fed this diet. Protein digestion in vitro in contents from the three sections of the small intestine was generally related to the prewincubation protein concentrations. Differences in protein digestion in contents from the three sections were reduced when these values were statistically adjusted for the pro-incubation protein levels in each section (Table 21). Protein digestion per unit of trypsin activity was greatest in contents from the upper third and least in contents from the lower third of the small intestine (P<:.001). Protein digestion per unit of chymotrypsin activity was not significantly different (P2>.05) in digesta from the three sections, but tended to be less in that from the lower than that from the upper or middle small intestine. The reduction in protein digestion per unit of enzyme activities in contents from the upper to the lower small intestine was less when calves were fed the highpsoy diet than when fed the other three diets. The high-soy diet contained an equivalent of 6.17 mg soybean trypsin inhibitor (SBTI)/g of airmdry diet, compared with 0.15 and 0.10 mg/g in the all-milk and promosoy diets, respectively (Table 22). This was a 00-fold difference in trypsin inhibitor activity. Differences of 89 Table 22. Trypsin inhibitor activity in three milk substitute diets, and in abomasal and intestinal contents from calves fed these diets. SBTI equivalent activity16 Source of Die fed trypsin inhibitor High-soy All-milk Promosoy Diet2 (mg/g) 6.17 0.15 0.14 Diet (mg/mg protein3) 1.56 0.046 0.052 Abomasal contents2 (mg/ml) 0.502 0.012 0.010 Abomasal contents (mg/mg proteina) 0.009 0.001 0.002 Small intestinal contents“ - upper (mg/ml) 0.2005 -- 0 - upper (mg/mg protein6) 0.033 -- 0 - middle (mg/ml) 0.301 -- e- - middle (ms/mg protein) 0.032 -- -- - lower (mg/ml) 0.129 -- -- - lower (mg/mg protein) 0.019 -- -- 1Trypsin inhibitor activity of extracts compared with t sin inhibitor activity of crystalline soybean trypsin inhibitor (SBTI)? 2Fractionation according to Garlich and Nesheim (66) to yield the equivalent of soybean whey solution. 3Trypsin inhibitor activity/mg protein present in the extract assayed for trypsin inhibitor. 4Fractionation according to Alumot and Nitsan (1). 5Average of intestinal contents from 7 calves fed the high-soy diet. 6Trypsin inhibitor activity/mg protein present in the intestinal contents before incubation. 90 similar magnitude in trypsin inhibitor were found in abomasal contents from calves fed these diets. No dietary differences were evident in the protein content of the extracts which were assayed for trypsin inhibitor. Consequently, the SBTI equivalent/mg of protein in extracts from the highpsoy diet or of abomasal contents from calves fed this diet was still approximately 00 times greater than that present in extracts of the all-milk or promosoy diets or abomasal contents from calves fed these diets. Competition between dietary protein in the extracts and the synthetic substrate (TAME) for the active sites of trypsin, therefore, would not account for the high trypsin inhibitor content of the high-soy diet. Fractionation of abomasal contents from calves fed the high-soy or promosoy diets by the method of Alumot and Nitsan (1) resulted in almost the same trypsin inhibitor values as those obtained by the procedure reported by Garlich and Nesheim (66). Thus the marked difference in trypsin inhibitor found in abomasal con- tents of calves fed the highpsoy vs. the promosoy diet was verified. Abomasal contents from calves fed whole milk contained no detectable trypsin inhibitor. Intestinal contents were treated according to the procedure of Alumot and Nitsan (1), in which trypsin inhibitor was precipitated by ammonium sulfate and dissolved in pH 7.6 Tris. buffer. Upper intes- tinal contents from calves fed the promosoy diet retained trypsin activity originally present in the contents in this fraction and there- fore no free trypsin inhibitor could be detected. Considerable free trypsin inhibitor was present in extracts of intestinal contents from calves fed the high-soy diet (Table 22). Contents from the lower small intestine contained less free trypsin inhibitor than contents 91 from the upper and middle sections. The amount of free trypsin inhibitor per milligram of protein in pre-incubated contents was equal in contents from.the upper and middle sections of the small intestine. A correlation coefficient of -0.28 (P:>.05) was found between free trypsin inhibitor and in vitro digestion in intestinal contents from calves fed the high. soy diet. The procedure of Alumot and Nitsan (1) does not give the amount of trypsin inhibitor combined with trypsin (or chymotrypsin) in the intestinal contents. The latter may be more closely related to inhibition of proteolysis than the level of free trypsin inhibitor. Exaggiment 0. Interfer substance 8 in testinal 0 en 8. Calf abomasal or intestinal contents, steer gall bladder bile, or purified bile salts interfere with assays for esterase activity (73). An increase in absorbancy occurred which was not due to hydrolysis of the synthetic substrate esters TAME and BTEE by trypsin and chymotrypsin, respectively. The extent of this reaction was determined by following the spectrophotometric assay procedures for trypsin and chymotrypsin, except that the TAME and BTEE solutions were replaced by water and water- methanol, respectively. The reaction was characterized by a very rapid nonlinear initial increase in absorbancy which diminished with time, and was dependent on the presence of calcium in the Tris buffers (Fig. 6). In the presence of CaCl2 a precipitate formed in the cuvette, and similar changes in absorbancy were noted at 300 as well as at 207 mu. Aqueous supernatant fluids of the all-milk, high-soy and promosoy diets contained interfering substance(s), and were retained in the fat-free diets. This apparent esterase activity associated with chymotrypsin assays at 256 mp was diminished markedly by lowering the final concen- tration of methanol in the assay solution from 26 to 0.7%, v/v cease one“. 5 «See on . 8.5 ”~85 59o ,\.eo.e.ees .na. to me on 9.33280“... .m $.33 3.5. on See es 5...... «mm: erases. ensessee defines: me he... fiesta .4 and... 0.55 coated one announce anonymous.“ hex glued Ease caduceus mo oonaeuaopmH .0 .mg w a 3 - N . 35 may 2885 .- m w e m . _ . . 1 no. co. mo. NH. .3. ma. «N. (1“ 4+1?) IONVHHOEV 93 (Table 23). Values obtained with 0.7% methanol were nearly equal to those obtained for the trypsin assay in the absence of methanol. Table 23. Measurement of interfering substances in calf intestinal contents as affected by methanol concentration in the chymotrypsin assay and treatment of contents with calcium chloride in the presence or absence of ammonium.sulfate and pH adjusted to 8. A nt esterase activit I— Treatment of TrypsinE Cfiiiptiiisin :ss;§2 intestinal assay Final methanol v v gontegts 26_ 59.0 0.7 (15A7min) Non-treated 0.020 0.060 0.032 --- 0.012, pH 8 0.0024 0.046 0.014 --- 0.012, (NHu)2504, pH 8 0.0003 0.046 0.012 ... 0.012, (Nflh)230h, pH 8“ 0.0027 --- ..- 0.0031 1Increase in absorbancy of blank reactions of intestinal contents from calves fed the promosoy diet, neglecting the first 3 min. 2TAME replaced by water. 3BTEE replaced by waterumethanol. hValues for intestinal contents from calves fed the promosoy diet, excluding those used to compare 26 and 9.0% methanol. Sodium glycocholate and Na desoxycholate but not Ha cholate and Na taurocholate interfered'with esterase enzyme assays. This interfer- .ence‘was related to precipitate formation whenc.,012 was added to the two former but not the two latter bile salts. Sodium.taurocholate was the only bile salt studied which was not precipitated at pH 0 or in 5% trichloroacetic acid. Both Na glycocholate and Na taurocholate absorbed very strongly at 280 mu or shorter wavelengths, while the non-conjugated Na cholate and Na desoxycholate did not absorb in this spectral range. The four bile salts studied, as well as steer gall bladder bile, were 90 also soluble in 70% toluene. Since bile salts are relatively small molecules (mol wt about 500 g) they should be retained on a Sephadex G~25 column which.would allow separation from larger protein molecules. The appearance of Na glycocholate and pancreatic juice protein in the eluant could be detected in the spectrophotometer since both absorbed strongly at 280 mu. Pancreatic juice protein was eluted as one major peak, with a second very broad minor peak (Fig. 7). The mixture of pancreatic juice and Na glycocholate produced a much larger peak at the same elution volume as pancreatic juice alone, with a second minor peak, indicating no separation of the bile salt from the protein. Intestinal contents which contain interfering substance(s) must be treated to remove the factor(s) reacting with CaClz to form a precipitate, or corrections made in the esterase enzyme assays. Otherwise the trypsin and chymotrypsin activities obtained would be greater than the actual values due to enzymatic hydrolysis of the synthetic substrates. A correction for interfering substance(s) was made by subtracting the change in absorbancy of blank reactions con- taining diluted supernatant fluids of intestinal contents without TAME or BTEE from the absorbancy change in the presence of these substrates. Considerable error is involved in such a procedure, since the reactions with and without the specific enzyme substrates tend to be curvilinear. The error was minimized by computing the (SA/min of both reactions after 3 to 0 minutes had elapsed. Increases in absorbancy of blank reactions containing intestinal contents from calves fed the promosoy diet were significantly reduced (P < .005) by incubating the contents at 37 C for 2 hr. Absorbancy increases of blank reactions were ABSORBANCY (280 mp) 95 2.0 - 1.0 '- Pancreatic juice plus <(/////’~ Na glycocholate Pancreatic juice 1 l L i J 0 2 0 6 8 10 TIME (min) Fig. 7. Sephadex Ge25 column chromatOgraphy of pancreatic juice and Na glycocholate. 96 greatest in contents from the middle small intestine and least in con, tents from the lower section. There were no changes in the absorbanay of blank reactions containing lower intestinal contents from several calves. Absorbancy increases in blank reactions were not linearly related to the extent of dilution of intestinal contents with 0.15M NaCl. A 1:1 dilution would reduce the AA/min several times more than the expected 50% reduction compared with undiluted contents. Trypsin and chymotrypsin assays should be determined on intestinal contents diluted to a maximum at which enzymatic hydrolysis of the synthetic substrates can be accurately recorded, if the interfering factor(s) cannot be satisfactorily removed. The trypsin and chymotrypsin activities of intestinal contents from several calves fed the promosoy diet were reduced approximately 30$‘by subtracting the absorbancy changes of blank reactions from those obtained in the presence of the enzyme substrates. Values for intestinal contents from one calf fed the high-soy diet were reduced by 50% using this procedure. Chymotrypsin activities were generally reduced more by correcting for blank reactions than were those for trypsin. Several extraction or purification procedures were applied to intestinal contents to remove certain bile salts. These procedures were based on the results obtained with purified bile salts. Acidifi- cation of the supernatant fluid of intestinal contents from calves fed the high-soy diet precipitated most of the factor(s) contributing to apparent esterase activity. Dialysis of the acid supernatant fluid completely removed interfering substance(s). Dialysis of the super» natant fluid of intestinal contents before pH adjustment was ineffective 97 in removing interfering substance(s). A major disadvantage of adjust- ing intestinal contents to pH 0 was precipitation of most of the protein and incomplete recovery of trypsin and chymotrypsin activity in the acid supernatant fluid. Approximately 70% of the enzyme active ities originally present in the supernatant fluid of intestinal contents were recovered in the acid(HCl) supernatant fluid (Table 20). Dialysis of this fraction against 0.001 N HCl resulted in little or no further loss of enzyme activity. There was a trace of interfering substance(s) in the intestinal contents before acidification. Correce tions were made by running blank reactions as previously described. The acid supernatant fluid contained no interfering substance(s). Acidification (pH 0 with 2 N HCl) of the supernatant fluid of rat intestinal contents resulted in recovery in the acid supernatant fluid of only 171 of the trypsin and 78% of the chymotrypsin activity orig- inally present before lowering the pH (top section Table 25). However, full enzyme activity was recovered in the acid precipitate when dise solved in 0.15M NaCl. No interfering substance(s) were detected before or after acidification of rat intestinal contents. Sephadex G~25, silicic acid or Amberlite IRA-000 added to calf intestinal contents to form a slurry did not remove interfering sub- stance(s) from the contents. Activated charcoal and Amberlite IRE-50 removed a portion of the substance(s) from intestinal contents which produced the apparent esterase activity. Preliminary trials were carried out with toluene (70% final concentration), to precipitate protein and enzymes from intestinal contents. Difficulty arose, however, in quantitative recovery and redissolving of the precipitate. Adjustment of intestinal contents to pH 8 and addition of CaClz was an attempt to simulate the spectrophotometric assay conditions for 98 Table 20. Trypsin and chymotrypsin activity of intestinal contents from a mature bovine as influenced by acidification to pH 0 and dialysis of the acid supernatant fluid against 0.001N HCl for 12 hours. Treatment of intestinal contents Trypsin C t sin . 111.15.17.15 (unitsESml) A. Control supernatant fluid 30.5 20.5 (no pH adjustment) B. Acid supernatant fluid 23.6 15.1 c. i recovery in acid supernatant fluid (B/A x 100) 68.0 73.6 D. Dialyzed acid supernatant fluid 22.8 15.2 E. $ recovery in D (D/B x 100) 96.0 101 ——w 1One unit equals hydrolysis of 1 umole TAME/min, corrected for blank reaction. 2One unit equals hydrolysis of 1‘umole BTEE in 26% methanol (v/v)/min, corrected for blank reaction. trypsin and chymotrypsin. The addition of solid CaClz and 2N NaOH to the supernatant fluid of intestinal contents from calves fed the promosoy diet until a constant pH near 8 was achieved resulted in precipitate formation. The supernatant fluid after the above treat- ment contained 1/10 the original amount of interfering substance(s) (Table 23). The blank reaction for the trypsin assay was further diminished by the addition of (NH0)2300 to 0.1M concentration followed by CaClZ and adjustment to pH 8. The presence of 0.1M.(NHu)2804, and excess CaClZ at pH 8 in intestinal contents (treated) from calves fed the promosoy diet enhanced (P < .000 5) the combined trypsin and chymotrypsin activities in the supernatant fluid compared with that of the untreated intestinal supernatant fluid (Table 26). Trypsin activity in the supernatant fluid 99 Table 25. Recovery of trypsin and chymotrypsin from the supernatant fluid of rat intestinal contents acidified to pH 0 or adjusted to pH 8 and excess CaClz added. Treatment T sin - C t sin (unitsI7ml) (units/10 n14) (unitsjfiml) (units710 n15 A Cent 1 supernatant fluid (no pH adjust- ment) 91.4 914 51 .3 513 Acidification to pH 0 B Supernatant fluid 15.6 156 39.9 399 C Precipitateu -- 700 -- 119 D Total (B + c) 900 518 Recovery in supernatant fluid (B/A x 100) 17 78 Total recovery (D/A x 100) 98 101 CaClz, pH 8 E Supernatant fluid 91.0 910 50.1 501 F Precipitateu ._ 67 -- 26 Recovery in supernatant fluid (E/A x 100) 100 98 Total recovery (E/A x 100) 107 103 1 One unit equals hydrolysis of 1 noble TAME/min. 2Total volume of 10 ml used. 3One unit equals hydrolysis of 1 uncle BTEE (26% methanol, v/v/min). “Dissolved in 0.15MlNaCl prior to enzyme assays. 100 Table 26. Trypsin and chymotrypsin activity in the supernatant fluid of calf intestinal contents from the upper, middle and lower third of the small intestine before and after the addition of ammonium sulfate, and simultaneous dition of emcess calcium chloride and adjustment to pH 8. Treatment of Ratio non-centrifuged Enzyme Treated C at sin tes in ontents activit Untre ted T sin W Untreated 4.993 0.70 Tm.t°du 8002 1061 00% P diff.5 (.0005 <.005 Interactions ntestin se ion t atme Upper - untreated 3.18 0.92 - treated 5.1“ 1.62 0.71 Middle - untreated 5.60 0.62 - treated 10.60 1.89 0.23 Lower - untreated 6.28 0.53 - treat“ 8.08 1029 0038 P diff. N.S.6 N.S. Enzxee ; tzeeteent (trypsin) Untreated 3.32 -- Treated 5.90 1.78 -- (chymotrypsin) Untreated 1.67 we Treated 2.12 1.27 -a P diff. ‘(.025 1Data for intestinal contents from six calves fed the promosoy diet. e unit equals hydrolysis of 1 umole TAME or BTEE (4.7f methanol, v/v) for trypsin and chymotrypsin, respectively, per milliliter of the supernatant fluid, corrected for absorbancy changes of blank reactions. Average activities of trypsin and chymotrypsin combined. “Ammonium sulfate (0.1M), excess CaClz and pH adjusted to 8, prior to centrifugation of intestinal contents. ee footnote 5, Table 18. 6See footnote :6. Table 18. 101 was augmented by an average of 78% and that of chymotrypsin by 27$. The ratios of chymotrypsinutoutrypsin were 0.70 and 0.00 in the superb natant fluids of the untreated and treated intestinal contents, respec- tively. The difference in these ratios reflected the differential effect of the treatment of intestinal contents on the trypsin and chymetrypsin activity. The section of the small intestine from which the contents were derived tended to influence the degree to which enzyme activity was enhanced by (NHu)ZSOu and.CaCl2 at pH 8. The com- bined trypsin and chymotrypsin activities in the supernatant fluid of treated intestinal contents from the upper. middle and lower intestine were increased by 62. 89 and 29$, respectively compared.with those from the supernatant fluid of untreated intestinal contents. Several cases were noted in which treatment of contents from.the lower third of the intestine had no effect on enzyme activity. Treatment of intestinal contents only with (N3u)25°u (0.1M. no pH adjustment) or CaClZ at pH 8, were nearly as effective in augmenting the enzyme activity of the super- natant fluid as both treatments combined. However, (NHu)2504 alone did not remove interfering substances from the digesta. The addition of excess Ca.C12 and pH 8 adjustment of rat intestinal contents produced no increase in the trypsin and chymotrypsin activity of the supernatant fluid, compared with that in the supernatant fluid of untreated contents (lower portion, Table 25). In this case, however, the digesta was centrifuged to remove solid material prior to treatment with CaCl2 and pH adjustment. Similar results were obtained when only the soluble portion of bovine intestinal contents were treated with.CaC12 and.pH adjustment. V. DISCUSSION 1. In vitro synthesis Ed secretion of enzmes from eancreetic tissee sliees The extensive loss of tissue dry matter into the incubation media might be attributed in part to discharge of enzymic and non- enzymic constituents from the tissue into the incubation media. Small tissue fragments were also freed during the incubation. Therefore, it was imperative to record tissue weights prior to incubation. The release of amylase and proteolytic enzymes into the incubation media, independent of a supply of oxygen. metabolic inhibitors, or'parasym- pathomimetic stimulation can be ascribed to passive discharge or leak- age from the tissue, rather than active extrusion requiring a source of energy (111, 112). Schramm et al. (237) related passive enzyme leakage to cold exposure of tissue slices prior to incubation. due to contraction of lipid components in the zymogen granules. Enzyme‘leak- ‘80 was prevented by the addition of longochain fatty acids to the media in the cold, or maintaining the tissue at body temperature prior to 111 vitro incubation (237). The linear log-dose response between level of pancreozymin in the incubation media and release of proteo-s lytic enzymes from tissue slices confirmed earlier data with dog Pmcreases in vivo (162). The apparent in vitro synthesis of amylase and proteolytic "minus from bovine pancreatic tissue slices reported here and in a Preliminary communication (71) can be attributed wholly. or in part. to the method of homogenizing the tissue and assaying only the oupematant 102 103 fluid for eneymes. Homogenization of nonaincubated tissue slices in 0.15M NaCl followed by centrifugation removed intact zymogen granules from the supernatant fluid which was then used for enzyme analysis (73). Incubation of tissue slices resulted in release of enzymes from.the zymogen granules into the media. Centrifugation of saline homogenates (including incubation media) of these tissues resulted in almost com- plete recovery of enzyme activity in the supernatant fluid coapared with that in the original homogenate. An apparent net increase in enzyme activity due to incubation of the tissue slices was then evident. Little or no net increase in trypsin and chymotrypsin activities of calf pancre- atic tissue slices incubated in Krebs media could be detected when the slices were homogenized in saline containing Triton X-100. There appeared to be a smell net increase in total trypsin and chymotrypsin activity during incubation of mature bovine pancreatic tissue slices in amino acid supplemented media. These increases were small, however, compared to apparent increases of 20 to 240% of total proteolytic activ- ity, when the supernatant fluids of saline tissue homogenates were ana- lyzed (Appendix. Fig. 1). A net loss of trypsin and chymotrypsin activity in tissues incubated in Krebs media with or without inhibitors might indicate destruction of preformed enzymes under these conditions. The presence of supplementary amino acids either prevented this apparent destruction of enzymes or more than made up for their loss by stimulat- ing de novo synthesis. Proof of de novo synthesis would require the use of isotOpe dilution experiments and isolation of newly formed enzyme molecules (18). In vitro synthesis of pancreatic enzymes has been reported, using pigeon pancreatic tissue slices or whole mouse pancreases (18, 205, 234). Synthesis of amylase and proteases were 100 stimulated by supplementing the incubation media with amino acids. including asparagine, glutamine or their peptides (105, 109, 234). Incomplete extraction of enzymes from.non-incubated tissue slices may account for at least part of the reported increases in these enzymes during in vitro incubation. The importance of such a phenomenon, however. may be negligible since amylase synthesis in pigeon pancre- atic tissue slices was decreased by metabolic inhibitors and anaero- biosis (10h). More consistent in vitro results were obtained.with pigeon and mouse pancreases by depleting these glands of preformed digestive enzymes by injections of pilocarpine or carbamylcholine about one hour before sacrificing the animals (108, 111). This pro- cedure may also be beneficial in studying in vitro enzyme synthesis in the bovine pancreas. 2. Mile eroeein vs2 soybean protein for celves The prevalence of diarrhea in calves fed the high-soy and to a lesser extent the allmmilk diet may be associated with the relatively high.pH and lack of curd formation in abomasal contents from these calves, compared with those from calves fed whole milk. Shoptaw et al. (249) suggested that the diarrhea produced in calves fed a soybean flour diet was due to lack of curd formation and rapid passage of the liquid abomasal contents into the duodenum. Diarrhea presumably occurred from overloading the small intestine with incompletely digested dietary constituents. The above factors apparently did not produce diarrhea in calves fed the promosoy diet. Heat treatment of milk in the manufacture of milk products used in the allumilk diet has been shown to delay coagulation time in the abomasum (187), by reducing the availability of calcium (6). Addition of extra calcium 105 to milk substitutes has partially offset this heating effect (131). Differences in the pH of abomasal contents from calves fed the differ- ent diets may be related to gastric acid secretion, or buffering capac- ity of the diet. Abomasal contents from.calves reared on whole milk were more acid than those from calves fed the other diets. The chyme ' entering the duodenum from the abomasum.was also at a lower pH. The pH of upper intestinal contents from calves fed whole milk and sacri- ficed 1 to 1.5 hr postsprandial (pH 5.8) was in the same range as that reported in contents obtained from calves fitted.with reentrant duodenal cannulas (189a). The pH of abomasal contents passing through the pylorus of calves just before feeding was found to be 2.0 to 2.8, and increased to 0.5 to 6.2 one-half hour after feeding whole milk (189a). Secretion of total volume and enzymes by the sheep pancreas into the duodenum has been shown to increase as the pH of contents entering the duodenum decreased (170). The greater total trypsin and chymotrypsin activities of intestinal contents from calves fed whole milk than those from calves fed the other diets might be explained, in part, by the lower pH of abomasal and upper intestinal contents from calves receiving whole milk. Calves fed a diet containing 86% of the total protein from soybeans (promosoy) grew at a rate comparable to those fed whdle milk, confirming previous trials with certain isolated soybean proteins (17. 208). Lack of growth on the high-soy diet supported previous results with milk substitutes containing soybean meal or flour (17, 6h, 151, 283). Porter and Hill (208) reported low digestibility and nitrogen retention of soybean meal and soya flour by young calves, but diges- tibility of certain isolated soybean protein was equal to casein. The 106 proportion of dietary protein intake that was digested by incubating intestinal contents ranged from 3 to 7% for calves fed the high-soy diet to 20 to 30$ for calves fed the other diets (Table 27). Diges- tion of promosoy protein appeared to be less than milk protein in intestinal contents from calves killed after 1 week of feeding. but was nearly equal after 5 weeks of feeding. The estimated intestinal digestion of protein was low comared with whines of 75 to 8 5i in digestibility trials (208). Protein digestion may occur in other parts of the digestive tract besides the small intestine. In vitro incubation of intestinal contents also may not adequately simulate conditions in vivo. The positive correlation between in vitro protein digestion and protein concentration of intestinal contents would indi- cate reduced.proteolysis due to limiting substrate. The poor perfor- mance of calves fed the high-soy compared with those fed the promosoy diet may be attributed to: 1) the higher protein content and conse- quently lower carbohydrate content of the promosoy supplement (70 vs. 50% crude protein), and 2) a 00-fold greater amount of trypsin inhibi- tor in the highpsoy diet. Since 40% of the protein in the highpsoy diet was supplied by skim milk powder, and total protein was increased by 5%, levels of the most limiting amino acids in soybean protein, particularly methionine (20)o were nearly equal in the highpsoy and all-milk diets (Table 8). The deleterious effect of the highpsoy diet on pancreatic juice secretion was evident after 2 to 3 days on this diet. There was a non- specific effect on the release of trypsinogen and chymotrypsinogen, since the ratio of chymotrypsinato-trypsin activity was not altered. Since pancreatic amylase as well as the specific activities of trypsin 107 Table 27. Proportion of dietary protein digested during incubation of intestinal contents from calves fed four different diets and killed after one or five to six weeks of feeding. Dieteez intake In vitro Estimated Feeding protein 2 protein Diet duration Diet1 Protein di estion di estibilit (weeks) (g724 hr) (g524 hr) (i of dietary protein) whole milk 1 3790 1313 26.4 20 5 5900 204 58.8 29 All-milk 1 495 95 24.7 26 5 990 190 44.0 23 Highpsoy 1 439 106 7.8 7.4 5 608 147 4.8 3.3 Promosoy 1 472 92 12.5 14 5 1080 211 46.6 22 Average amount of diet fed for two or more days before the calves were killed. 2Calculated from total protein digested during a two hour incubation of digesta from.the entire small intestine. 3Calculated from the average protein concentration (3.46%) of whole milk from the Michigan State University dairy herd assayed during 1965. and chymotrypsin were suppressed by the highpsoy diet, a larger prepare tion of the pancreatic juice protein from calves fed this diet must be in the form of other enzymes such as procarboxypeptidases which may constitute up to 30% of protein in mature bovine pancreatic juice (134) or non-enzymatic protein. Pekas et al. (204) reported that a 45-day- old pig on a soybean meal diet secreted 1.73 times the volume of pan- creatic juice and 5 to 6 times as much enzyme output as another pig on 108 a casein diet. The results obtained in calves with pancreatic duct cannulas were supported in part by lower enzyme concentrations in the pancreas and intestinal contents from calves fed the high-soy diet compared with those from calves fed whole milk, all-milk and promosoy diets. Since no corrections were made for interfering substance(s) in intestinal contents from calves fed the high-soy diet, the actual levels of trypsin and chymotrypsin present in these contents may be 50% less than those shown in Table 19. Application of this correc- tion, in addition to treatment of intestinal contents with CaClZ and (1099230,“ to release enzymes bound or complexed with insoluble particles might improve the low correlation between growth rate of calves and proteolytic enzyme activity of intestinal contents. In vitro digestion of intestinal protein was closely related to calf performance on the various diets. There was five to seven times more protein digested during a two hour incubation of intes- tinal contents from calves fed whole milk, all-milk or promosoy diets compared with that of intestinal contents from calves fed the high- soy diet. There was also greater destruction of trypsin and chymo- trypsin and less protein digested per unit of trypsin or chymotrypsin during incubation of intestinal contents when the high-soy rather than the other diets was the source of nutrients for calves. The very high levels of soybean trypsin inhibitor in the highasoy diet and abomasal and intestinal contents from calves fed this diet provide a logical explanation for the limited in vitro proteolysis in the intestinal contents. Alumot and Nitsan (1) reported a negative correlation between proteolytic activity and antitrypsin level of chick intestinal contents. Milk substitute diets in which ground parched soybeans or soybean oil meal supplied 40% of the protein resulted in 0 to 11% dry 109 matter digestibility and 0.03 to 0.13 g daily nitrogen retention in calves from 15 to 19 days of age (195a). Reduced in vitro stability of trypsin and chymotrypsin and protein digested per unit of enzyme activity in contents from calves fed the high-soy diet compared with those from calves fed the other diets may also be associated with soybean trypsin inhibitor(s). The following evidence suggests that the soybean trypsin inhibitor might be destroyed or inactivated as digesta traversed the small intestine of the calf. There was greater in vitro protein digestion and less destruction of trypsin and chymotrypsin during incubation of contents from the lower compared with that from the upper small intestine from calves fed the high-soy diet. These results were opposite to those found in contents from the different sections of the intestine from calves fed the other diets. Further- more, lower intestinal contents from calves fed the high-soy diet contained less free trypsin inhibitor than that found in upper and middle intestinal contents. This might be explained by more trypsin inhibitor complexing with trypsin in the lower tract, but would contra- dict the enhanced in vitro protein digestion which was found in contents from this section compared with that in upper intestinal contents. One or more of the following mechanisms may be responsible for the poor growth and hyposecretion of pancreatic enzymes by calves reared on the high-soy diet: 1) rapid passage of chyme through the upper small intestine due to constant diarrhea, 2) limited proteolysis due to the presence of soybean trypsin inhibitor, and 3) deficiency of essential amino acids for protein and enzyme synthesis. Intestinal contents from calves suffering diarrhea when sacrificed irrespective 110 of diet, contained very low levels of proteolytic enzymes. Under these conditions the release of secretin and pancreozymin which trigger the secretion of exocrine fluid.and digestive enzymes from the pancreas (10, 93) might be limited. This mechanism would also serve to prevent loss of endogenous protein secretions in the feces. The levels of trypsin and chymotrypsin in the pancreas from one diarrheic calf were similar to those in the pancreases of normal calves. Previous reports indicate that a certain degree of gastric digestion must occur to obtain a pancreatic response to protein (149). An adequate duodenal stimulus for the release of pancreozymin may require the release of sufficient quantities of certain stimulatory amino acid residues from the dietary protein (275). The absence of proteolysis in digesta from the upper small intestine of calves fed the highpsoy diet could therefore fail to trigger the release of pancreozymin in sufficient quantities.) Finally, inhibition of intestinal proteolysis would limit the supply of amino acids for synthesis of digestive enzymes as well as all other body proteins. The specific effects of purified soybean trypsin (or growth) inhibitors (216, 217, 218) on biochemical changes in the calf pancreas and intestinal proteolysis have not been studied. If soybean trypsin inhibitor was of major importance in producing the results obtained in calves fed the high-soy diet, it failed to evoke pancreatic hypertrophy and hypersecretion of digestive enzymes, as it did in rats and chicks (66. 90. 184. 216, 236). Soybean trypsin inhibitor per so may trigger the release of secretin and pancreozymin from the duodenal mucosa of 'rats (138). Intestinal proteolysis in rats was not inhibited by say- bean trypsin inhibitor (163), whereas practically no intestinal proteo- lysis occurred during the first 4 hr after feeding chicks raw soybeans (1). 111 The response of swine to raw soybeans has also been reported to differ from that commonly found in rats and chicks. The size and nitrogen content of the pancreases were reduced, while there was either no change or a decrease in pancreatic enzymes in the pancreases and intes- tinal contents of baby pigs fed raw soybeans compared with those fed cooked soybean oil meal in the diet (114). Protein secretion by the cannulated pig pancreas was also reduced from two pigs fed a diet containing raw soybeans compared with that from two pigs fed a diet containing processed soybean meal (201a). These results tend to corroborate those presently found with calves. Further research is essential to elucidate the specific effects of trypsin inhibitors and other growth factors in raw soybeansion young calves. 3. S ze d t sin and t sin activ t f cal c ses t stin teol c enz s s uenc . A Pancreas size, in proportion to body weight, generally increased with age at least to 6 weeks. The value of 0.75 g/kg body weight at 6 weeks approached 1 g/kg found in mature sheep.15 Huber et al. (117) reported pancreas weights ranging from 0.6 to 1 g/kg in 1 to 44-day-old calves, with little effect due to age or diet. However, data were obtained from only two calves at each age. Pig pancreases comprised 1.5 to 2 g/kg body weight (114), and two-week-old chick and 9-monthpold rat pancreases were 4 and 4.6 g/kg body weight, respectively.16 The possibility exists that the relatively reduced pancreas size in mature ruminants in relation to other species may reflect the importance of 1 5A. D. L. Gorrill, J. W. Thomas, and D. E. Bauman, unpublished data. 16 - M. G. Yang, and A. D. L. Gorrill, unpublished data. 112 pregastric digestion in ruminants compared with that in monogastric animals. Concentrations of trypsin, chymotrypsin and total proteolytic activity of the pancreas did not change appreciably from 1 week to several years of age (Appendix Fig. 1), in accordance with previous studies (51, 71, 117, 207). Augmented trypsinogen.synthesis, relative to chymotrypsinogen synthesis, from 10 to 41 days of age was implied by lowered ratios of pancreatic and intestinal cbymotrypsin-to-trypsin activities, but was not evident in pancreatic juice collected at 4 vs. 21 days of age. Increased flow rate of pancreatic juice from 3 to 21 days of age confirmed previous results with young calves (167, 261). These results were substantiated by the greater enzyme activity of intestinal contents from calves sacrificed at 41 vs. 10 days of age. ‘Large animal variations in pancreatic secretions were observed. Highly irregular and unpredictable changes*in flow rate of pancreatic juice have been recorded from the cannulated pig pancreas (202). The average protein concentration of pancreatic juice from calves fed the all-milk diet (12 mg/ml) was in the lower range of 4 to 41 mg/ml reported for the mature bovine (79. 135, 136). Reductions in protein and.proteolytic enzyme content of pancreatic juice from calves fed the all-milk diet from.4 to 21 days of age were partially offset by enhanced volume secretion. Nevertheless, total output of pancreatic juice protein, relative to dietary intake of protein, decreased about threefold during the period from.7 to 21 days of age (Table 28). Possibly pancreatic secretion does not increase linearly with increased food intake. The effects of the surgical operation per se, or the possible development 113 Table 28. Comparisons of dietary protein intake and total protein output in pancreatic juice collected from calves at different ages and fed two different diets. Ratio Dietary Pancreatic 2 re ic i e e et e to 1 e tein e e days g 24 hr g 24'hr All-Milk 3'5 5"" 2 o 29 0 e 042 7 ‘5“ 30% 0e073 14 81 3.77 0.047 21 108 2.65 0.025 High-soy 3-5 68 1.47 ‘0.022 7 68 0.97 0.014 14 102 0.76 0.007 21 136 0.55 0.004 w— —— 1Calctglated from.amount of diet fed and fl crude protein (Kjeldahl N x .25 . 2Calculated from.average protein concentration and flow rate of pancreatic juice collected before and 1, 6 and 12 hr after feeding. of a secondary pancreatic duct system (277) on post-operative pancre- atic secretion are not know. Infection of the pancreas could alter its function. Since only a small sample of pancreatic juice was obtained (1'2 n1) at each collection, the loss of pancreatic secretion from the duodenum would be small compared with that lost in total collection studies, where the enzyme content of pancreatic juice diminished with time (47, 170, 264). The absence of a marked increase in pancreatic juice flow rate in response to food intake in this report followed the same trend reported in mature ruminants (102). This would suggest a similar physiological response in the non-ruminating calf and mature ruminant. Stimulation of pancreatic secretion in calves has been noted, however, in response to feeding, drinking, and while rumin- ating (167). Total output of protein, trypsin and chymotrypsin in the Pancreatic juice of calves in this study were generally greater one 114 hour after feeding than those just before feeding. Maximal flow of chyme from the abomasum.to the duodenum.has also been reported during the first hour after feeding calves whole milk (189a). The presence of larger quantities of chyme in the duodenum at this time would be expected to increase flow rate and enzyme output by the pancreas, mediated via secretin and pancreozymin. A small transient increase in amylase output by the sheep pancreas occurred after feeding (264). The pancreatic response to feeding and exocrine secretion in ruminants, however, does not approach that found in non-ruminant species such as the dog (206, 212). ‘ Total in vitro protein digestion in contents from.the entire small intestine of calves, corrected for body weight changes, increased threefold in calves killed at 41 days of age compared with that in 10-day-old calves (0.074 vs. 0.025 g/2 hr/kg body weight). During this same age period, the total trypsin and chymotrypsin activity in the intestinal contents from these calves also increased two to threefold. Protein digestion per unit of trypsin or chymo- trypsin activity was equal in the intestinal contents from 10- or’41- day-old calves. Therefore, increased in vitro protein digestion in intestinal contents was most closely related to higher secretion rates of trypsinogen and chymotrypsinogen from the pancreas. Digestibility trials with calves have also illustrated increased utilization of milk replacers containing 30 to 40% soybean flour at 26 to 38 days of age compared with that at 10 to 14 days of age (196). 4. s cal ch mi e c ro erti s 0 al es in onto ts re s - in es in errel t o shi s a en es e s eetivity. 115 Intestinal contents from the middle third of the small intestine of all calves, except those fed the high-soy diet, contained the highest concentration of protein and proteolytic enzymes. Transport of contents from the upper to lower section of rat intestines has been used to explain higher enzyme concentrations in the middle and lower sections (156, 205). The low correlation between pancreatic trypsin and chymoe‘ trypsin and the corresponding enzyme activity in the intestinal contents may limit the usefulness of pancreatic enzyme data reported here and elsewhere (51, 117, 207). The best estimates of exocrine pancreatic function are the amounts of enzymes released into the duodenum, and their activity and stability in hydrolyzing dietary nutrients. Pancre- atic enzymes are secreted into the duodenum in response to pancreozymin released from the duodenal mcosa by cl-yme entering from the abomasum (166). The zymogen granule content of the pancreas may reach an equi- librium and not be altered appreciably by the quantities of enzymes secreted. The much greater stability of trypsin than chymotrypsin during in vitro incubation of calf intestinal contents supports data with rat intestinal contents (205, 253, 254), but not with human duodenal juice (285). The recovery of 80 and 50% of the trypsin and chymotrypsin activities, respectively, following incubation of intestinal contents from calves fed the all-milk or’promosoy diets at 37 C for two hours can be compared to recoveries of 56% trypsin and 18$ chymotrypsin from rat intestinal contents subjected to the same treatment (205). Destruc- tion of pancreatic enzymes in the lower digestive tract has been attributed to self-digestion in the absence, or diminished supplies of dietary protein (251, 253, 254), or due to the action of microbial 116 proteases (25). Stability of trypsin and chymotrypsin activity during incubation of intestinal contents in this study was not adversely affected by almost a 50$ reduction of protein concentration in contents from the lower small intestine or changes in pH. In vitro digestion any not have reached a point where dietary protein was markedly limited, since less than 50% of the total protein was hydrolyzed. Apparent increases in enzyme activity of upper intestinal contents due to in vitro incubation may be due to incomplete enterOpeptidase acti- vation of trypsinogen and chymotrypsinogen in the duodenum, or to changes in the amount of interfering substance(s), when the esterase enzyme assays were not corrected by blank reactions. Addition of exogenous enteropeptidase to sheep upper intestinal contents had no effect on trypsin and chymotrypsin activity.15 The pH of calf upper intestinal contents (6.02) was near the pH optimum of 6.2 for entero- peptidase activity (147). Activation of trypsinogen and chymotryp- sinogen in.vitro can occur in 10 min at body temperature, if relatively high concentrations of these enzyme precursors and low calcium ion concentrations are present (73). Rapid activation of trypsinogen and chymotrypsinogen by enteropeptidase probably occurs in the upper duodenum, prior to marked dilution of pancreatic juice with chyme and other intestinal secretions. The role of calcium in this activation in vivo is not known. However, the presence of ionic calcium in the intestinal contents would protect these active enzymes from self- digestion (34, 46, 70, 73). The concentrations of calcium.in digesta have been reported to be less in the lower than the upper smell intes- tine of calves (290). This might partially explain the greater destruction of trypsin and chymotrypsin in the lower compared with that in the upper digestive tract (251). 117 Rate of in vitro protein digestion of intestinal contents was more closely related to the initial level of protein in the contents than the activity and stability of trypsin and chymotrypsin. Reduced protein digestion in contents from the lower small intestine was largely due to a lower protein concentration. Decreased protein digestion per unit of trypsin or chymotrypsin activity in the lower compared with the upper intestinal contents may indicate a reduction in the more readily hydrolysable protein molecules as the contents traverse the small intestine. A.pH change from.approximately 6 in the upper portion to 7 or higher in the lower portion may also be involved. Ratios of pancreatic chymotrypsin-to-trypsin in calves (0.15 to 0-22) Ind sheep :(0.3“)15 differed markedly from ratios of two to three reported in pancreases and intestinal contents from.rats (252). Pre- liminary studies with chick pancreases16 indicated chymotrypsin-to- trypsin ratios of one to two, using the same enayme substrates and procedure as used with calves. Rabbit pancreatic juice chymotrypsin- to-trypsin ratios have been reported to be 0.1“ to 0.17 (226). Compari- sons of chymotrypsin-to-trypsin ratios involving different substrates and techniques are limited by the extreme dependence of BTEE concentra- tion and level of methanol on the apparent chymotrypsin activity (73, 132). Incomplete recovery of trypsin and chymotrypsin activities in the acid (pH b) supernatant fluid of intestinal contents implied that these enzymes were complexed or bound with protein or other molecules which were precipitated at this pH. The enaymes were recovered by dissolving the acid precipitate in 0.15H saline. Snook and Meyer (25“) 118 also reported incomplete recovery of trypsin and chymotrypsin in the supernatant fluid of acidified (pH 0) rat intestinal contents. Recoveries of these enzymes in the.acid supernatant fluid, however, were greater when intestinal contents were obtained from rats fed a 15% protein diet compared with those from rats fed a protein-free diet. Centrifugation of rat intestinal contents and chick feces has also reduced the enayme activities of the remaining supernatant fluid (155, 205). Lepkovsky et .1. (155) suggested that the ensymes in chick feces existed partly as insoluble complexes and could not be released by various solvents or pH changes. The ability of c.c12 or (NHa)ZSOu to increase the enayme activities of the supernatant fluid of calf intestinal contents several fold may be associated with disruption of insoluble enzyme-particle complexes. Centrifugation would then remove insoluble particles but not trypsin and chymotrypsin. Trypsin activity was increased more than chymotrypsin by treatment of intestinal contents with (NHu)25°u and CaClz. The apparent deviation in chymotrypsin-to-trypsin ratios of calf intestinal contents (0.3“) from those of pancreatic juice or pancreas (0.15 to 0.22) might be reduced or eliminated by treatment of intestinal contents withCaCl2 or (NRh)280u, or both. This procedure would also alter the calculated relative and total amounts of protein digested per unit of trypsin and chymotrypsin during incubation of intestinal contents. Comparisons of protein secretion in the pancreatic juice of calves with dietary protein intake are presented in Table 28. Since volume secretion and protein concentration of pancreatic juice were available for only short periods, the estimated 2n hr protein output may contain considerable error. Nasset (191) reported almost a 9-fold 119 dilution of exogenous with endogenous protein in the digestive tract of rats. Enzyme secretion by the stomach and pancreas was estimated to equal ingested protein. The ratios of pancreatic juice protein-to- dietary protein for calves fed the all-milk diet were only 0.025 to 0.073, compared with 0.00“ to 0.022 for calves fed the high-soy diet. Differences due to experimental errors, species, or both, are apparent between the results with calves and those reported by Nasset. Interfering substance(s) in calf intestinal contents which produced an apparent esterase activity can be attributed to certain non-lipid dietary constituents and possibly certain bile salts. Limited data suggest interfering substanceQ)of low molecular weight, complexed with larger molecules (dialyzable in acid supernatant fluid but not in untreated intestinal contents). The absorbancy change was due to precipitate formation with CaClz, causing reduced transmittance in the sample. The interfering substance(s) were apparently degraded by intestinal ensymes or bacteria, or both during in vitro incubation or passage from the upper to lower small intestine. Intestinal micro- organisms are known to convert conjugated bile salts into non-conjugated forms (89, 28“). This phenomenon would reduce the precipitation of Na glycocholate and possibly other glycine conjugates by CaCl2 in intes- tinal contents. The absence of interfering substance(s) in rat compared with calf intestinal contents may be due to greater dilution of the former contents required for enzyme assays, dietary or species differ- ences. Intestinal and cecal contents from other rats in which the dilutions with saline were much lower than in the present study, hows ever showed no evidence of interfering substance(s).16 Corrections for interfering substance(s), involving blank enzyme reactions introduced considerable error due to the curvilinear 120 nature of the reactions. This could be overcome by using appropriate blanks in a double beam spectrophotometer, although extraction of interfering substance(s) from intestinal contents would be ideal. A combination of 0.1M (NHu)ZSOu, CaClz and pH 8 was generally effective in precipitating most of the interfering compound(s), with the added advantage that the trypsin and chymotrypsin activity of the super» .natant fluid was increased several fold. The fact that one or more bile salts are soluble in trichloro- acetic acid and absorb strongly at 200 to 300 mu should be considered if a spectrophotometric assay of trichloroacetic acid soluble amino acids from intestinal contents was developed. Since certain other bile salts are precipitated by trichloroacetic acid, a certain degree of error is also involved in determining the protein content of the precipitate by Kjeldahl nitrogen. VI. INTEGRATED DISCUSSIOR AND CONCLUSIONS Comparisons of enzyme activities in pancreatic tissue slices and media before and after incubation, to be used as an index of in vitro synthesis of these enzymes, are dependent on complete release of enzymes from the zymogen granules prior to enzyme analysis. A relatively large apparent in vitro synthesis was observed when assays were performed on the supernatant fluid of saline tissue homogenates. The addition of’Triton 1.100 to the saline appeared to correct this error in procedure. Careful consideration must also be given to acti- vation of chymotrypsinogen and trypsinogen, to obtain maximum activity. Activation by enteropeptidase in the presence of CaClz at h C has been shown to produce maximum activity and stability of trypsin and clums- trypsin (73). Dietary and age effects on rates of in vitro synthesis of enzymes from pancreatic tissue slices might be detected, by emloy- ing the improved homogenization and activation procedures reported here. The high-soy diet had a marked deleterious effect on exocrine pancreatic function and intestinal proteolysis in calves, compared.with those fed whole milk, the all-milk or promosoy diets. A reduction of pancreatic juice secretion was observed in calves after being fed the high-soy diet for only two to three days. Growth rate of calves was related to in vitro proteolysis in contents from the small intestine. Limited.proteolysis in intestinal contents from calves fed the highpsoy diet would indicate amino acid deficiencies for>protein and enayme synthesis. This could explain the reduction in rate of pancreatic enzyme synthesis and secretion. However, minimal duodenal release of 121 122 pancreozymin due to rapid passage of chyme and limited proteolysis in the upper small intestine, may be a more important control mechanism in reducing pancreatic enayme secretion. In vitro studies with.pancre- atic tissue slices, in the presence or absence of supplementary amino acids, might indicate dietary differences in supplies of endogenous amino acids. Studies involving incorporation of radioactive labelled amino acids may be essential. The physiological and biochemical alterations in calves fed the high-soy diet appeared to be closely linked.with the high soybean trypsin inhibitor content of the diet. The exact meohanismuwhereby the trypsin inhibitor produced its effect was not elucidated. Hyperb trophy of the pancreas and hypersecretion of pancreatic enaymes produced in rats and chicks by feeding raw soybeans, was not observed in calves. Limited intestinal proteolysis in calves was, no doubt, closely associated with the soybean trypsin inhibitor. The trypsin inhibitor may be destroyed as digesta traverses the small intestine. Lower intestinal contents contained less free trypsin inhibitor, and in vitro protein digestion was greater than that in upper intestinal contents. Growth and in vitro proteolysis of intestinal contents from calves fed the promosoy diet were comparable to those in calves fed whole milk. The promosoy diet contained little or no soybean trypsin inhibitor. This might explain the marked difference in performance of calves fed the high-soy and promosoy diets. Greater attention must be directed to more completely remove soybean trypsin inhibitor from raw soybeans in the manufacture of soybean flour for inclusion in milk substitute diets for very young animals. Based on in vitro digestion 123 of protein in calf intestinal contents, it would appear that calves can efficiently utilize soybean flour free of trypsin inhibitor as the sole source of dietary protein at an early age. Limiting amino acids in soybean protein for optimum.growth of calves have not been determined. The digestive function of the pancreas, in proportion to body weight, increased with age. Pancreas size, except from.calves fed the promosoy diet, increased from 0.5 up to 0.75 g/kg body weight at 10 vs. #1 days of age. Concentrations of trypsin and chymotrypsin in the pancreas did not change, but total activities of these enzymes both in the pancreas and intestinal contents, increased during this age period. Total secretion of enzymes in pancreatic juice was greater at 21 than at 3 days of age. Trypsin.synthesis appeared to increase at a faster rate than chymotrypsin, since the chymotrypsinpto-trypsin ratios in the pancreas and intestinal contents were less at #1 than at 10 days of age. In vitro synthesis of trypsinogen and chymotrypsinogen appeared to be somewhat greater in pancreatic tissue slices from mature cows than in those fromayoung calves. The latter procedure may or may not be sensi- tive enough to detect small age differences in rate of pancreatic enzyme synthesis. A threefold increase in total in vitro protein digestion per kg body weight in contents from.the small intestine of #1 vs. 10 days oldpcalves was related to enhanced intestinal trypsin and chymotrypsin activities, rather than to a change in the amounts of protein digested per unit of trypsin or chymotrypsin activity. Proteolytic enzymes, other than trypsin and chymotrypsin may also be important in altering the rate of intestinal protein digestion in calves of different ages. The upper and middle sections of the small intestine appeared to be more important than the lower section in digestion of dietary 12b protein, except when the high-soy diet was the source of nutrients. Intestinal contents from the middle section of the small intestine from calves fed whole milk, all-milk or promosoy diets generally contained the most protein, and trypsin and chymotrypsin activities, whereas contents from the lower section contained the least. In vitro protein digestion was greatest in middle intestinal contents, but protein digested per unit of trypsin and chymotrypsin was greatest in upper intestinal contents, and least in lower intestinal contents. Proteins in lower intestinal contents were apparently less susceptible to hydrolysis by proteolytic enzymes, either due to the nature of the protein or changes in pH. Chymotrypsin was less stable during incubation of intestinal contents than was trypsin. The ratios of chymotrypsin-to-trypsin before and after in vitro incubation of contents was 0.3“ and 0.19, respectively. In vitro stability of these enzymes tended to be less in contents from the lower than the upper or middle small intestine, although the reverse was observed in calves fed the high-soy diet. Interfering substance(s) in milk substitute diets and contents from calves fed these diets which produced an apparent esterase active ity were found to be non-lipid in nature and of low mdlecular weight. They were precipitated by CaClz or acid pH (pH 4), and in the absence of large acid precipitable molecules, passed through a dialyzing mem- brane° The exact nature of the substance(s) reacting with CaClz were not determined. Degradation of the substance(s) occurred during in vitro incubation of intestinal contents, or as the digesta traversed the small intestine. Treatment of intestinal contents with (NHu)zSOu, CaClz and adjustment to pH 8 removed most of the interfering substance(s) from 125 the resulting supernatant fluid. This treatment of intestinal contents also augmented the trypsin and chymotrypsin activities of the super- natant fluid compared with those of the supernatant fluid of untreated contents. The mechanism of the enhanced enzyme activity may be due to release of trypsin and chymotrypsin complexed with insoluble particles which are removed by centrifugation. When the soluble portion (super- natant fluid) of rat or bovine intestinal contents were treated with CaClz and (NHQZSOM the trypsin and chymotrypsin activities in the final supernatant fluid were not altered. 2. 2a. 3. 7. 10. 11. VII. REFERENCES Alumot, E., and Z. Nitsan. 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Laskowski. Action of the naturally occurring trypsin inhibitors against chymotrypsinsoc and 6 . J. Biol. Chem. 213: 609-619. 1955. Wu, I. V., and H. A. Scheraga. Studies of soybean trypsin inhibitor. I. lesicochemical properties. Biochem. 1: 698-705. 1962. Iamashina, I. The action of enterokinase on trypsinogen. Acta Chem. Scand. 10: 739-743. 1956. Yang, M. G., and J. W. Thomas. Absorption and secretion of some organic and inorganic constituents and the distribution of these constituents throughout the alimentary tract of young calves. J. Nutrition, 87: 444-458. 1965. Younathan, E. S., E. Frieden. Studies on anylase synthesis by pigeon pancreas slices. J. Biol. Chem. 220: 801-809. 1956. APPENDIX VIII. on.~ :u: :1: mm.- .lmao. o o «co. N1. Hm.o o~.HH on.~ om.HH HeH. «no. owe. mHH. e He.~ no.m. oo.m mm.mH NNH. nno. oHH. omo. H 1:: se.: nn.n om.oH o moo. AHN. “NH. 0 Hmo- oe.o no.3 oo.- mm.m cow. nnH. nno. one. NH mm.n mm.~ on.HH mm.m New. coo. nee. «OH. 0 om.n n~.e se.-H ne.HH emH. nHH. emH. ooH. H Ne.~ o-.~ on.» co.e men. neH. moo. moo. o omen -.H om.m om.n om.a ANH. amH. oHH. “no. NH mH.H om.n om.e om.mH ooH. onH. n-H. «pH. e «m.H oo.- on.m me.-H ooH. anH. o-H. nNH. H oH.e se.: n-.~ om.o Heo. moo. oHH. “NH. o amm- OOdH Oh owl: m-.m on.nH mm.eH :1: «mo. mac. nNH. o NH mm.m mH.oH oe.- oe.oH moo. oeH. non. omo. e oH.m om.m o~.- oo.mH com. com. an. omH. H no.e om.eH .n: oH.~ emu. noH. 0 “NH. o amen me.n on.e~ “H.- o~.mH HnH. n-H. con. moH. NH mm.oH oH.m on.mH o-.e emu. ANH. omH. me. e oa.m o-.- mn.oH on.eH mam. onH. moH. owe. H me.e ne.- o~.e m-.oH an. mam. an. mac. 0 mac- oe.nH on.- oH.mH ne.mH an. own. cum. omH. NH mm.e on.oH no.n m-.o~ HHc. man. now. onH. e nm.nH oo.~H om.on o~.- Hem. «ma. non. ooH. H om.m nn.oH on.eH on.nH mmH. com. mNH. meH. o omen H0 H 8 I3 HHa-mav HcHaNHev Hue-commaeeom one saw] new in mum Hm eH -, wrn‘ coHHooHHoo HHeo “when” ew< Aehmmp.ew< mdevomm esswmw, 3053.58 032.. 03388..- no soap-3338, nae-end .95 Sad:- .H can: 83.-0&4 148 149 N00 Hp\> .Hocee-ea ueNV cHa\mmHm eHoao.H Ho aHeaHoeoao .Heooe -Hce econ cHa\mz wanna-em. ,A .336 Hoeoaoeo‘osaseefiflnfidaqa .ndu sacs: be.- eea-H-e scam ease-see Heed-n35 .925 we seduce-50:3 fine-cam on.- ma .88-op .>H cane.- Nausea-.2 152 .ecH-ae-sH .. o N.- ma.e eN.- H.HH n.NH Ne.e om.m mm.e nN.N nc.- Hm.- Ne.H ne.N mo.n Na.- em.- Na.- om.o om.- eo.n ea.- NH.- HN.- Nn.- c.- -.an n.-n e.H m.HH m.n H.HN E! '3 4’ 53 E3 0 D .hHebuaoemeeu .eeHnmee veueneoma one Ave-enoosdunosv Homoseo accession H one u r. on» no . . N N.oN n.aN N.-N H.-N H.mN H.mN c.n N.eH o.NN N.nN m-.H ea.N mm.“ on.- e.mN m.eN mm.n ms.- -.6N e.HN eH.- mm.- N.eH e.-H o.-n m.Ns n.cH -.oH e.mn n.He cHoooei m age 9 O ”O‘NB e .» Hose o.oH N.«N N.nH m.m 0.5" o.b QB e e O Nah-3 @0103 GB Nv-IN H 9.2 M {x occocorn N. N e.mH m.- N.©H m.nH N.om m.fia COIN» 0 PC I 02610“ ON“) ON NHN o .w ob.- mm.- mH.- 0e.- no.- «N.- Hu.n mu.- 1 we.- no.- on.- no.- me.- He.- .- B O as U\O\b- c-uxua O ~o\6\o\6 \6\O\o \oxo 0 same N.- mH.e en.- mN.e oH.e No.- mm.e \nm wed . 0 com HO\H e e e 0000 6mm we “HO e 30mm 424: use 0 .vam .aengs eA-_seau ease-coo Heed-eeucd accession .a on .D N chased-venues o H ..N oNN 4N oHH neN ONN amN n RH 8N 8N «H- n 8N N- no: «on m onH own o-H «AN n onH oeH mmH H eHneh.Ndv:emg< Trypsin and chymotrypsin activities of intestinal contents before and after in vitro Appendix Table V. incubation from calves fed four different dietsand sacrificed.at three different ages. LC sin Mi t ND units ml UC I LC (units‘fml UC1 Calf Feeding eriod wk Whole milk N83 OOH 11A 22A 26A 1 1 1 mmmm 153 use. O\U\(\N 0.29 0.93 1.78 0.85 1.17 5 3.00 5 8M 0 e 0 0H0 O\N H5- e-le-IO (“\0 09:0 923133 060 0.32 0.83 d'Ox Na eee GHQ 3033?: 0H0 1.03 1.82 Ga?“ O\HO NM# (“mm 0.40 2.01 9.49 0.51 2.95 11.91 1e 12.88 15-55 1. 13-33 16.67 0e96 2.95 1.70 3.01 5.36 4.88 33 47 M 0.000000000000000 .AopuSIHocdnpme was .0303 ha. 0000.32 0.33 as use as £0.23 50 93.30.90 0008.3 mo 03.000800 H0030? 50 momfino .00.“ 003090.000 00.00.050.000 0000005.: .n0a\0>\> .000000oa 0000 0000 00030.0 00 000000000: 00000o 00:0 0000 .00a\0z<0 00051.0 00 0000000000 000000 00:: 0000 .>H 0.30.0. 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K000080300. 155 00.0 00.0 00.0 0 0 00.0 00.00 00.00 0 0 00 00.0 00.0 00.0 0 0 00.0 00.00 00.00 0 0 0: 00.0 00.0 00.0 0 000. 00.0 00.0 00.0 0 000. 00 00.0 00.0 00.0 0 0 00.0 00.00 00.00 0 0 00 00.0 00.0 00.0 0 0 00.0 00.00 00.0 0 0 0: 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 000. ~00. 00 0000 00.0 00.0 00.0 0 0 00.0 00.0 00.0 0 0 00 00.0 00.0 00.0 0 000. 00.0 00.00 00.0 0 0 .0: 00.0 00.0 00.0 0 000. 00.0 00.0 00.0 0 000. 00 00.0 00.0 00.0 0 0 00.0 00.0 00.0 000. 000. 00 00.0 00.0 00.0 000. 000. 00.0 00.00 00.0 000. 000. 0: 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 000. 000. 00 <00 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 000. 000. 0: 000 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 0 000. 0: 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 0 000. 000 000 “H0033? “5.0 00000 “0000000800 00040000000 0 0 0 0 0 0 000 0 0 0 0 0000000 0000 00000 00000000 0000000.0 00000 00000 n=;000000wmw. 00mmmm00uummmmm 00000 fimagamo 0.0de9 ImovGH .w mm on 0285005000 020 030.0030 8:00.000 6000.00.50 8000:0053 0.3.03 00.089080 0000. .00 #:0030095 .3091 00:0 0.3ch 00.20 000050.00 9.3 new 090.000 08.0.0 000000.080 030000.00ch .00 00000.0.“ 0000000006900 05. 5.0 03.03.0000. 9009000000020 U20 00.009000 .H> OHDQH Nameconmmdw 156 .:oapan:0:0 0000> :0 0:0: 030 a 00000 0:0 000009 00:00:00 #:0000000 H 0:0 0 .0H0>0000m000 .0:00000:0 Hausa 000 no upanu_u0soH 0:0 0vafis .0000: 0:» 8000 00:00:00 H0:00000:0 0:000:000 A.v:0 z .50 .:AE\A>\> .Ho:dg00: $0.3v mmam 0H0E1.0:o mo mfithOHUA: madsvo 000:: 0:0m .>.00000 00000000 .0 00000000 000 0 .w mm 00 0kuoazo Edaoado 0:0 opQMHum 5:0:0820 £003.0000000 00:00:00 Hu:0pmop:0 mo vfinam 0:000:0000mn .00:00:00 H0:00000:0 vopaoupcn mo vwaah 0:000:00mzmm .mmem no mz¢a m:0:00¢:00 0:0000000 you 000000 0800 0:00 0:» 0:0000 :0E\0000:0U Haowuno :0 00:0:0 0A» 800% 0090000950 0003.000000 can no :08 0 on m Scum :0E\0000:0v duowamo :0 00:000 one .000000000000 000000.000000000000 000 0000000 000 0>\>0 00000000 000 000 00000 00 00000000.0000 000 02000 00.0 00.0 00.0 000. 000. A00 00.0 00.0 000. 000. 00.2» 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 000. 000. 00 00.0 00.0 00.0 000. 0 000. 00.0 00.0 00.0 000. 000. 00 00.0 00.0 00.0 000. 0000. 00.0 00.0 00.0 000. 000. 02 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 000. 000. 00 000 00.0 00.0 00.0 0 0 00.0 00.0 00.0 0 0 00 00.0 00.0 00.0 0000. 0000. 00.0 00.0 00.0 0 0 H: 00.0 00.0 00.0 0 0 00.0 00.0 00.0 0 0 00 00.0 00.0 00.0 0 0 00.0 00.0 00.0 0 0 00 00.0 00.0 00.0 0000. 0000. 00.0 00.00 00.0 0000. 0000. 02 00.0 00.0 00.0 000. 000. 00.0 00.0 00.0 0 0 00 000 00300003 JG? \0000 10000300006 0000303 000 0 0 0 0 000 0 0 0 0 0000000 0000 00000 :100000000 00000000 00000 00000 .. 000000001- 00000000.0 00000 00000 mmmmummmmxmw .mmwnwmmr, I000:H AU0::0p:oov .H> 0H909 NHU:0QQ< 157 Appendix.Table VII. Release of proteolytic enzymes from.bovine pancreas tissue slices during in vitro incubation and total enzyme activity in tissue plus media as influenced by pancreozymin. In tro c b tiS" Secretion Pancreo- Non-incubated ( Hedi; ) Apparent 2 z in tissue med Tissue Media t ssu s thesis pg ml units proteolytic activity o 270“ 212 186 . 53 1 .47 1 270 206 150 .58 1.32 10 270 218 116 .65 1.2a _;gp 270 221 98 .0. __.70 1,22 1Tissue homegenised in 0.1514 NaCl and activity determined in supernatant fluids 2Ratio of enzyme activity in incubated tissue plus media to non-incubated tissue. 3One unit equals release of 1‘pmole tyrosine equivalent (OD -1-.OD3/2 at 280 mu)/min/g fresh tissue. “All data average of tissue slices from.“ calves. Appendix Table VIII. Total proteolytic activity in bovine pancreas tissue slices and incubation media before and after in vitro incubation as influenced by amino acid supplementation.1 Casein Non-incubated Incubated Incubated tissue +'gggia dro s to tissue t ssu + medi Non-‘n b ted t ssue units proteolytic activit o 1743 zua 1.39 .1 17h 250 1.38 .2 174 250 1.39 .# 0f17h 2H8 1.4h 1800 footnote 1. Appendix Table VII. 2One unit equals ilnmole tyrosine equivalent (OD +.0D3/2 at 280 mp). released/min/g tissue. 3Average of tissue slices from 3 calves. 158 Appendix Table IX. Total trypsin and chymotrypsin activity in the incu- bation media and incubated pancreas tissue slices from calves fed four diets at three ages and the 1 effect of amino acid supplementation of the media. TEL—sin M Source and Krebs + Casein Krebs + Casein treatment of 2 Total hydr, Total dr. tissue sl ces N tivit r be t vi Krebs innitsBSmg tissue {unitsgamg tissue dry matter) dry matter) Incubation media Krebs 26 4.215 -- .92 -- Krebs + casein hydr. 26 4.53 1.08 .94 1.02 Interactions Diet x incubation media Whole milk + Krebs 9 5.24 -- 1.18 -- + Krebs + casein hydr. 9 5.60 1.07 1.21 1.03 All-milk replacer +-Krebs 8 3.88 -- .85 -- + Krebs + casein hydr. 8 4.27 1.10 .89 1.05 High-soy replacer + Krebs 9 3.47 -- .72 ~- + Krebs + casein hydr. 9 3.69 1.06 .71 .99 Age x incubation media 1 week + ' Krebs 10 4.21 -— .98 -- + Krebs + casein hydr. 10 4.51 1.07 .96 .98 3 weeks + Krebs 7 4.05 -- .96 -- + Krebs + casein hydr. 7 4.15 1.02 .97 1.01 6 weeks + Krebs 9 4.33 -- .82 -- 4-Krebs + casein 1r____gyg;. 9. 4.84 1.1g_» .90 1.10 See footnote 1. Appendix Table VII. Number of values/mean. 2000 unit equals hydrolysis of 1 00010 TAME/min. One unit equals hydrolysis of 1‘umole BTEE in 26% methanol (V/V)/min. 5N0 signigicant differences (P<fi.05) between incubation media and inter- actions with diet or age. ‘ k i m .03 .J -g. {murmulmmmm‘ we “a...“ t 159 .oflmmwu vovsnfiofiinoc 3% mad 25mg. bandages“ 5m hugwpou ashaso mo capes an vopmHsoHuo mamongshm 090w» sH .mooaam odmmwp msososem onfi>on ha weakens owthooponm mo mfimonpshm onufi> ca weekends so ems mo uoommm .H .wwm Naonomg¢ mu< mm