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Absorption of amino acids and B vitamins from the rumen Smith, Frederick Dabell 1959-12-31

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ABSORPTION OF AMINO ACIDS AND B VITAMINS FROM THE RUMEN by FREDERICK DABELL SMITH M.A», University of British Columbia, 1951 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE3REE OF DOCTOR OF PHILOSOPHY in the Department of Zoology W© accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1959 i i i ABSTRACT The purpose of this investigation was to provide direct experimental evidence for or against the suggestion that amino acids and B vitamins are among the nutrients absorbed from the rumen of the ruminant. The investigation was subdivided into two parts: (l) six ruminal fistula experiments and (2) nine blood-sampling experiments. The goat (Capra) was the experimental animal. In the ruminal fistula experiments, solutions of amino acids, B vitamins, and usually propionic acid were added to an empty rumen for 120 or 150 minutes. "Propionic acid, a substance known to be absorbed from the rumen, was included in most of the solutions to provide a positive- control for ruminal absorption. In addition, in a l l but the first two experiments, polyethylene glycol was added to the solutions in order to provide a marker substance for differential water movement into or out of the rumen. Controls for the experiments included the taking of a sample from the solution in the rumen at zero time and incubating the sample for the duration of the experiment in a water-bath held at 37°Co The ruminal and control solutions were sampled at periodic intervals during the experiment. The results demonstrated not only marked decreases in the concentrations of propionic acid in the ruminal solutions but also, in most cases, marked decreases in the concentrations of the amino acids and the B vitamins assayed: tryptophan, methionine, tyrosine, glycine, lysine, riboflavin, nicotinic acid, and pantothenic acido In contrast, the concentrations of these constituents in the control solutions usually demonstrated either no decrease or a small increase. In the first three blood-sampling experiments, the concentrations of amino acids and nicotinic acid in the plasma draining the rumen were compared with the concentrations in the peripheral plasma. The results demonstrated that seldom were the concentrations of an amino acid or nicotinic acid higher in the plasma draining the rumen than in the peripheral plasma. The results did suggest, however, that there had "been slight increases in the plasma concentrations of the amino acids with time. Accordingly, the next six blood-sampling experiments were designed to determine i f time-related increases in the blood concentration of alpha amino nitrogen occurred after the addition of amino acids to the rumen. Blood samples were removed periodically from anesthetized goats, both before and after the addition of a solution of amino acids to an empty rumen. The results demonstrated time-related increases in the blood concentrations of alpha amino nitrogen. These results and those obtained for the ruminal fistula experiments are interpreted as supporting the suggestion that amino acids and B vitamins are absorbed from the rumen* In the General Discussion, a preliminary attempt is made to answer three questions! 1) What is the manner of the passage of amino acids and B vitamins across the ruminal epithelium? 2) Does this passage of amino acids and B vitamins occur under normal feeding conditions? 3) How does this passage of amino acids and B vitamins across the ruminal epithelium f i t into the scheme pictured for the metabolism and final fate of these compounds in the ruminal contents? As a part of the attempt to answer the first question, literature reviews are presented on the manner of absorption of amino acids and B vitamins in V the small intestine, kidney, placenta, and other tissueso Then, after a literature review on the manner of absorption of substances other than amino acids and B vitamins from the rumen, the data of the present investigation are examined for evidence as to the manner of absorption of amino acids and B vitamins from the rumen. The examination led to the following statements the movement of amino acids across the ruminal wall of the goat is determined by a summation of the effects of chemical, electrical, and possibly metabolic potentials. Unfortunately, other than to suggest that the process of simple diffusion is involved, the nature of the data for the B vitamins was not such as to yield much information on their manner of absorption* In the attempt to answer the second question, the fulfillment or non-fulfillment of the following conditions is consideredj (l) the demonstration of the presence of amino acids and B vitamins in the rumen, (2) the demonstration of the relation of the concentrations of individual amino acids and B vitamins in the ruminal liquor to their concentrations in the blood plasma, and (3) the demonstration of the absorption of individual amino acids and B vitamins from an ingesta-filled rumeno Although fulfillment of the third condition was not obtained, fulfillment of the first two conditions was obtained and led to the statement that a strong possibility exists that the ab sorption of amino acids and B vitamins occurs from the rumen under normal feeding conditions. In the attempt to answer the third question, a number of factors are discussed in turni (l) the environmental conditions of the rumen, (2) the influence of the diet on the composition of the population of microorganisms in the rumen (3) the physical and chemical nature of the dietary constituents, (4) the relative stability of v i amino acids and B vitamins in the ruminal ingesta, and (5) the physiological state of the animal. Evidence is presented to demonstrate that each of these factors probably exerts an important influence on the content of amino acids and B vitamins in the rumen. It is suggested that, when the interaction of the many factors is such as to produce higher concentrations of amino acids and B vitamins in the rumen, the proportions of amino acids and B vitamins that pass from the rumen by absorption through its wall will bs an important pathway for these nutrients • To conclude the General Discussion, the possible nutritional implications of the absorption of amino acids and B vitamins from the rumen are considered.. One suggestion made is that a general absorption of nutrients from the rumen would permit the simultaneous arrival at the liver, or elsewhere in -Hie body, of volatile fatty acids, amino acids, B vitamins, and inorganic ions. This should promote more efficient utilization of each of the nutrients. Another suggestion made is that ruminal absorption of amino acids and B vitamins places the host ruminant in a better competitive position relative to its contained microbial population in the competition for nutrients. Finally, a suggestion is made that the action of the ruminal wall in absorbing amino acids helps to explain the results that have been obtained for certain nitrogen retention experiments in ruminants. Faculty of Graduate Studies P R O G R A M M E O F T H E FINAL ORAL EXAMINATION FREDERICK DABELL SMITH B. A., B. S. A., British Columbia, 1947 M. A., Briiish Columbia, 1951 IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING SATURDAY, SEPTEMBER 26th, 1959 AT 9:30 A. M. FOR THE DEGREE OF DOCTOR OF PHILOSOPHY C O M M I T T E E I N C H A R G E DEAN G. M. SHRUM: Chairman A . J . WOOD W. D. KITTS D. C. B. DUFF H. COPP W. S. HOAR I. McT. COWAN J. HALLAMORE B. A. EAGLES External Examiner: Dean E . C. STONE Washington State College ABSORPTION OF AMINO ACIDS AND B VITAMINS FROM THE RUMEN • ABSTRACT The purpose of this investigation was to provide direct experimental evidence for or against the suggestion that amino acids and B vitamins are among the nutrients absorbed from the rumen of the ruminant. The investigation was subdivided into two parts: (1) six ruminal fistula experiments and (2) nine blood-sampling experiments. The goat (Capra) was the experimental animal. In the ruminal fistula experiments, solutions of amino acids, B vitamins, and usually proprionic acid were added to an empty rumen for 120 or 150 minutes. Propionic acid, a substance known to be absorbed from the rumen, was included in most of the solutions to provide a posiiive control for ruminal absorption. In addition, in all but the first two experiments, polyethylene glycol was added to the solutions in order to provide a marker substance for differential water movement into or out of the rumen. Controls for the experiments included the taking of a sample from the solution in the rumen at zero time and incubating the sample for the duration of the experiment in a water-bath held at 37 °C. The ruminal and control solutions were sampled at periodic intervals during the experiment. The results demonstrated not only marked decreases in the concentrations of proprionic acid in the ruminal solutions but also, in most cases, marked decreases in the concentrations of the amino acids and the B vitamins assayed: tryptophan, methionine, tyrosine, glycine, lysine, riboflavin, nicotinic acid and pantothenic acid. In contrast, the concentrations of these constituents in the control solutions usually demonstrated either no decrease or a small increase. In the first three blood-sampling experiments, the concentrations of amino acids and nicotinic acid in the plasma draining the rumen were compared with the concentrations in the peripheral plasma. The results demonstrated that seldom were the concentrations of an amino acid or nicotinic acid higher in the plasma draining the rumen than in the peripheral plasma. The results did suggest, however, that there had been slight increases 'in the plasma concentrations of the amino acids with time. Accordingly, the next six blood-sampling experiments were designed to determine if time-related increases in the blood concentrations of aloha amino nitrogen occurred after the addition of amino acids to the rumen. Blood samples were removed periodically from anesthetized goats, both before and after the addition of a solution of amino acids to an empty rumen. The results demonstrated time-related increases .in the blood concentrations of alpha amino nitrogen. These results and those obtained for the ruminal fistula experiments are interpreted as supporting the suggestion that amino acids and B vitamins are absorbed from the rumen. In the General Discussion, a preliminary attempt is made to answer three questions: 1) What is the manner of the passage of amino acids and B vitamins across the ruminal epithelium? 2) Does this passage of amino acids and B vitamins occur under normal feeding conditions? 3) How does this passage of amino acids and B vitamins across the ruminal epithelium fit into the scheme pictured for the metabolism and final fate of these compounds in the ruminal content? To conclude the General Discussion, the possible nutritional implications of the absorption of amino acids and B vitamins from the rumen are considered. PUBLICATIONS Smith, Frederick D. Suffield Technical Paper No. 10 (Classified Material Relating to the Use and Care of Laboratory Animals in Biological Warfare Experiments), Defence Research Board of Canada, December 31, 1951. GRADUATE STUDIES Field of Study: Zoology (Physiology) Experimental Zoology W. S. Hoar Comparative Physiology W. S. Hoar General Physiology J. Allardyce General and Cellular Physiology L. B. Kirschner (Wash. State Univ.) Principles and History of Biology W. A. Clemens Special Advanced Course W. A. Clemens Other Studies: Biochemistry B. Eagles Advanced Animal Nutrition A. J. Wood Calculus J. E. A. Parnall Veterinary Pharmacology P. A. Klavane (Wash. Sta e Univ.) T H E S T A T E C O L L E G E O F W A S H I N G T O N P U L L M A N , W A S H I N G T O N C O L L E G E DF VETERINARY MEDICINE DEPARTMENT OF ANATOMY October 1, 19^ 9 Director of Library, University of British Columbia, Vancouver 8, Canada. Dear Sir: About a month ago your assistant checked over my thesis for library acceptance. At that time he suggested that the Abstract was too long for publi cation. Although I have not altered the length of the Abstract in the thesis, I am enclosing a shorten ed form of the Abstract that can be used for publi cation purposes. Yours sincerely, FDS:jh Frederick B. Smith Assistant Professor. TO MY WIPE In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ,—j-<9-Q-i Q- The University of B r i t i s h Colombia, Vancouver Canada. Date J l ^ ^ P w - ^ W V 3 / ? S % TABLE OF CONTENTS Page GENERAL INTRODUCTION The Activities of the Microbial Population in the Ruminant Stomachj A Brief Re-view Microbial Population 2 Symbiosis between the Ruminant and its Microbial Population • • • • • • • 7 The Ruminant Microbial Population Summary Absorption from the Rumens a Review General • 11 Substances Absorbed . . . . . 12 Comment • • • • • • • • • . • • . • . * . . 13 Amino Acids • • • • • • • . . . . . . . . 13 Vitamins . . . . . . . . . . . . . 15 Comment EXPERIMENTATION Part I t Ruminal Fistula Experiments Introduction Method Experimental Procedure . . . . . . . . . . . . X9 General Experiment Wl Experiment W2 Experiment W3 Experiment W4 Experiment W5 Experiment 1176 Listing of Individual Constituents for Experiments Wl to W6 Assay Procedures • • • • • • • • • • • • • • • • • • • • • • • • Pectin . Polyethylene Glycol Propionic Acid Amino Acids B Vitamins Results Pectin Polyethylene Glycol Propionic Acid • . Amino Acids Alpha Amino Nitrogen • • • • 66 B Vitamins . . . * . . . . . » . . . . . . . . . . . . . 70 Temperature and pH • • • • • • • • • • • • • 86 Discussion Stability of Amino Acids and B Vitamins During Freezing Storage 89 Evidence for Instability Evidence for Stability. Fluctuations of Constituents* Concentrations • • • • • • • • • • • • • • 96 Factors Causing Concentration Increases Factors Causing Concentration Decreases Comment Part II: Blood-Sampling Experiments Phase A: Comparison of the Concentrations of Amino Acids and Nicotinic Acid in Peripheral and Portal Blood Introduction Page 31 36 43 AA. Page Method 102 107 Results Discussion Phase Bj Increase in the Concentrations of Amino Acids in Peripheral and Portal Blood with Time Introduction Method Experimental Procedure • • • • • • • • • • • • • • • • • • • • • • • • 119 General Experiment B4 Experiment B5 Experiment B6 Experiment B7 Experiment B8 Experiment,B9 Assay Procedure • •* Results Discussion Fluctuations in the Concentrations of Amino Acids in the Blood • * . • Comparison between the Results of Annison and Those of the Present Investigation • • • • • • • • • • • • • • • • • • • • • GENERAL DISCUSSION Experimental Procedure General Experiment Bl Experiment B2 Experiment B3 Assay Procedure 123 143 145 Part At Amino Acids Manner of Absorption of Amino Acids from the Rumen Literature Review on the Manner of Absorption of Amino Acids . 151 X Page Small Intestine Kidney and Placenta Other Tissues Summary and Comment Literature Review on the Manner of Absorption of Substances from the Rumen . . . . . . . . . . . . . 166 General Comment Evidence from the Present Experiments as to the Manner of Absorption of Amino Acids from the Rumen 172 Introduction Effect of Initial Electrochemical Gradient on Rate of Absorption Comparison Among the Five Amino Acids of their Rates of Absorption Transfer of Amino Acids Against an Electrochemical Gradient Summary and Conclusions Amino Acid Absorption from the Rumen under Normal Feeding Conditions Introduction 206 Condition l i Demonstration of Amino Acids in the Rumen . . . . . . . . 207 Condition 2» Demonstration of an Electrochemical Gradient . . . . . . 209 Condition 3: Demonstration of Absorption from an Ingesta- Filled Rumen 210* Comment • 211 The Effect of Ruminal Absorption on the Fate of Amino Acids Present in the Rumen Introduction • 213 Influence of Environmental Conditions in the Rumen on the Fate of Amino Acids in the Rumen 216 Temperature pH Carbon Dioxide and Oxygen Influence of Diet on the Composition of the Population of Micro organisms in the Rumen and, therefore, on the Fate of Amino Acids in the Rumen . . . . . . . . . . . . . . . . . . . . 220 Page Discontinuous Variations Adaptive Enzymes Population Variations Comment Influence of the Physical and Chemical Nature of the Dietary' . Constituents on the Fate of Amino Acids in the Rumen . . . . . . 224 Influence of the Relative Stability of the Amino Acids on their Fate in the Rumen • • • 225 Special Comment Influence of the Physiological State of Activity of the Animal on the Fate of Amino Acids in the Rumen . . . . . . . . . . . . 228 Comment 230 Part B: 3 Vitamins Manner of Absorption of B Vitamins from the Rumen Literature Review on the Manner of Absorption of B Vitamins from the Small Intestine • 231 Introduction Riboflavin Thiamine Other B Vitamins Comment Evidence from the Present Experiments as to the Mechanism of Absorption of B Vitamins from the Rumen . . . . . . . . . . . . 238 Introduction Effect of Initial Electrochemical Gradient on Rate of Absorption Comparison Among the Three B Vitamins of their Rates of Absorption Transfer of the B Vitamins Against an Electrochemical Gradient Comment B Vitamin Absorption from the Rumen Under Normal Feeding Conditions Introduction 249 Condition l i Demonstration of B Vitamins in the Rumen . . . . . . . . 250 Condition 2j Demonstration of an Electrochemical Gradient . . . . . . 255 x i i Condition 3s Demonstration of Absorption from an Ingesta- x a ° e Filled Rumen 256 Comment . • • o * « * > * « » » e * e « « > « « * « « * * » . • 257 The Effect of Ruminal Absorption on the Fate of B Vitamins Present in the Rumen Introduction. • • • • • 259 Influence of the Diet on the Composition of the Population of Microorganisms in the Rumen and, therefore, on the Fate of B Vitamins in the Rumen • • • • • • • • • • • • • • • 262 Comment • « • • • « . * • < • • « . • • • • • • • • • • • • • • 266 . Part Cs The Possible Nutritional Implications of the Absorption of Amino Acids and B Vitamins from the Rumen. Introduction • • • • • • • • • •••••<>••••• 267 The Time Factor* • . • » » « « « » « e * o * » a » s » « e o » 267 The Competitive Factor • • o * . • • • • • • • • • • • < > • • • 270 Introduction Competitive Actions of the Microbial Population for Amino Acids and B Vitamins Competitive Actions of the Host for Amino Acids and B Vitamins The Beneficial Effect of Ruminal Absorption to the Host in its Competitive Actions for Amino Acids and B Vitamins Special Comment: The Application of the Concept of Ruminal Absorp tion of Amino Acids to Explain Certain Results of Nitrogen Retention in Ruminants » • • * • • < > • • « • • • • • • • 275 CONCLUDING COMMENT APPENDIX Ruminal Fistula Operation Carotid Loop Operation Cannulation of Ruminal Veins Operative Procedure for Experiments B3 to B9 Polyethylene G-lycol Assay Propionic Acid Assay Microbiological Assay of Amino Acids and B Vitamins Assay of Amino Acids as Alpha Amino Nitrogen LITERATURE CITED xiv LIST OP TABLES Table Page 1 « The results of an experiment by Annison on ruminal absorption of amino acids • • • • • • < > * a a a a a » » . 1 6 2 a Composition of the experimental solution for experiment Wl • • • a a • 2 3 3 a Composition of the experimental solution for experiment W4 a a a a a a a a a a a a a 2 8 4 a Composition of the experimental solution for experiment W 5 . a . . a * . . a a » . a . a a a e a a a a 3 0 5 a Complete listing of the constituents present in the experimental solutions » » m m . . » » , 3 2 6 « Ruminal fistula experiments: changes in weights of polyethylene glycol precipitates ••m»»»»»»mm 3 7 7 a Ruminal fistula experiments: changes in concentration of propionic acid 4 5 8 a Ruminal fistula experiments: changes in concentration of tryptophan . . . . a a 4 9 9 a Ruminal fistula experiments: changes in concentration of methionine , 5 2 1 0 a Ruminal fistula experiments: changes in concentration of tyrosine . ....•......<>... 5 5 1 1 a Ruminal fistula experiments: changes in concentration of glycine 5 8 1 2 a Ruminal fistula experiments: changes in concentration of lysine . a a . a » a . « . . . . a . * a 6 2 1 3 a Ruminal fistula experiment W 6 » changes in concentration of alpha amino nitrogen a a a a a a o a e c 6 7 1 4 a Ruminal fistula experiments: changes in concentration of riboflavin a a . a a • a a 7 1 1 5 a Ruminal fistula experiments: changes in concentration of nicotinic acid . . a . a * . . . . . . a 7 7 XV Table Page 16. Ruminal fistula experiments: changes in concentration of pantothenic acid . . . . . . . . . . . . 81 17. Ruminal fistula experiment si temperature and pH measurements • . . . . . . . 87 18. Ruminal fistula experiments: a comparison between the calculated and analytically-determined concentrations in the original experimental solutions • 91 19. Blood-sampling experiment Blj concentration of lysine • 109 20. Blood-sampling experiment B2: concentration of alpha amino nitrogen, nicotinic acid, and glycine . . . . . . . 110 21. Blood-sampling experiment B3» concentration of alpha amino nitrogen • • • • • . . . 113 22. Blood-sampling experiment B4j changes in concentration of alpha amino nitrogen . « • • • • • • • • 125 23. Blood-sampling experiment B5» changes in concentration of alpha amino nitrogen • 128 24. Blood-sampling experiment B6j changes in concentration of alpha amino nitrogen . . . . 131 25. Blood-sampling experiment B7j changes in concentration of alpha amino nitrogen . . . . . . . . . . . . . 134 26• Blood-sampling experiment B8» changes in concentration of alpha amino nitrogen . . . . . . . . . . . . 137 27. Blood-sampling experiment B9: changes in concentration of alpha amino nitrogen . . . . . . . . . . . . . . . . . . 140 28. Comparison between the blood-sampling experiments of Annison and those of the present investigation • • • • 146 29. Ruminal fistula experiments: a tabulation, for the amino acids, of the i n i t i a l electrochemical potential differences, of the absolute decreases of concentration, and of the decreases of concentration relative to the i n i t i a l electrochemical potential differences 180 30. Concentration of five amino acids in the plasma of ruminants and man • « . . . . . « 182 xvi Table Page 3 1 * Ruminal fistula experimentsi a tabulation, for propionic  acid, of the i n i t i a l concentrations, of the absolute decreases of concentration, and of the decreases of concentration relative to the i n i t i a l concentrations a a • 1 8 9 3 2 a Ruminal fistula experiments: a tabulation of data that are utilized to make a prediction of the rank-order for the rates of ruminal absorption of the amino acids a a a a . 1 9 2 3 3 a Ruminal fistula experiments: estimated chemical and electrochemical gradients existing between the ruminal solutions and plasma of the ruminal veins when each amino acid reached its lowest concentration in the ruminal solutions a 2 0 1 3 4 a Ruminal fistula experiments: a tabulation, for the B vitamins, of the i n i t i a l electrochemical potential differences, of the absolute decreases of concentration, and of the decreases of concentration relative to the ini t i a l electrochemical potential difference • • a . . • 2 4 1 3 5 a Ruminal fistula experiments: a comparison, for the B vitamins, between the lowest-found concen trations in the ruminal solutions and the concentrations in the plasma and blood . . . . . . . . . 2 4 3 3 6 a Ruminal fistula experiments: a tabulation of data that are utilized to determine the influence of the molecular size of the B_ vitamins on their rates of ruminal absorption . 2 4 6 3 7 a Concentrations of the B vitamins in the ruminal ingesta and plasma of the sheep and cow 2 5 1 3 8 « Ruminal fistula experiments: a comparison, for the B vitamins, between the lowest-found concen trations in the ruminal solutions and the concentrations in the ruminal ingesta • • • • a ' 2 5 8 3 9 a Weights of polyethylene glycol complex precipitated from control solutions a a a a . 2 9 9 4 0 a Analytically-determined concentrations of propionic acid in a control solution . . . . 3 0 2 4 1 a Analytically-determined concentrations of alpha amino nitrogen in control samples . • T~a . . . . . . . 3 0 7 LIST OP FIGURES Figure Page 1. The ruminal and reticular cavities, viewed from the left 3 2. Sheep stomach, viewed dorsally and partly from the left 5 3. Ruminal fistula experimentst changes in weights of polyethylene glycol • • • • * • • • • • • • • • • • • • • 38 4. Ruminal fistula experiments: changes in concentration of propionic acid • • • • • • • • • • • • • • • 46 5. Ruminal fistula experiments: changes in concentration of tryptophan • 50 6. Ruminal fistula experiments: changes in concentration of methionine 53 7. Ruminal fistula experiments: changes in concentration of tyrosine . . . . . . . . . . . . . . . . . . 56 8. Ruminal fistula experiments: changes in concentration of glycine 59 9. Ruminal fistula experiments: changes in concentration of lysine 63 10. Ruminal fistula experiment W6: changes in concentration of alpha amino nitrogen • • • • 68 11. Ruminal fistula experiments: changes in concentration of riboflavin . . . . . . . . . 74 12. Ruminal fistula experiments: changes in concentration of nicotinic acid . . . . . . . . . . . . . . 79 13. Ruminal fistula experiments: changes in concentration of pantothenic acid • • 83 14. Venous drainage of the ruminant stomach, viewed from the right 103 15. Blood-sampling experiment B3: changes in concentration of alpha amino nitrogen H4 x v i i i Figure Page 16. Blood-sampling experiment B4j changes in concentration of alpha amino nitrogen 126 17. Blood-sampling experiment B5s changes in concentration of alpha amino nitrogen . . . . . . . . . . . . . . . . . 129 18* Blood-sampling experiment B6s changes in concentration of alpha amino nitrogen •• 132 19. Blood-sampling experiment B7s changes in concentration of alpha amino nitrogen • • • • • • • • • • • 136 20. Blood-sampling experiment B8s changes in concentration of alpha amino nitrogen • 138 21• Blood-sampling experiment B9s changes in concentration of alpha amino nitrogen 141 22• The principal reactions of nitrogenous compounds in the rumen 214 23. The principal reactions of the B vitamins in the rumen . . . . 260 24. The position of the ruminal fistula. 280 25. Ruminal fistula operation* the i n i t i a l skin incision . • • • 283 26. Ruminal fistula operations exposure of the ruminal wall . . . 283 27. Ruminal fistula operations suturing of the ruminal wall to the skin . . . . 285 28. Ruminal fistula operations excision for removal of the ruminal wall . • • 285 29. Ruminal fistula operations fistula plug inserted . . . . . . 285 30. Carotid loop operations the approach for finding the common carotid artery . . . . . . . . . . . . . . . . . . 288 31. Carotid loop operations exposure of the common carotid artery and the vagosympathetic nerve • • 288 32. Carotid loop operations exposure of the common carotid artery free from other structures . . . . . 288 33. Carotid loop operations formation of the band of skin . • • • 288 34. Carotid loop operations placement of the common carotid artery in the band of skin 291 xix Figure Page 35• Carotid loop operation: formation of skin tube containing the common carotid artery and suturing of skin on the neck 291 36• Carotid loop operation: skin tube after the completion of the suturing 291 37. Carotid loop operation: position of the skin tube or "carotid loop" on the neck of the goat 291 ACMOWLEDGMEITTS Although, i t was done under the auspices of the Department of Zoology, most of the work described in this dissertation was conducted in the Laboratory of Animal Nutrition, Division of Animal Science, University of British Columbia (U«B.C.) and in the College of Veterinary Medicine, Washington State University (W.S.U.). I express my gratitude to the people in these institutions for their willing cooperation* Among the many people who tendered equipment and facilities, I especially mention Dr. A« J» Wood (Division of Animal Science, U.BoC), Dr. J. A. McCurdy (Chairman, Depart ment of Veterinary Anatomy, W.S.U.), Dr. G-. H. Keown (Department of Veterinary Clinical Medicine and Surgery, W.S.U.), Dr. R. D. Conrad (Depart ment of Veterinary Microbiology, W.S.U.), Dr. D. M. Fluharty (Department of Veterinary Pathology, W.S.U.), and Dr» L» B. Kirschner (Department of Zoology, W»S«U»)o I also express my gratitude to Drs. W. Hawkins, Ro Otto, and R. Loan for their assistance, while they were students in the College of Veterinary Medicine (W.S.U.), in the surgical procedures; to Mr. R. L« Perkins, a student in the College of Veterinary Medicine (W.S.U.), for his execution of Figures 1, 2, 14 and 24 and for his production of India ink copies of the other figuresj to Mr. R. Ediger, also a student in the College of Veterinary Medicine (W.S.U.), for his insertion of the lettering in the figures; and to Mrs. L. Stratton for her efforts to type this dissertation within a very limited period of time* To Dr. W. A. Clemens (Head Emeritus, Department of Zoology, U.B.C.), to Dr. I. MoT. Cowan (Head, Department of Zoology, U.B.C.), and to Dr. J. A. McCurdy (in whose Department of Veterinary Anatomy I have worked as an instructor since 1952) I express my appreciation for the latitude xxi and freedom that they allowed me in f u l f i l l i n g the dissertation requirement for the doctorate degree. To Dr. G-. H. Keown I also express my apprecia tion for his counsel and, in the early stages, assistance i n the surgical procedures. To Dr. L. B. Kirschner I express my appreciation for many f r u i t f u l discussions. Finally, to Dr. A. J . Wood I state my deep gratitude for his perceptive suggestions, for his many helpful and stimulating discussions, and most of a l l for his awareness, understanding, and wisdom in knowing that I needed time and solitude to work out, by myself, the many complex problems of this dissertation. T H E S T A T E C O L L E G E O F W A S H I N G T O N P U L L M A N , W A S H I N G T O N J9 C O L L E G E OF VETERINARY MEDICINE DEPARTMENT OF ANATOMY December 21, 1959 4- fr <>' Director of the Library, University of B. C , Vancouver 8 , Canada. Dear Sir, I recently completed my thesis for the doctorate degree in Zoology at your university. The thesis has been accepted and i s i n your possession. Unfortunately, in going through my own copy of the thesis, I now find that there are s t i l l three small errors: 1) page 196, line 6 from the bottom should have "or" replaced by "over" and should read as case the force favouring glycine over tryptophan, tyrosine, or lysine ; 2) page 2U7, footnote C should have the table and page number changed from "35" to n3hu and "I96" to " 2 i i l " , respectively, and should read as 5lhese:.:data are taken from Table 3J4 on page 2kL.} 3) page 303* line 6 from bottom should have the word "his" inserted before the last two words in the sentence and should read as perhaps the growth obtained by Barton- Wright i n his supposedly biotin- . Could you please have these errors corrected for me. I do not know what procedure you follow i n these cases and, therefore, I give to you the right to make the appropriate changes i n whatever manner that you think i s the most suitable. lours sincerely, Frederick D. Smith Assistant Professor. FDS:jh 1. GENERAL INTRODUCTION The bovids provide the only satisfactory intermediary by which grass can be used as a contributor to human l i f e * This, with their peaceful and gregarious disposition, has made them become our most important commensal* If there were no Bovidae there would be fewer human beings in the world, and our social organization would be very different* J. Z. Young in The Life of Vertebrates (419)* The term ruminant is a common name for a group of mammals designated by Simpson (341) as Peoora, an infraorder within the order Artiodactyla* The infraorder Pecora is further subdivided by Simpson into three super- families* Cervoidea, G-iraffoidea, and Bovoidea* These superfamilies are typified by the deer, the giraffe, and the ox, respectively* The goat, the animal used in the present investigation, belongs to the superfamily Bovoidea, family Bovidae, subfamily Caprinae, tribe Caprini* genus Capra* The ruminants are characterised by possessing a stomach of four com partments. The compartments are designated, in the order of their anatomical sequenoe, as rumen, reticulum, omasum, and abomasum* The mucosal surface of the first three compartments is lined by stratified squamous epithelium and is devoid of glands* The fourth compartment, the abomasum, is glandular and is homologous to the simple glandular stomach of most non-i*uminants* Sisson (347)" gives the average capacity of the stomach as being 30 to 40 gallons in the ox and about 4 gallons in the s h e e p T h e total volume of the stomach in the adult ox is partition ed among the four compartments according to the approximate percentages ABy way of comparison, the capacity of the stomach in adult man is between 2 to 3 pints or about 1/4 gallon (149)* 2. that follow (347, 227); rumen 80$ reticulum 5% omasum 7% abomasum ••••• ••••••••••••• 8$ The relative oapaoities of the stomaoh compartments in the sheep and goat approximate to the relationship described for the ox« Figures 1 and 2 outline schematically the anatomy of the ruminant stomach (see also Fig. 14, p. 103). The Activities of the Microbial Population in the Ruminant Stoma oh i A, Brief Review Microbial Population During the growth of the ruminant, the rumen acquires a population of microorganisms from the wide range of microbial species consumed with grass, hay, soil, and other materials that normally constitute or are associated with the diet of the young ruminant (49, 86, 174, 193, 195, 305). This acquired population, consisting of a complex mixture of many species of bacteria and protozoa (23, 154, 176, 282, 386), usually is large* The protozoa exhibit counts as high as 2 x 10^ cells per m i l l i  l i t e r of rumen contents (387); the bacteria exhibit counts as high as billions (109) of cells per mi l l i l i t e r (154). Hastings (154) has stated that "the protozoa and bacteria represent a density of population that is never met in vitro and probably not elsewhere in nature** This large population *of microorganisms not only exhibits interdependence among its members but exists in a symbiotic relationship with its ruminant host (280, 311, 376),2 'As lambs and goats that have been freed of protozoa do not demon-Fig. 1. - The ruminal and reticular cavities, viewed from the left (adapted from figure 121 of Martin and Schauder, Lehrbuoh der Anatomie der Haustiere, vol. 3, third edition, 1938) [ The left walls of the rumen and the reticulum have been removed. A, reticulum; B , posterior orifice of the oesophagus; B 1 , eosophagusj C, reticular groove connecting posterior orifice of the oesophagus with retiouloomasal orifice; D, ruminoreticular fold; E, anterior dorsal sao; F, anterior pillar; G, G1, right longitudinal pillar split into two parts; H, cross-section of posterior pillar; I, dorsal coronary pillar; J, posterior dorsal blind sao; K , dorsal sao; L, ventral sac; L', anterior ventral blind sac; M, ventral coronary pillar; N , posterior ventral blind sac; 0, abomasum; P, diaphragm. Fig* 2* - Sheep stomach, viewed dorsally and partly from the left (adapted from figure 771 of Ellenberger-Baum, Handbuch der Yergleichenden Anatomie der Haustiere, eighteenth edition, 1943) The stomach has been opened by removal of parts of the walls of the rumen and reticu lum* A, reticulum; B, oesophagus; C, the two lips of the reticular groove; D, rumino- reticular fold; E, anterior dorsal sao; F, anterior pillar; G, ventral sac; H, poster ior ventral blind sac; I, posterior pillar; J , posterior dorsal blind sac; K, dorsal ooronary pillar; L, ventral ooronary pillar; M, M', right longitudinal pillar split into two parts; N, beginning of left longitudinal pillar; 0, omasum; P, abomasum; Q, beginning of duodenum* SymM.gs.is between the Ruminant and its Microbial Population The Ruminant*- The beneficial activities of the ruminant host to ward the members of its microbial population are: 1) to provide living space, water, and food in the form of grasses, hays, leaves, and other fibrous materials; 2) to provide a temperature of approximately 39°C. and a carbon dioxide-rich atmosphere of anaerobiosis (50, 51, 401), conditions which are favourable for microbial fermentation; 3) to accelerate the microbial fermentation by: a) masticating the food, both when the food is ingested and when it is regurgitated, thereby increasing enormously the surface area of the food that is accessible to enzymic attaok by the ruminal microorganisms; b) mixing the ruminal ingesta through strong, rhythmic contrac tions of the ruminal pillars and sacs; 4) to maintain a range of hydrogen ion concentration that is favourable for microbial fermentation by: a) buffering the ruminal contents with a constant inflow of a bicarbonate-phosphate-rich saliva (102, 242) and a secretion by the ruminal wall of large amounts of phosphate and bicarbonate buffers (106, 214, 284, 349, 350); b) increasing the rate of ruminal absorption of volatile fatty acids when the pH of the ruminal contents begins to decrease (88, 138, 230, 294); strate signs of nutritional deficiency, the protozoa are considered to be commensals rather than symbionts (57, 282, 311)* 5) to remove the gaseous and dissolved end products of the micro bial fermentation by eructation, absorption through the wall of the stomach (139, 234, 235, 299), and passage to the more distal parts of the digestive tract (102)• Microbial Population.- The beneficial activities of the microorgan isms toward the ruminant host are the result of their fermentation of the cellulose-containing plant material that is ingested by the host. The ruminant itself does not possess a oellulase (154, 229, 311). Were i t not for the presence in the rumen of cellulase-possessing microorganisms (175, 311), the ruminant would be unable to utilize the cellulose of the plant cells. Further, as the cellulose forms a part of the cell wall of the plant cells, the ruminant also would be unable to utilize the carbo hydrates, proteins, amino acids, vitamins, and other nutrients contained within the cells (23, 374). The great benefit to the ruminant of its microbial population is that, by their fermentation of cellulose, they permit the ruminant to utilize plant material that otherwise would be unavailable. Specific examples of the beneficial activities of the ruminal micro organisms ares l) to ferment cellulose and other carbohydrates to form volatile fatty acids—mainly acetic, propionic, and butyric (23, 108, 141, 142, 295, 301, 374, 386)—which, on being absorbed from the stomach, provide a major contribution to the energy requirement of the ruminant (59, 295, 335); the magnitude of this fermentation is indicated by Phillipson's (295) suggestion that 40 to 50 per cent of the carbohydrate in common rations may be converted to fatty acids in the rumen; 2) to produce a dynamic system of nitrogen compounds (55, 62, 92, 240, 281, 287), in which protein is being both hydrolyzed and synthesized, bys a) attacking proteins in the ruminal contents with protease (92, 367), deaminase (110, 400), and decarboxylase (326) enzymes— the possible value of this hydrolysis to the host ruminant will be present ed in the general discussion (see p« 267); b) utilizing the nitrogen of compounds such as ammonium salts and urea (33, 147, 151, 152, 220, 250, 351, 352) and, in some cases, the sulfur of compounds of inorganic sulfate (13, 42, 43, 103, 220) to synthesize amino acids^ or protein—because of its microbial utilization, urea can serve as a source material for approximately one third of the amino acid requirements of the ruminant (102, 250, 287, 319); 3) to synthesize vitamin K and most i f not a l l the B-complex group of vitamins, including vitamin B 1 2 ( l , 31, 76, 78, 102, 135, 199, 200, 201, 226)—because of this synthesis, the ruminant can be independent of exogenous sources of vitamin K and the B vitamins (201; see also 102)• Loosli et al (220) conducted an experiment in which sheep and goats were kept in positive nitrogen balance and gained weight on a diet con taining no source of nitrogen other than urea or glycine. Assay of the hydrolyzed ruminal contents for the presenoe of amino acids demonstrated that the ten amino acids "essential" (an "essential amino acid" is one that must be supplied to an animal in its diet because the animal cannot synthesize the amino acid or cannot synthesize i t rapidly enough to meet the total requirement of the tissues of the animal for the amino acid) for the rat were synthesized in large amounts (see also 5, 47, 103, 114, 170, 241, 248, 283). Black et al (35) have presented evidence that the requirements of the ruminant for the essential amino acids are the same as for the dog and rat (see also 38)• Summary* - The ruminant apprehends, masticates, and swallows plant material* This material passes from the oesophagus into the warm, moist environment of the rumen. Here, the strong contractions of the ruminal walls rhythmically mix the ingesta and the enzyme-rich microorganisms of the rumen actively attack the fragments of plant material* 4 At intervals, the ruminant regurgitates portions of the fermenting ingesta for remastica tion and then reswallows these portions for renewed fermentation* A constant inflow into the rumen of large volumes of bicarbonate-phosphate- rich saliva and ruminal secretion helps to maintain a pH favourable for fermentation, as viell as to provide a basal medium favourable for micro biological activity* This activity results in the formation of volatile fatty acids from cellulose, amino acids and other nitrogenous compounds from proteins, amino acids and proteins from urea or ammonium salts, and the syntheses of vitamin K and the B-complex of vitamins. The volatile fatty acids are absorbed from the first three compartments of the rumen— this absorption not only makes readily available to the ruminant a major source of its energy, but also assists in maintaining a favourable pH in the rumen for fermentation. The synthesized proteins, amino acids, and vitamins are assumed to pass along to the small intestine where they are digested and absorbed (18, 23, 92, 154, 200, 201, 228, 317)* The flow of these and other nutrients out of the rumen and reticulum occurs when the ingesta becomes fine enough to pass from the reticulum to the omasum* The omasum absorbs water, as well as bicarbonate and phosphate ions, from the ingesta before it passes on to the abomasum (106, 129)* Beyond this Recently, Holtenius (l7l) has obtained evidence which indicates that the enzyme systems of the ingested plant material can contribute to the fermentation process in the rumen* point, the aotion of the digestive tract on the ingesta usually is assumed to he similar to the action of the digestive traot in many non-ruminant herbivores (62, 102, 343)• The above brief review has attempted to point up the salient feature of ruminant nutrition that is illustrated aptly in the following state ment (176)i The first step in ruminant nutrition is microbial nutrition* Mioroorganisms are first fed and they in turn feed the host* Absorption from the Rument a Review General Por a long time, absorption studies on the rumen were discouraged because of its luminal lining of stratified squamous epithelium (see the review on this in 377). The same type of epithelium forms the outer layer of the epidermis—a structure considered to be relatively impermeable to water and water-soluble substances. The inferenoe was that the rumen likewise would be impermeable to water and water-soluble substances. Comparative studies (27, 99, 377) on the epithelia of the epidermis and the rumen have demonstrated, however, that important structural differences exist between the two epithelia. In contrast to the epidermal epithelium, the ruminal epithelium lacks in many areas a stratum granulosum, has a relatively thin stratum oorneum, and is not covered with sebaceous secre-- tion. Further, the ruminal epithelium possesses a rich blood supply, an ^The results of Larson and Stoddard (209), however, do not appear to support this statement. They placed a oomplete non-ruminant ration (a hog ration) into the abomasum of yearling cattle. After six to ten days on this diet, the animals were near death. In contrast, i f the same ration was placed in the rumen, the animals did not sicken. extensive network of capillaries being intimately associated with the basal epithelial oells. These results prompted one group of workers ( 2 7 ) to state that i t is not surprising that the rumen absorbs soluble nutrients that are continually in contact with its epithelium* Indeed, much experi mental evidence now has been accumulated to support the belief that the ruminal absorption of water and of many water-soluble substances does occur* Substances Absorbed Trautmann reported in 1 9 3 3 ( 3 7 7 ) on absorption experiments in which drugs such as pilocarpine and atropine were introduced into the stomach compartments of goats and sheep. From the results of these experiments, Trautmann proposed that the rumen, as well as the reticulum and omasum, rapidly absorbs water and substances dissolved in water. Rankin ( 3 1 5 , 3 1 6 ) demonstrated in 1 9 4 0 the absorption of glucose, potassium iodide, pilo carpine, strychnine, and sodium cyanide from the rumen. Beginning in 1 9 4 2 , Baroroft et al ( 2 6 , 2 7 ^ 2 3 4 , 3 0 1 ) published a series of papers that demonstrated the absorption of the lower volatile fatty acids—acetic, propionic, and butyric—from the rumen, as well as from the reticulum and omasum, of sheep. More recently, higher volatile fatty acids—isobutyrate, n-valerate, isovalerate, and alpha-methyl butyrate—also have been reported to be absorbed from the rumen ( 2 9 7 ) * Some of the other substances which have been reported to pass across the ruminal wall into the portal blood are sodium ( 8 8 , 2 8 6 , 2 9 8 ) , potassium ( 2 8 6 , 3 5 9 ) , magnesium ( 3 6 1 ) , chloride ( 9 7 ; 2 8 4 , 3 5 9 ) , bromide ( 3 5 9 ) , sulfide ( 1 3 ) , phosphate ( 2 1 4 , 2 8 5 , 3 3 0 , 3 5 9 , 4 1 8 ) , lactate ( 1 7 2 ) , bicarbonate ( 2 9 9 ) , and different gases—e.g., ammonia ( 4 7 , 9 5 , 2 3 7 , 2 3 8 ) , carbon dioxide ( 1 0 2 ) , and methane ( 1 0 2 ) * Some of these substances also have been demonstrated to pass from the 13. blood across the ruminal wall into the rumen. This is true for sodium (286), chloride (230, 286), bicarbonate (230, 286, 298), and phosphate (215, 285, 330, 349, 350). There exists, then, considerable evidence for the permeability of the ruminal epithelium and the ability of the rumen to absorb many of the constituents of the ruminal contents. Comment The foregoing experimental results substantiate Trautmann's original suggestion that the rumen absorbs water and water-soluble substances (377). More recently, phillipson and Cuthbertson (299) have stated "that anything in solution in the rumen probably penetrates the rumen epithelium unless the mass is too great." As amino acids and B vitamins are in solution in the rumen and as the range of their masses is of the same order of magni tude as of the masses of substances known to be absorbed from the rumen, i t is possible that also amino acids and B vitamins are absorbed from the rumen. A. few experiments and several suggestions have been reported in the literature on the absorption from the rumen of amino acids and B vita mins. A brief review of these experiments and suggestions is presented in the next two sections. Amino Acids As far as this author is aware, no one has obtained direct evidence as to the permeability of the ruminal epithelium to amino acids. Several workers (216, 319, 375, 411) have suggested that amino acids are absorbed from the rumen. Chalmers and Synge even have suggested (62) that amino acids may pass from the blood into the ruminal contents: It is ••• quite possible that free amino acids and urea of the blood may pass into the rumen through its wall. As far as °Akkada and El-Shazly (7) recently have stated that they are u t i l i z  ing radioactive isotopes to determine i f amino acids can pass into the we are aware, this possibility has not been investigated experimentally* The concentration gradients between blood and rumen liquor are [sometimes?] such that substantial quantities could pass by simple diffusion. The entry of nonprotein [including amino acids] into the rumen from the animals own body with subsequent conversion to protein could assume importance under star vation conditions, and may be decisive for the survival of wild ruminants during winter and drought conditions when the only available food is the equivalent of badly leached hay. Annison (15), in two experiments, unsuccessfully attempted to demonstrate the absorption of amino acids from the rumen. The premise underlying the procedures in these two experiments was that i f amino acids are absorbed into the blood draining the rumen, that because of the removal by the liver of amino acids from the portal blood (389), the concentration of amino acids would be higher in the blood of the portal vein than in the blood of the jugular vein or common carotid artery. In the first experi ment, Annison added byway of a ruminal fistula 100 grams of casein hydro- lysate to the ingesta-filled rumen of a sheep. The animal immediately was anesthetized with sodium pentobarbitone. Ninety minutes after the addi tion of the hydrolysate, Annison removed blood samples from the portal and jugular veins. As the concentration of alpha amino nitrogen in the blood of the portal vein was lower than that in the blood of the jugular vein, he did not obtain evidence in this first experiment for ruminal absorp tion of amino acids. In the second experiment, Annison added by way of a ruminal fistula 70 grams of oasein hydrolysate to the ingesta-filled rumen of a sheep. The animal was not anesthetized. Annison removed blood rumen through its wall* 7 See page 209 where i t is demonstrated that the concentration of alpha amino nitrogen in the ruminal liquor can be higher than in the blood. samples from the sheep by using a portal vein cannula and an exteriorized Q common carotid artery. The results of the experiment are given i n Table 1. As for the results of the f i r s t experiment, the results record ed i n Table 1 do not demonstrate a greater concentration of amino acids in the portal blood than in the carotid blood; hence, Annison was unable to obtain evidence in either of his two experiments to support a hypothesis that amino acid absorption oocurs from the rumen. Vitamins A few experiments and some comments have been i n the literature on the question of ruminal absorption of B vitamins* In 1941 Hunt et a l (179) suggested the possibility of riboflavin absorption from the rumen of cat t l e . In 1944, Lardinois et a l (208) made the same suggestion for thiamine. Both suggestions were made on the basis of indirect evidence, the evidence being dependent upon a comparison of the concentration of the vitamin i n the feed with the concentration of the vitamin in the rumen. Unfortunately, the method of experimentation did not exclude the observed decrease of concentration being the result of vitamin destruction. In 1947 Kon and Porter (201) discussed the absorption of B vitamins from the rumens Further work on vitamin absorption also i s required. It has usually been assumed that absorption is mainly from the abomasum and small intestine, but since the demonstration by Marshall and Phillipson (1945) that v o l a t i l e fatty acids are absorbed from the rumen, the possib i l i t y exists that v i t a  mins also may be absorbed from i t . Contrary to the expectation of the above quotation, Frenoh workers (322, °See the appendix for a description of the techniques for the cannulation of veins (p.290) and the exteriorization of the common carotid artery (p. 286). TABLE 1. - The results of an. experiment by Annison (15) to determine i f , after the addition of casein hydrolysate to the ingesta-filled rumen of a sheep, there was a greater concentration of alpha amino nitrogen in the blood of the portal vein than in the blood of the common carotid artery Time after adding amino acids (hr.) Alpha amino Kf (mg./lOO ml.) Ruminal contents Portal blood Carotid blood 0 0.86 3.84 3.04 1 83.7 3.18 2.94 .1.5 • • • 2.54 2.91 4.5 18.8 2.02 1.93 373) were unable to demonstrate ruminal absorption of B vitamins. They did obtain indirect evidence that nicotinic acid was absorbed from the reticulum and that some of the other B vitamins were absorbed from the omasum. On the other hand, a personal communication to Phillipson and Reid (302) by Kon and Porter states that they have demonstrated thiamine to be absorbed from the rumen* Although meager and even oonflicting, the evidenoe to date suggests that B vitamin absorption may occur from the rumen* Comment The previous review of the literature presents the ruminant as an animal that possesses a stomach with a highly developed capacity for absorbing certain nutrients* Direct experimental evidence exists for the function of the rumen in the absorption of volatile fatty acids and many inorganic ions. Indirect evidenoe has led several authors to suggest that the rumen functions in the absorption of amino acids and B vitamins* The present investigation, divided into two parts, was designed to provide direct experimental evidenoe for or against the suggestion that amino acids and B vitamins are among the nutrients absorbed from the rumen of the ruminant* 18, EXPERIMENTATION One aspect of ••• this type of work which is not generally appreciated, • *• , is the physioal difficulty of carrying out experimental work on ruminantst the difficulty of anesthesia, difficulties due to their peculiar anatomy, and many others* A physiologist working with experimental animals such as sheep and goats has problems which a person working on small laboratory animals never encounters* Dr* F* Alexander (10)* Part I t Ruminal Fistula Experiments Introduction The first approach to the detection of ruminal absorption of amino aoids and B vitamins was to demonstrate a deorease in the concentration of these substances when they were added in solution to an empty rumen* The use of goats with ruminal fistulas not only enabled the rumen to be emptied of its contents, but also facilitated the addition to the rumen of an experimental solution and the sequential sampling of this same solution* Pectin or polyethylene glyool was added to the experimental solutions* As neither of these substances is absorbed from the rumen (138, 181, 360), their addition provided a marker substance for any water movement into the rumen through the ruminal wall or by saliva inflow and for water movement out of the rumen through the ruminal wall* The measurement of water move ment was considered necessary in order that concentration changes of amino acids and vitamins due to water movement could be differentiated from concentration ohanges of these substanoes due to ruminal absorption* Propionic acid also was added to the experimental solutions of nearly a l l the experiments* The addition of this substanoe, known to be absorbed from the rumen, was made to provide a positive control for ruminal absorption* The selection of the particular amino acids and B vitamins to be studied and the selection of the method of their analysis were determined by several considerations* The amino acids and B vitamins were chosen on the basis oft (l) their susceptibility to microbiological assay* (2) their diversity of structure, (s) their variation of molecular volume* The method of assay, the microbiological assay, was ohosen on the basis ofi (l) the existence of excellent facilities for this type of assay, (2) the relative simplicity and accuracy of the assay when i t is carried out by an individual experienced in microbiological procedures, (3) the applicability of this one method of assay to a large number of amino acids and B vitamins* All these considerations, together with the limita tions of time and money, determined the choice of the particular amino acids and B vitamins to be studied and the method of their assay* Method Experimental Procedure General*- This series of experiments was carried out using goats . . 9 whioh previously had been provided with a ruminal fistula (see. p* 279)• The first step of the experimental procedure was to immobilize the goat on a milking stand* The contents of the rumen and reticulum were removed manually via the fistula* The rumen and reticulum then were rinsed with warm tap water* This rinsing procedure was repeated several times until the ruminal wall appeared to be clean* In the first two experiments, some difficulty was experienced in rinsing the reticulum adequately* In ^The details of the surgical and analytical procedures utilized in the present investigation are presented in the appendix* the other experiments, a one-third horsepower vaouum pump facilitated the rinsing procedure "by drawing most of the rinsing solution into a large suction flask that was inserted in the intake line of the pump* The thoroughness of the rinsing could be checked by observing the cloudiness of the solution in the suction flask* The solution that was added to the rumen was prepared on the day previous to the experiment. After first warming this solution to a temperature of 37° C*, a sample was taken from i t and labelled experi mental solution control* In the f i r s t two experiments, the experimental solution control was subdivided* One part was placed for the duration of the experiment in a hot air' oven that was kept at approximately 37° C* The other part of the solution was placed in a refrigerator that was held at 4 to 10° C**^ In the rest of the experiments, the experimental solu tion control was not subdivided, a l l of i t being plaoed in a refrigerator*** Two to four liters of the experimental solution now were added through the fistula to the rumen* The amount of solution added was enough to f i l l the rumen to within one or two inches of the level of the anterior pillar (see Figs* land 2, pp* S and 5 )• This restriction on the f i l l was imposed, not only in order to oonfine the absorption studies to the rumen, but also to reduce the possibility of contamination of the experimental solution in the rumen—now called the ruminal solution—by ingesta from the reticulum or the omasum* This reduction of the possibility of contamin ation was the reason for also removing the ingesta of the reticulum* 1 0 I n the first two experiments (Wl and W2), the experimental solution controls served to measure the effect of temperature on the concentration of the constituents when they were contained together in solution and held at 57° C* for two hours* 1*In these experiments (W3 to W6), the experimental solution controls Immediately after the addi t ion of the solution to the rumen, the experi mentalist passed his hand through the fistula and vigorously mixed the contents* This period of mixing "was judged to be about SO seconds* At the end of the mixing period, samples were taken from the solution in the rumen* The pH of the samples was determined electrometrically using a glass electrode**** The samples then were filtered through four layers of cheesecloth. In the first two experiments, the filtered samples were labelled zero minute samples and plaoed in the refrigerator. In the rest of the experiments, these samples first were subdivided. One part, labelled zero minute sample, was placed in the refrigerator and the other 13 part, labelled ruminal solution control, was placed in a water-bath that was held at 37° C* Immediately after the solution in the rumen had been sampled, the fistula either was draped with a heavy towel or closed with a fistula-plug. At periodic intervals, more samples were taken from the solution in the rumen. Af ter pH measurements of the samples had been made, they were placed in a refrigerator* Each time the solution in the rumen was sampled, the ruminal solution control in the water-bath also was sampled* The temperature of the solution in the rumen was cheoked at frequent intervals by inserting a glass thermometer through the fistula. At the end of the experiment, as much as possible of the solution was collected served to provide samples for the determinstion of the concentration of the experimental solution constituents before they were placed in the rumen. 12 A Beckman pH meter was used* " i n these experiments (W3 to HIT6), the ruminal solution controls served to measure not only the effect of temperature, but also the effect of possible contamination by ruminal ingesta on the concentration of the constituents when they were contained together in solution and held at 37° C. for two and one half hours* 22 from the rumen and the volume measured. A l l samples were frozen at about -17° C. In the first two experiments, the samples were kept frozen until their assay. In the other experiments, the samples were kept frozen for two to four weeks and then were transferred by car five hundred miles to another institution. For the transfer, the frozen samples were packed in ice; as, however, the journey occurred during warm weather and lasted about twelve hours, the samples lacked refrigeration for a part of the journey. At the end of the journey, the samples were re-frozen at about -13° C. More specific details for each of the experiments now are presented. Experiment Wl»- 1. Animal history: A young adult female goat weighing 98 pounds and of mixed breeding was used. Seven months prior to the experiment, the left common carotid artery was exteriorized to form a "carotid loop" (see p.286)» Four months prior to the experiment, the goat was provided with a ruminal fistula. The operation was carried out in two stages, the second stage taking place about 10 days after the f i r s t . At the time of the experiment, the animal apparently was in good health. 2. Composition of the experimental solution: Table 2 presents the composition of the experimental solution. 3. Sampling procedure: Two liters of the experimental solution were placed in the rumen of the goat. Single samples were removed from the solution in the rumen. The sampling times were 0, 40, 60, 95, and 120 minutes after the i n i t i a l 30 second period of mixing. Single samples were removed from the experimental solution control at the 0 and 120 minute • sampling times. The total volume of solution removed by the sampl ing procedure from the solution in the rumen was 570 mil l i l i t e r s . The TABLE 2. - Composition of the experimental solution for experiment Wl Constituent6 Milligrams per Liter Calo ium-d-pant othenat e ......... •• 20 L-glutamio acid.... 441 Glycine 225 L-leuoine. • •• 394 L-lysine • 439 L-ciethionine•...••••••••••••»•••••••••• 448 Nicotinic aoid. 20 Riboflavin. 20 L-tryptophan. 613 aIn addition, 3.75 grams per liter were added of pectin (Nutritional Biochemical Corporation. Cleveland, Ohioi Pectin, N.F.). The pH of the solution was adjusted to 6.75 by making i t 0*067 molal in sodium orthophosphate, mono-hydrogen (Na2HP04) • volume of solution recovered from the rumen at the end of the experiment was 1200 milliliters. This volume together with the volume removed by the sampling procedure accounts for 1770 of the original 2000 milliliters placed in the rumeno At least some of the volume not accounted for was lost as a small but unknown volume of spillage through the fistula during restless movements of the goat. Also, not recoverable at the end of the experiment was a small but unknown volume of the solution lost in the folds and depressions of the ruminal wall. This loss occurred in the other experiments as well. Experiment W2<- 1. Animal history: An adult male goat weighing 100 pounds and of mixed breeding was used. About 9 months prior to the experiment, the spermatic cords were crushed to cause a reduction in the odour of the animal. Eight months prior to the experiment, the goat was provided with a ruminal fistula in the same manner as for the goat of experiment Wl« About 7 months prior to the experiment, the left common carotid artery was exteriorizedo At the time of the experiment, the animal apparently was in good health. 2. Composition of the experimental solution: The amino acids were supplied in the form of a casein acid-hydrolysate solution*** One hundred milliliters were used in each liter of total solution. Riboflavin, pectin, sodium orthophosphate (mono-hydrogen), and potassium orthophosphate (di-hydrogen) were added to the experimental solution in the same concen tration as in the solution for experiment Wl. Nutritional Biochemical Corporation, Cleveland, Ohio: "Vitamin-Free Casein Hydrolysate-Acid (10$) solution." 25. 3* Sampling prooedure* Three and one half liters of the experi mental solution were placed in the rumen of the goat. Single samples were removed from the solution in the rumen. The sampling times were 0, 65, 95, and 125 minutes after the i n i t i a l 30 second period of mixing. Single samples were removed from the experimental solution control at the 0 and 120 minute sampling times. The total volume of solution removed "by the sampling procedure was 522 m i l l i l i t e r s . The volume of solution recovered from the rumen at the end of the experiment was 600 m i l l i l i t e r s . This volume together with the volume removed by the sampling procedure accounts for only 1122 of the original 3500 milliliters placed in the rumen. At least some of the volume of 2300 milliliters not accounted for was lost as a large but unknown volume of spillage through the fistula. Most of this spillage occurred during the later part of the experiment and was oaused by unexpected jumping-movements of the goat. Experiment W3.- 1. Animal history: A young adult female goat weighing 110 pounds and of Saanen breeding was used. Two and one half months prior to the experiment, the left common carotid artery was exteriorized and the first stage of the ruminal fistula operation was performed. One month prior to the experiment, the second stage of the fistula operation was performed. Three weeks prior to the experiment, the goat aborted. After the abortion, the goat ceased to eat and exhibited an elevation of body temperature. Treatment in the form of ruminal ingesta transplants* 5 and A The contents of the rumen and reticulum in the sick goat had lost their normal smell, colour, and consistency: being foul-smelling, brown, and watery. After first removing the foul contents and rinsing the compart ments, large transplants of ingesta—enough to h a l f - f i l l the rumen—were transferred from a healthy fistulated goat to the sick goat. Such a antibiotic therapy was begun and maintained for four days* The animal resumed eating and the body temperature returned to normal* 2* Composition of the experimental solution! The amino acids were supplied in the form Of a casein acid-hydrolysate solution* Twenty milliliters were used in eaoh liter of total solution* For each l i t e r of solution, there also were added 40 milligrams of glycine, 50 milligrams of nicotinic acid, 5 grams of sodium propionate, 1*625 grams of sodium ortho- phosphate (di-hydrogen), and 2*5 grams of polyethylene glycol* The pH of the solution was adjusted to 6*5 with sodium hydroxide* 3* Sampling procedure! Three liters of the experimental solution were placed in the rumen of the goat* Single samples were re moved from the solution in the rumen* The sampling times were 0, 10, 30, and 70 minutes after the i n i t i a l 30 second period of mixing* Single samples were removed from the ruminal solution control at the 0 and 70 minute sampling times* The total volume of solution removed by the sampl-. ing procedure was 565 milliliters* The volume of solution recovered from the rumen at the end of the experiment was 2320 milliliters* This volume together with the volume removed by the sampling procedure accounts for 2885 of the original 3000 milliliters placed in the rumen* Spillage of the solution through the fistula was not observed to occur* procedure not only supplied the sick goat with a new population of actively fermenting ruminal microorganisms but also probably supplied the goat with pre-formed volatile fatty acids and other nutrients* The author's person al experience was that the use of such large transplants usually proved to be beneficial to fistulated goats that had "gone off feed"* In a series of papers (77, 161, 305, 307, 308, 309), Pounden, Hibbs, and Conrad have demonstrated the nutritional benefits that result from the successful transplantation of ruminal microorganisms into oalves by means of cud inoculations from adult oows* 16 Carbide and Carbon Chemicals Company, New York, N*Y*i Carbowax 4000* 27. Experiment W4.- 1* Animal history! The goat of experiment W3 was used also for this experiment. At the time of the experiment, the goat s t i l l appeared in a weakened, emaciated condition and had required several ruminal ingesta transplants to keep i t eating. There was not, however, any elevation of body temperature. 2. Composition of the experimental solution* Most of the amino acids were supplied in the form of a casein acid-hydrolysate solution. Forty milliliters were used in each li t e r of total solution. Table 3 presents the constituents that were added to complete the composition of the experimental solution. 3. Sampling procedure* Three and seven tenths liters of the experimental solution were placed in the rumen of the goat. Duplicate samples were removed from the solution in the rumen. The sampling times were 0, 10, 30, 70, and 150 minutes after the i n i t i a l 30 second period of mixing. Duplicate samples were removed from the ruminal solution control at the same times as for the solution in the rumen. The total volume of solution removed by the sampling procedure was 1300 milli l i t e r s . The volume of the solution recovered from the rumen at the end of the experi ment was 2130 mi l l i l i t e r s . This volume together with the volume removed by the sampling procedure accounts for 3430 of the original 3700 m i l l i  liters placed in the rumen. At least some of the volume of 270 milliliters not accounted for was lost as an unknown volume of spillage through the fistula. Experiment W5«- 1. Animal history* A young adult female goat weighing 90 pounds and of Saanen breeding was used. Three months prior to the experi-TABLE 3* - Composition of the experimental solution for experiment W4 Constituent6 Milligrams per Liter L-cystine 100 Calcium-d-pantothenate•••••••••••••••••• 100 Glycine....... ••••• •••••• 100 Nicotinic aoid 100 Riboflavin ••• • 100 D L - s e r i n e * 1 0 0 L-tryptophan • 100 aMost of the amino acids vere supplied in the form of oasein acid-hydrolysate solution (1C$), 40 milliliters in 1 l i t e r * Other constituents that were added to each l i t e r of solution were 5 grams of sodium propionate, 2*5 grams of polyethylene glyool, and 1*625 grams of sodium orthophosphate, di-hydrogen (tfaHgPO^)* The pH of the solution was adjusted to 6*5 vith sodium hydroxide* merit, the left common carotid artery was exteriorized and the first stage of the ruminal fistula operation was performed. One and one half months prior to the experiment, the second stage of the fistula operation was performed. At the time of the experiment, the animal apparently was in good health* 2* Composition of the experimental solutioni Most of the amino acids were supplied in the form of a casein acid-hydrolysate solution* Twenty milliliters were used in each liter of total solution* Table 4 presents the constituents that were added to complete the composition of the experimental solution* 3* Sampling procedureJ Three liters of the experimental solu tion were placed in the rumen of the goat* The number of samples removed and the times of sampling were the same as for experiment W4* The total volume of solution removed by the sampling procedure was 1300 milliliters* The volume of the solution recovered from the rumen at the end of the experiment was 800 milliliters* There was, however, s t i l l fluid left in the arterior ventral blind sac of the rumen (see Fig* 1, p* 3 ) which could not be reoovered* The volume recovered together with the volume % removed by the sampling procedure accounts for 2100 of the original 3000 milliliters placed in the rumen* In addition to the unknown volume of solution left -in the rumen, at least some of the volume of 900 m i l l i  liters not aocounted for was lost as an unknown volume of spillage through the fistula. Experiment W6*» 1* Animal historyi The goat of experiment W5 was used also for this experiment* At the time of the experiment, the animal apparently was in good health* TABLE 4* - Composition of iihe experimental solution for experiment W5 Constituent6 Milligrams per Liter L-cystine..»...••••••• ••••••••••<• 50 Caloium-d-pantothenate. 50 G l y c i n e * . . . . . . . . . . . . . 5 0 Nicotinic acid • 50 Riboflavin.. 50 DL-serine....••••••» •••••••••• 50 ]>bryptophan............................ 50 aMost of the amino acids were supplied in the form of a casein acid-hydrolysate solution (10#), 20 milliliters in 1 l i t e r * Other constituents that were added to each l i t e r of solution were 5 grams of sodium propionate, 2*5 grams of polyethylene glycol, and 1*625 grams of sodium orthophosphate, dihydrogen (NaHjjPO^ L.)". The pH of the solution was adjusted to 6*5 with sodium hydroxide* 31. 2. Composition of the experimental solution! The solution for this experiment was prepared in the same way as for the experimental solution of experiment W4. 3* Sampling procedure! Three liters of the experimental solu tion were placed in the rumen of the goat. The number of samples removed and the times of sampling were the same as for experiment W4. The total volume of solution removed by the sampling procedure was 1300 mil l i l i t e r s . The volume of solution recovered from the rumen at the end of the experi ment was 880 milli l i t e r s . There was, however, fluid s t i l l left in the rumen which could not be recovered. The volume recovered together v/ith the volume removed by the sampling procedure accounts for 2180 of the original 3000 milliliters placed in the rumen. In. addition to the un known volume of solution .left in the rumen .(estimated as 300 mi l l i l i t e r s ) , at least some of the. .volume of 820 milliliters not accounted for was lost as an unknown volume of spillage through the fistula. Listing of Individual Constituents for Experiments Wl to ¥6.- Table 5 presents, for experiments Wl to W6, the concentrations of the individual constituents in the experimental solutions. Assay Procedure The details of these procedures are presented in the appendix. Listed here are the substances assayed, with a brief statement as to the method of their assay. Pectin.- The method of pectin assay was adapted from the procedure of Nanji and Norman (269). Basically the method involves precipitating the pectin as calcium pectate and weighing the preoipitate. Polyethylene Glycol.- The gravimetric procedure of Sperber et al (360) was used. TABLE 5. - Complete listing of the constituents present in the experimental solutions, the concentrations8, being expressed as milligram-moles per liter Constituent Experiment TO W2 W3 W5 W4 and W6 Amino acids Alanine • • • 2*13 0.43 0*43 0.85 Arginine • • • 2.18 0*44 0.44 0*87 Aspartio acid • • • 3*08 0*62 0*62 1*25 Cystine • • • 0*13 O.OS 0.24 0*47 Glutamic aoid 3*00 14*83 2*97 2.97 5*93 G-lyeine 3*00 0.67 0*80 0.80 1.60 Histidine • • • 1*23 0.25 0*25 0.49 Hydroxyproline • • • 0.15 0*03 0*03 0.06 Isoleuoine • • • 3.90 0.78 0.78 1.56 Leucine 3.00 11*28 2*26 2.26 4.51 Lysine 3.00 4*05 0.81 0.81 1.62 Methionine 3.00 i 2.00 0.40 I 0.40 0.80 TABLE 5 - Continued Experiment UwUBvJlvUWiv Wl W2 W3 W5 W4 and W6 Phenylalanine • • • 2.38 0.48 0.48 0.95 Proline • • • 7.30 1.46 1*46 2.92 Serine • • • 0*38 0.08 0*56 1.11 Threonine • • • 3.88 0.78 0*78 1.55 Tryptophan.. 3.00 ... ... 0.25 0*49 Tyrosine • • • 2.88 0*58 0*58 1.15 Valine • • • 4.45 0.89 0*89 U78 Vitamins Nicotinic aoid 0.16 • . • 0*41 0.41 0*81 Pantothenic acid 0.04 ... ... 0.11 0.21 Riboflavin 0.05 0.05 ... 0.14 0.27 Other Peotinb 1 1 ... ... ... Polyethylene glycol ... • . • 0.63 0.63 0.63 TABLE 5 - continued Constituent Experiment Wl W2 W3 W5 W4 and W6 Potassium orthophosphate, di-hydrogen 0.067 0.067 • • • • • . • • • Sodium hydroxide 0 • • • • • • ? i m 1 Sodium orthophosphate, di-hydrogen ... . • . 11.77 11.77 11.77 Sodium orthophosphate, mono-hydrogen 0.067 0.067 ... • • . • • . Sodium propionate ... ... 52.04 52.04 52.04 Total 18.384 67.084 78.94 80.13 95.67 ^he se concentration data are constituted of composite figures based oni 1. The manufacturer's (Nutritional Biochemioal Corporation) estimate of the concentrations of the constituents in the oasein acid-hydrolysate solution; 2. the weighed-out amounts of the constituents added in making up the experimental solutions. b I t is not possible to calculate a molal concentration for pectin,as pectin i s a mucilagenous vege table carbohydrate of unknown molecular weight (323). °A record was not kept of the amount of sodium hydroxide added to the experimental solutions; however, enough was added to adjust the pH to 6.5. Propionic Acid.- An adaptation of the steam distillation procedure of Wish (271) was utilized. Amino Acids.- The amino acids were assayed by use of two procedurest the microbiological procedures of Barton-Wright (30) and the Van Slyke manometric determinations of alpha amino nitrogen (148, 59l). B Vitamins— The microbiological procedures of Barton-Wright (30) were used. RESULTS Peotin This substance was added to the experimental solutions of experi ments Wl and W2 to serve as a marker for water movement. Unfortunately, the assay for pectin was carried out improperly. There is not a record, therefore, of the water movements that may have occurred during these two experiments. The osmotic pressure for each of the two solutions, 17 however, can be calculatedt for experiment Wl, 0.5 atmospheres; for experiment W2, 1.7 atmospheres. The osmotic pressure of goat's blood averages about 8 atmospheres at 37° C. (9). At the time of being added to the rumen, the solutions of experiments Wl and W2 were, therefore, strongly hypotonic relative to the blood draining the rumen. Parthasarathy and Phillipson (286), working with sheep, found that the rumen absorbed 70 to 305 milliliters of water in one hour from solutions only slightly hypotonic to the blood draining the rumen. This should mean that water was absorbed when the hypotonic solutions for experiments Wl and W2 were TT" mRT, where TT is "the osmotio pressure in atmospheres, m is the molality, R is the gas constant (0.08205 liter-atmospheres per degree per mole), and T is the absolute temperature. placed in the rumen of the experimental goats* Polyethylene Glyo ol This substance was added to the experimental solutions of experiment W3 to ¥6 to serve as a marker for water movement. The results are pre sented in Table 6 and Figure 3. The data were analyzed statistically by applying analysis of variance to determine the significance for the divergence from a zero gradient of the slopes of linear ttbest-fitn lines* Although the best-fit lines are not drawn in Figure 3, the statements of significance for the best-fit lines are entered in the figure. In experi ment W5, for example, there is a statement of significance given for a concentration change in the rumen that reads ast .005 (0 30)• This statement means that the data of the 0, 10, and 30 minute samples were used to calculate a best-fit line; further, that the divergence from a zero gradient of the slope of this best-fit line is significant at or at less than the 0.5 per cent level. When a statement of significance is not recorded on the figure for a particular part of the data, this means that the divergence of the slope of the best-fit line for this part of the data was not significant at even the 10 per cent level. This method of presenting the statements of significance is used also for the data that are recorded in Figures 4 to'20. Figure 3 indicates that only in experiment W5 was there in the rumen a change of concentration that was significant at the 10 per cent level or less. In experiment W5, the data for the 0 to 30 minute interval yield a best-fit line whose positive slope is significant at the 0.5 per cent level or less. These results can be interpreted to indioate that only in experiment W5 did demonstrable water movement occur. In this experiment, the increase of polyethylene glycol concentration should TABLE 6. - Ruminal fistula experiments W3, W4, W5, and W6» changes in the weights (milligrams from 3-milliliter samples) of polyethylene glyool preoipitates (the data recorded are means and standard devia tions, with numbers of assays in parentheses) Experiment Time (min.) W3 14 W6 Ruminal solution Control* Ruminal solution Control 8 Ruminal solution Control 8 Ruminal solution Control 8 Experimental solution" • • • • 51.5+2.1 (2) . . . . 55.5+2.0 (6) . . • . . . . . . . • . . . . . 0 54.0±4.2 (2) 54.0+4.2 (2) 49.8+2.7 (4) 49.8±2.7 U) 40.512.4 (4) 40.5+2.4 (4) 51.4+3.0 (5) 51.4+3.0 (5) 10 50.5+4.9 (2) . . . . 52.5+3.3 (4) 49.8+3.8 (4) 46.5+2.9 (4) 45.5+1.7 (4) 51.513.9 (4) 52.8+3.5 (4) 30 52.5+2.1 (2) . . . . 50.0+4.6 (4) 48.7+2.9 (3) 50.Q+3.2 (4) 45.8+2.1 (4) 50.5±2.1 (4) 47.5+4.8 (4) 70 49.0+4.2 (2) 57.5+3.4 (4) 48.9+4.3 (4) 53.3+4.0 (4) 49.0+4.3 (4) 49.3+2.9 (4) 51.0+2.9 (4) 54.7+1.3 (3) 150 . . . . . . . . 48.9+4.7 (4) . * . . 50.5+2.4 (4) . . . . 52.6+2.9 (5) . . . . Except for the data of the experimental solution, the word "control" here designates the data of the ruminal solution control (see p. 21). Analysis of the ruminal solution before i t was placed in the rumen. o o TIME (MINUTES) F i g . 3 '(-Fii/St part) C O UJ Z Ul — A - I 70 T I M E ( M I N U T E S ) P i g . 3 (Second part) Pig. 3. - Ruminal fistula experiments W3, W4, W5, and W6j changes in weights of polyethylene glycol in the ruminal solution A) and in the ruminal solution control (Q Qj) • The inscriptions such as .10 (10 ^30) are statistical statements of significance, the explanation of which is given in the text* 41 mean that water was absorbed from the rumen. Ordinarily, this would lead to correcting the data of the amino acids, B vitamins, and propionic acid for the concentrating effect of the water movement. The polyethylene glycol concentration changes in the control, however, complicate the interpretation of the polyethylene glyool concentration changes in the rumen. Between 0 and 10 minutes, there is in the control a concentration . increase that is significant at less than the 2.5 per cent level. Between 30 and 70 minutes, there is another concentration increase that is signif icant at less than the 10 per cent level. Although these concentration " increases in the control do not match those in the rumen, i t can not be said with certainty that a l l the inorease of polyethylene glycol concen tration in the rumen is due to water absorption. If the polyethylene glyool concentration changes in the control are real changes and not just reflections of sampling error, then the same factor that effected the change of concentration in the control also may have effected a corresponding change of concentration in the rumen. If, indeed, such/.a change of concentration was effected in the rumen, then the use of the polyethylene glycol concen tration changes to estimate the absorption of water from the rumen would result in an overestimation. As there appears to be no way to determine i f the control changes were the result of the effect of some factor or merely sampling error, the data for the constituents in experiment W5 18 w i l l be presented both corrected and uncorrected for 7fater movement. To correct the data for the constituents—i.e., propionio acid or any one of the amino acids or B vitamins that was determined—, each measured concentration was multiplied by its own correction factor. Each correction factor was the ratio of the polyethylene glycol concentration in the 0 minute sample to the polyethylene glycol concentration in one of the subsequent samples; thus, the correction factor for the measured concentration of a constituent in the 10 minute sample from the rumen is 40.5/46.5 = 0.8709. Tables 7 to 25—tables that present the results for propionic aoid, the amino acids, and the B vitamins—have two rows of data for each sampling time in the columns for experiment W5* The top row presents the data uncorrected for water movement; the bottom row presents the data correct ed for water movement* Likewise, Figures 4 to 12 present the data of experiment W5 as both uncorrected (W5-U) and corrected (W5-C) concentra tions* Except for the 0 minute sample concentration, the corrected concentrations of the different constituents are a l l lower than the uncorrected concentrations* The corrected data indicate a greater decrease of concentration than do the uncorrected data* The uncorrected data of experiment W5 represent, therefore, a more conservative estimate of the constituents' concentration ohanges* By utilizing both sets of data, i t is hoped that the true concentration ohanges w i l l be bracketed within upper (uncorrected data) and lower (corrected data) limits* Actually* comparison of the uncorrected and corrected data for the different constituents reveals that only for nicotinic acid, pantothenic acid, and tyrosine does the interpretation differ much between the two sets of data* Even for these three constituents, the differences in interpreta tion are minor* The polyethylene glycol results of experiments W3, W4, and W6 require further discussion* In none of these experiments was there in the rumen a concentration change that showed significance at the 10 per cent level or less* These results were interpreted as indicating that demonstrable water movement did not occur* These results do not mean necessarily, however, that there was no water movement; only that, according to the methods employed, water movement did not occur that showed significance at the 10 per oent level* A visual method was devised to check on the possibility that perhaps real but undetected (undetected by significance testing) water movements could produce concentration changes of the amino acids and B vitamins* This method consists of plotting the data for polyethylene glyool on the graphs of the data for each the other constituents (e.g., see Fig* 4, p*46)* In each graph, the scale of concentration changes of polyethylene glyool is 19 adjusted to the soale of concentration changes of the constituent oonoerned* In this way, a constituent's concentration changes that result only from water movement will be indicated in the figures; thus, i f the slope of the line drawn between the 0 and 10 minute concentrations for polyethylene glycol is parallel to the slope of the line drawn between the 0 and 10 minute concentrations for an amino acid, then the concentra tion ohange of the amino acid in this time interval is probably the re sult of water movement alone* Conversely, any significant divergence of the slopes ©^f two such lines should be an indication that the concentra tion change of the amino acid is the result of more than just the effeots of water movement* This type of comparison between the concentration changes for polyethylene glycol and the concentration changes for a constituent is made for propionic acid and each the amino acids and B vita mins that was measured* Propionic Acid The results of the steam-distillation assay for propionio acid are To adjust the scale of concentration changes of polyethylene glycol to the scale of concentration changes of a constituent, each of the polyethylene glycol concentrations was multiplied by a conversion factor* Each conversion factor is the ratio of the constituent's concen tration in the 0 minute sample to the polyethylene glycol concentration in the same sample; thus, the conversion factor to be used to convert the polyethylene glycol concentrations for plotting on the graph of propionio acid in experiment W3 is 50*6/54*0 • 0*9370* presented in Table 7 and Figure 4. Examination of Figure 4, taking note of the statements of significance, reveals that in a l l the experiments there was a strong deorease of propionic acid concentration in the ruminal solution during at least a part of the experiment* At the same time, the concentration in the control solution either remained constant or increas ed. In addition, a comparison of the slopes of the lines for the adjusted polyethylene glycol data and the propionic acid data demonstrates that, with one possible exception, the concentration changes of propionic acid in the ruminal solution were not a reflection of a dominant influence of water movement. The one possible exception is in experiment W3. In this experiment, i t appears that at least a part of the concentration changes could have resulted from the effects of water movement. With the possible exception of these data for experiment W3, the results of the propionic acid experiments indicate ruminal absorption of propionic acid. As the rumen already is known to absorb propionic acid, the results of the propionic acid experiments provide controls which indicate that the rumens of the goats in experiments W3 to W6 were exhibiting absorption. Amino Acidsi Tryptophan, Methionine, Tyrosine, Glycine, and Lysine The results of the microbiological assay for these compounds are presented in Tables 8 to 12 and Figures 5 to 9. Examination of the figures, taking note of the statements of significance, reveals that in a l l the experiments, except one, there were strong decreases in the concen trations of the amino acids in the ruminal solution during at least a part of the experiment. The one exception is the uncorrected data for tyrosine. Even in this experiment, however, there were decreases of concentration that were significant at the 10 per cent level. At the same time, the concentrations of the amino acids in the control solution TABLE 7 e - Ruminal fistula experiments 7/3, Y i T 4 , W5, and w 6 : changes in concentration (milligram-moles per liter) of propionic acid (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a second set of data is given: this set results from multiplying the first set of data by polyethylene glycol correction factors) Experiment Time (min.) W3 W4 W5 W6 Ruminal solution Control a Ruminal solution Control 6 Ruminal solution Control 6 Ruminal solution Control 6 Experimental solution • • • • 47+8 (2) 9 • • • 69±9 (4) • » O O 72±13 (4) • * e c 77+16 (4) 0 51±3 (2) 5lt3 (2) 68+1 (4) 68±1 (4) 53+4 53+4 (4) 53+4 53+4 (4) 65+8 (4) 65+8 (4) 10 4616 (2) • • • • 63+5 (4) • • • • 70+4 61+3 (4) • t • • • • O f ) 66±2 (4) • tt • 0 30 45+7 (2) • • « d 61+5 (4) • O « • 59+4 48+3 (4) • • • • •> • • • 58+5 (4) • • 0 o 70 41+2 (2) 54+0 (2) 51+5 (4) 0 * • • 51+3 42+3 (4) • • • e • • • 0 41+11 (4) • • • • 150 o • • • • * • • 39±3 (4) 68+1 (2) 35+2 28+2 (4) 73+11 • • a a (2) 28+3 (4) 69+5 (2) The same as for Table 6. TIME (MINUTES) Fig. 4 (First part) o> TIME (MINUTES) Fig. 4 (Second part) Pig. 4 . - Ruminal fistula experiments W3, W4, W5, and W6i changes in concentration of propionic acid in the ruminal solution and in the ruminal solution control TjQ- Q) • The symbols W5-U and W5-C designate the data of experiment W5, uncorrected for water movement and corrected for water movement, respectively. The dotted lines (• •) in the figure demonstrate the concentration changes of polyethylene glycol. The reason for including the polyethylene glycol data in the figure is presented in the text. TABLE 8. - Ruminal fistula experiments W4, W5. and w6» changes in concentration (mioro- gram-moles per 100 milliliters) of tryptophan (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a second set of data is given i this set results from multiplying the first set of data by polyethylene glyool correction factors) Experiment W4 W5 W6 Ruminal solution Control* Ruminal solution Control* Ruminal solution Control* Experimental solution** • • • • 18.5+0.8 (3) • . 5.4+0.7 (3) • • . . 9.7+1.6 (s) 0 13.5+1,1 (12) 13.5+1.1 (12) 1.2+0.2 1.2+0.2 (12) 1.2+0.2 1.210.2 (12) 7.9+0.9 (12) 7.9+0.9 (12) 10 13.9±1.3 (11) 12.4+0.9 (11) 0 0 (12) 1.4+0.2 1.3+0.2 (12) 0.8+0.2 (12) 13.4+1.4 (12) 30 12.6+0.7 (12) 12.3+0.8 (12) 0 0 (12) 1.6+0.2 1.4+0.2 (12) 0.8+0.1 (12) 12.911.4 (12) 70 8.1+0.9 (12) 11.2+0.7 (12) 0 0 (12) 1.810.2 1.5+0.2 (6) 0.2+0.2 (12) 12.3+1.0 (12) 150 3.9+1.5 (12) 9.5+1.6 (12) 0 0 (12) • . . . .... 1.5+0.3 (12) 11.0+0.5 (6) **tlThe same as for Table 6. CJ1 o 15 _ to U J W4 O O rr u o. < x a o DIV., 0-M50: p <.005 10 5 _ W5-U -.005(0-10) 30 . 0 0 5 ( 0 - 7 0 ) 70 10 30 70 70 TIME (MINUTES) _ 55.3 36.9 18.4 150 # « 168.8 84.4 A o 150 150 o 0) o -n TJ o m H X •< r m z m o o o 5 m g H —H m Fig. 5. Fig. 5. - Ruminal fistula experiments W4, W5, and W6i ohanges in concentration of tryptophan in the ruminal solution (A A) and in the ruminal solution control (Q Q)» plus changes in concen tration of polyethylene glycol (• • • • -m) in the ruminal solution. In the graph for experiment W4, there is recorded the inscription, MDiv., 0 >150t P (. .005.M This type of inscription is entered in those graphs for which any doubt exists that a negative gradient, of the best-fit line for the data of the ruminal solution control might not diverge.significantly from the negative gradient of the best- f i t line for the data of the ruminal solution* The inscription reoorded in the graph for experiment W4 indioates that the divergence of those two best-fit lines (not drawn in the graph) which are based on the 0 to 150 minute data, inclusive, is significant at the 0*5 per oent level or less. The rest of the symbols in this figure have the same meaning as previously described in Figures 3 and 4. TABLE 9* •Ruminal fistula experiments W4, W5, and W6i changes in concentration (micro* gram-moles per 100 milliliters) of methionine (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a seoond set of data is given t this set results from multiplying the first set of data by polyethylene glyool correction factors) Experiment Time W4 W5 W6 (min*) Ruminal solution Control* Ruminal solution Control* Ruminal solution Control* Experimental solution1* • • • • • . • • .... .... .... .... 0 21.312.0 (8) 21.3+2.0 (8) 7*4+1*2 7.4+1.2 (8) 7.4+1.2 7.4+1.2 (8) 18.811*5 (8) 18.8+1.5 (8) 10 20*6±1*7 (8) 22.2+1*8 (8) 8*9+1*9 7.7+1*6 (8) 10.8+2.5 9*6±2.2 (8) 15.4+1.3 (8) 20.9+2.1 (8) 30 19.0+0.9 (8) 22.5+2.3 (8) 5.2+0.7 4.2+0.6 (s) 10.5+2.1 9.311.9 (7) 15.8+0.5 (8) 21.5+3.1 (8) 70 16.6+1.1 (8) 21*6±1.7 (8) 4.4+1.2 3.6+1.0 (8) 10.8+1.2 8.9+1.0 (7) 8.9+2.5 (8) 20.9+2.9 (8) 150 13.5±0.9 (8) • • • . 4.0+1.1 3.2+0.9 (8) • • . . .... 8.0+1.3 (8) .... •^ The same as for Table 6. Fig. 6. - Ruminal fistula experiments W4, W5, and Vf6i changes in concentration of methionine in the ruminal solution (A A ) and in the ruminal solution control (Q 0), plus changes in concen tration of polyethylene glyool (••••••) in the ruminal solution. The symbols in this figure have the same meanings as previously described in Figures 3 and 4. TABLE 10* - Ruminal fistula experiments W4, W5, and W6t ohanges in concentration (mioro- gram-moles per 100 milliliters) of. tyrosine (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a second set of data is given t this set results from multiplying the first set of data by polyethylene glycol correction factors) Experiment Time (min*) W4 W5 W6 Ruminal solution Control* Ruminal solution Control* Ruminal solution Control 6 Experimental solution1' • • • • 21.511.0 (2) . . . . . « . . . . . . . 18.2+2.9 (2) 0 21.3+2.7 (12) 21.312.7 (12) 3.6+0.7 3.6+0.7 (8) 3.6+0.7 3.6+0.7 (8) 14.0+1.6 (12) 14.0+1.6 (12) 10 22.411.8 (12) 20.5+1.5 (12) 2.9+0.8 2.5±0.7 (8) 5.2+0.3 4.610.3 (5) 10.811.8 (12) 16.9+1.5 (12) 30 21.312.3 (12) 21.0+1.9 (12) 3.6+0.4 2.9+0.3 (4) 5.0+0.8 4.4+0.7 (6) 13.0+2.2 (12) 18.3+2.0 (10) 70 19.7+U9 (12) 20.2+1.8 (12) 3.2+0.2 2.6+0.2 (8) 5.3+0.6 4.4+0.5 (3) 11.311.7 (12) 16.511.2 (12) 150 16.7+1.6 (12) . . . . 3.0+0.3 2.410.2 (8) . . . . . . . . 11.0+1.9 (12) . . . . *»tThe same as for Table 6 . cn cn TIME ( MINUTES) Fig. 7 . Fig. 7. - Ruminal fistula experiments W4, W5, and W6t changes in concentration of tyrosine in the ruminal solution 0^ and in the ruminal solution control (G O), plus changes in concen tration of polyethylene glycol (• - - - - •) in the ruminal solution* In the graphs for experiment W4, there is recorded the inscription, wDiff., 0 >150j P < • 0 0 5 T h i s type of inscription is entered in those graphs for which any doubt exists that there is not a significant difference be tween the i n i t i a l (0 minute) and final (150 minute) concentrations of a constituent in the ruminal solution* To make the test of significance, the slope of a line joining the mean concentration of the 0 minute sample to the mean concentration of the 150 minute sample was tested for its diverg ence from a zero gradient* The inscription recorded in the graph for experiment W4 indicates that the difference between the i n i t i a l and final concentrations is significant at the 0*5 per cent level or less* In the graph for experiment W5-U, there is added to one of the statements of significance the sign, ro • This sign is used in this case to indicate that the level of significance is only slightly higher than the 10 per cent level* The sign is used again with the same type of meaning in the graph for experiment W6* The rest of the symbols in the figure have the same meaning as previously desoribed in Figures 3 and 4* TABLE 11• - Ruminal fistula experiments W4, W5, and W6t ohanges in concentration (mioro- gram-iaoles per 100 milliliters) of glycine (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a second set of data is given * this set results from multiplying the first set of data by polyethylene glyool correction factors) Experiment Time (min.) W4 W5 W6 Ruminal solution Control* Ruminal solution Control* Ruminal solution Control* Experimental solution* • • • • 189.2+22.4 (8) . . . . 86.4+16.9 (8) • • • • . 201.4+13.1 (7) 0 172.5+18.9 (8) 172.5+18.9 (8) 60.1+9.7 60.1+9.7 (8) 60.1+9.7 60.1+9.7 (8) 193.6+18.4 (8) 193.6+18.4 (8) 10 185.8115.2 (8) 180.5+20.6 (8) 82.2+11.5 71.3+10.0 (8) 84.6+15.1 75.5+13.5 (8) 172.9+14.0 (8) 193.6+14.5 (8) 30 183.0+5.9 (8) 188.0+11.7 (8) 78.9+6.6 63.8±5.3 (8) 92.2+5.9 81.7+5.2 (8) 178.0+5.9 (8) 193.2+9.7 (8) 70 176.4+10.3 (8) 193.8+6.7 (8) 72.2+9.4 59.2+7.7 (8) 91.218.8 75.1+7.2 (8) 155.2+21.0 (8) 196.7+5.9 (8) 150 156.5+17.9 (8) 190.2+25.6 (8) 69.7+11.9 55.8+9.5 (8) 85.7+13.7 . . . . (8) ^_________ 130.2+12.5 (8) i 200.5+9.7 (8) a» tThe same as for Table 6. 70 Tl ME (MINUTES) Fig. 8 . (First part) Fig. 8. (Second part) Pig. 8* - Ruminal fistula experiments W4, W5, and W6» changes in concentration of glycine in the ruminal solution (A A,) and in the ruminal solution control (Q O), plus changes in con centration of polyethylene glycol (• •) in the ruminal solution* The symbols in the figure have the same meaning as previously described in Figures 3, 4, and 7* TABLE 12* - Ruminal fistula experiments W4, W5, and W6t change in concentration (mioro- gram-moles per 100 milliliters) of lysine (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5f a seoond set of data is given t this set results from multiplying the first set of data by polyethylene glyool correction factors) Experiment Time (min*) W4 ; W5 W6 Ruminal solution Control* Ruminal solution Control* Ruminal solution Control* Experimental solution1* • • • • 187.6+5.5 (3) . . . . 62.0+3.5 (3) . . . . 177.8+4.4 (3) 0 185.918.4 (12) 185.9+8.4 (12) 59.9111.3 59.9+11.3 (12) 59.9+11.3 59.9+11.3 (12) 176.4tl4.9 (8) 176.4+14.9 (8) 10 174.0+12.5 (12) 183.5+14.0 (12) 72.614.8 63.0+4.2 (12) 76.3+6.5 68.115.8 (12) 163.1+19.8 (8) 175.8110.7 (8) SO 170.5+15.4 (12) 192.3+14.8 (12) 68.9+3.6 55.7+2.9 (12) 77.7+4.3 68.9+3.8 (12) 156.6+4.3 (8) 180.7+13.3 (8) 70 166.2112.5 (12) 184.2+10.7 (12) 64.2+3.4 52.6+2.8 (12) 79.0+4.7 65.113.9 (12) 147.3+8.3 (8) 181.7+13.2 (8) 150 144.2+10.3 (12) 183.7+16.3 (12) 56.3+5.6 45.1+4.5 (12) 78.1+5.4 . . . . (11) 128.1110.2 (8) 184.0112.0 (8) *'l3The same as for Table 6. Fig. 9 (First part) TIME (MINUTES) Fig. 9 . (Second part) Fig. 9. - Rundnal fistula experiments W4, W5, and W6t changes in concentration of lysine in the ruminal solution (__ __) and in the ruminal solution control ^ 0 ) » P*us changes in concentration of polyethylene glycol (• a) in the ruminal solution. The symbols in the figure have the same meaning as previously described in Figures 3, 4, and 7. usually increased or remained constant* The data of the control solution for tryptophan in experiment W5 (Fig. 5), however, exhibited a progressive decrease of concentration. The decreases of tryptophan concentration in the ruminal solution were, nevertheless, s t i l l greater than those in the control solutions; and, the best-fit lines for the concentrations in the ruminal and control solutions exhibit a divergence that is significant at the 0,5 per cent level or less. A comparison of the slopes of the lines for the adjusted polyethylene glycol data and the amino acid data demon strates that, with one exoeption, the concentration changes of the amino acids in the ruminal solution were not a reflection of a dominant influence of water movement. The one exception is for lysine in experiment YT4 (Fig. 9). In this experiment, a large part of the concentration changes could have resulted from the effects of water movement. There is s t i l l a part of the concentration decrease, however, for which water movement can not account. These results support the hypothesis that the amino acids can be absorbed from the rumen. Alpha Amino Mitrogen The results of the Van Slyke assay for alpha amino nitrogen in experiment W6 are presented in Table 13 and Figure 10. Examination of Figure 10, taking note of the statements of significance, reveals a large decrease in the concentration of alpha amino nitrogen in the ruminal solution. At the same time, there is an over-all increase in the concen tration of alpha amino nitrogen in the control solution. A comparison of the slopes of the lines for the adjusted polyethylene glycol data and the alpha amino nitrogen data demonstrates that the concentration changes of alpha amino nitrogen in the ruminal solution were not a reflection of a dominant influenoe of water movement. These results for alpha amino nitrogen support the hypothesis that a l l or some of the amino aoids can TABLE 13• - Ruminal fistula experiment W6» ohanges in concentration (milligrams per 100 mill i l i t e r s ; of alpha amino nitrogen (the data reoorded are means and standard deviations, with numbers of assays in parentheses) Time (min*) Experiment W6 Ruminal solution Control* Experimental solution*3 • • • •• .... 0 37.110.9 (4) 37.110.9 (4) 10 34.711.3 (3) 41.511.6 (4) 30 34.112.0 (4) 39.213.1 (3) 70 32.211.9 (4) 57.910.6 (4) 150 29.111.5 (4) 40.112.0 (4) a»°The same as for Table 6. Fig. 10. - Ruminal fistula experiment W6j changes in concentration of alpha amino nitrogen in the ruminal solution (& Aj a J 1^ i * 1 ruminal solution control (Q Q), plus changes in concentration of polyethylene glycol (• aO in the ruminal solution. The symbols in the figure have the same meaning as previously described in Figures 3 and 4. 05 CD "be absorbed from the rumen. B Vitamins> Riboflavin, Nicotinic Acid, and Pantothenic Acid The results of the microbiological assays for these substances are presented in Tables 14 to 16 and in Figures 11 to 13. Examination of the figures, taking note of the statements of significance, reveals that in a l l the experiments, except one, there were decreases in the concen trations of the B vitamins in the ruminal solution during at least a part of the experiment. At the same time, the concentrations of the B vitamins in the control solution either exhibited increases, or remained relatively constant, or exhibited slight decreases. A slight concen- tration decrease occurred in the control solution for riboflavin in experiment W5 (Fig. l l ) . The decrease of riboflavin concentration in the ruminal solution was s t i l l greater than that in the control solution; and, the best-fit lines for the concentrations in the ruminal and control solutions exhibit a divergence that is significant at the 1 per cent level or less. The one exception to the previous statement—that i s , about the decreases in the concentrations of the B vitamins in the ruminal solution—are the results for pantothenic acid in experiment W5 (Fig. 13). In this experiment, there was not in the ruminal solution a concentration decrease that was significant at the 10 per cent level or less. In experiments W3 to W6, a comparison of the slopes of the lines for the adjusted polyethylene glycol data and the B vitamin data demon strates that the concentration changes in the ruminal solution were not a reflection of a dominant influence of water movement. In experiments Wl and W2, the absence of polyethylene glycol or pectin data does not permit this type of comparison to be made. As, however, the solutions in these two experiments were strongly hypotonic (see p. 35), i t is likely TABLE 14a. - Ruminal fistula experiments Wl and W2» changes in concentration (microgram-moles per 100 milliliters) of riboflavin (the data recorded are means and standard deviations, with numbers of assays in parentheses) Experiment Time (min.) Wl W2 Ruminal solution Control* Ruminal solution Control* Experimental solution • • • • 5.9+1.8 (8) a • • . 4.511.1 (9) 0 5.6+1.6 O) 5.6+1.6 (9) 3.5+0.7 (8) 3.5+0.7 (8) 40 3.2+0.9 0) • • • • . . a . .... 60 4.5+1.0 (8) . . • • .... .... 65 . • • • • • • . 2.3+0.6 (9) .... 95 2.7+0.8 (9) .... 2.7+0.6 (8) .... 120 3.1+0.6 (8) 5.0+0.7 (9) . • • . .... TABLE 14a - continued Time (min • ) Exper iment Wl W2 Ruminal solution Control 8 Ruminal solution Control 8 125 • • t • • • • • 2 •8+0.7 0) 4.3+1.4 (9) aThe word "control" here designates the data of the experimental solution control. Data for the ruminal solution control are not avail able for these two experiments (see p. 20). The same as for Table 6. TABLE 14b. - Ruminal fistula experiments W4, W5, and W6» changes in concentration (micro- gram-moles per 100 milliliters) of riboflavin (the data recorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a second set of data is given i this set results from multiplying the fi r s t set of data by polyethylene glyool correction factors) Experiment Time (min*) W4 W5 W6 Ruminal solution Control* Ruminal solution Control* Ruminal solution Control* Experimental solution** • • • • 18.0+4.2 (8) . . . . 7.9±2.0 (8) . . . . 10.4+2.1 (4) 0 14.2+3.1 (8) 14.2+3.1 ," (8) 5.9+0.8 5.9+0.8 (8) 5.9+0.8 5.9+0.8 (8) • • • • . . . . 10 18.0+2.6 (8) 19.2+2.8 (8) 7.7+1.0 6.6+0.9 (8) 7.7+1.4 6.9+1.3 (8) 12.4+1.5 (8) 11.411.5 (8) 30 16.9+2.9 (8) 20.1+3.4 (8) 7.6+1.1 6.1+0.9 (8) 8.9+0.8 7.9+0.7 (8) 13.1+2.1 (8) 11.8+1.8 (8) 70 14.9+2.7 (8) 19.4+3.5 (8) 7.4+0.7 6.0+0.6 (8) 8.5+1.0 7.0+0.9 (8) 12.8+1.6 (8) 13.5+1.1 (8) 150 15.2+1.9 (8) 19.6+3.5 (8) — 5.710.9 • 4.5+0.7 8.1+0.9 . . . . (8) 10.7+2.0 (8) 14.811.8 (8) a»bThe same as for Table 6. 10 - CO Q_ _) r- 5^ — — — I ^ I -mi*?'in) - • Wl A -J -J r\ • , DIFF., 0*120: P C005 | 1 s 0 40 60 95 120 o 2 5. or a. > < - i u. O CO o CO bJ _ l o I s < or o o OC o x .10(65*125) W2 DIFF., 0*125: P <.I0 _ l . _ L 10 W4 DIFF., 0*150: P>.I0 10 30 70 10 o > (A o "O o m H X -< 70.1 __ m -452.6 g o 35.1 ^ m 68.6$ 150 x t \ 0 0 5 ( 0 * I 0 ) opsjc^g -g—. — — — — • - __5r3cr__) ^ q . 0 0 5 ( 1 0 - 1 5 0 ) W5-U JL DIFF.,0-150: P>.I0 I DIV., 10*150: P <.0I J4.3ro 10 30 70 TIME (MINUTES) 150 Fig. 11. (First part) - 3 I 0 i - to £ 51 2 .005(0*30} _ Q £5(30-70) v , ^ ^ * - & — -OOS(/o-.|50) — 2 W5-C I 1 DIFF., 0-150: P -C.005 1 -YCO I o 2 0 r O cr 15 UJ O. 10 10 30 70 .005(I0-I50L_ 83.« » , 2 8 -o •< 62.31} ™ m r 41.5 10 30 70 T I M E ( M I N U T E S ) 150 F i ? . 11. (Second part) Fig. 11. - Ruminal fistula experiments Wl, W2, W4, and W6» changes in concentration of riboflavin in the ruminal solution (A f_) and in the ruminal solution control Q), plus changes in concentration of polyethylene glycol (• •) in the ruminal solution. The symbols in the figure have the same meaning as previously described in Figures 3, 4, 5, and 7. TABLE 15a. - Ruminal f i s t u l a experiment Wl: changes in concentration (microsram-moles per 100 m i l l i l i t e r s ) of nicotinic acid (the data recorded are means and standard deviations, with numbers of assays i n parentheses) Experiment Wl Time (min.) Ruminal solution Control a Experimental solution* 3 • • • • 14.S+2.4 (3) 0 12.2±2.2 (3) 12.212.2 (3) 40 8.3+0.5 (3) . . . . 60 8.210.8 (3) . . . . 95 6.910.2 (3) . . . . 120 5.7+0.2 (2) 13.7+2.5 (3) aThe same as for Table 14a. The same as for Table 6. TABLE 15b. - Ruminal fistula experiment W5 and W6s changes in concentration (miorogram-moles per 100 milliliters) of nicotinic aoid (the data reoorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5 a second set of data is given t this set results from multiplying the first set of data by polyethylene glyool correction faotors) Experiment Time (min*) W5 W6 Ruminal solution Control* Ruminal solution Control* Experimental solution'1 • • • • 29.9*7.2 (12) . . . . 77.9+7.5 (12) 0 24.7±3.6 24.7±3.6 (12) 24.713.6 24.713.6 (12) . . . . . . . . 10 34.3+6.2 29.815.4 (12) 32.9+3.2 29.312.8 (12) 71.3+5.0 (12) 76.9+5.5 (12) SO 33.616.3 27.1+5.1 (10) 33.412.3 29.612.0 (12) 72.6+11.1 (12) 72.6+5.2 (12) 70 29.5+6.1 24.314.9 (12) 35.8+6.1 29.5+5.3 (12) 64.315.4 (12) 73.3+5.4 (12) 150 30.012.1 24.0+1.7 (12) 36.7+7.3 . . . . (12) 58.1+5.9 (12) 73.6+5.4 (12) •^ The same as for Table 6. « 5 L. c f V ^ ^ 005(0*10). £ 5 ( _ 1 0 _ - * I 5 0 ) 05 ( /0->7m DIFF, 0-150: p<.005 30 70 30 20 80 1 o 65.6 J CO 32.8 g m 150 ^>^r 5 / - —.005(0*10) — 1 DIFF., 0*150: P>.I0 _ 0 10 30 70 150 I -< r m 2 m o o o 70 T I M E (MINUTES) F i g . 12. Fig« 12» - Ruminal fistula experiments Wl, W5, and W6i changes in concentration of niootinio acid in the ruminal solution A) and in the ruminal solution control (Q Q)» plus changes in concentration of polyethylene glycol (•»••• -•) in the ruminal solution* The symbols in the figure have the same meaning as previously described in Figures 3, 4, and 7» TABLE 16a* * Ruminal fistula experiment Wit changes in concentration (miorogram-moles per 100 milliliters) of pantothenic acid (the data recorded are means and standard deviations, with numbers of assays in parentheses) Time (min*) Experiment Wl Ruminal solution Control* Experimental solution0 • • • • 5.8+0.2 (2) 0 5.7±0.6 (3) 5.710.6 (3) 40 3.9+0.1 (3) . . . . 60 5.910.5 (3) . . . . 95 3.8+0.1 (3) . . . . 120 4.1+0.2 (3) 5.710.4 (3) *The same as for Table 14a. t>The same as for Table 6. TABLE 16b* - Ruminal fistula experiments W4, W5, and 7/6: ohanges in concentration (micro- gram-moles per 100 milliliters) of pantothenic acid (the data reoorded are means and standard deviations, with numbers of assays in parentheses; in experiment W5, a second set of data is given t this set results from multiplying the first set of data by poly ethylene glycol correction factors) Experiment Time (min*) W4 W5 W6 Ruminal solution Control* Ruminal solution Control* Ruminal solution Control* Experimental solution • • • • 17*4+2*9 (12) • * * • 7.7+1*4 (12) • . . • 16.114*1 (12) 0 20*6±7.2 (12) 20*6±7*2 (12) 7.2+0.8 7.210.8 (12) 7.2+0.8 7 .2+ 0.8 (12) 10.8+-1.3 (12) 10.8+1.3 (12) 10 20.8+5.6 (12) 18*3+3*7 (12) 8*9+0*9 7*7±0.8 (12) 8.910.9 7.9+0.8 (12) 10.0+^ .0 (12) 16.8±2«4 (9) 30 16.2+4.0 (12) 16.212.0 (12) 9*9+2*4 8.0+1.9 (12) 9.9+1.4 8.8+1.2 (12) 9.1+1.3 (12) 10.612.8 (12) 70 13.8+2.3 0) 19.7+3.9 (12) 9.0+1*7 7.4+1*4 (12) 9*511*2 7*8+1.0 (12) 8*410*7 (12) 11.412*6 (12) 150 13.6+3*4 (12) 18.5+4.0 (12) 8.811.6 7*0+1.3 (12) 9.6+1.6 . * . • (12) • . • • 9.912.3 (12) a» bThe same as for Table 6. s o DIFF., 0-120: P <-025 40 60 95 120 o > 53.2 2 CO SSgtlO-TQ) 41.1 o -n o I DIFF..0-150; P <.0I 29.0 m 150 70 TIME (MINUTES) Fig. 13. (First part) X z _ o CO U l _J o 5 < cr o o rr o CO cc LU 18 - 2 13 U 0. < 00S(0-»7O) -< o _ , 85.7O 38. l £ 70 TIME (MINUTES) 150 m o ~t j> CO o TJ a m -t x -< r-m z m F i g . 13. (Second part) Fig. 13. - Ruminal fistula experiments Wl, ¥4, W5, and W6: changes in concentration of pantothen  ic acid in the ruminal solution (A A) and in the ruminal solution control (Q 0, plus changes in concentration of polyethylene glycol (• •) in the ruminal solution. The symbols in the figure have the same meaning as previously described in Figures 3, 4, and 7. that water was absorbed from the rumen* Suoh absorption, i f not accompanied by a more rapid absorption of the other constituents in the ruminal solu tion, would produce increased concentrations of these other constituents* The fact that the decreases in the concentrations of the B -vitamins occurred in spite of the concentrating effeot of the water absorption indioates that the actual decreases were probably greater than the recorded decreases* These results for the B vitamins, excluding the results for pantothenic acid in experiment W5, support the hypothesis that the B vitamins can be absorbed from the rumen* Temperature and pH Table 17 presents the temperature and pH measurements made for the six ruminal fistula experimentsi experiments Wl to W6* Examination of the table reveals that, throughout experiments Wl and W2, the temperature of the solutions in the rumen approximated to the body temperature of the 20 goat* The examination also reveals that the pH of the solutions re mained slightly acidic* The same two statements apply to experiment W3, except that the temperature of the solution was several degrees lower than the body temperature during the fi r s t part of the experiment* In experiments W4 to W6, the temperatures of the solutions in the rumen were several degrees below body temperature throughout the experiment* The pH of the solutions for these experiments did not remain acidic, but became basic (pH 7*3 to 7*6) between 30 to 60 minutes after the beginning of the experiments* The following quotation (344; see also 88, 230) is pertinent 2°Rectal temperatures for the goat have been recorded within the range of 38*7 to 40*7° C*, with an average temperature of 39*9° C. (102)* TABLE 17. - Ruminal fistula experiments Wl, W2, W3, W4, W5, and W6i temperature (°C*) and pH measurements for the ruminal solutions (the data in the upper left-hand corner of the squares are pH measurements] the data in the lower right-hand corner are temperature measurements) aThis is the ruminal solution before being placed in the rumen* 88. to the pH changes that occurred in experiments W4 to W6j Any solution in the rumen tends to change to a pH of 7.2 - 7.4, whether acid or alkaline originally, and this is due to a differential passage of ions across the epithelium in both directions. - continued on p. 89 -Discussion •The results of the experiments for propionic acid, the amino acids, and the B vitamins may be interpreted to support the hypothesis that these constituents can be absorbed from the rumen* There remain, however, several details that require discussion* Stability of Amino Acids and B Vitamins During Freezing Storage 21 As there was a relatively long period of storage before the samples of the ruminal f i s t u l a experiments were assayed, the question can be raised as to the st a b i l i t y of the constituents during this period of stor age of the samples* In each of the experiments, before the experimental solution was added to the rumen, a sample (designated as experimental solu tion control) of the experimental solution was removed* Unfortunately, this control sample was not assayed at the time of i t s removal; rather, i t was frozen and stored along with the other samples taken during the experiments* The assays of the control sample were performed at the end of the storage period, along with the assays of the other samples. There are not, therefore, any control data for the original concentrations—i*e., the concentrations at the beginning of the storage period—of the constituents in the experimental solution* The ava i l a b i l i t y of such control data, together with the data of the assays at the end of the storage period, would have permitted A l l the samples, including those for the experimental solution controls, were frozen and stored together. Between one and six weeks after the beginning of the storage period, the samples were thawed period i c a l l y to permit subsamples to be taken for vitamin assays* Between two and six months after the beginning of the storage period, the samples again were thawed periodically, this time to permit subsamples to be taken for amino acid assays* In addition to these periods of thawing to permit subsampling, the samples also thawed out during their transfer five hundred miles by car from^the University of British Columbia to the Washington State University (see p» 22)• a determination of the stability of the amino acid and vitamin molecules during their storage at freezing temperatures. In place of the missing control data, the calculated concentrations recorded in Table 5 for the constituents of the experimental solutions can be utilized. Table 18, listing only those constituents that -were assayed, presents a comparison between their calculated concentrations at the beginning of the storage period and their analytically-determined oonoentrations at the end of the storage period. By the use of this table, evidence may be gained as to the stability of the constituents between the time of their use in the experiments and the time of their assay* Evidence of Instability.- Examination of Table 18 reveals that for some of the constituents—tryptophan, tyrosine, probably methionine, and riboflavin in experiments W4 to W6—the calculated concentrations do not lie within or close to the 95 per cent confidence intervals for the analytically-determined concentrations. These results may indicate poor stability of these constituents under the storage conditions to which they were exposed in the present investigation. There are, however, several other suggestions that can be made to explain these significant discrepan cies between the calculated and the analytically-determined concentrations* 1* The calculated concentrations of the constituents listed in Table 18 consist of composite figures that are based ont (a) the manu facturer's estimate of the concentrations of the amino aoids in the casein acid-hydrolysate solution, (b) the weighed-out amounts of those constituents that were used in addition to the casein hydrolysate in preparing the experimental solutions* The manufacturer's estimate of the amino acid concentrations did not include confidence intervals. There is a possibility that at least some of the 95 per cent confidence intervals of the calcu-TABLE 18• - Ruminal fistula experiments Wl to W6t moleoular compounds that were examined for absorption from the rumen; a comparison between their calculated8- and analytioally-determined0 concentrations in the original experimental solutions 0 o x s a a Experiment Moleoular Wl W2 W3 W4 W5 W6 oompound h h k k h •a © ta r H -rj a $ o r j "S •8 r H - r j a) © o t t •H >H £ l r H «> a) 4> •% tfl r H TJ <t © O {3 rr j © •8 r H >rj s i *H «H % •8 r H T j 01 © o tt © ta r H T J «) g> o a u J , r 1 Q i H g &1 w r H g w r H § ID r H g & © el 4 i 43 u# r H g W r H g £§ r H aS O 4 l r H at O r H a) o 4 i r H al o r H o •~j w l-H « O 1* Amino aoids d Glycine • • • • • • * .* * • • • • • • • • • 1*60 1*89 + 0*19 0*80 0*86 + 0.14 1*60 2*01 + 0.12 Lysine • • • •• • • • • • • • • • • • • • 1*62 1.88 + 0.14 0,81 0*62 •+ 0*09 1*62 1.78 0.11 Methionine6 • •• ••• • •• • •• • • • • •• 0.80 0.21 + 0.02 0.40 0.07 0.01 0.80 0.19 + 0.02 Tryptophan • •• • • • • •• • • • • • • 0.49 0.19 ± 0.02 0*25 0.05 ± 0.02 0.49 0.10 + 0.04 Tyrosine • • • ••• ... ... • • • • 1.15 0.22 + 0.09 0.58 ... 1.15 0.18 -+• 0.26 1 TABLE 18 - continued Molecular oompound Experiment Wl •_ o "8 rH 3 O rH « C3 rH rH XI o d S I rH 0 «> 4 3 4 l W2 i" •8 o rH O rH rH TJ w © O CJ •rl rH O 4 l ws © •8 rH O rH 0 O I rH • •H rH O 41 W4 •s rH O r H cri O a * rH © a) 4J 4 l •W5 W6 © s o rH at U I rH « d § s •rl •H M at +» 41 •a © •8 r H O rH at a Vitamins Nicotinic aoid Pantothenic acid Riboflavin Other Sodium propionate 0.16 0,04 0*05 0.15 0*06 0*06 0.02 0.06 ± 0.02 0*05 0*05 + 0.01 0.81 0.21 0*27 52 47 68 52 0.17 ± 0.02 0.18 + 0.04 69 + 14 0.41 0.11 0.14 52 0.50 +• 0.05 0.08 + 0.01 0.08 4- 0.02 72 + 21 0.81 0.21 0*27 52 *The calculated data are based ont 1. the manufacturer's estimate of the concentrations of the amino acids hydrolysate solution; in the casein aoid-1 • 2. the weighed-out amounts of the constituents added in preparing the experimental solutions. "The data for the analytically-determined concentrations are presented as means with their 95 per cent confidence intervals designated* °The ruminal solution before i t was placed in the rumen. ^Comparative data for alpha amino nitrogen exist only in experiment ¥6. In this experiment, the calculated concentration of alpha amino nitrogen in the experimental solution is 42.6 milligrams per 100 milliliters (from Table 13, p. 67). Unfortunately, the experimental solution was not analyzed for :its content of alpha amino nitrogen; however, the analytically-determined concentration of alpha amino nitrogen in the 0 minute sample of the ruminal solution—that i s , the experimental solution after being placed in the rumen—is 37.1±1.4 milligrams per 100 milliliters. This concentration is expressed as the mean with its 95 per oent confidence interval. eThe experimental solutions of experiments W4, W5, and W6 were not assayed for their methionine concentrations; therefore, the methionine oonoentrations in the 0 minute samples of the ruminal solu tions for these experiments are substituted as possible approximations. lated concentration means may have overlapped the 95 per cent confidence intervals of the analytically-determined concentration means. This suggestion can not apply to tryptophan, as supposedly the only tryptophan present in each of the experimental solutions was that which was added during the preparation of the solutions* 2* The manufacturer's casein acid-hydrolysate solutions were stored at room temperature on an open shelf for an unknown length of time. Decreases in the concentrations of some of the amino acids may have occurred during this time of holding because of molecular destruction or precipitation* 3. The effect of poor stability in the period of freezing storage may have been combined with the effects of one or both the factors in the above two suggestions* Unfortunately, there is not enough evidence available to decide whether poor stability of some of the constituents during their storage period or one of the other factors discussed is responsible for the observed discrepancies* Fortunately, the decision as to which of the factors i t was that caused the significant discrepancies between the calculated and analytic ally-determined means is not critical for the previously-made proposal that the results of the present ruminal fistula experiments support the hypothesis of ruminal absorption of amino acids and B vitamins; that i s , the existence of the discrepancies does not alter the fact that concen tration decreases of a l l the assayed constituents occurred in the rumen* Knowledge of the cause of the discrepancies becomes important, however, when an attempt is made to compare the oonoentrations of the constituents in the ruminal solution with their concentrations in the blood (see p* 2Ds)* If the discrepancies are in part or wholly the result of the effect of poor stability of the constituents during the storage period of the samples, then such a comparison may be meaningless—because the concen trations of the constituents in the ruminal solution samples at the end of the storage period would be less than the concentrations of the constit uents in the ruminal solution at the time of the experiments* If the discrepancies are the result of the effects of the factors in the first two suggestions, then a comparison between the concentrations in the ruminal solution samples and the concentrations in the blood samples probably would be valid—because the discrepancies would have been effect ed before the experiment began* As a comparison will be made in the General Discussion between the concentrations of the experimental constit uents in the ruminal solutions and the concentrations of the same constit uents in the blood, the question of the discrepancies wil l be discussed again* Evidence for Stability*- In contrast to the constituents that were discussed in the previous section, the calculated concentrations of the other oonstituents—glyoine, lysine, nicotinio acid, pantothenic acid, sodium propionate, and riboflavin in experiments Wl and W2—usually l i e within or just outside the 95 per cent confidence intervals for the analytically-determined concentration means* There also is evidence (see footnote d to Table 18) that many of the other amino acids in the experi mental solution controls would exhibit, upon assay, the same close agree ment between the calculated and the analytically-determined concentration means. This evidence derives from a comparison in experiment W6 of the calculated concentration of alpha amino nitrogen for the experimental solution control with the analytically-determined concentration mean of alpha amino nitrogen for the 0 minute sample*5*5 from the rumen* The calou* lated concentration of 42*6 lies not far above the upper limit, 38*5, of the 95 per cent confidence interval for the analytically-determined concentration of 37*1* The relative closeness of these two concentra tions probably indicates that the calculated concentrations for most the individual amino acids in the experimental solution controls would l i e close to, or within, the 95 per cent confidence intervals for the analytically-determined concentrations. These results can be interpreted to mean that, in addition to the apparent stability of nicotinic, panto thenic, and propionic acids, stability also was a property of glycine, lysine, and most of the other amino acids* Fluctuations of Constituents1 Concentrations Examination of the figures (Figs* 5 to 13) for the ruminal fistula experiments reveal that concentration fluctuations occurred for the constituents in both the ruminal solutions and the ruminal solution controls* The concentration decreases of the constituents in the ruminal solutions oan be explained as resulting from the absorption of the constituents through the ruminal wall* There may be, however, addition al factors whose effects cause concentration deoreases or increases of the constituents* Suggestions are made below as to the possible identity of these factors* Factors Causing Concentration Increases*- The increase of a constituent's concentrations in the ruminal solution and in the ruminal Unfortunately, the experimental solution control was not assayed for alpha amino nitrogen* The analytically-determined concentration for the 0 minute sample of the solution in the rumen is substituted, there fore, as an approximation* solution and in the ruminal solution control may be the result of micro b i a l activity—fermentation, synthesis, autolysis* Although the rumen and reticulum were rinsed with warm tap water prior to the addition of the experimental solution to the rumen, th i s rinsing procedure would s t i l l leave an "inoculum1* of mioroorganisms and ingesta on the walls of these compartments* Upon the addition of the experimental solution to the rumen, the mioroorganisms conceivably could have begun to grow and i n  crease in number* Growth and multiplication of these microorganisms, with their attendant metabolic a c t i v i t i e s , might account for a l l or some of the constituents' concentration increases i n the ruminal solution and in the ruminal solution control* A concentration increase that results from microbial activity in the rumen might mask partly, or even completely a concentration decrease that i s occurring because of ruminal absorption* The present ruminal f i s t u l a experiments may present examples of such a masking effect. Figures 7, 8, 11, 12, and 13 record, for the 150 minutes of the experiments, statements of significance for the net decreases of concentration (reoorded as, for example, Di f f . , 0—>150j P < *0l) i n 24 the rumen* Some of these net decreases do not demonstrate significance at the 10 per cent level or less. The graph for experiment W5-U i n Gross contamination apparently occurred in experiments W2, W4, and W5* The 70 or 150 minute samples from the ruminal solutions in these experiments exhibited either a content of brown sediment or a darker, greener colour than did the samples of the original experimental solutions ?4 fi The slope of the line joining the mean concentration of the 0 minute sample to the mean concentration of the 150 minute sample was tested for the significance of i t s divergence from a zero gradient* This significance test was made whenever any doubt existed that the concentra tion means for the 0 and 150 minute samples might not exhibit a difference significant at the 10 per cent level or less* 9 8 . . Figure 13 demonstrates that there was even a net increase in the concen tration of pantothenic acid in the rumen and that this net increase was significant at less than the 0.5 per cent level. This failure of some of the constituents in some of the ruminal solutions to exhibit, for the 150 minutes of the experiment, net deoreases of concentration that were significant at the 10 per cent level or less occurred in spite of the fact that deoreases of concentration that were significant at the 10 per cent level or less did occur during a part of the experiment* This apparent contradiction could be a reflection of the simultaneous occurrence of microbial activity producing concentration increases and ruminal absorp tion produoing ooncentration deoreases* This suggestion is supported by the fact that concentration inoreases oocurred in many of the ruminal solution controls. The results of these experiments that do not exhibit significant net decreases of concentration s t i l l can be interpreted, then, as supporting the hypothesis of ruminal absorption of amino aoids and 5 vitamins* It is unfortunate that size determinations of the microbial population in the samples were not made* Such determinations, i f reveal ing a continual increase in the size of the miorobial population, could provide further evidenoe to support the validity of the suggestion that miorobial aotivity produced inoreased concentrations of the constituents in the rumen. Factors Causing Concentration Decreases*- Two suggestions can be made to explain the decreases of constituent concentrations in the ruminal solution oontrols* The first suggestion is that decreases of concentra tion may result because of an uptake of constituents by microorganisms. The seoond suggestion is that decreases of concentration may result beoause of microbial or physical-chemical breakdown of the constituent molecules* Moreover, decreases of concentration may be the result of the simultaneous action of both microbial uptake and moleoular breakdown of the constituent molecules* If these factors that produce decreases of concentration were opera tive in the ruminal solution controls, then the same faotors also should have been operative in the ruminal solutions* An effectuation of a concentration decrease of a constituent i n the ruminal solutions by micro b i a l uptake or molecular breakdown would have supplemented any decrease in concentration of the same constituent effected by ruminal absorption* The value of the ruminal solution controls, then, is to provide a compari son between the concentration changes occurring in the control solutions and the concentration ohanges occurring i n the ruminal solutions* By means of this comparison, the concentration decreases effected by ruminal absorption can be identified among the concentration decreases effected by miorobial uptake or physical-chemical breakdown of the constituent molecules* In eaeh the figures of the present experiments (Figs* 5 to 13), this identification can be made by noting a more negative slope for the . lines joining the means of the ruminal solution data as contrasted to the slope of the lines joining the means of the ruminal solution oontrol data* Comment*- In the previous discussion, the factors that may produce increases of constituent concentration have been considered separately from those that may produce decreases of constituent concentration* This method of presentation was adhered to i n order to f a c i l i t a t e the analysis of the effects that might be produoed by the various faotors* Actually* both sets of faotors may be operative simultaneously* In fact, the assump tion made in the present investigation was that, with one exception, a l l the factors effecting increases or decreases of concentration were acting 100 both in the ruminal solutions and in the ruminal solution controls. The exoepted factor-** is ruminal absorption, whose effect is confined to the rumen. It i s , however, the solutions in the rumen, not the control solu tions, that almost consistently exhibited the marked decreases of constit uent concentration. It is proposed, therefore, that the differential decrease in the concentration of the assayed constituents in the rumen reflects the action of ruminal absorption. Acceptance of this proposal means that, even although the possible effects on constituent concentra tion by factors other than ruminal absorption be admitted, the results of the present ruminal fistula experiments s t i l l can be interpreted as supporting the hypothesis of ruminal absorption of amino acids and B vita mins* - continued on p. 101 - To be accurate, also the factor of water movement acted only in the ruminal solutions and not in the ruminal solution controls. In experi ments Wl and W2, there were not any corrections made for water movement. It is known, however, that the solutions in the rumen were strongly hypotonic to the blood (see p. 35). This means that the concentration decreases which took place in these experiments occurred in spite of probable water absorption, a process whose effect would be to concentrate the constituents of the ruminal solutions. In experiments W3 to W6, the effect of water movement was corrected for by the use of the polyethylene glycol data. 101. Part II: Blood-Sampling Experiments The second approach to the detection of ruminal absorption of amino acids and B -vitamins was to demonstrate the passage of these substances into the blood that drains the rumen. There are two phases to this approach. Phase A» Comparison of the Concentrations of Amino Acids and Nicotinic Acid in Peripheral and Portal Blood Introduction Expected higher concentrations of amino acids and nicotinic acid in the -venous blood that drains the rumen were to be compared with expected lower concentrations of amino acids and nicotinic acid in peripheral venous or arterial blood. The lower concentrations of these substances in the peripheral blood would result supposedly by a partial removal of the absorbed amino acids and nicotinic acid from the ruminal venous blood as it filtered through the liver. This experimental approach has been used by several workers to demonstrate the ruminal absorption of volatile fatty acids (e.g. 225, 230, 234) and ammonia (238). The selection of the particular amino acids and B vitamin to be studied and the method of their assay were determined by the same con siderations as given for the ruminal fistula experiments (see p. 19); however, in contrast to the ruminal fistula experiments, the Van Slyke manometric method for alpha amino nitrogen was used more extensively than the microbiological assay. This was done because, although the Van Slyke method only measures the collective concentration of amino acids, it is more sensitive than the microbiological assay for detecting small differences in concentration. Method Experimental Procedure General*- As the procedures for the three experiments of phase A differed somewhat from one another, a general description of the experi mental procedures is not given here; rather, the complete experimental procedure for each experiment w i l l be described separately* Experiment Bl*- In this experiment, blood samples were removed from 26 an anesthetized goat* The blood vessels that were sampled were the left jugular vein and the right ruminal vein (see Fig* 14). Appropriate surgi cal intervention***5 was made to enable the removal of blood samples from the right ruminal vein of the anesthetized goat* 1* Animal history! An adult female goat weighing 103 pounds and of mixed breeding was used* The goat had not been subjected to previous surgical intervention* Food but not water was withheld from the animal for the 24 hour period that preoeded surgical intervention* At the time of the experiment, the animal appeared to be healthy* 2* Composition of the experimental solution. An experimental solution was not added to the rumen in this experiment* 3* Sampling procedure* Ten milliliter samples of blood were withdrawn from the blood vessels. The sequence of sampling of the blood vessels is indicated in Table 19 (p*109). Each blood vessel was sampled as soon as possible after the preceding vessel had been sampled* Experiment B2«- In this experiment, blood samples were removed from a goat, not only while i t was anesthetized, but also on several days The details of the method of ariesthezia and of the surgical proced ures for a l l the experiments of Part II are given in the appendix* Fig. 14. - Venous drainage of the ruminant stomach, viewed from the right (adapted from figure 166 of Martin and Schauder, Lehrbuch der Anatomie der Haustiere, vol. 3, third edition, 1938). The present author traced out the venous drainage of the ruminant stomachs in several sheep and goats and found i t to agree with the description presented by Martin and Schauder (see also 334)• Normally the liver is related ventrally to the retioulum, omasum, and abomasum, but in the illustra tion the liver has been reflected dorsooraniad* Also, the spleen has been elevated from its normal position of lying on the left faoe of the rumen. A» liver; B, spleen; C, oesophagus; D, dorsal sac of rumen; E, posterior dorsal blind sac; F, posterior ventral blind sac; G-, ventral sac of the rumen; H, retioulum; I, omasum; J, abomasum; K, duodenum; a, portal vein; b, mesenteric vein; c, gastro- splenio vein; d, splenic vein; e, right ruminal vein; e', branohes of right ruminal vein on left faoe of rumen; f, left ruminal vein; f', left ruminal vein on left faoe of rumen; g, reticular vein; h, left gastric vein. 105. following the first day of sampling. The blood vessels sampled were several peripheral veins and the right and left ruminal veins (see Fig. 14). To enable the removal of blood samples from the ruminal veins of the anesthetized goat to be made, appropriate surgical intervention was per formed. To enable these same veins to be sampled in the non-anesthetized goat on the days that followed the day of surgical intervention, poly ethylene oannulas were inserted in the veins and the free ends of the cannulas were passed through the dorsolateral part of the body wall in the lumbar region. The insertion of the polyethylene cannulas was carried out as a part of the procedure of surgical intervention (see p. 290 )• 1. Animal historyi An adult made goat weighing 137 pounds and of mixed breeding was used. The goat had not been subjected to previous surgical intervention. Food but not water was withheld from the animal for the 24 hour period that preceded surgical intervention. At the time of the experiment, the animal appeared to be healthy. 2. Composition of the experimental solutiont An experimental solution as such was not used in the present experiment. Usually, how ever, concentrate pellets were fed to the goat prior to the sampling periods. On one occasion, casein hydrolysate powder also was fed. The details as to the quantity of feed given and the time of feeding are presented in the footnotes of Table 20 ( see p. 110). The composition of 27 the concentrate pellets is as followst Alfalfa meal...... 460 Whole corn....... • • 640 Deer Pellets U.B.C. 15-55, 1/4 inch pellets. 106. Wheat bran.. 280 Beet pulp 200 Molasses..... •• 140 Soyabean meal. • 140 Caloium carbonate. 20 Iodized salt 20 Linseed meal • 100 2*555 5. Sampling prooedurei Ten milliliters samples of blood were withdrawn from the blood vessels. The days of sampling and the sequence of sampling of the blood vessels are indicated in Table 20. Each blood vessel was sampled as soon as possible after the preceding vessel had been sampled. The time required to take each day's series of samples ranged from 15 to 30 minutes. Experiment B5.«- In this experiment, blood samples were removed periodically from an anesthetized goat after the addition to the rumen of a solution of amino acids. The solution, previously warmed to 37° C , was added to the rumen by means of a stomach tube. The addition was made just after the goat had been anesthetized. The blood vessels that were sampled were the oommon oarotid artery and the jugular, portal, and right ruminal veins. To facilitate the removal of blood samples during the experiment, appropriate incisions were made to allow cannulation of the blood vessels (see p.294). Upon completion of the cannulation, the incisions were closed with temporary sutures for the remainder of the experiment. 1. Animal history* A young adult male goat weighing 90 pounds and of mixed breeding was used. About one year prior to the experiment, the goat had a section, about three inches in diameter, of 29 the "wall of the dorsal sac of the rumen removed* The goat was on a diet of alfalfa hay and oats prior to the experiment. At the time of the experiment, the animal appeared to be healthy. 2. Composition of the experimental solution: The amino acids were supplied in the form of a 10 per cent casein acid-hydrolysate solution. The pH of the solution was adjusted to 7.0 with sodium hy droxide. 3. Sampling procedure: One hundred milliliters of the experi mental solution were added to the rumen of the goat. Duplicate 10 m i l l i  liter samples of blood were removed from the blood vessels, the first sample being removed from the common carotid artery 15 minutes after the addition of the amino acids to the rumen. The sampling times for each of the blood vessels are recorded in Table 21. Assay Procedure 30 Plasma was obtained from the heparinized blood samples of the * three experiments. The substances for whose content each of the plasma samples was assayed are indicated in the tables that reoord the results of each experiment (see below). Lysine, glycine, and niootinio acid were assayed by the microbiological procedures of Barton-Wright (30). Alpha amino nitrogen was assayed by the Van Slyke manometric method (148, 391). This section of rumen wall was removed for the purposes of another investigation that was not connected with the present investigation. 3 0The plasma samples were prepared on completion of the experiment and then frozen until their assay was begun. 108. Results The results of experiments Bl, B2, and BS are presented in Tables 19, 20, and 21, respectively. Figure 15 is a graph of the results for experiment B3» Examination of the data in Tables 19 to 21 x reveals that seldom were the oonoentrations of a constituent higher in the venous plasma that drained the rumen than in the peripheral plasma. In fact, the concentrations were often higher in the peripheral plasma than in the plasma that drained the rumen. These results of experiments Bl to B3 do not provide evidence, then, for ruminal absorption of amino acids or B vitamins* Discussion Perhaps applicable to the results of the present experiments is the explanation that was given by Fisher (117) for the apparently contradic tory results that were obtained by different groups of research people in their attempts to demonstrate amino acid absorption from the small intestine* Some workers (e.g., see 119 and 393) could not demonstrate an increase of amino acid concentration in the portal blood during protein digestion in the small intestine. Other workers (118, 119, 392) readily were able to demonstrate such an increase, both during protein digestion and after the placement of a relatively large amount of an amino acid in an intestinal loop. Fisher (ll7j see also 392, 393) ^"The alpha amino nitrogen concentrations that are recorded in these tables for goat plasma approximate to the concentrations that are re corded by Annison (15) for sheep plasma (see p. 209 ). TABLE 19• • Blood-sampling experiment Bit ooncentra tion (miorogram-molea per 100 milliliters) of lysine in plasma samples taken from a goat during surgical inter- vention (the data recorded are means and standard deviations* vith numbers of assays in parentheses) Blood vessel Lysine sampled* Left jugular vein •••••••••••••••• • 13*5±0*1 (3) Eight ruminal vein • ••• •••••• 11*6+3.0 (3) Left jugular-vein • • •••••••• 9*1+1*1 (3) aThe blood vessels are listed in the order that they vere sampled* TABLE 20. - Blood-sampling experiment B2« concentration of alpha amino nitrogen, niootinio acid, and glycine in plasma samples taken from a goat during and on days subsequent to the day of surgical inter vention (the data recorded are means and standard deviations, with numbers of assays in parentheses) Blood vessel sampled* Alpha amino nitrogen (milligrams per 100 milliliters) Niootinio aoid (miorogram-moles per 100 milliliters) Glycine (microgram-moles per 100 milliliters) b 0 d e e f S Left jugular vein 3.73 (1) 4.18 (1) 0.69+0.03 (3) 0.3610.01 (3) 63.9119.3 (3) 73.7+26.9 (3) «... Left jugular vein • • • • 4.30+0.45 (2) • • • « .... .... .... • . . . Left lateral saphenous vein • • • • .... • • • * • . • . . • • . .... 46.2117.3 (3) Right ruminal vein 3.66 (1) 3.74+0.48 (2) 0.58+0.03 (3) .... .... .... .... Right ruminal vein 3.59 (1) 4.29+0.28 (2) .... .... .... .... Left ruminal vein .... .... 0.58+0.04 (3) 0.34+0.00 (3) 72.3+8.9 (3) 64.9119.3 (3) 43.6+17.2 (3) Left ruminal vein .... .... 0.63+0.07 (3) 0.37+0.01 (3) 63.0117.7 (3) 61.3113.9 (3) .... Left jugular vein 4.57 (1) 3.7910.69 (2) 0.72+0.09 (3) 0.3910.03 (3) 75.5+18a (3) .... .... TABLE 20 - continued Blood vessel sampled* Alpha amino nitrogen (milligrams per 100 milliliters) Nicotinic acid (mi or og ram-mol e s per 100 . milliliters) Glycine (microgram-moles per 100 milliliters) b o d e e f g Left jugular vein • • • • 3.63 (1) • • • • • • • • • • • • • • • • . • • . Left lateral saphenous vein • • • • • • • • • • • • • • • • • • • • 74.2+16.9 (3) . • • . Left cephalic vein • • • • • • • • • • • • • • • • • • • • • • • • 61.7+15.5 (3) The blood vessels are listed in the order that they were sampled. The blood samples were taken the first day after the day of surgery. The animal was fed 200 grams of concentrate pellets 15 minutes prior to the taking of the first sample. °The blood samples were taken the seoond day after the day of surgery. The animal was fed 200 grams of concentrate pellets 15 minutes prior to the taking of the first sample. ^ h e blood samples were taken' during surgery. The animal had a l l food but not water withheld for 28 hours prior to surgery. eThe blood samples were taken the third day after the day of surgery. The animal had been fed 200 grams of oonoentrate pellets 15 minutes prior to the taking of the first sample. TABLE 20 - continued The blood samples -were taken the sixth day after the day of surgery* The animal was fed 200 grams of concentrate pellets 15 minutes prior to the taking of the first sample* Slhe blood samples were taken the seventh day after the day of surgery* The animal was fed 240 grams of concentrate pellets and 30 grams of casein hydrolysate powder 10 minutes prior to the taking of the first sample* TABLE 21. - Blood-sampling experiment B3i concentration (milligrams per 100 milliliters) of alpha amino nitrogen in plasma samples taken from a goat after the addition at 0 minute time of an amino aoid solution* to the ingesta-filled rumen (the data recorded are means and standard devia tions, with numbers of assays in parentheses) Right common carotid artery Right jugular vein Right ruminal vein Portal vein Time (min*) Nitrogen (ngO Time (min*) Nitrogen (mg*) Time (min*) Nitrogen (mg*) Time (min.) Nitrogen (ng») 15 3.4010*29 (4) 19 3*67+0*12 (3) 22 3.40+0.08 (4) 25 3*5010.14 (4) 34 3*50+0*27 (3) 42 3.4010.14 (4) ... • . . . . .... • . . . 59 3*78±0*17 (4) 62 3.5510.25 (4) 50 3.43+0.32 (3) 55 3.70+0.14 (4) a0ne hundred milliliters of a 10 per cent casein aoid-hydrolysate solution were added. I—1 if* z o r- _: < E U J o 4.0„ V05(/9-»42) 3.01 J. A 20 30 40 50 60 20 TIME (minutes) 30 40 50 60 Fig. 15. Fig. 15. - Blood-sampling experiment B3: changes in concentration of alpha amino nitrogen in goat plasma samples taken from the jugular vein (iff 0) > common carotid artery (4% portal vein (• -•), and right ruminal vein (£ 0^ • The plasma samples were taken after the addition (0 minute time) of an amino acid solution to the ingesta-filled rumen of the goat comments on this situation as follows* If there is good reason to suppose that a substance of a particular class is being added to the blood at a particular time, and i f the concentration in the blood of this class of substance rises at this time, the rise may be interpreted as a consequence of the known process of addition. But the ab- senoe of a detectable rise in such circumstances could not be  taken as meaning that there was absence of addition (underlining mine]. When King and Rapport (1933) administered as much as 5 g. of tyrosine to a dog by intravenous injection, l i t t l e or none of the tyrosine could be found in the blood five minutes later, in any of the forms of amino-nitrogen, phenol, non- protein-nitrogen or urea. In order to obtain this amount of tyrosine from a protein meal i t would be necessary to absorb the produots of digestion of 100-240 g. of protein. A change in concentration in the blood merely signalizes a change in relation between the totality of processes adding the substance to the blood and the totality of processes re moving i t . Apparently, under certain feeding conditions, the quantity of amino acids being absorbed per unit time from the small intestine is quite small relative to the large volume of blood that carries the amino acids away per unit time (also see 91, 397). An actual increase in the concentra tion of the amino acids in the portal blood may be too small to be de tected by the classical assay methods. The unsuccessful attempt in experiments Bl to B3 to demonstrate increased concentrations of amino acids and nicotinic acid in the blood draining the .rumen might be a reflection of the same type of relationship that was found in the work with the small intestinej that i s , ruminal absorption of amino acids and nicotinic acid actually may have occurred, but the quantities absorbed may have been so small, relative to the volume of blood passing through Cummins (85) has expressed the same concept more briefly* Studies of systemic blood concentrations of in gested materials reflect utilization, rate of storage, excretion, and circulatory competency as well as absorption. the ruminal veins, that increased concentrations were not detected by the 33 assay methods that were employed. Re-examination of Tables 19 to 21 leads to the suspicion that, although differences between the concentrations of the amino acids and nicotinic acid in the peripheral blood and their concentrations in the portal blood may not have been demonstrated, there may have been slight increases in their concentrations with time. Accordingly, the data of experiment B3 (the only one of these three experiments for which a record of the sampling times was kept) were analyzed statistically for the significances of the concentration increases with time. The method of statistical analysis already has been explained (see p. 36 )• Figure 15 (see p. 114), a graph of the data for experiment B3, reoords the results of the statistical analysis. Except for the plasma of the jugular vein, the plasmas of the different blood vessels exhibit a slight increase with time in the concentration of alpha amino nitrogen. The increase is significant at the 5 per cent level for the common carotid artery, non significant at the 10 per cent level for the ruminal vein, and signifi cant at the 10 per cent level for the portal vein. These results, indicating slight support for ruminal absorption of amino acids,-led the author to attempt to demonstrate larger, time-related increases in the blood concentration of alpha amino nitrogen. This attempt yielded the experiments of phase B. 33 i \ A group of French workers (322; have suggested a similar explana tion for their failure to demonstrate directly B vitamin absorption from various parts of the alimentary tract of sheep. The method that was employed by these workers was to compare the blood concentrations of the individual B vitamins in the appropriate branches of the portal system with the blood concentrations in the jugular vein. They were unable to demonstrate significantly higher concentrations of the B vitamins in the blood of the portal system. Phase B: Increase in the Concentrations of Amino Acids in Peripheral and Portal Blood with Time Introduction In this phase of the second part of the experimentation, time-related increases in the blood concentrations of amino acids were demonstrated 34 after their addition to the rumen of a goat. These increases were contrasted wi th the blood concentrations that were recorded before the addition of the amino aoids. A. similar experimental approach has been used by many workers to demonstrate the intestinal absorption of amino acids. The selection of the method of assay for this phase of the experi mentation was based upon considerations of sensitivity and upon availability of equipment and fa c i l i t i e s . Especially applicable to the experiments of this phase is a method of assay that utilizes amino acids tagged with radioactive carbon. The primary reason for not utilizing such a method of assay was the lack of equipment and facilities for handling radioactive isotopes. As an alternative method of assay, the Van Slyke mano- metrio method for alpha amino nitrogen was selected. Although this method measures only the collective concentration of amino acids, i t was chosen because of its sensitivity in the detection of small changes of total amino acid concentration in the blood. It readily i s acknowledged, however, that the use of a method of assay that utilized isotope-tagged amino acids (and B vitamins), either in addition to or in place of the methods used, would have facilitated the experimentation, not only in 'This procedure was not attempted with the B vitamins. this part, but in a l l parts of the present investigation* Method Experimental Procedure General.- In this series of experiments, the blood samples were removed periodically from anaesthetized goats, both before and after the addition of a solution of amino acids to the empty rumen. Appropriate incisions (see p. 294) were made to enable the contents of the rumen and reticulum to be removed. These two compartments were rinsed in the same manner a s for the ruminal fistula experiments ( see p. IS). After plac ing a tight-fitting cotton plug in the reticulo-omasal orifice, a ligature was tied around the reticulo-omasal sphincter, care being taken not to obstruct the large blood vessels that occur in the area of the sphincter. This ligating procedure was performed in order to isolate the rumen and 35 the reticulum from the remainder of the digestive tract. The addition of the experimental solution, previously warmed to 37°C, was made to the rumen 0 0 through the incised opening in its wall. After this addition, the opening to the rumen was sutured closed. The blood vessels that were sampled are indicated in the tables and figures that record the results for each of the experiments (see T ables 22 to 27 and Figures 16 to 21). To facilitate the removal of blood samples during the experiment, appropr riate incisions were made in the body wall of the goat in order to allow In experiment B4, additional ligatures were tied around the pyloris of the abomasum and around the cervical part of the oesophagus. 36 As, in this series of experiments, each goat lay on its right side during the experiment, i t was not possible to ensure confinement of the experimental solution to the rumen. It is possible, therefore, that a small amount of the experimental solution may have occupied the reticulum during the experiment. 120. cannulation of the blood vessels (see p. 294). Upon completion of the 37 cannulation, the incisions were closed with temporary sutures for the remainder of the experiment. Experiment 334.- 1. Animal history* A young adult female goat weighing 6.7 pounds and of mixed breeding was used. About one year prior to the experi ment, the goat had a section, about four inches in diameter, of the wall 38 of the dorsal sac of the rumen removed. Food but not water was with held from the goat for the 72 hour period that preceded the experiment. At the time of the experiment, the animal appeared to be healthy. 2. Composition of the experimental solution* The amino acids were supplied in the form of a solution whose composition was 40 m i l l i  liters of casein acid-hydrolysate solution (lO per cent); 1.63 grams of sodium orthophosphate, dihydrogen (NaB^ PO^ .); 2.5 grams of polyethylene 39 glycol ; enough sodium hydroxide to adjust the pH of the solution to 6.5; and enough water to make a final volume of one l i t e r . 3. Sampling procedure* One l i t e r of the experimental solution was placed in the rumen of the goat. Five milliliter samples of blood were withdrawn from the blood vessels. The times of sampling are indioated in Table 22 (see p. 125). The volume of the experimental solution left in the rumen at the end of the experiment was not measured. As the goats were euthanized with an overdose of sodium pento barbital at the end of the experiments, only temporary sutures were used. 38 See footnote 29 on page 107. 3®This substance for checking on water movement was added to the experimental solutions because, originally, i t was intended that the concentration changes of the constituents in the experimental solutions be measured. Experiment B5>- 1* Animal historyi A young adult male goat weighing 60 pounds and of Saanen breeding was used. The goat had not been subjected to previous surgical intervention. Food but not water was withheld from the goat for the 48 hour period that preceded the experiment. At the time of the experiment, the animal appeared to be healthy. 2. Composition of the experimental solution! The composition of the solution was the same as the composition of the solution for experiment B4. 3. Sampling proceduret One liter of the experimental solution was placed in the rumen of the goat. Duplicate 10 mi l l i l i t e r samples of blood were withdrawn from the blood vessels. The times of sampling are indicated in Table 23 (see p. 128 ). The volume of the experimental solution recovered from the rumen at the end of the experiment was 940 mill i l i t e r s . Experiment B6.- 1. Animal historyi A young adult male goat weighing 90 pounds and of mixed breeding was used. About 18 months prior to the experiment, the spermatic cords were crushed to cause a reduction in the odour of the animal. Food but not water was withheld from the goat for the 96 hour period that preceded the experiment. At the time of the experiment, the animal appeared to be healthy. 2. Composition of the experimental solution» The amino acids were supplied in the form of a solution whose composition was 500 m i l l i  liters of casein acid-hydrolysate solution (10 per cent); 1.8 grams of sodium orthophosphate, dihydrogen (NaH2P04); '2.5 grams of polyethylene glycol; enough sodium hydroxide to adjust the pH of the solution to 6.7; 122* and enough water to make a final volume of one liter * 3* Sampling prooedure* One li t e r of the experimental solution was placed in the rumen of the goat. Duplicate 10 mi l l i l i t e r samples of blood were withdrawn from the blood vessels* The times of sampling are indicated in Table 24 (see p* 131 )• The volume of the experimental solution recovered from the rumen at the end of the experiment was 800 milliliters** Experiment B7*- 1* Animal history* A young adult female goat weighing 66 pounds and of mixed breeding was used* The goat had not been subjected to previous surgioal intervention* Food but not water was withheld from the goat for the 96 hour period that preceded the experiment* At the time of the experiment, the animal appeared to be healthy* 2 * Composition of the experimental solution! The composition of the solution was the same as the composition of the solution for experiment B6, except that polyethylene glycol was omitted* 3* Sampling procedure* One liter of the experimental solution was placed in the rumen of the goat* Duplicate 10 milliliter samples of blqod were withdrawn from the gastrosplenie vein (see Fig* 14, p*l03)* The times of sampling are indicated in Table 2 5 (see p* 134)* The volume of the experimental solution recoverable from the rumen at the end of the experiment was not measured* Experiment B8*» 1* Animal history* A young adult female goat of mixed breed ing was used. Through negligenoe, the weight of this goat was not record ed* The goat had not been subjected to previous surgical intervention* Food but not water was withheld from the goat for the 96 hour period that 123. preceded the experiment. A t the time of the experiment, the animal appear ed to he healthy. 2. Composition of the experimental solution! The composition of the solution was the same as the composition of the solution for experiment B6, except that polyethylene glycol was omitted. 3. Sampling procedure! The description for experiment B7 also applied here. The sampling times for the gastrosplenic vein are indicated in Table" 26 (see p. 137). Experiment B9.- 1. Animal history! A young adult male goat weighing 62 pounds and of mixed breeding was used. The goat had not been subjected to previous surgical intervention. Food but not water was withheld from the goat for the 96 hour period that preceded the experiment. At the time of the experiment, the animal appeared to be healthy. 2. Composition of the experimental solution! The composition of the solution was the same as the composition of the solution for experiment B6. 3. Sampling procedure! Nine hundred milliliters of the experi mental solution were placed in the rumen of the goat. Duplicate 10 mill i l i t e r samples were withdrawn from the blood vessels.. The times of sampling are indicated in Table 27 (see p. 140 )• The volume of the experimental solution recovered at the end of the experiment was 825 millil i t e r s . Assay Procedure The heparinized blood samples were assayed for alpha amino nitrogen by the Van Slyke manometrio method (148, 391). The assays were begun within an hour of the completion of the experiments* Results The results for experiments B4 to B9 are reoorded in Tables 22 to 27 4 0 and Figures 16 to 21. The data were analyzed statistically for the significances of the concentration increases. The method of statistical analysis already has been explained (see p. 36 )• The results of this analysis are reoorded as a part of Figures 16 to 21. Increases in the concentration of alpha amino nitrogen, significant at the 10 per cent 41 levral or less, occur in most of the blood vessels. The increases of concentration are large in experiments B6 to B9. In these experiments, 4%he alpha amino nitrogen concentrations that are recorded in these tables for the whole blood of the goats at zero or pre-zero sampling times (5.2 to 7.8 milligrams per 100 milliliters) f a l l within the range of concentrations that are reoorded in Specter*s Handbook of Biological Data (358) for the whole blood of sheep (4.6 to 8.0 milligrams per 100 mil l i l i t e r s ) . Although the goats in these experiments were fasted for 72 or 96 hours prior to the experiment, the fact that their blood concentra tions f a l l within the range of concentrations for non-starved sheep is in agreement with the results of White et al (406). These authors found no decrease in blood amino aoid nitrogen for sheep that had been subject ed to decreasing levels of feed intake followed by starvation. It also is of interest to note here the effect of anesthesia on the concentration of amino acids in the blood. Folin and Lyman (120) found no change in the concentration of non-protein nitrogen in the blood of cats that had been anesthetized for as long as three hours with ether. Peters and Van Slyke (290) cite work which indicates that the blood amino acids f a l l slightly during anesthesia with pentobarbital or ether. Since in the present experiments anesthesia was induoed with pentobarbital and main tained with ether, i t is unlikely that the observed increases in the concentration of alpha amino nitrogen in the present experiments were the result of the anesthesia. 41 It is to be noted that these increases in concentration of alpha amino nitrogen occurred in spite of the fact that the goats had been starved for 72 to 96 hours (48 hours in experiment B5) prior to the experiments. Although no measurements were made, i t is probable that these animals were in the state of negative nitrogen balanoe at the time of the experiments ( l l , 131, 366). In suoh a state i t might be expected that the tissues would take up more rapidly from the blood the amino TABLE 22• - Blood-sampling experiment B4t . changes in concentration (milligrams per 100 milliliters) of alpha amino nitrogen in blood samples taken from a goat before and after the addition (0 minute time) of an amino acid solu tion* to the empty rumen (the data recorded are means and standard deviations, with numbers of assays in parentheses) Time (min*) Right jugular vein (fflg») Right common carotid artery (mg») -142 6.13+0.55 (4) • » • • -152 5*85±0*15 (4) • • •• • - 3 • • • • 5.95+1.15 (4) + 30 • • • •' 5*68+0.73 (4) + 51 . . • . 7•60+1.27 (4) E^he estimated concentration of alpha amino nitrogen in the solution was 37 milligrams per 100 milliliters. z o 7 . 5 — S£g 6 . 5 z *v U l cn 2. £ O u 5 .5 -150 -140 -130 -10 0 10 TIME (minutes) 2 0 3 0 4 0 5 0 6 0 Fig. 16. Fig. 16. - Blood-sampling experiment B4« changes in concentration of alpha amino nitrogen in goat blood Bamples taken from the jugular vein (ft #) and common oarotid artery (A - - - - -A). The blood samples were taken before and after the addition (0 min ute time) of an amino acid solution (estimated concentration of alpha amino nitrogen* 37 milligrams per 100 milliliters) to the empty rumen. 128 TABLE 23. - Blood-sampling experiment B5i ohanges In concentration (milligrams per 100 milliliters) of alpha amino nitrogen in blood samples taken from a goat before and after the addition (0 minute time) of an amino acid solutiona to the empty rumen (the data record ed are means and standard deviations, with numbers of assays in parentheses) Right jugular vein Right common carotid artery PSrtal vein Time (min.) Nitrogen (mg«) Time (min.) Nitrogen (ag«) Time (min.) Nitrogen (*g«) -145 5.15+0.64 (4) . . . . . . . . . . . . . . • • • . . . . - 3 5.73+0.51 (4) - 6 5.23+0.89 (4) . . . « . . . + 37 6.06+0.62 (4) + 32 6.03+0.27 (4) . . . . . . . + 64 5.6310.62 (4) + 60 5.9810.31 (4) . . . • . . • + 97 6.53±0.40 (4) +92 6.18+0.68 (4) aThe estimated oonoentration of alpha amino nitrogen in the solution was 37 milligrams per 100 milliliters. 2 O r - £ § ui >» 0 d» 1 E 7 . 0 , - 6.0 5.0 • 150 -I4i 10 .051-6-92)- 10 20 30 40 TIME (minutes) 50 60 70 80 90 100 Fig. 17. Fig. 17. - Blood-sampling experiment B5i changes in concentration of alpha amino nitrogen goat blood samples taken from the jugular vein ( # ), common carotid artery - . . - A.) $ ~ and portal vein (• • The blood samples were taken before and after the addition (0 minute time) of an amino acid solution (estimated concentration of alpha amino nitrogen! 37 milligrams per 100 milliliters) to the empty rumen. TABLE 24• - Blood-sampling experiment B6i ohanges in concentration (milligrams per 100 milliliters) of alpha amino nitrogen in blood samples taken from a goat before and after the addition (0 minute time) of an amino acid solution 8 to the empty rumen (the data re oorded are means and standard deviations, with numbers of assays in parentheses) Bight jugular vein Right common carotid artery G-astrosplenic vein Time (min.) Nitrogen (mg») Time (min.) Nitrogen (mg.) Time (min.) Nitrogen (mg.) -133 5.48+0.61 (4) • a • • a a • ... a a a a • • • • . a • - 106 5.63+0.81 (4) ... .... ... .... - 52 5.28+0.95 (4) - 55 5.6510.79 (4) . . • a a a . - 3 5.90+0.72 (4) - 6 6.9010.54 (4) • . • . . . a +• 10 6*66+0.61 (4) + 7 7.3510.56 (4) .... .... + 34 7.3810.86 (4) -r 31 9.5010.22 (3) • • . • • • • + 63 8.45+0.78 (4) + 61 9.7010.71 (2) r • . a • a a a + 92 8.50+0.53 (4) +- 90 11.00+0.86 (4) • . . a . . . + 124 10.43+0.63 (3) + 121 11.45+0.66 (4) ^he estimated concentration of alpha amino nitrogen in the solution was 462 milligrams per 100 m i l l i l i t e r s . 03 ro «2 r I I r - o i l UI -S. O di Z E O O 10 9 8 0 ' / f &• ... .0 .0 j L •150 -130 -HO -90 -70 -50 -30 -10 0 +10 T I M E (minutes) 30 50 70 J L 90 110 130 Fig. 18. Fig. 18. - Blood-sampling experiment B6s ohanges in concentration of alpha amino nitrogen in goat blood samples taken from the jugular vein ( % ), common carotid artery (A A)» and. gastrosplenic vein (Q Q) • The blood samples were taken before and after the addition (0 minute time) of an amino acid solution (estimated concentration of alpha amino nitrogenj 462 m i l l i  grams per 100 milliliters) to the empty rumen. TABLE 25* - Blood-sampling experiment B7? ohanges in concentration (milligrams per 100 milliliters) of alpha amino nitrogen in blood samples taken from a goat before and after the addition (0 minute time) of an amino acid solu tion* to the empty rumen (the data reoorded are means and standard deviations, with numbers of assays in parentheses) G-astrosplenio vein Time Nitrogen (min.) (mg») - 4 6.98+0.93 (4) + 2 6.73+1.18 <4) + 31 8.20+0.87 (4) + 63 ... 8.40+0.80 (4) + 93 • 9.0510.64 (4) +122 13.5310.25 (4) E^he estimated concentration of alpha amino nitrogen in the solution was 462 milli grams per 100 milliliters* 0 / * / / pi 41 Oil gi / 0 — -20 0 20 40 60 80 feo" fej 140 TIME (minutes) Fig. 19. Fig. 19. - Blood-sampling experiment B7» changes in concentration of alpha  amino nitrogen in goat blood samples taken from the gastrosplenic vein. The blood samples were taken before and after the addition (0 minute time) of an amino acid solution (estimated concentration of alpha amino nitrogenj 462 milligrams per 100 milliliters) to the empty rumen. TABLE 26* - Blood-sampling experiment B8» ohanges in concentration (milligrams per 100 milliliters) of alpha amino nitrogen in blood samples taken from a goat before and after the addition (0 minute time) of an amino aoid solution* to the empty rumen (the data record ed are means and standard deviations, with numbers of assays in parentheses) Grastrosplenio vein Time Nitrogen (min.) (mg.; - 4 6.03±0.83 (4) -+• 1 8.50+0.46 (4) + 38 7.73+0.75 (4). -r 61 7.5810.51 (4) + 90 8.48+0.47 (4) -1-120 7.5010.80 (4) *She estimated concentration of alpha amino nitrogen in the solution was 462 mllliS grams per 100 m i l l i l i t e r s . CO 90 < E 80 U O o> 70 60 -20 I X 20 40 60 80 TIME (minutes) 100 J 120 140 Fig, 20, Fig. 20. - Blood-campling experiment B8i ohanges in concentration of alpha  amino nitrogen in goat blood samples taken from the gastrosplenic vein. The blood samples were taken before and after the addition (0 minute time) of an amino acid solution (estimated concentration of alpha amino nitrogeni 462 milligrams per 100 milliliters) to the empty rumen. TABLE 27. - Blood-sampling experiment B9i ohanges in concentration (milligrams per 100 milliliters) of alpha amino nitrogen in blood samples taken from a goat before and after the addition (0 minute time) of an amino aoid solution to the empty rumen (the data reoord ed are means and standard deviations, with numbers of assays in parentheses) Bight common oarotid artery (Jastrosplenio vein C ommon me sen— terio vein Time (min*) Nitrogen (mg.) Time (min.) Nitrogen (mg.) Time (min.) Nitrogen (mg.) -96 6.3810.47 (4) . . . . . . . . . . . . . . . -81 7.2010.96 (4) . . . . . . . . . . . . . . -47 6.50x0.74 (4) . . . . . . . . . . . . . . • • • . . . . -29 5.50+0.83 (4) . . . . . . . • • • . . . . . . . . . . . -11 6.53+0.82 (4) - 5 6.9311.18 (4) — 3 7.80+1.62 (4) - 0.5 6.5310.22 (4) + 19 7.00+1.25 (4) +-16 9.68+0.52 (4) . . . . a . . +•33 6.7811.43 (4) + 30 9.13+0.84 (4) . . . . a a a + 53 8.3310.75 (4) + 51 11.4310.83 (4) + 48 9.0810.67 (4) + 68 7.73+0.47 (4) + 63 10.73+0.17 (4) +•66 10.10+0.52 (4) + 96 8.8811.42 (4) + 90 10.63±0.44 (4) +-93 9.83±0a79 (4) ^he estimated concentration of alpha amino nitrogen in the solutions was 462 milligrams per 100 milliliters. I-1 Fig. 21. Fig. 21. - Blood-sampling experiment B9j changes in concentration of alpha amino nitro gen in goat blood samples taken from the common carotid artery (A A), gastrosplenic vein 0 ) » and mesenteric vein (• Ej)» The blood samples were taken before and after the addition (0 minute time) of an amino acid solution (estimated concentration of alpha amino nitrogenj 464 milligrams per 100 m i l l i l i t e r s ) to the empty rumen. 143. the concentrations of the amino acids in the experimental solutions were 12.5 times the concentrations of the amino acids in the solutions for experiments B4 and B5. The heights attained by the concentrations of alpha amino nitrogen in the blood of the different vessels that were sampled in experiments B6 to B9, especially when contrasted to the pre- zero time concentrations in the blood of these vessels, appear to support the hypothesis of ruminal absorption of amino acids. Discussion Although the results of experiments B4 to B9 provide evidence for the ruminal absorption of amino acids, two topics remain that require discussion. Fluctuations in the Concentrations of Amino Acids in the Blood One striking feature of the post-zero time data of this series of experiments is that, even although there may have been a general increase in the blood concentrations of alpha amino nitrogen, there were s t i l l strong fluctuations in these blood concentrations. There also were some fluctuations in the pre-zero time concentrations; and, in experiments B6 and B9, the blood of the gastrosplenic vein even exhibited concentration fluctuations that were significant at the 5 per cent level. These fluctuations may represent merely sampling error; the fluctuations are, acids that were absorbed from the alimentary tract ( l l , 389). It is suggested, therefore, that the increases in the blood concentrations of alpha amino nitrogen that did occur in these experiments may have been more dramatic i f the goats had been in positive nitrogen balance (see also the quotation of Fisher on p. 116 and footnote 32 on the same page, both of which point out that the rate of uptake of a constituent from the blood is one of several factors which determine the constituent's concentration in the blood. however, similar to fluctuations of amino acid concentrations observed by Hartmann et al (153)• These workers carried out experiments on the intestinal absorption of individual amino acids from a mixture of amino acids that were placed in intestinal loops of 16 dogs* Detection of amino acid absorption was made by noting the increase in the concentration of individual amino acids in the mesenteric blood draining the loops of intestine* By plotting the nmean value curves11 (Mittelwertskurven) of the concentration data for each of the amino acids investigated, Hartmann and co-workers observed periodical maxima and minima in the curves* For some of the amino aoids, the minimal concentrations were often almost as low as the i n i t i a l concentrations* In experiments of about two hours duration, they observed three Resorptionsmaximat the first maxima occurr ed at about 10 minutes from the beginning of the experiment, the second between 30 and 35 minutes, and the third between 50 and 60 minutes* The authors of the paper reporting on this investigation were unable to state the cause of this periodicity of maxima and minima in the wmean value curvesn* They did suggest, however, that the rhythm of the amino acid concentrations in the mesenteric venous blood may reflect a periodi city in the activity of an intestinal wall mechanism that is concerned in the absorption of amino aoids from the intestinal lumen* The same type of suggestion can be made to explain the post-zero time fluctuations of alpha amino nitrogen concentrations in the present blood-sampling experi- 42 ments for the goat* •- *2soarisbrick and Ewer (330) found the amount of absorption of inorganic phosphate from the rumen of sheep to be quite variable, "vary ing considerably from time to time*1* These results may be a reflection of the same phenomenon that is described by Hartmann et al (see above in text) for the small intestine* Comparison between the Results of Annison and Those of the Present Investi  gation In contrast to the blood-sampling experiments of Annison (see p. 14), the results of the blood-sampling experiments in the present investigation support the hypothesis that amino acids are absorbed from the rumen* A comparison of some of the conditions in the two sets of investigations might help to explain the difference in the results obtained* Table 28, a record of some of the conditions in the two investigations, has been constructed in order to facilitate such a comparison* One important point that is not brought out by the information in Table 28 is the criterion that was used in each experiment to judge whether or not ruminal.absorption of amino acids had occurred* The criterion used by Annison was based upon a comparison between the concen tration of alpha amino nitrogen in the blood of the portal vein and the concentration of the same substance in the blood of the jugular vein or common carotid artery* A concentration of alpha amino nitrogen in the blood of the portal vein that was higher than the concentration in the blood of the jugular vein or common carotid artery would be considered 43 evidence of ruminal absorption of amino acids* This criterion of Annison also was used for experiments Bl to B3 of the present blood- sampling experiments* In experiments B4 to B9, another oriterion was adopted as the basis for detecting ruminal absorption of amino acids* Measurements were made, both before and after the addition of amino aoids to the rumen, of the oonoentration of alpha amino nitrogen in the blood of a vein or artery* The ruminal absorption of amino acids was See p*101 for the deduction of this statement* TABLE 28. - Comparison between the conditions and results for the blood-sampling experiments of Annison ( 15 ) and the conditions and results for the blood-sampling experiments of the present investigation Experiment* (1) Feeding prior to experiment (2) Ruminal contents (3) Initial concentration of casein hydrolysate (gm. per liter) (4) Blood vessels sampled (5) Absorption of amino acids from the rumen (6) 1-Annison 1 present (o»33) portal vein none demonstrated 2-Annison ? present (/U23) portal vein none demonstrated B 3 oats given 30 min* prior present. (/U 3) portal and right ruminal veins none demonstrated B 4 72 hr. fast removed 4 common oarotid artery questionable B 5 48 hr. fast removed 4 portal vein questionable B 6 96 hr. fast removed 50 gastro- splenio vein demonstrated B 7 96 hr. fast removed 50 gastro- splenic vein demonstrated TABLE 28- continued Feeding Initial Blood Absorption Experiment6 prior to Ruminal concentration vessels of experiment contents of oasein sampled amino aoids hydrolysate^5 from the (gm* per liter) rumen (1) (2) (3) '<*) (5) (6) B 8 96 hr* removed 50 gastro- demonstrated fast splenio vein B 9 96 nr. removed 50 gastro- demonstrated fast splenic vein aSheep were used for the experiments of Annisonj goats were used for the experiments of the present investigation* ^The data that are recorded in parentheses for the first three experiments listed in the table are only approximations* In these experiments, the casein hydrolysates were added to ruminal ingestas of unmeasured volumes* From the data that are presented in Ellenberger-Baum1s Yergleiohenden Anatomie der Haustiere (420) for the oapaoity of the sheep and goat rumens, volumes of 5 liters were assumed for the ruminal ingestas* The assumption at least should allow the correct order of magnitude of the casein hydrolysate concentrations to be calculated* considered to have been demonstrated i f , after the addition of amino acids to the rumen, a concentration increase occurred which was significant at the 10 per cent level or less and which was maintained. It should be noted that the design of the second experiment of Annison (see p. 14 ), in which he sampled the portal vein at intervals after the addition of amino acids to the rumen, allows the criterion applied to experiments B4 to B9 also to be applied to his data. By this criterion, Annison*s results are s t i l l negative for the demonstration of ruminal absorption of amino acids. Finally, in experiments B6 and B9, the use of a more con centrated experimental solution in the rumen apparently has provided, even by the criterion of Annison, positive results—a higher concentration of alpha amino nitrogen in the blood that was draining the rumen via the gastrosplenic vein than in the peripheral blood of the common oarotid artery (see Figs. 18 and 21)• Examination of the experimental conditions reoorded in Table 28, together with a consideration of the two criteria just discussed, can lead to the conclusion that the demonstration in the present investiga tion of ruminal absorption of amino acids depended on a l l or some of the following conditions. 1. The removal of the ruminal contents prior to the addition of the amino acids to the rumen—this procedure would reduce or eliminate competition for the amino aoids by the microbial population* 2* The use in experiments B6 to B9 of higher i n i t i a l concentrations of amino aoids in the rumen—this would produce a higher, downward concentration gradient in the direction from the ruminal solution in the rumen to the blood in the ruminal veins; such an increased gradient would accelerate the rate of diffusion of amino acids aoross the wall of the 149. rumen and into the blood. 3. The ohoioe of the criterion to be used for the detection of ruminal absorption of amino acidsi an increase in the concentration of alpha amino nitrogen in the blood of an artery or vein after the addition of amino acids to the rumen—this criterion is probably a more sensitive test than the criterion of Annison. 4. The use of the gastrosplenic vein rather than any other blood vessel for the application of the criterion just cited—this use of the 44 gastrosplenic vein enabled samples to be taken of the blood that was draining from the ruminant stomach before this blood had mixed with the blood that was draining from the intestines and other organs; this avoid ance of a large dilution effect provided a more sensitive test for the detection of any increase in the concentration of alpha amino nitrogen in the blood that was draining from the rumen. All four of the above conditions, conditions that stand in contrast to those of the experiments of Annison, were a part of the experimental design in experiments 56 to B9. These are the experiments that provide strong evidence for the ruminal absorption of amino acids. It is suggested that the difference between the design of the experiments of Annison's investigation and the design of the experiments of the present investiga tion accounts for the difference between the results of the two investi gations. Most of the blood in the gastrosplenic vein comes from the ruminant stomach, with a small amount coming from the spleen (see Fig. 14, p.103). GENERAL DISCUSSION Just as chemistry could not have developed without test tubes to hold reacting substances, so organisms could not have evolved without relatively impermeable membranes to surround the cell constituents* This barrier between the inside and the outside, the inner and external world of each living unit, has been and always must be considered one of the fundamental structures of a cell* No one can f a i l to be impressed with the great difference in properties of living and dead cells. The dead are completely permeable to dif fusible substances, while the living retain one material and pass another. This difference, selective permeability [underlining mine] , is so marked that i t becomes the surest test to distinguish the living from the dead, holding where al l other methods f a i l . It can truly be said of living cells, that by their membranes ye shall know them. E. Newton Harvey in Permeability of Natural Membranes by Davson and Danielli (90). If we can assume, on the basis of the results of the present investi gation, that at least some of the amino acids and B vitamins can pass across the epithelial lining of the rumen, then several questions on this phenomenon naturally arise. 1) What is the manner of this passage of amino aoids and B vitamins across the ruminal epithelium? 2) Does this passage of amino acids and B vitamins across the ruminal epithelium occur under ordinary feeding conditions? 3) How does this passage of amino acids and B vitamins across the ruminal epithelium f i t into the scheme pictured for the metabolism and final fate of these compounds in the ruminal contents? An attempt will be made to answer each of these questions in turn. To simplify the presentation, however, the discussions for the amino acids and B vitamins will be presented separately. To conclude the General Discussion, the passage of amino acids and B vitamins across the ruminal epithelium will be discussed from the point of view of the nutritional implications. 151 Fart At Amino Acids Manner of Absorption of Amino Acids from the Rumen In recent times, considerable work has been carried out on the passage of amino acids across the epithelial linings of the small intestine and kidney tubule. Prom such studies, theories have been proposed on the manner of absorption of amino acids. It is not unreasonable to assume that the passage of amino acids across the epithelial lining of the rumen may occur in the same manner as the passage of amino acids across the epithelial lining of the small intestine or kidney tubule. A review will be made, therefore, of the literature dealing with the manner of absorption of amino acids in these other structures. Literature Review on the Manner of Absorption of Amino Acids Small Intestine.- Por many years, i t was undecided as to whether the absorption of amino acids from the small intestine was by means of 45 simple diffusion or by some other process. The investigations of Hober and Hober in 1936 and 1937 (165, 166) provided strong support that more than simple diffusion was concerned in the absorption of amino acids. Previously, i t had been shown that the diffusion coefficients of molecules tend to vary inversely as the molecular volumes (164). The The term "simple diffusion" is used here to designate that net transfer of molecules from one site to another that results because of a difference in the chemical potential of the molecules in the two sites. Some authors write of a difference in the concentrations of the molecules in the two sites and this is adequate i f the other components (e.g., temperature) that help to make up the chemical potential are equal in the two sites. results of Hober and Hober, however, demonstrated that amino acids were absorbed from the small intestine of the rat far more rapidly than many other compounds of oomparable molecular volume* This behaviour of amino acids in the small intestine was the more remarkable in that earlier work had shown amino acids to possess abnormally-low permeation rates for diffusion through certain natural and a r t i f i c i a l porous-type membranes* The following quotation (164) describes the results of this earlier work and contrasts them with the results of work on the small intestine* It is conspicuous from old observations of Overton con cerning the osmotic properties of frog muscles, that amino- acids, like glycine or alanine, enter with great slowness, i f at a l l , although, because of their smaller molecular size, one could expect them to exceed, for instance, erythritol* S t i l l more unexpected is their inertia in penetrating the cell surface of the sulfur alga Beggiatoa, which is remarkable for its outstanding behaviour as a molecular sieve and in which the permeation rates of several amino-aoids have been found to be abnormally low* The reason for this slowness is not of a physiological nature, since the slowness is evident also in diffusion experiments with collodion membranes, and can be explained as being due to the ampholyte character of the amino-aoids, which, probably due to the formation of a shell of water dipoles around the ampholyte ions, brings about an enlarge ment of the moleoular volume. Consequently, a porous mem brane, such as the intestinal wall, would be expected to be passed comparatively slowly also* But the contrary is true* In comparison with acid amides, with erythritol, with xylose, the amino-acids actually pass the intestinal wall much faster than was anticipated from their diffusion rates• The results of Hober and H'ober also did not agree with the results to be expected i f the rate of amino acid absorption by the rumen is in accord ance with Pick's law of diffusion* Hober (164) has stated this law in terms that are relative to the absorption of substances from the small 46 intestinei Aocording to Pick's law, when a substance is administered in a 'This statement can be derived from the following equation which 155. loop experiment at different concentrations, the absolute amount entering by passive penetrations Q that i s , by diffusion"] would bear a linear relationship £ s i c , direot proportionality3 to the concentration, and the relative amount would be constant, • • • • Such substanoes as polyhydric alcohols, aliphatic acid amides and certain "non-physiological" sugars—e.g., mannose, xylose and arabinose—are absorbed from the small intestine in accordance with Fick 1s law (164, 166)* In oontrast, the amino acids do not disappear from the lumen of the small intestine in accordance with Fiok's law. For i n i t i a l amino acid concentrations ranging between 6.5 and 71 millimoles per m i l l i l i t e r of solution in the intestinal lumen, Hober and Hober (164, 166) found that, with increasing i n i t i a l concentrations, the absolute rate of disappearance of the amino aoids did not inorease linearly but remained constant and the relative rate did not remain constant but deoreased. Significantly, these results of Hober and Hober for the amino acids are the same type of results that have been obtained for the "physiologioal sugars"— glu- ccse, galactose, and f r u c t o s e — , sugars whose absorption i s known to states Fick»s law (162)t / \ noted, however, that, i n the statement of Hober, the concentration of the substance in the intestinal loop apparently i s assumed to approximate to the concentration difference (dc) or concentration gradient (dc/chr) that existed across the intestinal wall. Such as assumption i s v a l i d when the concentration of a substance in the lumen of the intestine i s very much larger than the concentration of the substance in the plasma. As the range of concentration of the amino acids added by Hober and Hober (166) to the intestinal loops was between 5.5 to 71 milligram-moles per m i l l i l i t e r and as the magnitude of the concentration of amino acids in the plasma i s of the order of 10-5 to 10-3 milligram-moles per m i l l i l i t e r , the intestinal concentrations of the amino aoids in these loop experiments closely approximated to the concentration differences or gradients that existed across the wall of the small intestine. involve more than simple diffusion (164)• Hober and Hober (166) con cluded that the absorption of amino acids from the small intestine like wise involves more than simple diffusions "Their absorption is not like a diffusion, but is a process complicated by the presence of an acceler ating faotor, the effect of which becomes more visible with lower than with higher concentrations•" Stated again, the conclusion of these workers is that the intestinal absorption of amino acids is the result of the effects of both an accelerating factor and the process of simple diffusion, the latter being of more importance at higher concentrations (see also the modern work of Fridhandler and Quastel (123)). In more reoent times evidence has been accumulating as to the possible nature of the accelerating factor that was described by Hober and Hober as being ooncerned in the absorption of amino acids. Several groups of workers (2, 71, 133, 207, 232, 270, 338, 414, 415) have demonstrated a stereochemical specificity on the part of this faotor in the absorption of amino acids from the small intestine. The ]>isomer, as compared to the D-isomer, disappears from the intestinal lumen more rapidly and appears in greater concentration in the blood vessels draining the small intestine. Although not necessarily so, this stereochemical specificity on the part of the accelerating factor suggests that one or more enzymes m 47 are involved in its action. The L-isomers of the neutral amino acids , in contrast to the D-isomers that have been examined, even oan be absorbed The neutral amino acids are the mono-amino, mono-carboxylic amino acids such as glycine and phenylalanine, in contrast to the dicarboxylio amino acids such as aspartic acid and glutamic acid or the diamino amino acids such as lysine and arginine. 155 from the small intestine against a concentration gradient (2, 414, 415, 416). This indicates that the accelerating factor is one which can utilize metabolic energy to perform work. When the absorption experi ments have been conducted using solutions containing equimolar amounts of the L-isomers of several of the neutral amino acids, the individual amino aoids have demonstrated marked differences among themselves in their rates of disappearance from the lumen of the small intestine (4, 153, 190, 276, 277, 303, 416). The rank order of these different rates of disappearance is not in accordance with the molecular volumes or 4R concentration gradients of the amino acids ; rather, the rank order for the rates of transfer of the individual neutral amino acids from a mixture of neutral amino aoids generally is the reverse of the rank order found when the rates of transfer are measured for solutions containing single 49 m amino acids • This reversal of rank order for the rates of absorption of the neutral amino acids from a mixture of amino acids is believed to be a reflection of competitive inhibition among these acids for a common cellular mechanism that brings about their absorption (416, see also 190). The preferential transfer of L-isomers also has been demonstrated to be inhibited by potassium cyanide (2, 3), oxygen lack (123, 273, 413), cell cooling (231, 232), 2l4 dinitrophenol (2, 3, 123, 340), but not by Only by the use of a very high concentration of a particular amino acid were Hartmann et al (153) able to demonstrate any consistency in the effeot of concentration gradient or molecular volume on the rate of transfer of the amino acid from a mixture of amino acids in the small intestine. 49 / The results of Wiseman (416) provide an example for illustration. He found that the rapid transfer against a oonoentration gradient of either glycine, L-proline, or L-histidine was inhibited completely in the presence of the more slowly absorbed L-methionine. 156, phlorhizin (123, 222; see also 166). 5 0 Sinoe, except for 2:4 dinitro phenol5^" and phlorhizin, a l l these compounds or conditions are known to inhibit cellular respiratory (aerobic) metabolism, i t appears that this is further evidence that the aotion of the accelerating factor has a requirement for metabolic energy;also, that the requirement is relatively large, since the energy yield of a non-respiratory (anaerobic) metabolism apparently is too small to meet this requirement. Further, since 2:4 dinitrophenol inhibits the utilization of the energy of the respiratory oxidative reactions for the generation of the high-energy phosphate bonds of adenosine triphosphate (219), the inhibition of the preferential transfer of the L-isomers of certain amino acids by 2»4 dinitrophenol Sinoe phlorhizin has been demonstrated to exert an inhibiting action on glucose transfer (122, 166, 222), this non-inhibiting action of phlorhizin on amino acid absorption has led several authors to state that the mechanism for the preferential absorption of amino acids is not the same as that for glucose. Singer et al (342) have obtained results, however, that the authors suggest may be indirect evidence of competition between amino acids and glucose for the use of a phosphorylation mechanism present in the intestine wall. They found that when gelatin was fed with gluoose the blood amino aoid and blood glucose curves were depressed, as oompared to the curves when gelatin or glucose was fed alone. On the other hand, Agar et al (2) found no evidence for the existence of any competition between glucose and amino acids during their selective absorptions from the small intestine. Furthermore, Fridhandler and Quastel (123) found that only relatively high concentrations of glucose would inhibit the rate of transfer of L-alanine from the small intestine. Since similar high concentrations of sorbose or sodium chloride also had a similar retarding effect on the rate of transfer of L-alanine, these authors suggest that the inhibition of the absorption of L-alanine by glucose merely is due to an osmotic effect and not due to their competing for a common mechanism of transfer. Yet these same authors have found the presence of 0.01 M deoxypyridoxine to inhibit the absorption of D-L alanine, gluoose, and fructose from the small intestine. They suggest that deoxypyridoxine, a pyridoxine analogue which can compete with pyridoxine for sites of meta bolic action, exerts its action by the "inhibition of a mechanism con trolling active absorption in general." ^^Dinitrophenol does not inhibit respiration except in high concen trations (219). 157, indicates that adenosine triphosphate (ATP) is concerned direotly or 52 indirectly in the absorption of these amino acids. Just how ATP is conoerned in the absorption process remains, however, unknown. T r i - antaphyllopoulos and Tuba (380) have made one suggestion. It has been demonstrated that a soluble cell fraction from rat liver contains enzymes which, in the presence of ATP* catalyze a carboxyl activation of specific amino acids (163). The compound that i s formed is suggested as being an amino acid/Uadenosine monophosphate (AMP) complex, wherein extra bond energy is retained in the linkage between the carboxyl group of the amino acid and the phosphate group of the AMP. Triantaphyllopoulos and Tuba suggest that this same carboxyl activation may occur in the preferential absorption of the L-isomers of amino aoids from the small intestine. The function of ATP, then, would be to aid in this carboxyl activation of the amino aoids. There remains, however, another possibility, that of phosphorylation of amino acids by transphosphorylation. This follows from the demonstration that rich amounts of alkaline phosphatase are present in the epithelium of the small intestine (396j see also 124, 379), especially in the striated border whioh faces on the lumen (233, 262). The feeding of casein (212, 382) or of the L-isomers of individual neutral amino acids (380) oauses an inorease in the alkaline phosphatase concen tration in the intestinal cells. Now this intestinal enzyme has been demonstrated to be capable not only of hydrolyzing and synthesizing phosphomonoesters (124, 254) but also of transphosphorylating ability (124, Perhaps in confirmation of this is the recent work of Shishova (340). This worker found greater absorption of amino acids when inorganic phosphorous and ATP were included in the mixture of amino acids added to the small intestine. 158, 144, 255, 264). Meyerhof and Green (255), for example, have demonstrated that, when phosphate compounds of higher bond energies than that of the acceptor phosphate oompound were present, intestinal alkaline phosphatase was able to tring about transphosphorylation to the common biological alcohols (e.g., glucose, glycerol). It may be that in the preferential absorption of the amino acid L-isomers that a transphosphorylation occurs between ATP and the L-isomers. Lipmann (213aj see also 124) has suggested that just such a reaction may be the i n i t i a l step in the synthesis, of protein* ". . . the cell might first prepare the acyl phosphate with adenyl pyrophosphate £ATP] as the source of energy-rich phosphate groups." There remain other types of amino acid-phosphate compounds that also might be formed* in the laboratory, the amino and the hydroxyl groups of amino acids are known to form compounds with phosphate (l-3~4$,4l3av 419a); in the animal, the amino group in the guanidino part of arginine and the hydroxyl group of serine are known to form compounds with phosphate (124). Finally, of considerable interest to the general problem under discussion are the recent results of Shishova (340). This worker has demonstrated the presence of "labile phosphorous compounds of amino acids" in the intestinal wall during the absorption of amino acids from a solution containing amino aoids, inorganic phosphate, and AIP* Dinitrophenol prevented the formation of these phosphorous compounds. Shishova suggests that most, i f not a l l , amino acids are phosphorylated during their absorp tion. The results do not indioate, however, the type of chemical reaction that brought about the formation of the amino aoid-phosphorous compound. Although the nature of the postulated amino acid-phosphorous oompound remains unknown, i t does appear as possible that the accelerating faotor of the Hobers is actually the cellular arrangement of enzymes that brings about the formation of some type of phosphorous compound with the L-iso mers of at least some of the amino acids* Since diffusion i s a relatively slow means of conveying substances from the surrounding f l u i d into the interior of a c e l l (70), the phosphorous-linking of the amino acids would provide a chemical mechanism for increasing the rate of transfer of .the amino acids from the lumen of the small intestine. Complementary to the previous description of the Hobers1 accelerating factor i s the work of Agar et al (3, 4). They have published results that may be of great significance for an understanding of the manner of transfer of amino acids across an epithelial l i n i n g . Working with intestinal epithel ium, Agar et a l found that the uptake or accumulation of L-histidine and L-phenylalanine by the cells of the intestinal epithelium could occur against a concentration gradient. The uptake occurred even when the concentration of the amino acid i n the epithelial cells was ten or more times the concentration of the amino acid in the solution bathing the c e l l s . Very low concentrations of cyanide or 2:4 dinitrophenol inhibited the uptake of L-histidine. Equimolar concentrations of the L-isomers of amino acids with large non-polar side chains—members of the neutral amino acids—inhibited the uptake of L-histidine strongly; equi molar concentrations of the L-isomers of amino acids with polar side chains—not members of the neutral amino acids—inhibited the uptake of L-histidine weakly or not at a l l . Wow these features for the uptake of amino acids by the cells of the intestinal epithelium are strikingly similar to those features cited previously (p. 154)-for the transfer of amino acids across the intestinal epithelium. Prom this similarity, Agar et a l (4) propose that the concentrative uptake of amino aoids by the cells of the intestinal epithelium i s the f i r s t step in the intestinal transfer of amino acids against a concentration gradient* As Agar et al found the loss of amino acids from the cells apparently not to require energy of respiration—that i s , the loss was not inhibited by cyanide or 2»4 dinitrophenol—, they suggested the seoond step to be the diffusion of the accumulated amino acids "down a concentration gradient" to the subepithelial tissues* The diffusion of the amino acids from the sub epithelial tissues to the venous blood which drains from the intestinal wall would be the third step (44)* Thus, the continual accumulation of amino acids in the cells of an epithelial lining by means of the Hobers* accelerating faotor and the continual removal of amino acids by blood draining the area of the epithelial lining provide an overall mechanism for the conoentrative transfer of amino acids aoross the epithelial l i n i n g . 5 3 Kidney and Placenta.- Besides the small intestine, evidence exists for the conoentrative transfer of amino aoids across two other epithelial membranes. It is well known that the tubules of the kidney transfer amino acids against a concentration gradient, from the tubular lumen to the blood of the renal capillaries (164, 139). Recently, Kamin and Handler (189) have demonstrated that individual amino acids compete with one Perhaps i t should be emphasized here that the above discussion has been oonoerned with the conoentrative transfer of amino aoids from the small intestine. As i t was indioated previously (see p.154), when the concentration gradient is favourable there also is a transfer of amino acids from the small intestine by the non-seleotive action of simple diffusion. Although apparently not mentioned in modern work, there also exists the possibility that the transfer of amino aoids aoross the intest inal wall may be acoelerated by the presence of a favourable electrical gradient (see footnote 62 on p.176). 161. another for the cellular mechanism of tubular absorption. The strongest evidenoe for this competition was among the neutral amino acids. These results suggests the -probable presence of a cellular conoentrative mechanism.in the epithelial cells of the kidney tubules. The results of Crampton et al (81, 82) indicate that this cellular mechanism is a stero- chemically specifio mechanism for the reabsorption of L-amino acids. In the guinea pig, rabbit, and human, Christensen and Streicher (68) have found that amino acids are transferred across the placenta, from the maternal circulation to the foetal circulation, and this transfer occurs even against a concentration gradient. Crumpler et al (84) have confirmed these results in the human and have demonstrated the results to be the same for a l l the amino acids commonly found in plasma. Both groups of workers proposed that the placenta acts as an amino acid "pump". Other Tissues.- If the concept—that a concentrative uptake of amino acids by the individual c e l l s of the intestinal epithelium is the first step in the concentrative transfer of amino acids across the intestinal epithelium—proposed by Agar et al is true, then the results of the large amount of work that has been conducted on the uptake of amino acids by the cells of non-epithelial tissues may be applicable to the uptake of amino aoids by the oells of epithelial tissues. If the results of this other work are applicable, then they should contribute to a better under standing of the manner of transfer of amino aoids across epithelial membranes. A brief summary of some of this other work is presented below. The amino acid concentrative process has been studied in greatest detail by Christensen andco^workerSj, who used the isolated cells of mouse ascites tumors. These cells possess a high conoentrative activity for amino acids. Strong evidence was found for the existence of a special oellular "carrier-like" mechanism that transports amino acids into the cells* This carrier mechanism for the uptake of amino aoids is character ized, in part, by the followingi 1* The pH of the solution influences the magnitude of the uptake of at least some of the amino acids* For glycine and histidine, the maximum concentrative uptake occurs between pH 7*3 and 7*9 (63, 65)* 2* The uptake of amino acids by the cells is reduced by cyanide, 2»4 dinitrophenol, malonate, iodoacetate, fluoride, physostigmine, and anaerobiosis (64, 157)* Other work (67, 324) indicates that these sub stances or conditions do not inhibit direotly the carrier mechanism; rather, they appear to act upon that part of the cellular metabolism which supplies the energy requirements for the mechanism* 3* The carrier mechanism has a limited capacity* The existence of this limitation was deduced from an analysis of the relation between the rate of amino acid uptake by the cells and the i n i t i a l concentration of amino acids in the solution bathing the oells* By use of the Michaelis- Menton type of analysis (124, 256), a rate-limiting step was demonstrated* The occurrence of this rate-limiting step is proposed as reflecting a limited capaoity of the carrier mechanism (156, 157)* 4* The oells concentrate both the L- and the D-isomers, the L-form far more strongly than the D-form* D-isomers oan compete to some extent with the L-isomers for the carrier mechanism (65)* Christensen (63) statesl "A carrier which wil l combine with either isomer is visualized with the advantage of the L-form s t i l l to be explained 5* The oellular absorption of both the L- and the D-isomers of any of the neutral amino aoids may be inhibited competitively by the presence of one or more of the other amino acids (65, 69). Christensen (63, 65) 163 e makes the suggestion that the inhibition among the neutral amino acids probably indicates the existenoe of one or a very few carriers of relatively low specificity. This supposition of low specificity is supported by the fact that many synthetic amino acids of abnormal structure can be concen trated in the cells (65, 66, 325). 6. The magnitude of the concentration gradient that is attainable for the straight-chained amino acids is found to decrease as the length of the aliphatic side chain increases. Illustrative distribution ratios for the concentration gradient between the cell and the bathing solution aret glycine, 7; alanine, 4; valine, 1.4; leucine, 1.1 (63). 7. The presenoe or absence of certain chemical groupings on particu lar sites of the amino acid molecule produce some striking effeots on the rate of amino acid uptake by the cells. Two examples of this effeot of ohemical structure are the inhibitory action of a seoond carboxyl group (63, 65) and the strong stimulatory effect of a seoond amino group attaohed to the beta or gamma carbon atom of an amino acid (65,^66, 325) On the basis of suoh results, Christensen andcso_-wor'kBns make the following general statement about the relation between the cellular carrier-mechanism TIetenyi and Winter (160) have found the selective absorption of ' glycine by the small intestine to be abolished by the loss of either the carboxyl or amino group, as neither glycine-amide nor sarcosine was absorbed selectively. Each of these latter substances was absorbed by simple diffusion alone. Also, proline, which lacks a free amino group, was absorbed by simple diffusion alone. On the other hand, beta-alanine was absorbed selectively and the authors suggest that this may mean that the selective mechanism is independent of the intramolecular position of the carboxyl and amino groups. The results of Schofield and Lewis (338), however, indicate that the rate of this selective absorption from the small intestine is dependent on the intramolecular position of the carboxyl and amino groups. and the structure of the amino acids (65): The concentration which a given acid undergoes probably is a consequence of the compositional, steric and configura- tional details of its structure which determine its reactiv ity with the carrier. Leucine presents the poorest, alpha, gamma-diaminobutryate the best approach, so far, to the structure most favorable for concentration* Other mammalian tissues have been demonstrated to contain amino acids in cellular concentrations that are many times the concentrations in the plasma* The ratio of the concentrations of amino nitrogen in several mammalian tissues to the concentrations of amino nitrogen in the plasma was found by Van Slyke and Meyer (393, 394) to range between five and ten (see also the modern work of Astrup et al (20))* Similar results were obtained by Christensen and coworkers and, in addition, these workers found that competitive inhibition oocurred among the amino acids during their conoentrative uptake by the tissues (63)* The uptake of glycine by liver and muscle cells, for example, could be inhibited by the presence of any one of a large number of amino acids* The strongest inhibition was among the neutral amino aoids (69). The parallelism of these results to those obtained for the small intestine and the ascites cells suggests the existence of the same type of "carrier mechanism" in a variety of mammalian tissues* Summary and Comment*- Among the previously discussed structures—epi thelial membranes of the small intestine, of the kidney tubule, and of the placenta: normal tissue cells; and neoplastic oells of mouse ascites tumors—, there are several common features in their behaviour toward the amino acids* A l l the structures concentrate amino acids, presumably by means of a cellular mechanism that is present close to the surface of the cell* The concentrating mechanism usually has been demonstrated to either favour the L- to the D-isomer or not measurably concentrate the D-isomer at a l l . The concentrative ratio is affected by temperature, pH, and respiratory inhibitors. The pattern of competitive inhibition among the amino acids during their uptake is similar for the different types of tissues. In particular, the widespread occurrence of mutual interference among the neutral amino aoids is to be noted. Christensen, on the basis of this similarity of behaviour between the epithelial membranes and the non-membranous tissues, suggests that the conoentrative transfer of amino acids across epithelial membranes is brought about by a "oarrier mechanism"• He comments on this suggestion as follows (63)» A l l that is required to permit a cell-layer £i*e., an epithel i a l membrane"| to act in this way is that the conoentrative process be weaker on one surface than on another. Conoeivably such oells instead may have their own distinct transfer appa ratus, but a priori this appears less likely* Although some yet-to-be-explained differences occur among some of the different types of mammalian oells in their behaviour toward amino acids (123, 417), the above review of the literature indicates a develop ing conoepti the ubiquity among different types of mammalian oells of the same or similar mechanisms for the concentrative accumulation of amino acids. With suoh a conoept developing, i t is to be wondered i f also the epithelial cells of the rumen possess a similar mechanism for concen trating amino acids. The structural arrangement of these cells in forming an epithelial membrane between the ruminal contents and the ruminal blood could favour the concentrative transfer of amino acids across the wall of the rumen. Some of the experiments reported in the literature will be cited to demonstrate that, in faot, the ruminal epithelium does possess an ability to transport at least some substances against a concentration gradient* 166. Literature Review on the Manner of Absorption of Substances from the Rumen General*- Although simple diffusion is probably the manner of trans fer of the gases, ammonia and methane (74, 100), the passage of most molecules across the ruminal epithelium does not appear to be by simple diffusion alone* It might be suggested, for example, that the three lower volatile fatty acids—acetic, propionio, and butyric—, because their concentrations in the ruminal contents of normally feeding sheep are always much higher than their concentrations in the blood, are absorbed from the rumen by simple diffusion alone* Parthasarathy and Phillipson (286) have found, however, that not only is the ruminal absorp tion of acetate ions reduced in the presence of "0*002 M-mercurie chloride", but also that this ruminal absorption of acetate ions is more efficient at low concentrations than at high concentrations. These results are not to be expected i f the absorption of acetate ions occurs only by simple diffusion* The authors themselves suggest that these "are good reasons for believing that the absorption of acetate * . • is influenced by the activity of the epithelial cells lining the rumen*" Although the situation for propionic acid and butyric acid may be the same 55 as for acetic acid, this point has not been investigated. Moreover, there is a complication in the study of the manner of absorption of pro pionio and butyric acidst the cells of the ruminal epithelium metabolize It is perhaps of significance for the ruminal studies of fatty acid absorption that Smyth and Taylor (354, 355) have found the intestinal mucosa to be capable of transferring short-chain fatty acids against a conoentrative gradient. The transfer was "greatly reduced anaerobically, in the absence of glucose or in the presence of 10~4 M 2«4 dinitrophenol*" 167 appreciable amounts of the two aoids, especially of butyric (288, 289). It i s , however, for the ruminal absorption of the simple ions of chloride and sodium that strong evidence has been accumulated for more than simple diffusion being involved. Both ions have been shown to be absorbed from the rumen against a oonoentration gradient (96, 286, 359)• In seeking the mechanism of movement of chloride and sodium ions against a concentration gradient, Dobson and Phillipson (96, found, in normally feeding sheep, the existence of two foroes. One of these foroes was an electrical potential difference across the ruminal wall. The potential differences ranged between +25.4 and + 44.0 millivolts, with the ruminal contents being negative with respect to the blood of the ruminal veins. The other force, that which is exerted on molecules by a concen tration difference, also can be expressed in millivolts. The algebraic sum of the measured number of millivolts for the electrical and the chemi cal potential differences gives an estimate of the electrochemical potent i a l difference acting on a single ion to transfer i t across the ruminal epithelium (384). This relation i s expressed more concisely in the 56 following equations e-cp.d. = B J In ^4 + zCVJ-Vl whereC-Cp-cLis the electrochemical potential acting on a single ion and is expressed in millivolts,*R is the gas constant,T is the absolute tempera ture, F is the faraday, C, and are the respective concentrations of the ion in two solutions which are separated by a membrane and which are 56This is the equation used by Dobson and Phillipson (-96°), but with a slight rearrangement in order to contrast more effectively the contribu tion of each the chemioal and electrical potentials to the total potential of the ion. 168. denoted by the subscripts I and a. ,-p. arid "fare the respective activity coefficients of the ion in the two solutions, 21 i s the ion charge, and Vfand\4 are the respective potentials i n the two solutions* The f i r s t term on the right-hand side of the equation represents the chemical potential of the ion, the second term represents the ele c t r i c a l potential • of the ion. err Dobson and Phillipson (&?•» 98, 298) found that the net flux of chloride ions across the ruminal epithelium always corresponded with that to be expected from the effect of the estimated electrochemical potential 5 7 difference. For sodium ions, however, Dobson (96) found that the net flux was against the electrochemical potential difference. The move ment of sodium ions against the electrochemical gradient occurred even when i t was as high as 80 m i l l i v o l t s . These results indicate the existence of another foroe which can move sodium ions against an electrochemical gradient whose magnitude can be larger than 80 m i l l i v o l t s . Dobson suggests that this force i s energy supplied by the ruminal epithelium; and, that i t i s this metabolically-supplied energy that "drives" the sodium ions from the ruminal contents into the ruminal blood. Such a 57 The ions of chloride, bicarbonate, sodium, and phosphate have been demonstrated to pass i n both directions across the ruminal epithelium (see p. 12 and 13). 5 8 I t i s interesting to note that also the kidney (28) absorbs sodium ions against a concentration gradient. Both the epithelial cells of the kidney tubules and the basal columnar cells of the ruminal epithelium have the same arrangement and concentration of mitochondriat densely packed mitochondria which often are orientated in the long axis of the oells (9S)• As Bartley and Davis (28) have suggested that the mitochondria play a significant role in the transport of sodium i n the kidney tubules, this parallelism of arrangement and concentration of mitochondria in the two epithelial membranes provides indirect evidence that the mitochondria of the ruminal epithelial cells are concerned in the transport of sodium ions across the ruminal epithelium. A comment by Phillipson (298) i s of interest here: " . . . the animal has to work hard to obtain sodium and also to reabsorb muoh of the sodium which pours into 169. force of metabolic energy also appears to be involved in the movement of bicarbonate and inorganic phosphate ions from the ruminal blood to the ruminal contents. The movement of these ions has been shown to be against a strong electrochemical gradient (230, 298, 299). In the case of potassium ions, however, no evidence for any force other than that of chemical potential has been found to explain their transfer across the ruminal epithelium. In spite of an unfavourable electrical gradient for these positively charged ions, their concentration in the ruminal contents is high enough to move them into the blood by simple diffusion* Phillipson (298) comments on the absorption of potassium ions as follows» "The quantities absorbed . . . are small oompared with sodium. There is no evidence yet to suppose that any 'active' mechanism is concerned." Finally, reference now can be made to the statement previously made (p« 166) that the absorption of acetate ions is more efficient at lower than at higher concentrations of these ions in the rumen and, therefore, "is influenced by the activity of the epithelial cells lining the rumen." The electrical gradient that exists across the ruminal wall may be a manifestation of this "activity". Such a gradient, with the ruminal blood positive with respeot to the ruminal contents, would accelerate the transfer of acetate ions and a l l other negative ions across the ruminal wall (299). For aoetic and the other volatile fatty acids, the effect of the favourable electrical gradient would be added to the effect of the favourable concentration gradient. It i s , however, from the equation presented on page 167, obvious that the effect of an electrical gradient on the rate of transfer of ions across a membrane will be more apparent the rumen in the saliva in a similar manner to the kidney which has to work hard to reabsorb sodium to maintain the high concentration of sodium in the plasma." at lower than at higher concentrations of the ions. This follows from the fact that at the lower concentrations of the ions the electrical potential force would constitute a greater percentage of the total force acting on the ions; therefore, when the rate of transfer of the ions is compared at two different concentrations, the rate of transfer will appear to be more efficient at the lower ooncentrations--if only the effect of concentration gradient is considered. Although the phenomenon of more efficient absorption of the acetate ions at lower concentrations can be explained as an effect of the electrical gradient, this does not rule out additional forces acting on the ions, with the effect of these additional forces also being more apparent at lower than at higher concentrations of the ions. Suoh an additional force might be, for example, metabolic energy supplied by the cells of the membrane being traversed. The evidence available, however, does not permit any conclusion as to whether metabolic energy also is a force that moves the acetate ions across the ruminal wall* In conclusion, i t can be stated that, on the basis of the evidence available to date, the movement of molecules across the ruminal epithelium is usually not by simple diffusion alone; rather, the movement usually involves the interaction of the effect of at least two of the 59 following foroes t concentration gradient, eleotrieal gradient (if the molecules are charged), and metabolic energy. Comment.- It has been demonstrated for many different types of tissues, including epithelial membranes, that amino acids can be absorbed If the ruminal solution is not isoosmotic with the plasma of the ruminal capillaries, then the effect of an additional force may be present: the friotional foroe of migrating solvent molecules of water on the solute molecules (385). or transferred against a concentration gradient. It also has been demonstrated that the ruminal epithelium possesses the ability to transfer at least some substances against a concentration gradient. It is pro posed, teleologically, that a lack of ruminal absorption of amino acids against a concentration gradient would be an unusual exception. The question of whether or not such a movement of amino acids across the ruminal epithelium would be the result of the force of an eleetrical gradient, or of the force of metabolic energy, or both oan not be answered as yet. Of special interest to this question, however, are the results of Lofgreen et al (214), especially i f i t is remembered that considerable evidence has been accumulated to indicate that amino acids are phosphory- lated in*their absorption from the small intestine (see p.157 and on). Lofgreen et a l , upon determining the tissue distribution of injected p32 into oalves, found the rumen, omasum, and jejunum to have high phosphate exchange rates as compared with the other sections of the gastrointestinal tract. The authors suggest that the rapid rate of exchange of phosphates in these organs may be a reflection of the presence 60 of a rapid phosphorylating-dephosphprylating mechanism which could be important in the absorption of certain nutrients. Thus, i f selective absorption—-i.e., absorption that requires metabolic energy in order to Such a mechanism would require a source of metabolic energy. With respect to possible sources of this energy, a suggestion ..of Pennington (288) is of interest. He found ruminal, reticular, and omasal epithelial tissues to be capable of metabolizing considerable amounts of propionic and butyric acids. From these results Pennington statesi These epithelia are not glandular and hence would not require the energy.of oxidation of the fatty acids for secretory purposes. It is possible, however, that such energy may be utilized in connexion with 'active absorption' cf substances from the rumen. 172. occur—of amino acids from the rumen be demonstrated, perhaps this suggested phosphorylating-dephosphorylating mechanism (or transphosphory- latingl) would be the energy-requiring mechanism concerned in this selective absorption of the amino acids from the rumen. First, however, it must be determined i f amino acids are absorbed selectively from the rumen. The data of the present investigation now will be analyzed, therefore, for evidence as to the manner of absorption of amino acids from the rumen. Evidence from the Present Experiments as to the Manner of Absorption of  Amino Acids from the Rumen Introduction.- From the information provided in the literature review on the manner of absorption of amino acids from the small intestine (p.lSl), kidney (p. 160), and placenta (p. 160), i t is evident that several criteria have been used in the attempts to elucidate the manner of transfer of amino acids across epithelial membranes. In order both to summarize the previous review and to provide an introduction to the present discussion, some of the more important criteria are listed below. 1. The effect of varying the i n i t i a l concentration of the amino aoid on its absolute and relative rates of transfer—if the absolute rates are directly proportional to the i n i t i a l concentrations and the relative rates are constant, these relations are considered evidence for simple diffusion being the only manner of transfer; i f the relative rates are inversely proportional to the i n i t i a l concentration, this relation is considered evidence for simple diffusion not being the only manner of transfer! that there also is involved a oellular accelerating factor. 2. The effect of stereoisomerism of the amino acid on its rate of transfer—the more rapid rate of transfer of the L-isomer in contrast 173. to the rate of transfer of the D-isomer is considered evidence for simple diffusion not being the only manner of transfers that there also is involved a cellular accelerating mechanism which demonstrates a specificity for the L-isomer. 3 . The transfer of the amino acids against a concentration gradient- such a transfer is considered evidenoe for simple diffusion not being the only manner of transfer: that there also is involved a cellular mechanism which is able to do work. 4. The effect of the stereoisomerism of an amino acid on its transfer against a concentration gradient—the transfer of the L-isomer but not the D-isomer against a concentration gradient is considered evidence for simple diffusion not being the only manner of transfer: that there also.is involved a cellular mechanism which is not only able to do work but which also demonstrates a specificity for the L-isomer. 5 . The effect of various metabolic poisons or metabolioaily-unfavour- able conditions on the transfer of amino acids against a concentration gradient—the partial or complete inhibiting action by these substances or conditions on the transfer is considered evidence for simple diffusion not being the only manner of transfer: that there also is involved a cellular mechanism which is dependent on the energy of respiration. 6 . The competitive inhibition that exists among the L-isomers of the neutral amino acids when their transfer is from a mixture of amino acids—such competitive inhibition is considered evidence for simple diffusion not being the only manner of transfer: that there also is involved a cellular mechanism which, although demonstrating a relative specificity toward the L-isomers of amino acids, demonstrates a relative non-specificity toward the L-isomers of the neutral amino aoids. 7 * Although apparently not as yet applied to amino acid transport across epithelial membranes, a rate-limiting step has been found to occur in the uptake of amino acids by the isolated cells of the mouse ascites tumors—if the valid i t y be assumed of the proposition of Agar et a l ( 4 ) that the conoentrative uptake of amino acids by the cells of an epithel i a l membrane i s the f i r s t step in the transfer of amino acids across an epithelial membrane, then the demonstration of the existenoe of a rate- limiting step in the uptake of amino aoids by the oells of the epithelial lining would be considered evidenoe for simple diffusion not being the only manner of transfer* that there also would be involved a oellular mechanism with a limited capacity for the uptake of amino acids and, therefore, also a limited capacity for the transfer of amino acids across an epithelial membrane* The design of the ruminal f i s t u l a experiments i n the present investi gation was not such as to permit a l l the just-listed c r i t e r i a to be applied to them* Actually, the original intent i n oonducting the experiments that are reported herein was to demonstrate that ruminal absorption of amino aoids did or did not occur, not to demonstrate the manner of absorption* At the beginning of the investigation, the author had hoped, i f ruminal absorption of amino acids could be demonstrated to oocur, to conduct further experiments designed to elucidate the manner of th i s ruminal absorption of amino acids* The author, however, underestimated the time required to perform and analyze the experiments which were designed to demonstrate ruminal absorption of amino acids and, therefore, the investi gation of the manner of absorption was not oonducted* I t i s apparent, however, that the results of those experiments that were executed provide preliminary-type data which can be subjected to some of the c r i t e r i a used by various authors to determine the manner of absorption of amino aoids by epithelial membranes. In applying many of the above criteria to the ruminal absorption of amino acids, there also must be considered the effect on the amino acid molecules of the electrical gradient that has been demonstrated to exist across the ruminal wall. In the present experiments—i.e., in experiments W4, W5, and W6—the pH of the ruminal solutions ranged between 6.5 and 7.6 (see Table 17, p. 87). Within this range of pH, a l l the amino acids present in the ruminal solutions, with the exception of arginine, histidine, and lysine, should each exist as an equilibrium mixture of dipolar ions (Zwitterions) and anions. The excepted amino aoids should each exist as an equilibrium mixture of dipolar ions and cations. This follows from a consideration of the isoionic points (pl) of the different amino acidss arginine, pl = 10.76j histidine, pl • 7.64; lysine, pi = 9.47; and for 61 the rest of the amino acids, pi's a 2.98 to 6 . 3 (167). As the electri cal gradient across the ruminal wall is orientated with the blood side positive and the ruminal side negative, the movement of anions or cations in either direction across the ruminal wall wil l be accelerated or deaccelerated accordingly as the charge on the ions is orientated with or against the eleotrioal gradient. Of necessity, then, the effect of the electrical gradient across the ruminal wall must be considered in any analysis of the manner of transfer of amino acids across the ruminal wall. Effect of Initial Eleotroohemioal Gradient on Rate of Ahsorption.- Althpugh Hober and Hober determined the effect of the i n i t i a l concentra- AThe pl numbers for the five amino aoids that are discussed in the present experiments are (167)s tryptophan, 5.88; methionine, 5.74; tyrosine, 5.63; glycine, 6.06; and lysine, 9.47. 176. tions of the amino acids on their rates of absorption from the small intestine, the existence of an electrioal gradient across the ruminal wall 62 should indicate that, in the oase of ruminal absorption, not only is a foroe of concentration difference operative upon the ions of amino aoids, but also the parallel force of an electrical potential difference. As the rate of simple diffusion of ions should be proportional to the total force acting upon them (385), i t would seem more logical to determine the relation between the absolute rate of amino acid absorption and the algerbraic sum of the parallel forces of ohemioal and electrical gradients, rather than between the absolute rate of absorption and the chemical gradient alone. The appropriate relation can be written: n /u e-cpd. = l n ^ _ + z f a - V j , where h is the amount of amino acid absorbed in a given time and the other symbols have the same meaning as given on p. 167. This means that i f no force other than those of the chemical and electrical gradients are acting upon the amino acid ions, the absolute rate of absorption should be directly proportional to the electrochemical potential difference. If Modern workers who have studied the absorption of amino acids from the small intestine seem to have ignored the question of an electrical gradient. Although no direct experimental evidence appears to be avail able for the existence of an electrical gradient across the wall of the mammalian intestine, there is some indirect evidence in the older litera ture that such a gradient may exist. Verzar and McDougall (397), for example, cite the early work of Rosenthall who found the serosal side of the frog's intestine to be positive with respect to the mucosal or luminal side of the intestine. More recently, Phillipson (298) cites the work of Dobson who found a potential difference to exist aoross the oaecal wall of the 'sheep. This potential difference was of the same magnitude and direction as the potential difference across the ruminal wall. The existence of these just-cited electrical gradients are strongly suggestive that a similar gradient may exist across the wall of the mammalian small intestine. Such a gradient would influence the intestinal absorption of the ions of amino acids. 177. direct proportionality is not obtained, then the results may indicate the presence of one or more other forces which also are acting on the amino acid ions. One of these other foroes could be the frictional foroe.that is exerted upon solute molecules by water movements (385). Such water movements occur when there is a difference in the osmotic or hydrostatic pressures of two solutions separated by a membrane permeable to water. Another force could be that resulting from the active participation of the enzymes of the oells that constitute the ruminal epithelium in the The v/ord "may" is used advisedly for several reasons. In the first place, the equational calculation of the effect of concentration difference is based upon the premise of the applicability of the van't Hoff equation that P • RTC, where P is the osmotio pressure, R is the gas constant, T is the absolute temperature, and C is the concentration of a solution. As this equation of van't Hoff is a limiting one, that i s , the equation is strictly true only for solutions of infinite dilution, i t oan be of applicability only to very dilute solutions. The oloseness of the approximation to the true data of data which are obtained by use of the equation diminishes as the finite concentration in the solution increases. Jaoobs (183) has pointed out that the effects of frictional resistance and of attractive forces between molecules or ions will ohange as the concentration increases, thereby lessening the applicability of the equation for solutions of stronger concentrations. Another reason for the use of the word "may" is exemplified by a comment of Hober (164)• This author, in writing of the applicability of Fick» s law to absorption by- diffusion from intestinal loops, makes the following comment: . • •, i t should be anticipated that the method described does not permit an exact confirmation of Fick 1s law to be obtained, Impairment of the vitality of the delicate epithelium, through mechanical insults following the repeated f i l l i n g and emptying of the loop, uncontrollable changes of the blood circulation, the effect of narcotics, and other factors are unavoidable causes of irregularity. Hober's comment also would be true for absorption by diffusion from the rumen. Schambye (333, 334, 335), for example, found in acute experiments on anesthetized sheep a decrease of cardiac output and a slowing of the blood circulation as compared with chronic experiments on non-anesthetized sheep: the average portal blood flow for the anesthetized sheep (334) was about 29 milliliters per minute per kilogram of body weight and the aver age portal blood flow for the non-anesthetized sheep (335) was about 37 milliliters per minute per kilogram of body weight (see also 230, 292). Again, Shaw and Peterson (337) have demonstrated large blood volume uptake of amino acids. Such enzyme participation usually involves the utilization of ohemical energy of the epithelial cells to do the "•work" of moving nutrients from one solution to another. If it is assumed that no forces other than those previously discussed are involved in the absorption of amino acids and i f conditions in the two solutions are such as to preolude any water movement, then a lack of direct proportionality for the effect of the electrochemical potential differences on the absolute 64 rates of absorption may indicate the participation of cellular enzymes and metabolic energy in the transfer of amino aoids across the ruminal wall. The data of the present experiments now will be examined to see i f they oan be analyzed according to the terms of the above discussion. One point of question immediately becomes apparent: Hober and Hober (166), in their determination of the effect of i n i t i a l concentrations on the rates of absorption of an amino acid, considered the absorption of the individual amino acid only from a pure solution of the amino acid. In the present investigation, a l l the studies of amino acid absorption were made using a mixture of amino acids. This means that, because of the known existence of interionic forces of attraction and repulsion, the diffusion of each amino acid oould not have been an entirely free diffu sion. In the application of Fiok's law to the effect of concentration on rate of diffusion, one of the assumptions that is made is that there is free diffusion of the molecules. This is a necessary assumption, as the changes to occur in the mammary gland of the cow during its excitation. It is possible that a similar blood volume change occurs in the ruminant stomach i f the non-anesthetized animal is excited by the experimental procedure. 64 See footnote 63 on the previous page. 179, foroes of intermolecular or interionic attraction increase as the concen tration of the molecules increases. This would mean that, as the concen tration increased, the attraction of a slower moving particle for a faster moving particle also would increase and, thereby, slow down the faster moving particle from its free diffusion rate and speed up the slower moving particle from its free diffusion rate. Suoh an effect would add to or subtract from the effect of an increased concentration difference on the rate of diffusion of a molecule or ion, thus masking any possible relation of direct proportionality between the i n i t i a l concentration difference and the rate of diffusion of the molecule or ion. This effect of the intermolecular or interionic forces, plus the points discussed in footnote 63' (p«177), make i t doubtful, therefore, that a deviation of the results in the present experiments from direct proportionality between the electrochemical potential difference and the rate of absorption can be taken as evidence for the effect of forces other than the electrical and ohemical potential differences acting upon the amino acids. In spite of the doubtful significance—for the reasons just given—of any such calculation, a calculation of the effect of the i n i t i a l electro chemical potential differences on the rates of absorption has been made for the data of experiments W4, W5, and W6. Table 29 presents, for each the five amino acids whose concentrations were measured, estimates of the 65 i n i t i a l electroohemical potential differences, the absolute decreases ^I n contrast to the procedure of Hober and Hober (see discussion on page 153* as well as footnote 46 on the same page), who used in their cal culations the i n i t i a l concentrations of the amino acids, the initial, concentration differences were calculated for the present experiments. The initial concentrations used by Hober and Hober were so high, relative to the plasma concentrations, that the i n i t i a l concentrations actually approximated very closely the i n i t i a l concentration differences. In the TABLE 29. - Ruminal fistula experiments W4, W5, and W6i a tabulation, for each the five amino acids whose concentrations were measured, of estimates of the in i t i a l electrochemical potential differences (millivolts), of the absolute deoreases of concentration (microgram-moles per 100 milliliters), and of the deoreases of concentration relative to the in i t i a l electrochemical potential differences (per cent) Ex pe ri me nt  Glycine Lysine Methionine Tyrosine Tryptophan Ex pe ri me nt  • OS • •H O i s . a ? © Concen tration decrease* • r j -0 •ri p4 s . © Concen tration decrease* . rH TJ OS • •ri P.! £ • © Concen tration decrease* rH -d OS • •ri O, •s . © Concen tration decrease* Concen tration decrease* Ex pe ri me nt  © rH o W a. Re la ti ve  © rH O W Re la ti ve  <D •s rH O to Re la ti ve  © rH O W Re la ti ve  as . •ri a. s . rH Y © Ab so lu te  i R el at iv e 21.0 75.7 2.1 600 45.2 11.7 6.6 27.3 - 8.1 W5 to 15.9 to to 12.5 to to 5.3 to to 1.8 to to 1.5 35.8 44.4 18.0 69.4 63.9 8.2 17.7 10.2 - 2.0 51.3 84.8 30.0 114 69.4 17.3 42.0 12.3 41.3 29.3 W6 to 43.5 to to 34.4 to to 12.0 to to 5.2 to to 12.1 to 66.1 65.8 45.9 74.9 88.1 13.6 53.1 9.8 57.4 21.1 49*6 35.1 32.8 54.9 73.5 6.8 53.8 0.9 56.0 5.5 W4 to 17.4 to to 18.0 to to 5.0 to to 0.5 to to 3.1 to 64.4 27.0 48.6 37.0 92.2 5.4 64.9 0.8 62.1 5.0 The concentration decreases that are recorded for eaoh experiment are those decreases which occurred in the first 70 minutes of the experiment. of concentration, and the decreases of concentration relative to the i n i t i a l electrochemical potential differences. Unfortunately, neither the concentrations of the amino acids in the plasma nor the electrical gradients across the ruminal wall were measured. Lacking such data for the calculation of the i n i t i a l electrochemical potential difference, i t was decided to use data that are available in the literature; For the calculation of the electrical component of the electrochemical gradients, the data of Dobson and Phillipson (98) were utilized. These authors found, both in conscious sheep with normal rumen contents and in anesthet ized sheep with ar t i f i c i a l solutions in the rumen, electrical potential differences which averaged about 30 millivolts to exist across the ruminal wall. This number was utilized in the calculation of the electrochemical gradient. An obvious weakness of such a procedure is that the measured electrical potential differences will vary, not only in the same animal from time to time, but also among different animals. Further., the assumption is being made that the electrical potential differences aoross the ruminal wall of the goat are the same as those found for the sheep. For the calculation of the chemical component' of the estimated, i n i t i a l electrochemical gradients, there were utilized the i n i t i a l ruminal solu tion concentrations of the amino acids recorded in Tables 8 to 12 (pp. 49, 52, 55, 58 and 62.) and the plasma concentrations of the same amino acids recorded in Table 30. For tyrosine, glycine, and lysine, the plasma data given for the goat were used; for tryptophan, the plasma data for the sheep were used; and for methionine, the plasma data for man were present experiments, however, the i n i t i a l concentrations were of the same order of magnitude as the plasma concentrations and, therefore, the i n i t i a l concentrations could not be used as approximations of the concen tration differences. TABLE 30. - Concentration (miorogram-moles per 100 milliliters) of five amino aoids in the plasma of ruminants and man Amino aoid Goat . Sheep . Cow Man Tryptophan- • • • • 3.9 to 4.9a 3.9 to 5.9a 3.4 to 7.46 Methionine • • • • • • • . • • • . 2.0 to 4.0a Tyrosine 5,5 to 8.3a • • • • . • • . 4.4 to 12«la Glycine 43.6 to 75.5b • • • . • . . . 17.3 to 30.6a Lysine 8.9 to 16.4a 9.1 to 13.5° .... .... 14.4 to 26.0a ^hese data, from Albritton'e Standard Values in Blood ( 8 )» are the lower and upper limits of the 95 per cent range of concentrations that have been reported in the literature. ese data are the lower and upper limits of the data that are recorded in Table 20 (p.ilO). °These data are the lower and upper limits of the data that are recorded in Table 19 (p.109). used.^ In each case, both the lowest- and the highest -found plasma concentrations were utilized in order to calculate the upper and lower limits of possible oonoentration differences; hence, in Table 29 two numbers are given for the electrochemical gradient acting on each amino acid. The assumption made, then, is that the actual i n i t i a l electro chemical gradient that oocurred during the experiments was within the 67 range limited by the two numbers. °As the plasma concentrations of the amino acids might differ considerably between ruminants and non-ruminants, i t is realized that the use of plasma concentrations found in man for those found in the goat may be hazardous. Nevertheless, the procedure is followed in the belief that at least the probable order of magnitude of the amino acid concentrations in the goat plasma will be indicated. Although not for glycine, such a belief proves to be valid in the oase of tryptophan, tyrosine, and lysine; thus, comparison in Table 30 of the plasma concentrations for these three amino acids in man and the ruminant animals reveals a olose approximation of the concentration ranges. It must be noted that an assumption was made in calculating the chemical component of the eleotroohemical gradient; that i s , that* where is the concentration and is. the activity coefficient. The validity of this approximation rests upon the following considerationsJ 1) The osmotic pressure of the ruminal solution in eaoh of the experi ments W4 to W6 (see Table 6, p. 37) apparently was close to the osmotic pressure of the plasma; 2) Most of the osmotic pressure in the ruminal solutions (see Table 5, p» 32) or in the plasma was due to the presence of uni-univalent salts — predominantly sodium propionate for the ruminal solutions and sodium chloride for the plasma—; therefore, the approximation of the osmotic pressure in the ruminal solutions to the osmotic pressure in the plasma means approximate equivalence in the total numbers of univalent ions for the two solutions; 3) The ionic strength of a solution is calculated from the following equation (70)* where s the ionic strength, C is the molar concentration of each ion 67 The data in Table 29 for the absolute rates of decrease of amino acid concentrations were derived from the 0 to 70 minute time interval results that are presented in Tables 8 to 12, (pp. 49, 52, 55, 58, and 62 )• *n order that the rates of absorption reoorded in Table 29 would be in the solution,2 is the ion's oharge number, and £he sign of the summation ( H ) indicates that a l l the productsC21 are to be added; therefore, the approximation of the total number of ions (mostly univalent) in the ruminal solutions to the total number of ions (mostly univalent) in the plasma signifies that the ionic strengths of the two solutions also must approximate to one another; 4) The activity coefficient is proportional to the ionic strength of a solution, other conditions being heldconstant, because (70)t where "His the activity coefficient of an ion,<^j,is its charge number, andT^lis the ionic strength of the solution; therefore, the approxima tion of the ionic strengths-of the two solutions should mean that the activity ooeffioient of each the amino acids in the ruminal solutions approximated to the activity coefficient for the same amino acid in the plasma; 5) In consequence of the statement under 4-), the ratio ("(",/£) of the activity coefficient of an amino acid in the ruminal solution ( -pj ) to the activity coefficient of the amino acid in the plasma ( -f^  ) was assumed to be unity; therefore, in calculating the chemical component of the electrochemical gradient, it.was considered valid to substitute the Finally, in addition to the above considerations, there is the fact that the activity coefficients of the amino acids change only slightly, even when relatively large changes in the molal concentration occur. Mehl and Schmidt (252), for example, found the activity coefficient of glycine at 30° C. to change from 0.97 for 0.2 molal concentration to 0.86 for 1.0 molal concentration. This means that the relatively small differences that existed between the concentrations of each amino acid in the ruminal solution and in the plasma of the ruminal capillaries were of l i t t l e consequence in the previously-made assumption that the ratio of the activity coefficients is unity. 68 The longest possible time interval was selected in order to magnify any differences in the data caused by differences in rates of absorption. The data of the 0 to 150 minute time interval could not be used for the present calculations, however, as some of the necessary control data were missing. The data of the 0 to 70 minute time interval, with none of the control data missing, were used. more likely to reflect only those changes of concentration that resulted from ruminal absorption, the data from Tables 8 to 12 were corrected for the concentration changes that occurred in the control solutions. In making the correction, the concentration changes in the ruminal and control solutions were calculated for the 0 to 70.minute time interval. Positive or negative signs were attached to the concentration changes obtained! positive for a concentration inorease and negative for a concen tration decrease. The concentration ohange for each control solution then was subtracted algebraically from the concentration change for the corresponding ruminal solution. The difference, always a negative quant ity, was considered to be the concentration change that resulted because of ruminal absorption. This oorreotion procedure assumes, of course, that the concentration changes that occurred in control solutions also occurred in the ruminal solution, and to the same extent. Examination of the data in Table 29 for experiments W5 and Yf6 reveals that the absolute rates of absorption were proportional to the i n i t i a l electrochemical potential differences. It is not possible to determine i f the proportionality is direot or not, as the i n i t i a l concentrations only can be presented as being within a given range of concentrations. If the ranges for the relative rates of absorption between the two experiments are compared for each amino acid, i t is found that, for each the amino acids tyrosine, glycine, and lysine, there are overlappings of some of these ranges with one another. This means that direct proportionality between the absolute rates of absorption and the i n i t i a l electrochemical potential differences could have oocurred for these three amino acids, but does not mean that i t did occur. For methionine, there is no over lapping of the ranges for relative rates of absorption in experiments W5 186. and W6. The upper limit of the range in experiment W5 is quite olose, however, to the lower limit of the range in experiment W6j therefore, the possibility of direct proportionality between the absolute rates of absorption and the i n i t i a l electrochemical potential differences does exist for methionine also. For tryptophan, no comparison can be made because the absorption of tryptophan in experiment W5 was apparently against the electrochemical gradient. The data of Table 29 also can be examined for the relation between the relative rates of absorption and the ini t i a l electrochemical gradients. A comparison between the ranges for the relative rates of absorption in experiments W5 and W6 reveals that, for each the amino acids tyrosine, glycine, and lysine, the upper limit of the range in experiment W5 is higher than the lower limit of the range in experiment W6. For these three amino acids, then, the possibility does exist for their relative rates of absorption being inversely proportional to the in i t i a l electrochemical potential differences. Up to this point, the data for experiment W4 in Table 29 purposely have been ignored. This has been done because a comparison between the data for the absolute rates of absorption in experiments W4 and W6 reveals a strong possibility of the existence of an effect of animal variability. It is suggested that the manifestation of this effect is the much smaller absolute rates of absorption for a l l five amino acids in experiment W4 as contrasted with the rates in experiment W6. For tryptophan and tyrosine, the smaller absolute rates of absorption in experiment W4 occurred in spite of the fact that the estimated i n i t i a l electrochemical gradients for these amino acids in experiment W4 were ; either the same as or larger than the estimated i n i t i a l electrochemical gradients in experiments W6. For the other three amino acids, methionine, glycine, and lysine, as the ranges of their estimated i n i t i a l electro chemical gradients in experiments W4 and W6 overlap, the possibility does exist that the i n i t i a l electrochemical potential differences were higher in experiment W6 than in W4. For a l l five amino aoids, however, a compari son of the relative rate of absorption in the two experiments reveals that the relative rates of absorption were higher in experiment W6 than in experiment W4. This indicates that, even for the three amino acids methionine, glycine, and lysine, any possible higher i n i t i a l electro chemical potential differences in experiment W6 were not enough to account 69 for the much larger rates of absolute absorption in experiment W6. 6 9 I n the calculation of the data for Table 29, a number of 30 m i l l i  volts was assumed, in a l l three experiments, for the electrical component of the eleotrochemioal potential difference* Now i t is true that the electrical gradient can vary in the same animal and among animals (see p. 181 )• It might be suggested, therefore, that the abnormally low relative rates of absorption in experiment W4~that i s , considering the relation of the i n i t i a l eleotroohemical gradients in experiment W4 to those in experiment W6, abnormally low relative to the rates of absorption found to occur in experiment W6—might not appear abnormally low i f the previous assumption of the existence of an eleotrioal gradient in experiment W4 of 30 millivolts be changed to an assumption of the existence of a much smaller electrical gradient. This latter assumption would yield, for a l l the amino acids except lysine, an i n i t i a l eleotrochemioal gradient for experiment W4 that was muoh lower than the i n i t i a l electrochemical gradient for experiment W6; therefore, the relative rates of absorption in experi ment W4 might no longer appear to be abnormally low. The data for lysine, however, appear to rule out the assumption of a much lower eleotrioal gradient in experiment W4. The use of a much lower number for the electrical gradient in experiment W4 would make the i n i t i a l electrochemi cal gradient for lysine much higher than that which is reoorded in Table 29—because the eleotrioal gradient is orientated to oppose the movement of the positively charged lysine ions and a reduction in the electrical gradient would increase the effect of the chemical gradient—; yet, the absolute rate of absorption of lysine in experiment W4 is only about one- half the absolute rate of absorption of lysine in experiment W6. Table 31'u reveals a similar situation for propionio acid, the substance that was added as a positive control for ruminal absorption* The abso lute decrease in the concentration of propionic acid in experiment W4 was much less than that in experiment W6, this in spite of the fact that the 71 • in i t i a l concentration in experiment W4 was slightly higher than that in experiment W6. Noting that experiments W5 and W6 were carried out with one goat (see p. 29) and experiment W4 was carried out with another goat (see p. 27)» i t is suggested that the results of these experiments for the absorption of amino acids and propionic acid indicate the presence of the effect of animal variability on the rate of absorption from the rumen. It was in order to eliminate this apparent effect of animal variability that the previous examination of the effect of i n i t i a l electro chemical potential difference was restricted to the data of experiments W5 and W6—both carried out on the same goat* It now can be asked what interpretation can be placed upon the findings of the analyses just carried out* The following interpretations are suggested: 1. As, from the data of experiments W5 and W6, the absolute rates of The data for this table were corrected in the same manner as for the i n i t i a l concentration data of Table 29 (p. 180): in calculating the absolute rates of absorption, the data from Table 7 (p. 45) were corrected for the concentration changes that occurred in the controls. It was necessary, however, to use the 0 to 150 minute time interval, as the concentrations of propionic acid in the control solutions were measured only in the 0 and 150 minute control samples. 71 The i n i t i a l concentrations can be used in this case in place of the calculated i n i t i a l concentration differences. This could be done because the concentrations of propionic acid in the ruminal solutions were of such a high order of magnitude relative to the range of concentrations normally found in the blood draining the rumen: 0*7 milligram-moles per lit e r (calculated from data that are presented in 196). See also footnote 46, P«152* TABLE 51* - Ruminal fistula experi ments W4 and W6i a tabulation, for propionic aoid, of the initial concen trations (milligram-moles per liter), of the absolute deoreases of concen tration, and of the decreases of con centration relative to the initial concentrations (per cent) •p § Concentration Si § In it ia l aa ee nt ra ti  decrease* Ex pe ri  In it ia l aa ee nt ra ti  1 ! 1 O P4 W6 65* 41 63*0 m 68 29 42*6 aThe oonoentration decreases that are reoorded for each experiment are those deoreases which occurred during the 150 minutes of the experiment* absorption of the amino aoids were proportional to their estimated i n i t i a l electrochemical potential differences and as the electrical component of the i n i t i a l electrochemical potential differences is assumed to have been approximately the same in the two experiments—i.e., 30 mi l l i v o l t s — , the findings obtained are evidenoe that the ruminal absorption of these, amino acids is at least partly due to the effect of the force of chemical potential difference and that, therefore, simple diffusion forms at least a part of the mechanism concerned in this absorption. 2. As, from the data of experiments W5 and W6, the overlappings or approximations of the ranges for the relative rates of absorption of a l l the amino acids, except tryptophan, admit of the possibility of direct proportionality existing between the i n i t i a l electrochemical gradients and the absolute rates of absorption, there must be admitted, in turn, the possibility that the ruminal absorption of the four amino acids entirely was due to the effeot of the forces of chemical and electrical potential differences. On the other hand, as the upper limits of the ranges for the relative rates of absorption in experiment W5 for each the amino acids tyrosine, glycine, and lysine are higher than the lower limits of the ranges for each these same three amino aoids in experiment W6, there also must be admitted the possibility that the relative rates of absorption were inversely proportional to the i n i t i a l electrochemical gradients. This latter possibility would mean, in turn, that the absorp tion of the three amino acids was more efficient at the lower concentra tions. If such a relation 'oocurred, then i t would be similar, not only to the relation found by Hober and Hober for the intestinal absorption of amino acids (see p.154), but also to the results obtained by Parthasarathy and Phillipson for the ruminal absorption of acetic acid (see p. 166)• These two groups of workers considered such a relation as possibly indicative of the effect of cellular energy that is participating in the absorption of amino acids or acetic acid, with the effect of the cellular energy relative to the effect of the chemical gradient being more notice able at the lower chemical gradients. 3. As, from the data of experiment W5, there is movement of trypto phan against the electrochemical gradient, this is evidence for the effect of a force other than the chemical or the electrical gradients being con cerned in the movement of tryptophan across the ruminal wall. It can be suggested that this other force may have been that of metabolic energy which is supplied by the cells of the ruminal epithelium. The above interpretations indicate, then, that the movement of the five amino acids aoross the ruminal wall is at least in part the result of the process of simple diffusion, plus the effect of the electrical gradient. In addition, for tyrosine, glycine, and lysine, the possibility does exist that another force, cellular energy, may have been involved in their movement. For tryptophan, i t appears, indeed, that this other force of cellular energy was involved (but see page 94 for a comment on the validity of the recorded concentrations of tryptophan). Comparison Among the Five Amino Acids of their Rates of Absorption.- There is another way that the data of the ruminal fistula experiments can be examined in this attempt to determine the forces concerned in the absorption of amino acids from the rumen. The data of Table 29 have been rearranged in Table 32. Table 32 also includes for each amino acid its molecular weight, its apparent molecular volume and the square root of TABLE 32* - Ruminal fistula experiments ¥4, W5, and W6t a tabulation of data that are utilized in the text (p«194 ) to make a prediction of the rank-order for the rates of ruminal absorption of the five amino acids listed Experi ment Amino aoid Molec ular weight* Apparent molecular volume0 Diffusion coefficient 0 Estimated i n i t i a l electro chemical poten t i a l differ- Absolute concentration deoreased»e (miorogram-mole s y (lO* 7 em.2/seo.) enoed (millivolts) per 100. milliliters) Methionine 149*2 110 10.5 68 73.5 to 92.2 5.0 Tryptophan 204.2 144 12.0 61.4 56.0 to 62.1 3.1 W4 Tyrosine 181.2 123 11.1 62 53.8 to 64.9 0.5 Glycine 75.1 45 6.6 95.0 49.6 to 64.4 17.4 Lysine 146.2 109 10.4 69 32.8 to 48.6 18.0 Methionine 149.2 110 10.5 68 45.2 to 63.9 5.3 Glycine 75.1 43 6.6 95.0 21.0 to 35.8 15.9 W5 Tyrosine 181.2 123 11.1 62 6.6 to 17.7 1.6 Lysine 146.2 109 10.4 69 2.1 to 18.0 12.5 Tryptophan 204.2 144 12.0 61.4 - 8.1 to -2.0 1.5 TABLE 32 - continued Experi ment Amino aoid Molec ular weight* App mole volu arent oular me Diffusion coefficient0 Estimated initial electro chemical poten tial differ ence* (millivolts) Absolute concentration decrease*»e (miorog ram-moles per 100 milliliters) - / (10~7 o»m.2/8e<»«) Methionine 149.2 110 10.5 68 69*4 to 88.1 12.0 Glycine 75.1 43 6.6 95.0 51.3 to 66.1 43.5 W6 Tryptophan 204.2 144 12.0 61.4 41.3 to 57.4 12.1 Tyrosine 181.2 123 11.1 62 42.0 to 53.1 5.2 Lysine 146.2 109 10.4 69 30.0 to 45.9 34.4 aThese data, rounded off to one decimal place, are from the Handbook of Chemistry and Physios (167). These data are from references (73) and (204). °See footnote 72 on page 194. ^he se data are taken from Table 29 on page 180. 3 The concentration deoreases that are recorded for each experiment are those decreases which occurred in the first 70 minutes of the experiment. • this volume, and its diffusion coefficient* 72 This information is included because i t is known that the rate of free movement of a molecular species is not only directly proportional to the electrochemical gradient that exists for i t , but also is dependent upon its inherent rate of free diffusion* The information wi l l be utilized in a further attempt to elucidate the manner of absorption of the amino acids from the rumen* Examination of the descriptive data for each of the amino acids listed ih Table 32 enables a prediction to be made of the rank-order of their rates of absorption—if the effects of the forces here considered are the only ones concerned in the absorption, of amino acids from the rumen* The effect of the i n i t i a l electrochemical gradients and the effect of the inherent diffusion rates (as expressed by the diffusion The diffusion coefficients for glycine and tryptophan were experi mentally determined (310), those for the other amino acids were derived by the present author from a figure (Fig* 4, p* 1911) of Poison (310) that demonstrates the relation between the diffusion coefficients of amino acids and their molecular weights* . These derived data are at best only approximate data, because i t has been demonstrated that the diffusion coefficient of a molecule more properly is related to its volume and shape than to its molecular weight (252). It is for this reason that the square roots of the molecular volumes for the amino acids also are produced in Table 32* It is interesting to note that the diffusion coefficients that are presented in Table 32 do approximate to a direct.inverse proportion with respect to the square roots of the molecular volumes. This is not an unexpected relation, since the moleoular volume of a compound tends to vary as its moleoular weight. It is probable that the variations in the shapes of the five amino acids that are presented in Table 32 account for at least part of the deviation from an exact proportionality. Poison (310), for example, has demonstrated that the more stretched a molecule i s , the lower is its diffusion coefficient. He illustrated this by comparing the diffusion coefficients of tryptophan and arginine. Arginine, a stretched molecule, has a molecular weight of 174, a molecular volume of about 117, and a diffusion coefficient of 57.6 x 10-7 cm.2/sec. Tryptophan, a more nearly spherical molecule, has a molecular weight of 204, a molecular volume of .144, and a diffusion coefficient of 61.4 x 10~7 om.2/sec* 195. coefficients) -will be compared to determine the net effect upon the flow rate of the individual amino acids. To demonstrate the method of analysis employed, the data of experiment W4 w i l l be considered in detail. On comparing the ranges of electrochemical potential differences for the five amino acids in experiment W4, i t is seen that the lower limit of the range for methionine exceeds the upper limits of the ranges for the other four amino acids. On this basis alone, then, a preliminary rank-order can be predicted for the absolute rates of absorption: methionine^ tryptophan, tyrosine, glycine, lysine -— - ( l ) . Now, as the diffusion coefficient for methionine, 68, is larger than the coefficients for tryptophan, 61.4, and tyrosine, 62, the position of methionine in the rank-order, relative to the positions of tryptophan and tyrosine, is strengthened. Also, as the diffusion coefficient of methionine, 68, is almost the same as that for lysine, 69, the position of methionine in the rank-order, relative to the position of lysine, is not altered. The diffusion coefficient of methionine, 68, is smaller, however, than that of glycine, 95.0. In order, therefore, to determine the rank-order of these two amino acids, relative to one another, the ratio of the force favouring glycine over methionine will be compared with the various possible ratios of the force favouring methionine over glycine (the subscripts G and M refer to glycine and methionine. - continued on p". 196 -1 9 6 . respectively) t rates of diffusion i n i t i a l electrochemical gradients 9 5 Q > 6 8 M J 9 2 . 2 M t 4 9 . 6 & , or * 111 1 t 1 . 8 6 ^ s L j - (a) j 9&G- i 6 8 M f t 9 2 . 2 M • • 6 4 . 4 & , or 1 . 4 % t *M 1 t 1 . 4 3 M t % (b)j 9&G t 6 8 M . t * 7 3 . 5 M 8 4 9 . 6 G , or 1 . 4 % m * » 1 . 4 8 M t V ~ - ( o ) | 9 5 G t • 7 3 . 5 M t 6 4 . 4 G , or i % 1 J h (*). In (a) and (o), the force favouring methionine over glycine exceeds the force favouring glycine over methionine. In (b), the forces are approxim ately equal and, i n (d), the force favouring glycine over methionine exceeds that favouring methionine over glycine. The rank-order of these two amino acids i s expressed, therefore, ast methionine ^ or ^ glycine (2). U t i l i z i n g this same method of analysis, i t next i s found that i n every oase the force favouring glycine or tryptophan, tyrosine, or lysine exceeds the force favouring any one of these amino acids over glycine. Taking into account these relations and the relation expressed by rank- order (2), rank-order (l) now can be rewritten as. methionine > or <^  glycine ^ tryptophan, tyrosine, l y s i n e - ( 3 ) , 73 where the larger sign, \ signifies that, although methionine or glycine The larger sign indicates that a l l the amino acids which come before i t always w i l l be in a higher position in the rank-order than a l l the amino acids which come after i t . 1 9 7 may be lower than one another in the rank-order, the lower members of these two s t i l l will be higher than any of the other three amino aoids in the rank-order. Finally, ratio comparisons made among the remaining three amino acids, in the 6ame manner as just outlined, permit rank-order (3) to be rewritten as follows 1 experiment W4 a glycine^ or ^ methionine^ tryptophan^ or ^ tyrosin^or & lysine (4) • This rank-order listing is to be read in the following manner. For the amino acids, methionine and glycine, two possibilities exist* the first position will be occupied by methionine and the second position by glycine, or the first position by glycine and the second position by methionine, but neither of these two positions can be occupied by any of the other three amino acids* For the remaining three amino acids, several possibilities exist* the third position wi l l be occupied by tryptophan, the fourth position by tyrosine, and the f i f t h position by lysine; or, the third position wi l l be occupied by tyrosine, the fourth position by tryptophan, and the fi f t h position by lysine; or, the third position w i l l be occupied by tryptophan and the fourth position by both tyrosine and lysine; or, the third position wi l l be occupied by tyrosine and the fourth position by both tryptophan and lysine; or, tryptophan, tyrosine, and lysine oan occupy together the third position* Utilizing the same type of analysis as just given for experiment W4, the data for experiment W5 and W6 yielded, upon analysis, the following rank-orders t experiment W5* glycine^ or^methionine^ tyrosine^ or^lysine^ tryptophan-(5); experiment W6* glyoine^ or^methionine^tryptophan^or^tyrosine^or^lysine(6)« 198 The rank-order listing for experiment W5 is to he read in the following manner* For methionine and glycine, the same two possibilities exist as outlined previously for these two amino acids in experiment W4J the first position will be occupied by methionine and the second position by glycine, or the first position by glycine and the second position by methionine, but neither of these two positions can be occupied by any of the other three amino.acids. For tyrosine and lysine, two possibilities also existt the third position w i l l be occupied by tyrosine and the fourth position by lysine, or the third position will be occupied by lysine and the fourth position by tyrosine, but neither of these two positions will be occupied by tryptophan* For tryptophan, there is only one possibility: the fifth position in the rank-order is ocoupied by tryptophan. The rank-order listing for experiment W6 likewise is to be read in the following manner. For methionine and glycine, the same possibilities exist as outlined previously for these two amino acids in experiments W4 and W5 and, as before, neither of the first two positions can be occupied by any of the other three amino acids. For tryptophan, tryosine, and lysine, |_3_ or 6 possibilities exist: the.third position can be occupied by any one of the three amino acids, the fourth position by either one of the two remaining amino aoids, and the fi f t h position by the one remaining amino acid. Examination of the data of Table 32 reveals the rank-orders of the 74 absolute rates of absorption actually to be: experiment W4 • lysinesiglycine^ methionine^ tryptophan^ tyrosine—(7) i 7 4The rank-orders listed are based on data for the 0 to 70 minute time interval. The use of this time interval biases,, however, tryptophan downward in.its placement in the rank-orders for experiments W5 and W6. experiment W5 = glycine^ lysine^ methionine^ tyrosiney tryptophan (8); experiment W6 s glycine^ lysine^) tryptophanft^methionine^tyrosine -(9). A comparison now w i l l be made, for each experiment, between the experimentally-found rank-order and the predicted possible rank-orders* A comparison of rank-order (7), the experimentally-found rank-order for experiment W4, with rank-order l i s t i n g (4) reveals that, except for the position of lysine, rank-order (7) f i t s one of the predicted possible rank-orders* A comparison of rank-order (8), the experimentally-found rank-order for experiment W5, with the rank-order l i s t i n g (5) reveals that, except for the position of lysine, there i s a close matching of 'rank-order (8) to one of the predicted possible rank-orders. Finally, a comparison of rank-order (9), the experimentally-found rank-order for experiment W6, with the rank-order l i s t i n g (6) reveals that, except for the positions of lysine and tryptophan, there is a close matching of rank-order (9) to one of the predicted possible rank-orders. Now, as the predicted possible rank-orders were set up upon the basis that the absolute rates of absorption of the ami no aoids were dependent only on the action of the electrochemical potential differences, the above comparisons can be interpreted to indicate that the force of the eleotro chemioal potential gradient plays a predominating role i n the absorption of amino acids from the rumen. These results can not be interpreted, however, as ruling out that the absorption of the amino acids could have been due to the effect of other foroes—e.g», cellular energy—as well* This i s so because, in these two experiments, a l l or nearly a l l the tryptophan was absorbed in the f i r s t 10 minutes of the experiments (see Table 8, p. 49). On making a comparison, however, of 0 to 10 minute interval data, i t was found that there was no change in the position of tryptophan in the rank-orders. 200. Transfer of-Amino Acids Against an Electrochemical Gradient.- A comparison of the individual amino acid concentrations in the ruminal solutions with the concentrations present in the plasma of the ruminal veins, as well as a measurement of the electrical gradient existing across the ruminal wall, would enable, a calculation to be made of the magnitude and direction of the electrochemical gradient between the ruminal solution and the plasma of the ruminal veins. One value in making such a calculation- is that the demonstration of a net movement of an amino acid against an electrochemical gradient would be considered as strong evidence for the existence of one or more other foroes being concerned in the movement of the amino acid. Unfortunately, in the present experi ments, there is no record of the plasma concentrations for the individual amino acids or of the electrical gradient. The same kind of procedure will be followed here, therefore, as was carried out in the calculation cf the i n i t i a l electrochemical potential differences (see p. 18l)• For the calculation of the chemical oomponent of the electrochemical gradient, there were utilized the lowest-found ruminal solution concentrations of the amino acids recorded in Tables 8 to 12 (pp. 49, 52, 55, 58 and 62) and the plasma concentrations recorded in Table 30 (p. 182). The lowest- found amino acid concentrations in the ruminal solutions were used for this calculation in order to determine i f downward, plasma to ruminal solution concentration gradients existed during any part of the ruminal fistula experiments. Table 33 has been set up to record the calculated amino acid concentration gradients that existed between the ruminal solution and the plasma of the ruminal veins when the concentrations in the ruminal solution were at their lowest. Except for lysine in experiment W5, Table 33 records quantitatively only those concentration TABLE 33* - Ruminal f i s t u l a experiments W4, W5, and W6t estimated chemioal and eleotro-ohemi- cal gradients (millivolts) existing between the ruminal solutions and plasma of the ruminal veins when each amino acid reached i t s lowest concentration i n the ruminal solutions* Referenoe should be made to the text (p202 ) for an explanation of the meanings to be attached to the positive and negative signs recorded in the table Experiment Amino aoid W4 W5 W6 Chemioal gradient Eleotro- chemical gradient Chemioal gradient Eleotro- chemioal gradient Chemioal gradient Eleotro- chemical gradient Tryptophan + -80 .1 to - 8 6 . 3 a -30.6 to - 7 8 . 3 a -80.1 to -86*3 -30.6 to 78*3 Methionine + +- to - 6*0 +• + Tyrosine + -22*4 to -35*5 + to -27.5 + Glycine + +• + to - 8*2 +• to - 0.2 Lysine 4- +27.3 to+-43.8 •+• to -22.2 +• As the lowest-found concentration for tryptophan in the ruminal solution of experiment W5 was less than the concentration detectable by the microbiologioal assay method, the lowest-found . concentration was recorded in Table 8 (p. 49) as a zero concentration. It i s unlikely, however, that a zero concentration actually was reached. In the absence of a known concentration, the lowest- found concentration for tryptophan in the ruminal solution of experiment W6, 0.2 microgram-moles per 100 m i l l i l i t e r s , is assumed for the lowest-found concentration in experiment vV5. This assumption enables a calculation to be made of the chemical gradient in terms of m i l l i v o l t s . This is done, however, only with the understanding that the actual chemical gradient was probably larger than indicated by the data which are recorded i n the above table* gradients that were orientated downward from plasma to ruminal solution— these gradients are designated in the table by negative signs* This proced ure was adhered to because the only interest at this point is for the detection of the ruminal absorption of amino acids that occurred against an electrochemical gradient* The chemical gradients that were orientated downward from ruminal solution to plasma are recorded in Table 33 quali tatively only—these data are designated in the table by positive signs* The one exception to this procedure is for lysine in experiment W5« here, the gradient, even although i t is positive, is recorded quantitatively. This is done beoause the lowest-found lysine concentrations were low enough that they may have been overbalanced by the oppositely orientated electrical gradients that existed for lysine* Next, the data of Dobson and Phillipson (§8) for electrical potential differences were utilized in conjunction with the calculated chemical gradients to calculate the electro chemical gradients* The range found by these authors for the electrical potential differences between ruminal contents and blood in conscious sheep was 8 to 49*5 millivolts* It is assumed that the range for the electrical potential differences in the goat is similar to that for 75 JQ the sheep* By adding algebraically the chemical and electrical com ponents, the lower and upper limits were determined for the range of possible electrochemical gradients* These limits are reoorded as a part of 75 Dobson (-0$) found the electrical potential differences for a red poll, non-lactating heifer to be within the range found for the sheep* 76 The electrical potential difference across the ruminal wall is orientated with the ruminal vein blood negative with respect to the ruminal contents (see p* 167)* From the discussion that previously was given on page 175, this means that the electrical component is orientated positively for a l l the five amino acids except lysine and is orientated negatively for lysine* Table 33, qualitatively only when they are positive, quantitatively when they are negative. Examination of the data in the table reveals that . negatively orientated eleotrochemioal gradients existed against the move ment of tryptophan in experiments W5 and W6 and may have existed against the movement of tyrosine and lysine in experiment W5« This does not mean,, however, that reversals of the positively orientated electrochemical gradients oould not have occurred, especially i f there had been longer time intervals for absorption or lower i n i t i a l concentrations of the amino acids in the.ruminal solutions. This demonstration of the movement of tryptophan against an electrochemical gradient should mean that another foroe was involved in the movement of tryptophan across the ruminal wall. Likewise, another force also may have been involved in the movement of tyrosine and lysine. It is suggested that this other force may be that of the metabolic energy which is supplied by the epithelial cells of the ruminal wall. Unfortunately, there exists some doubt as to the validity of the above analysis, at least for tryptophan, tyrosine, and probably methio nine. In a previous discussion on the stability of the experimental solution constituents (see p. 94), i t was pointed out that molecular deterioration of the aforementioned amino acids may have oocurred during the storage time that occurred between the time of the experiment and the time of assay. Such molecular deterioration, i f large enough, could convert an actual concentration gradient that favoured a constituent's passage from ruminal solution to plasma into an apparent concentration gradient that opposed a constituent's passage from ruminal solution to plasma. If this did happen, then the chemical and electrochemical grad ients that are recorded in Table 33 would not be valid. Unfortunately, . there is no way to decide i f such molecular deterioration did occur during the storage of the samples* The validity of the deductions made for tryptophan and tyrosine from the data in Table 33 remain, therefore, in doubt. Only in the case of lysine, for which no evidence was found for molecular deterioration, are the deductions not affected by this doubt of the stability of the amino acids in their storage period. Summary and Conclusions.- The purpose of the previous discussion was to determine i f the data of the ruminal fistula experiments provided any evidence which was relative to an elucidation of the manner of transfer of amino acids across the ruminal wall. The examination of the data for the effect of the i n i t i a l electrochemical gradients on the absolute and relative rates of absorption led to interpretations that indicated that the foroes of ohemical and electrical gradients were involved in the movement of the five amino aoids—tryptophan, tyrosine, methionine, glycine, and lysine—across the ruminal wall. The same examination also indicated that, for tyrosine, glycine, lysine, and tryptophan, a possibility exists for their movement being assisted by the force of metabolic energy as well. An examination of the data by compar ing experimentally-found rank-orders of the absolute rates of absorption for the five amino acids with their predicted possible rank-orders led to the conclusion that the forces of the chemical and electrical gradients play a predominating role in the movement of the five amino acids across the ruminal wall. It also was noted, however, that this conclusion did not exclude the possibility of the force of metabolic energy being involved as well. Finally, an examination of the data for evidence of transfer of the amino acids against an electrochemical gradient led to the suggestion that the force of metabolic energy was involved in the movement of tryptophan across the ruminal wall, and that this also might he true for tyrosine and lysine. This suggestion was qualified, however, for tryptophan and tyrosine because of the uncertainty as to the cause of a suspected molecular deterioration of these two amino acids in the assayed samples. Taking into consideration a l l these various factors, plus a necessary prudence because of the many assump tions made in the discussions that led to the above interpretations, i t appears from the evidence available that the following statement can be made with some assurance of validityt the movement of the five amino acids—tryptophan, tyrosine, methionine, glycine, and lysine—across the ruminal wall of the goat is determined by a summation of the effects of the forces of ohemioal, electrical, and possibly metabolic potentials. The question raised at the end of the review section (p. 171) on the manner of absorption of substanoes by the ruminal epithelium—whether the movement of amino acids across the ruminal wall against a concentration gradient would be the result of the effect of the force of an electrical gradient or of the force of metabolic energy or both—can not be answered from the data that are available in the present ruminal fistula experi ments. All that can be said is that the data do not provide proof against the force of metabolic energy being involved with the force of the electri cal gradient, but neither do the data provide conclusive evidence for the involvement of the metabolic force. Of necessity, then, further experi mentation is required, especially of the type that has been carried out on the absorption of amino aoids from the small intestine (see p. 151to p. 160). It is hoped that the data of the present ruminal fistula experi ments and the examination of that data in the present discussion may at least serve the purpose of indicating some of the assumptions and considera-tions to be checked and taken into account in any future research on the 77 manner of absorption of the amino acids from the rumen. Amino Acid Absorption from the Rumen under Normal Feeding Conditions Introduction The question naturally arises as to whether the absorption of amino acids occurs in the rumen under normal feeding conditions. To be able to answer such a question, there is required a knowledge of the fulfillment or non-fulfillment of the following conditions! 1. the demonstration of the presence of amino acids in the rumenj 2. the demonstration of the relation of the concentrations of the individual amino acids in the ruminal liquor to their concentrations in the blood plasma! a rumen to plasma, downward electrochemical gradient would permit the possibility of amino acid absorption without requiring direct participation of metabolic energy, but would not rule out the possibility of metabolic energy also participating; a plasma to rumen, downward electrochemical gradient would limit any amino acid absorption to that accomplished directly or indirectly by the force of metabolic energy; One point that was not considered in .the above discussion is the possible influence of anesthezia, shock, or poor general condition (of the experimental animal) on any special selective mechanism that would accelerate the ruminal absorption of amino acids. Several authors (46, 79, 134, 146) have presented evidence to demonstrate that these experi mental conditions can reduce the selective absorption of glucose from the small intestine. Shock (146) has been demonstrated to reduce also the absorption of amino acids from the small intestine. The influence of these factors should be considered in any future research directed at determining i f amino acids are absorbed selectively from the rumen. 207. 3. the demonstration of the absorption of individual amino aoids from an ingesta-filled rumen, the concentrations of the individual amino acids at which this absorption occurs being of the same order of magnitude as those found in the ingesta fluid ordinarily. Each of these points will be discussed in turn. Condition lt Demonstration of Amino Acids in the Rumen Although originally McDonald (237, 240) was unable to detect the presence of free amino acids in the ruminal liquor of sheep fed on a hay diet, several authors (15, 62, 213) since have demonstrated the presence of appreciable amounts of amino aoids in the ruminal liquor of sheep. These positive results for the presence of free amino acids in the ruminal liquor axe not unexpected, especially as leaves, stems, tubers, roots, cane and beet molasses, and "fish solubles" are known to contain apprecia ble amounts of free amino acids (see the review by Chalmers and Synge, 62). Annison (15), for example, has reported for sheep alpha amino nitrogen concentrations that ranged between 10 and 15 milligrams per 100 milliliters 78 of ruminal liquor. These concentrations occurred during and after "These amounts are large enough to be interesting but give no indi cation of the daily turnover." This statement was made by Elsden and Phillipson (109) about the measured concentrations of volatile fatty acids in the rumens of ox and sheep. Elsden and Phillipson*s statement also applies to the data that are reported in the literature for the concentra tions of amino acids in the ruminal liquor. This point is emphasized here because too often the low concentrations of the amino acids in the ruminal ingesta are cited to support the implication that the ruminal absorption of amino acids, i f i t does occur, must be of l i t t l e nutritional significance. Such an implication takes no account of the possibility that the low con centrations of amino acids in the ruminal liquor may be, in part at least, a reflection of the rapid absorption of amino acids by the rumen. For example, the concentrations that were found by Blaizot and Raynaud (see p. 208) are relatively low; however, i t is to be noted that the measure ments were made on ruminal contents that had been removed from the animals 24 hours after the last feeding. Of particular interest at this point is the following statement of Almquist (12): In the digestion and assimilation of whole proteins i t is 208. the feeding of dried grass. 7 9 In addition, Blaizot and Raynaud (37) have demonstrated chromat©graphically that the majority of the amino acids are present as free acids in the ruminal liquor of the ox. They estimated that the concentrations of eight of the amino acids w^ ggs between 78 0.03 to 1 milligram per 100 grams of the i n i t i a l fresh ruminal contents. probable that enzymatic digestion is a relatively slow proc ess and that absorption across the intestinal walls is  relatively fast. Therefore, in disregard of known large differences in solubility, moleoular diffusion rates, etc., of the individual amino acids, they are absorbed almost as  soon as released from food protein and enter the circula tion in proportions closely related to those in the diet (underlining mine] . Wow i t must be admitted that the distance between the wall of the rumen and the center of the ruminal contents is much greater than the distance between the intestinal wall and the oenter of the intestinal contents. This means that a l l the ruminal contents may not be as readily exposed to the absorbing action of the ruminal wall as the intestinal contents are to the absorbing action of the intestinal wall. Nevertheless, there probably is exposure of much of the ruminal contents to the absorbing action of the ruminal wall because of the active churning of the ruminal contents by the contractions of the ruminal walls and this exposure also is over a much longer time period than i t is the case in the small intestine because the rate of passage of the ingesta through the rumen is relatively slow as compared with the rate of passage of the ingesta through the small intestine (102). Trautmann (377) has pointed out that these two conditions—i.e., the churning of the contents and its relatively slow passage—for the ruminal contents especially favour the absorption of the diffusible constituents that are present in the rumenj Zunachst sind die Bedingungen zur Resorption gunstig, denn der Inhalt verweilt hier lange und wird auch so durchmischt, dass immer wieder neues Material mit den Schleimhauten in Beruhrung kommt. With the action of these conditions just discussed to ensure a continuous contact of new ruminal material against the ruminal wall, i t again is suggested that the existence of a rapid rate of absorption of amino acids from the rumen, especially relative to the rate of formation of new amino acids by proteolysis, could be one of the main reasons for the finding of relatively low concentrations of amino acids in the ruminal liquor. Synge (369) states that "the making of silage results in conversion Condition 2t Demonstration of an Electrochemical Gradient- Annison (15), commenting upon the fact that young grasses contain 80 appreciable quantities of free amino acids, suggested that " i t is likely that when ruminants graze on lush pastures the concentration of free amino nitrogen in the rumen becomes appreciably greater than in the blood." Such concentrations of amino acids in the ruminal liquor as just cited on the previous page—that i s , between 10 and 15 milligrams of alpha amino nitrogen per 100 milliliters—are considerably higher than the concentra tions reported for the whole blood or plasma of sheep. In one sheep anesthetized with sodium pentobarbotone, Annison (15) found plasma concentrations of 4.11 in the portal vein and 4.27 in the jugular vein. In another sheep, conscious rather than anesthetized, Annison found plasma concentrations of 3.84 in the portal vein and 3.04 in the common carotid artery. A l l these concentrations are milligrams of alpha amino nitrogen per 100 milliliters of plasma. Spector's Handbook of Biological Data (358) reports amino acid nitrogen concentrations for the whole blood of sheep as 4.6 to 8.0 milligrams per 100 millil i t e r s . Prom these data, i t appears of much of the protein of grass into soluble nitrogenous compounds of lower molecular weight." It is to be wondered, therefore, how high the concentration of amino acids in the rumen would be after the feeding of grass silage. 80 Annison cited the work of Waite and Boyd (398, 399) in support of this statement. The present author could not find, however, any mention in the cited work to the concentration of free amino acids in grasses. Other work (see 173, 368, 369, 373) does exist to support the state ment that young grasses contain appreciable quantities of free amino acids. that, under certain conditions of feeding, a rumen to plasma, downward concentration gradient for amino acids can exist in the normal-feeding animal* ^his, in turn, means that a rumen to plasma, downward electro chemical gradient can exist, at least for those amino acids whose pi numbers are such that the amino acids form equilibrium mixtures of dipolar ions and anions in the normal ruminal contents (see previous discussion on p. 175)• Condition 3» Demonstration of Absorption from an Ingesta-Filled Rumen The existence of such rumen to plasma, downward concentration gradients as just described suggests the possibility of amino acid absorption from the rumen by at least simple diffusion in the normal-feeding animal* Annison, however, even when he had a concentration of 83.7 milligrams of alpha amino nitrogen per 100 milliliters in the rumen liquor (see Table 1, p« 16), was unable to demonstrate the absorption of amino acids from the ingesta-filled rumen of a sheep. Likewise, the ruminal absorption of amino acids from ingesta-filled rumens was not demonstrated in the present blood sampling experiments (see Table 28, p. 146). It is to be noted, however, both in Annison's and the present experiments, that the method of detection of amino acid absorption was by the making of a comparison between the concentrations of alpha amino nitrogen in the portal and systemic plasmas. The lack of sensitivity of this prooedure for the detection of substances that are absorbed continuously and in small amounts has been commented upon previously (see p. 116)• Only when relatively high concentrations of amino acids were placed in empty rumens was the method of comparing blood concentrations of alpha amino nitrogen successful in demonstrating amino acid absorption. By contrast, when there was utilized the more sensitive procedure of sampling at periodic inter-vals a solution that was placed in an empty rumen—that i s , as i t was done in the present ruminal fistula experiments—, the absorption of amino acids was demonstrated at relatively low concentrations. In experiment W6, this demonstration of ruminal absorption was made when the concentra tion of alpha amino nitrogen in the ruminal solution was as low as 30 milligrams per 100 milliliters (Table 13, p. 67)• Such a concentration is of the same order of magnitude as that found by Annison (15)—that i s , 10 to 15 milligrams of alpha amino nitrogen per 100 m i l l i l i t e r s — t o be present in the ruminal liquor of sheep during and after their ingestion of dried grass. Comment The previous discussion indicates that the amino acids are present in the rumen in.appreciable concentrations. Further, these concentrations often are large enough that rumen to plasma, downward electrochemical gradients can exist for many of the individual amino acids. These grad ients, when they occur, should allow amino acids to be absorbed from the rumen even i f the ruminal epithelium does not possess the ability to absorb amino acids against an electrochemical gradient. On the other hand, when the concentrations of the amino acids in the ruminal liquor are small enough that plasma to rumen, downward electrochemical gradients exist, then these gradients should allow amino acids to pass from the blood of the portal circulation into the liquor of the rumen, provided that the ruminal epithelium does not possess the ability to absorb amino acids against such gradients. The existence of such an ability for the ruminal epithelium would, in accordance with the capacity of this ability, either reduce or even eliminate any net passage of amino acids into the rumen--even under starvation conditions* Finally, although the previous discussion does not present direct evidence which demonstrates the occurrence of ruminal absorption of amino acids from an ingesta-filled rumen, evidence is presented which suggests that such a demonstration could be made i f suitable methods of experimentation were utilized* perhaps, by the use of radioactive-labelled amino acids, the ruminal absorption of amino acids from the ingesta-filled rumen into the portal circulation could be demonstrated* - continued on p. 213 - See the quotation from Chalmers and Synge on page 13 • The Effect of Ruminal Absorption on the Pate of Amino Acids Present in the Rumen Introduction The question can be raised as to how the ruminal absorption of amino acids fits into the pattern of the dynamic, nitrogen metabolism that is occurring continuously in the ruminal ingesta. The discussion of the present section wi l l attempt to answer to this question. Chalmers and Synge (62), as well as Phillipson and Cuthbertson (299), have presented a synoptic figure of the principal reactions of nitrogenous compounds in the rumen. Figure 22 is a reproduction of the figure of these authors, but with the addition of one more arrow to indicate the ruminal absorption of amino acids. Figure 22 reveals that the fate of amino acids that enter or are formed in the ruminal ingesta is manifold. 1. the amino acids are utilized in the synthesis of miorobial protein, or they are deaminated to form ammonia and nitrogen-free com pounds ; 2. the amino acids pass with the rest of the constituents of the ruminal ingesta into the more distal parts of the alimentary tract; 3. the amino acids are absorbed from the ruminal ingesta by passage through the ruminal wall into the portal circulation. It is to be expected that the distribution of each amino acid among the various possible pathways just described wi l l be influenced by the particular set of environmental conditions prevailing in the rumen, by the known influence of the diet on the composition of the population of microorganisms in the rumen, by the physical and chemical nature of the dietary constituents, by the relative stability of the amino acids in the In saliva, from body fluids -In food Entering rumen In rumen Leaving rumen to abomasum and intestines Fro tein Nitrate Free ami et no acids Ur c. ea < Food protein- Food protein >Free amino: acids etc. Protein of microorganisms Protein of microorganisms 4»Absorbed through rumen wall Absorbed through rumen wall Fig. 22. Fig. 22. - The principal reactions of nitrogenous compounds in the rumen (adapted from figures of Chalmers and Synge (62) and Phillipson and Cuthbertson (299), ruminal ingesta, and by the physiological state of the animal. In order to acquire some concept of the distribution of the amino acids among the pathways which are presented in Fig. 22, each of the influencing factors briefly will be discussed in turn. Influence of Environmental Conditions in the Rumen on the Fate of Amino  Acids in the Rumen Variations in the environmental conditions of the rumen will have an influenoe upon the fate of amino acids in the rumen. This follows from the considerable evidence that has been accumulated relative to the influence of temperature, pH, and so forth on the activities of micro organisms. Several examples will be given of the extent to which the environmental conditions of the rumen are known to vary. In each example, an attempt will be made to demonstrate how the variation of an environ mental condition probably can influence the fate of the amino acids in the rumen. Temperature.- Several workers (52, 87, 94, 205, 268, 378) have measured temperature variations in the rumen, both for different positions within the cavity of the rumen and for the effect of feed and water ingestion. During active fermentation in the rumen, the temperature in the rumen can increase as much as 2°C. (2;05, 378). After the drinking of water by sheep, Nangeroni (268) found the temperature of the ruminal ingesta to f a l l from about 40o down to about 37° in one experiment, down to about 33° in a second experiment, and down to about 30° in a third .experiment. Similar temperature changes have been recorded in the cow after the ingestion of water by this animal (94). Sometimes a period of time as long as two hours is required for the ruminal ingesta temperatures to return to their original level. Smaller temperature changes, of the order of magnitude of 1 to 2° C , have "been recorded after the ingestion by ruminants of various quantities and types of feed (52, 87, 378). It is likely that such temperature changes in the ruminal ingesta will influence the fate of the amino aoids. This statement follows from the demonstrated effect that even small changes in the incubation temperature of mioroorganisms oan have on their synthetic ability. Borek and Waelsch (45), for example, found that a temperature change of 2° C. can determine whether or not a Lactobacillus oan synthesize its own require ment of phenylalanine. Similar results were obtained for tyrosine and aspartic aoid. Borek and Waelsch also cite several authors who have found in other microorganisms this same type of temperature influence on their ability to synthesize amino acid requirements. It is significant to note that these temperature effects found by Borek and Waelsch were obtained using temperatures .that lie within the range of the temperatures which are found in the rumen. This implies, then, that the demonstrated variations of temperature that occur in the ruminal ingesta likely w i l l influence the amino acid requirements and the amino acid synthesis of the ruminal microorganisms and, therefore, will influenoe the fate of the amino acids in the rumen. pH.- It amply has been demonstrated that marked variations in the pH of the ruminal ingesta oocur. Recordings as low as pH 5.5 and as high as pH 7.5 or greater have been reported (88, 102, 113, 188, 259, 261, 265, 294, 296, 320, 353; see also the citations in 139). The more usual range of pH is 6.0 to 7.0 (102). Wow, G-ale (126) states that many of the bacterial enzymes which are involved in amino acid catabolism are 218 adaptive enzymes. The production of these enzymes will be influenced by the conditions of the growth medium and, apparently, the pH of the medium 82 is the main factor. determining whether the enzymes of deamination or decarboxylation will be produced. In general, microorganisms cultivated under acid conditions will produce decarboxylation systems. Sale suggests that the effective stimulus for the formation of the different 85 enzymes may be the ionic form of the amino acid s the cationic form of an amino acid stimulating production of decarboxylase enzymes and the anionic form stimulating the production of deaminase enzymes. At a pH of about 5.5, a culture of Escherichia coli demonstrated no effective decarboxylase or deaminase aotivity. On the acid side of this pH, the effective decarboxylase activity rapidly increased with increasing acidity. On the alkaline side, the effective deaminase activity increased slowly with increasing alkalinity and reached a near-to-maximum activity at a pH of about 7.5. These results imply, then, that the demonstrated OA variations of pH that occur in the ruminal ingesta likely w i l l influence on Temperature apparently is another factor that may have an influence on the formation of the adaptive anzymes of bacteria. Gale (126) states that, although l i t t l e work has been done on this effeot of temperature, i t is known that often decarboxylases are formed in greater amount when growth takes place at low temperatures than when growth takes place at 37° C. "°See page 175 for a discussion of the influence of the pH on the ionic form of amino acids. 84 Two examples are presented here to illustrate how the pH of the ruminal contents can vary, especially as i t is influenced by the diet. Elsden and Phillipson (10S), in writing of the influence of diet on the pH in the rumen, statej "The acidity is greatest . . . when sheep graze young summer grass; then the average pH is approximately 6, in contrast to an average figure of 6.5 with animals that are stall fed." Reid et al (320) found that the pH in the rumen declined below 5.0 when sheep were fed on rations which contained a high proportion of wheat starch. 219. the degree of decarboxylation and de ami nation., of amino acids in the ruminal ingesta and, therefore, -will influence the fate of the amino . . . ,. 85 acids in the rumen. Carbon Dioxide and Oxygen*- The percentage distribution of the ruminal gases, mostly carbon dioxide and methane, varies l i t t l e with different diets (102). The composition and total volume of the gases in the rumen can vary considerably, however, with the time that has elapsed after feeding (74, 313, 40l). During the first few hours after feeding, the percentage volume of carbon dioxide increases in the rumen: in one experiment, from approximately 48 per cent up to 65 per cent. The percentage volume of oarbon dioxide then gradually falls off until a relatively low level is reached: down to approximately 22 per cent at 20 hours after feeding. One of the gases which demonstrated a percentage volume increase in this period of carbon dioxide decrease was oxygen: from approximately 0 per cent at about 7 hours to approximately 7 per cent at about 21 hours. Broberg (50, 51) states that not only does oxygen enter the rumen during rumination, but that large quantities of the gas enter the rumen during the consumption of feed. Now i t is likely that these variations in the tensions of carbon dioxide and oxygen will have an influenoe upon the metabolic activities of the ruminal micro- 85j ust recently, Reis and Reid.(32l) have obtained results which appear to confirm this statement. They found, on incubating casein hydrolysate in ruminal liquor for four hours, that there was an optimum pH for the production of ammonia from the amino acids of the hydrolysate. The optimum pH varied between 6.0 and 6.7, the optimum being dependent upon the diet of the animal from which the ruminal liquor was obtained. On the acid side of the optimum pH, Reis and Reid found the production of ammonia to f a l l off rapidly. The authors suggest that these results are a reflection of the effect of pH on the production and activity of the microbial deaminase. As the present author has done (see p. 218), they cite work which has demonstrated that pH does affect the production and activity of microbial deaminases. organisms. For example, carbon dioxide has been demonstrated to affect the aforementioned (see p. 217) influence of temperature on the ability of mioroorganisms to synthesize their amino acid requirements. Borek and Waelsch (45) found that the incubation of the microorganisms in an atmosphere rich in carbon dioxide could, in many cases, reverse the effect that a temperature rise has in increasing the amino acid requirements of microorganisms. In the case of oxygen, the tension of this gas has been demonstrated to affect bacterial deaminase production (126) I the produc tion of those enzymes that utilize oxygen in their actions are suppressed by anaerobic conditions; the production of those enzymes that do not utilize oxygen in their actions is enhanced in anaerobic conditions. Further, increased oxygen tension has been demonstrated to inhibit amino acid decarboxylase synthesis in fecal cultures from rats (253). These results suggest that the demonstrated variations in the carbon dioxide and oxygen tensions in the rumen will influence the fate of the amino acids in the rumen. Influence of Diet on the Composition of the Population of Microorganisms in the Rumen and, therefore, on the Fate of Amino Acids in the Rumen It is suggested that there are at least three mechanisms by which variations in the diet of the ruminant oan affect the population of ruminal microorganisms: 1) by producing, within each species of ruminal microorganisms, the "discontinuous variations" of Dubos (lOl); 2) by influencing, within each species of ruminal microorganism, the production of the "adaptive enzymes" (101); 3) by producing, within the population of ruminal microorganisms, qualitative and quantitative variations. Discontinuous variations*- The "discontinuous variations" of Duhos refer to the property of a single strain of a microorganism to "adapt" or "train" to a new set of environmental conditions* Dubos cites as an example E. ooli mutabile, "a coliform organism which does not at first attaok lactose when seeded into a lactose broth medium, but which begins to ferment the sugar only after a lapse of days or even w e e k s T h i s type of adaptation is considered to be the result of the new lactose environment bringing about the natural selection of lactose-fermenting variants which occur under normal conditions in the parent culture* It is to be suspected that qualitative changes in the diet of the ruminant would produce such "discontinuous variations" within at least some of the members of the ruminal population of microorganisms* Adaptive enzymes*- The "adaptive" enzymes of a microorganism, in contrast to "constitutive" enzymes, depend for their production upon the presence of homologous substrate in the medium* "They appear without delay when the cells of the proper miorobial species start multiplying in a medium containing the specifio substrate; the specific enzymic activities reach their maximum development during growth of the very first transfer into the specific media, and the enzymes again f a i l to accumulate as soon as the cultures are transferred to media lacking the specific substrates" (lOl). It is to be suspected that qualitative ohanges in the diet of the ruminant would bring about the initiation or the oessation of adaptive enzyme formation among at least some of the members of the ruminal population of microorganisms* Population variations*- Many workers have presented evidence to demonstrate that the qualitative and quantitative distributions of the different kinds of ruminal microorganisms can vary with the type of diet (24, 53, 56, 127, 128, 168, 188, 228, 258, 306, 320, 373, 386, 412). Wan der Wath (386), for example, has demonstrated that different bacteria preferentially attack different substrates in the rumen at the same time; therefore, i f the ratio of the substrates in the diet is altered, the ruminal facies also should change. Moir (258) noted a similar influence of diet on the oharacter of the ruminal population. He attributed this influence to variations occurring in the chemical composition of graze material—especially, the variations of the protein content (see also 260). Williams et al (412) have demonstrated that the concentration and types of microorganisms in the rumen of sheep are affected by the levels of dietary protein and dietary starch, as well as by the proportion of these two dietary constituents to one another. On low-protein diets, the addi tion of more starch to the diet was found to cause a reduction in the number of ruminal microorganisms. There also was a change in the types of microorganisms present: from a predominance of gram-negative micro- 86 organisms to an increased proportion of gram-positive microorganisms • When the protein content of the diet was high no such effect was obtained. In diets containing starch, the addition of more protein caused the count of ruminal microorganisms.to increase. Raising the nitrogen intake from 2.3 to 9.5 grams per day more than doubled the bacterial count: from 21 6 6 x 10 oells to 44 x 10 oells per cubic millimeter. In antelope, Van der Wath and Myburgh (387) found eight times as many infusoria in the ruminal ingesta when the animal browsed as when i t grazed. The protein and carbohydrate content of the browse material was relatively high 8 6Van der Wath (386) has demonstrated the starch-digesting bacteria to be gram-positive microorganisms. 223. compared to the protein and carbohydrate content in the graze material. Although other examples could be cited, the previous material demonstrates the effect that variations in the diet of the ruminant can have on the qualitative and quantitative distributions of ruminal microorganisms. 87 Comment.- The previous discussion suggests that qualitative and quantitative variations in the diet of a ruminant can affect, by at least three mechanisms, the population of ruminal microorganisms. The result ing alterations of this population should cause, in turn, alterations i n the metabolism or fate of the amino acids in the rumen. Warner (400), for example, has demonstrated that the power of suspensions of ruminal microorganisms to produce deaminases varies with the composition of the dietj that the presence of deaminases is dependent on the presence of readily-attacked protein i n the diet of the animal from which the mioroorganisms were taken (see also 110, 213). Since Gale (126) suggests that deaminases are adaptive enzymes, t h i s experimental finding of Warner could be an example of diet variation affecting the population of micro organisms by the second mechanismt by influencing the production of adaptive enzymes. Actually, however, either one or both of the other two mechanisms also could have been operative i n his experiment. The. The three mechanisms that have been discussed i n the above text probably account for the demonstrated requirement of ruminants for a period of adaptation when their diet i s changed. Sheep, for example, need a week of adaptation to a diet that contains a relatively high proportion of starch in order to be able to digest i t with maximal efficiency (386j see also 168, 320). Sheep also require time to become adapted to a diet that contains a relatively high proportion of protein (168). Further, lambs have been demonstrated to require two to three weeks to become adapted to the maximum ut i l i z a t i o n of diets that•contain various non-protein nitrogen compounds (145). See also the discussion by Hoflund et a l (168). relationship that was observed may have been the result of the action of a l l three mechanisms: discontinuous variation, increased production of an adaptive enzyme, and qualitative and quantitative population variations* Influence of the Physical and Chemical Nature of the Dietary Constituents  on the Fate of Amino Aoids in the Rumen The physical and chemical nature of the dietary constituents should have an influence on the nitrogen metabolism of the ruminal ingesta. The solubility, 8 8 the surface area, and the type of the proteins in the diet will help to determine, for example, what proportion of the protein will be converted into amino acids and what proportion into ammonia. To illustrate, McDonald (240) found an increase in the ammonia concentra tion of the ruminal liquor when plant protein, in the form of meadow hay, and readily-soluble animal proteins, in the form of casein or gelatin, were fed. Similarly, El-Shazly (110) found that the ability of a washed- suspension of a mixed population of ruminal microorganisms to decompose a mixture of amino acids was "strikingly increased" by increasing the content of soluble protein in the diet of the animal from which the micro organisms were taken (see also 213, 400). On the other hand, McDonald (240) found that the ammonia concentration in the ruminal liquor did not 89 increase when relatively-insoluble animal protein, in the form of zein,. o o 0 f interest here is the fact that the solubilities of the proteins in leaves probably are affected by the drying of hay-making. Likewise, the solubilities of the proteins in blood, meat, and fish meals preMbly are affected by the heating procedure to which these substances often are subjected. In both cases the effect on solubility apparently is the result of the proteins undergoing denaturation (60, 62). 89 In faot, there actually was a decrease in the ammonia concentra tion. McDonald interpreted this result as indicating that the rate of proteolysis of zein by the ruminal population is slower than the capacity 225. was fed. These examples (see also 60, 61, 111, 128, 168, 287) indicate that the physical and chemical nature of the dietary constituents play a role in determining the fate of amino acids in the rumen. Influence of the Relative Stability of the Amino Aoids on theiff Fate in the Rumen G-ale (126) states that the ability of microorganisms enzymatically to attack amino acids varies widely with the species. He notes that many gram-positive microorganisms, such as lactobacillus, streptococcus, and staphylococcus, are quite limited in their ability to attaok or synthesize amino acids. *n contrast, many of the gram-negative microorganisms, such as proteus, pseudomonas, aerobacter, and escherichia, are able to attack a wide range of amino acids. Even these latter organisms, however, do not always attack amino acids. In the light of these results, the question can be raised as to the stability of the different amino acids when they are present in a mixed population of ruminal microorganisms. Such knowledge should help in determining what is the fate of the amino acids in the rumen. Several groups of workers (110, 213, 279, 345, 346) have investi gated the stability of amino acids in the presence of mixed cultures of ruminal microorganisms. Sirotnak and co-workers (345, 346) studied the degradation of amino acids, each amino aoid being incubated separately for 72 hours in vitro with mixed suspensions of ruminal microorganisms. The temperature and pH of incubation were 39° C. and 6.9, respectively. of the population to utilize the products of proteolysis and that already- available ammonia was used by the microorganisms as an additional source of nitrogen for their growth. 226 Of the 22 amino acids tested, only aspartie acid, glutamic acid, serine, arginine, cystein, and cystine were deaminated or decarboxylated. The rate of dissolution of aspartie acid was five times greater than for . the others. In the 72 hour period, the ammonia yields of aspartate, gluta- mate, serine, and cystein approximated to the theoretical possible yields. Arginine and cystine each demonstrated an ammonia production of only about 50 per cent of the theoretical. Sirotnak and co-workers further found that 5 micromoles was the maximum amount of aspartie acid that could be broken down in two hours; that i s , there was a maximum dissolution rate. Work of perhaps greater applicability to the conditions found in the rumen are the in vitro experiments of El-Shazly (110). This experimenter determined the dissolution activity of washed-suspensions of a mixed population of ruminal microorganisms on a mixture of amino acids that was in the form of casein hydrolysate. Such a procedure allows for the occurrence of interactions between the amino acids in the presence of microorganisms (124, 362), a definite possibility in the ruminal fermentations. The results demonstrated that at least some of the amino acids are susceptible to deamination and decarboxylation by a population of ruminal microorganisms. For incubation periods of 1 3/4 hours, El-Shazly found the dissolution of the amino acids to be between 16 and 22 per cent of the total alpha amino nitrogen present. For incubation periods of 3 hours, he found the dissolution to be about 70 per cent. In further experiments of the same type as just described, except that the 90 amino acid mixture was incubated with ruminal liquor instead of a 90The.term ruminal liquor—i.e., the * rumen liquor 1 of Pearson and Smith (286a)—designates a filtrate of ruminal contents that is obtained by the use of muslin or cheesecloth. The filtrate contains microorganisms and the smaller plant fragments. washed suspension of microorganisms, El-Shazly determined chromatograph- ically the disappearance or reduction in concentration of different amino acids over a period of 3 hours. He found tyrosine, phenylalanine, proline, alanine, threonine, glycine, serine, and aspartic aoid to disappear or decrease in concentration quite rapidly; glutamic acid was "considerably weakened while the remaining spots were relatively strong, although obviously weaker than the corresponding spots from C [^control not incubated^] These results, as well as the similar results of Lewis (213) and Otagaki et al (279), demonstrate that at least some of the amino acids are susceptible to deamination and decarboxylation by a population of ruminal microorganisms. This means that some amino acids are less stable than others in the presence of ruminal microorganisms and that this stability or lack of stability of the amino acids will influence 91 their fate in the rumen. Special Comment.- Of the amino acids used in the present ruminal fistula experiments—tryptophan, methionine, tyrosine, glycine, and lysine—, only tyrosine and glycine have been demonstrated to be utilized extensively by a mixed population of ruminal microorganisms. Of particu lar significance here is the fact that the other three amino acids—trypto phan, methionine, and lysine—were stable in the presenoe of ruminal microorganisms for at least as long as three hours. Further, the results of El-Shazly demonstrated only between 16 and 22 per cent dissolution of Perhaps i t is appropriate at this point to emphasize that the power of the ruminal microorganisms to cause the dissolution of amino acids probably will vary with the environmental conditions in the rumen (see discussion beginning on p. 216), as well as with the qualitative and quantitative nature of the diet (see discussions beginning on p. ^ ^and on p. 224)• a mixture of amino acids in 1 3/4 hours* These results mean that many of the amino acids present in casein hydrolysate demonstrate stability in the presence of ruminal microorganisms—at least, for a sufficiently long period of time to allow part of the amino acids to pass out of the rumen, either with the ingesta as i t passes to more distal parts of the 92 alimentary tract or by absorption through the ruminal wall* This interpretation lends support to the prediction that was made previously (p. 212)t that, with suitable methods of experimentation, the absorption of amino acids from the ingesta-filled rumen will be demonstrated* Influence of the Physiological State of Activity of the Animal on the  Fate of Amino Acids in the Rumen No attempt will be made here to present a detailed analysis of the influence of the physiological state of an animal on the fate of amino acids in the rumen. Rather, a brief delineation wi l l be presented to indicate the many factors involved* 1. the state of activity of the animal in the formation of its highly buffered saliva and in the secretion of bioarbonate and phosphate into the rumen—the products of these functions help to determine the pH of the ruminal ingesta and, therefore, the state of activity of the animal- in these functions influences indirectly the fermentative actions of the ruminal microorganisms on the amino acids; 2. the state of activity of the animal in the absorption from the rumen of the volatile fatty acids—this function helps to determine the 9 2Lofgreen et al (216) also have suggested that varying amounts of amino acids may be absorbed from the rumen before they can be.acted upon by the ruminal microorganisms. See also footnote 78 on p. 207. 229. pH of the ruminal ingesta and, therefore, the state of activity of the animal in this function influences indirectly the fermentative actions of the ruminal microorganisms on the amino acids; 3. the state of activity of the animal in moving the ruminal ingesta from the rumen to the more distal parts of the digestive tract—this function serves to remove the amino aoids from the fermentative and absorptive processes of the rumen and, therefore, the state of activity of the animal in this function influences the rate of passage of the ingesta from the rumen; 4. the state of activity of the animal in the ruminal wall contrac tions—this function serves to mix and churn the ruminal ingesta and, therefore, the state of activity of the animal in this function, by influencing the rate and vigor of the ruminal wall contractions, influences indirectly the fermentative actions of the ruminal microorganisms on the amino aoids; 5 . the state of activity of the animal in the blood and lymph flow through the capillaries of the ruminal wall—this function serves to remove the absorbed amino acids from the ruminal wall and, therefore, the state of activity of the animal in this function influences the rate of absorp tion of amino acids from the rumen. The states of activity just presented are a l l influenced, in turn, by the 93 physiologioal age of the animal, by the state of activity of the 9 3 P h i l l i p s and Gilder (293) have demonstrated in the rat that the rate of absorption of glucose from the small intestine decreases with increasing age of the animal. endocrine organs, and by the state of activity of the nervous system. These and other factors, each factor playing an important role in determin ing the physiological state of activity of the animal, in consequence influence directly or indirectly the fate of the amino acids in the rumen* Comment. In the previous presentation, an attempt has been made to demonstrate that the distribution of the amino acids in the rumen among the various possible pathways will depend upon the influences of many factors* Although, for ease of presentation, the influence of each factor was considered separately, in actual fact, i t is the summation of the inter actions of the many factors that determines the distribution of the amino acids* Even i f the previous presentation does not permit precise quantita tive predictions to be made of the amino acids' distribution in the rumen, the presentation does make i t evident that the proportions of the individ ual amino acids that are absorbed from the rumen will be greater when the interactions of the many factors are such as to produce higher concentra tions of the amino acids in the ruminal ingesta* Such higher concentra tions of amino acids, occurring together with a metabolically-active Desoxycorticosterone appears to influence the membrane permeability not only of the renal tubules but of tissues in general (34)* Yerz&r (396) has reviewed the considerable evidence which exists in support of his con tention that hormones of the adrenal cortex play a role in phosphorylating mechanisms, including those that may be concerned in the absorption of glucose from the small intestines and the kidney tubules* The administra tion of thyroxine to an animal increases the rates of absorption of the monosaccharides from the small intestine (124)* Finally, as insulin is known to promote the cellular synthesis of protein from the free amino acids in the blood (221), i t is likely that insulin, especially in conjunction with the action of the growth hormone of the anterior pitui tary gland, will have an effect on the rate of absorption of amino acids from the small intestine* 2 3 1 ruminal wall and a high, rate of flow of blood and lymph through the ruminal wall, should lead to an increased amount of amino acid absorption. This increased absorption of the amino aoids would be of great nutritional significance to the ruminant (see p. 267and on). Part Bi B Vitamins Manner of Absorption of B Vitamins from the Rumen If, on the basis of the present results, i t can be assumed that one or more of the B vitamins are absorbed from the rumen, then the question of the manner of this absorption naturally arises. Following the same approach utilized for the discussion of amino aoid absorption, i t is not unreasonable to assume that the passage of B vitamins aoross the ruminal wall might occur in the same manner as their passage across the intestinal wall. A brief review will be made, therefore, of the literature on B vitamin absorption from the small intestine. Literature Review on the Manner of Absorption of B Vitamins from the  Small Intestine IntroduotionAlthough i t generally is assumed that the B vitamins are absorbed from the small intestine (32, 201), l i t t l e evidence is available as to the manner of absorption of most these vitamins. Appar ently, this laok of evidence i s , in part, the result of the fact that many of the B vitamins have been discouvered and synthesized relatively recently. Only for riboflavin has there been accumulated any considerable evidence as to its manner of absorption. The evidence available for the other B vitamins is scanty and inconclusive. Accordingly, the following review will be confined largely to an examination of the literature that pertains to the manner of absorption of riboflavin from the small intestine* Riboflavin.- The early work on the manner of absorption of riboflavin adhered to the same approach that had been utilized to study the manner of absorption of the different sugars. Wilbrandt and Laszt (210, 408) had demonstrated indirectly the ability of a glyoerin extract of the mucous membrane of the rat's small intestine to phosphorylate the sugars glucose, galactose, and fructose, but not mannose and xylose. The phosphorylation 95 was shown to be inhibited by the addition of sodium monoiodoaoetate. Of special significance was the fact that each of the sugars that was oapable of being phosphorylated also had been demonstrated in earlier work to be selectively absorbed from the small intestine, this selective absorption being inhibited by sodium moniodoaoetate• Each of the two sugars that was not capable of being phosphorylated had been demonstrated to be absorbed only by the process of simple diffusion, this diffusion- type absorption being uninhibited by sodium monoiodoaoetate. Utilizing this same approach for the study of riboflavin, Rudy and Kuhn (206, 328) demonstrated the phosphorylation of lactoflavin (riboflavin). Two other flavins, both closely related chemically to lactoflavin but in effective as growth stimulants, were not phosphorylated. The implication of these results was that, perhaps, as for the selectively absorbed sugars, the lining epithelial cells of the small intestine were concerned in the seleotive absorption of riboflavin—and, by means of a phosphoryla- Many enzymes are inhibited irreversibly by iodoacetate, "and it is widely assumed that the inhibition involves a combination of the reagent with essential sulfhydryl groups on the enzyme protein" (124). tion process* *n support of this implication, Laszt and Verzar (211) found that, although the addition of sodium monoiodoaoetate to the food of young rats inhibited their growth, this inhibition could be prevented by the daily addition to the food of 20 micrograms of phosphorylated lactoflavin. Verzar and MoDougall (397), taking cognisance of a l l these results, stated that the parallelism of the behaviours of glucose and riboflavin strongly suggested that riboflavin, like glucose, was phosphory- lated in the intestinal mucosa. Suoh a phosphorylation would require the metabolio intervention of the lining epithelial cells—that i s , more than the process of simple diffusion would be involved in the absorption 96 of riboflavin by the small intestine. Although not concerned directly with the manner of riboflavin absorp tion, the results of recent work on the manner of glucose absorption (89, 116, 158, 159, 191, 266, 278, 381j see also 396) not only provide indirect support for the concept that riboflavin is phosphorylated in its absorp tion from the small intestine, but also indicate promising future lines of investigation on the manner of riboflavin absorption. To indicate the nature and scope of this recent work that has helped to elucidate the manner of absorption of glucose, some of the results of the work are reviewed briefly here. It has been demonstrated that the intestinal epithelium is the richest source of alkaline phosphatase in the body (396j see also 379). Tuba and co-workers (93, 380, 381, 383) have demonstrated that the concentration of this intestinal alkaline phosphatase is decreas- 96 Lust et al (223) have reported that fetal blood (at term) has about four times as much free riboflavin as maternal blood. From their results, they suggest that an active metabolic process is involved in the transfer of riboflavin from maternal to fetal blood by the human placenta. ed significantly after fasting and is increased significantly after the feeding of carbohydrates, fats and fatty acids, and amino acids. As alkaline phosphatase is known to function in a number of tissues either as a dephosphorylating (124), phosphorylating (254), or transphosphorylat- ing (124, 144, 255, 264) enzyme, Tuba and co-workers suggest that these changes in the concentrations of intestinal phosphatase reflect its partic ipation in a phosphorylation-dephosphorylation meohanism that is concerned with the selective absorption of certain substances by the small intestine. As i t is conceived by several workers (155, 397; see also previous discussion on p. 156). for the absorption of glucose, this phosphorylation- dephosphorylation meohanism would function in the following manneri glucose first is phosphorylated by an enzyme-catalyzed reaction as the glucose diffuses into the epithelial cells and, then, the glucose- phosphate compound is dephosphorylated before i t diffuses from the epithelial cells into the portal blood. The recent work of Ota and Shibata (278) provides experimental support for this concept. Their results suggest that the phosphorylation of the selectively absorbed sugars.occurs on the mucosal surface of the intestinal epithelial oells and that dephosphorylation occurs before the sugars pass from the cells into the portal blood. Further experimental support is provided by the results of Feher et al (116). They found that the administration of- enzyme poisons sharply reduced the concentration of adenosine triphosphate (ATP) in the intestinal mucosa i f , at the same time, gluoose was being absorbed from the small intestine. As the enzyme poisonB that were utilized—iodoaoetic acid, sodium fluoride, or 2.4 dinitrophenol— are those known to prevent directly or indirectly the resynthesis of AT? (see 89, 124), the implication of these results is that the decrease in the concentration of ATP was the result of its being utilized in the phosphory lation of glucose. These results, and other results not cited, provide strong support to indioate the presence of a phosphorylation-dephosphoryla- tion mechanism in the lining epithelial oells of the small intestine. In view of the previously demonstrated similarities between the mechanisms of absorption for glucose and riboflavin, there is a possibility that these additional results obtained on the manner of glucose absorption also may be of value for gaining an understanding of the manner of absorption of riboflavin. There does not appear to have been any attempt, however, to extend these sugar experiments to include the study of riboflavin absorp tion. The one exception to this latter statement is the work of Bourne (48). This author demonstrated, by histochemical methods, the dephos- phorylation of riboflavin and pyridoxal phosphates in various tissues, including the epithelial cells in the jejunal part of the small intestine. Bourne suggestsi It seems likely that we are dealing here with a phosphorylating/ dephosphorylating or transphosphorylating system which may be concerned with the process of synthesis of the phosphates of these vitamins and that under the conditions described in this paper the system dephosphorylates• This evidence of Bourne supports the oonoept that riboflavin is absorbed from the lumen of the small intestine by a phosphorylation process. Thiamine.- Very l i t t l e work has been carried out on the manner of intestinal absorption of thiamine. Verzar (395) proposed that thiamine is phosphorylated in its absorption from the small intestine. Several workers (327, 370, 37l) have demonstrated the in vitro phosphorylation of thiamine when they utilized dried powdered intestinal mucosa or glycerol extracts of the intestines. Ochoa (274), however, was unable to demon-236. strate such phosphorylation when he utilized an aoetone preparation of the intestinal mucosa. More recently, Magyar (224) has reported that the absorption of thiamine from the intestine greatly was impaired i f un- physiologically large doses of the other B vitamins or glucose were administrated suboutaneously before the absorption of thiamine was tested. Magyar's explanation of these results was that the prior administration of the B vitamins or gluoose in such large amounts produced exhaustion of the phosphorylating apparatus in the bodily tissues—including the epithelium of the small intestine. Such an effect on the phosphorylating apparatus in the intestinal epithelial cells probably would reduce the intestinal absorption of thiamine, i f the latter is absorbed in part or wholly by being phosphorylated. As, under the conditions of Magyar's experiments, the intestinal absorption of thiamine was reduced, his results support the concept that thiamine is phosphorylated during its absorption from the small intestine. Although scanty, most of the results obtained to-date do suggest, then, that more than the process of simple diffusion is involved in the absorption of thiamine from the small 97 intestine* Other B Vitamins.- Although i t generally is assumed that the other B vitamins gain entry into the body by being absorbed from the .small intestine (32, 327), almost no work has been done on the manner of absorption of these vitamins from the small intestine. Por pyridoxine, the work of Bourne (48), on the histochemioal demonstration of the de- phosphorylation of pyridoxine phosphate in the epithelial cells of the 97 Slobody et al (348) have reported that fetal blood (at term) has about twice as much thiamine as maternal blood. The authors suggest that perhaps the placenta selectively transfers thiamine to maintain this concentration gradient* jejunum, provides suggestive evidence for the manner of absorption of pyridoxine. For pantothenic acid, results have been obtained (80) which demonstrate the in vitro action of an intestine phosphatase preparation in the reversible synthesis of co-enzyme A and intermediate compounds. These results may be indirect evidence that also pantothenic acid is phosphorylated during its absorption from the small intestine. In fact, these results for pyridoxine and pantothenic acid f i t in with the suggestion that has been made by Beerstecher (32) as to the manner of absorption of the B vitamins other than riboflavin and thiamine. By analogy with the manner of absorption proposed for riboflavin and thiamine—i.e., primarily by means of a phosphorylation mechanism—, Beerstecher suggests that possibly those other B vitamins that can form phosphoric acid esters will undergo phosphorylation as a part of their absorption from the small intestine. This suggestion at least could apply to pantothenic acid, nicotinic acid, and the Bg group of vitamins—that i s , these vitamins or compounds containing them are known to be phosphorylated in the cells of the animal body (124). Comment.- The above review points up the scarcity of evidence that is available in the literature on the manner of absorption of the B vita mins from the small intestine. The fragmentary evidence available does provide, however, some support for a gradually developing concept: the involvement of a phosphorylation-dephosphorylation mechanism in the absorption of the B vitamins from the lumen of the small intestine. The following statement by Beerstecher (32) can serve as a comment upon the present state of knowledge of the absorption of B vitamins from the small intestine: The state of knowledge of the prooesses involved in the absorp tion of the individual B-vitamins is to a large extent a func tion of the time that the B-vitamin in question has been well recognized. The more explicit information which is available concerning thiamine and riboflavin absorption indicates clearly that the absorption of the B-vitamins cannot be regarded as a simple process, even though i t is frequently assumed to be for the more recently discovered vitamins. Passage of a metabolite aoross a living membrane seldom is a matter of passive trans fer or simple diffusion. This is certainly a good generaliza tion for the B-vitamins, even in view of the lack of muoh experimental data to verify the assertion. It is to be wondered i f the manner of absorption of some or a l l the B vita mins may not be the same in the rumen as in the small intestine. The data of the present ruminal fistula experiments will be examined, therefore, to see i f there is any evidence to indicate that the absorption of the B vita mins from the rumen involves more than the process of simple diffusion. Evidence from the Present Experiments as to the Mechanism of Absorption of B Vitamins from the Rumen Introduction.- From the information provided in the literature review on the manner of absorption of the B vitamins from the small intestine, i t appears that metabolic intervention by the lining epithelial oells of the intestinal wall may occur in the absorption of the B vitamins. Although the present experiments were not designed to elucidate the manner of absorption of the B vitamins from the rumen, nevertheless the data will be examined to determine i f any evidenoe exists for a similar metabolic intervention by the lining epithelial oells of the ruminal wall occurring in the absorption of the B vitamins. The same procedure of examination that was applied previously to the data for the amino acids (see p. 175 and on) will be applied to the data for the B vitamins. It is necessary, however, first to determine what 239. effect the demonstrated electrical gradient of the ruminal wall likely- w i l l have on the ruminal absorption of the B vitamins. In the present experiments—that i s , in experiments Wl to W6—the pH of the ruminal solutions ranged between 6.5 and 7.6 (see Table 17, p. 87). Within this range of pH, the B vitamins riboflavin, nicotinic acid, and pantothenic acid should exist, at least in part, as negatively charged ions. In the case of riboflavin, this statement follows from the fact that its iso electric point is at pH 6 (327). For nicotinic acid and pantothenic acids, the above statement follows from the fact that the carboxyl groups of these two acids form negatively charged prosthetic groups by ionization— 98 . arid the degree of ionization becomes greater with increasing. pH. As the eleotrioal gradient across the ruminal wall is orientated with the blood side positive and the ruminal side negative, the movement of the B vitamin anions across the ruminal wall should be accelerated by the force The ionization constants of nicotinic and pantothenic acids are 1.4 x 10-5 (167) and approximately 3.9 x 10"5 (409, 410), respectively. Such ionization constants signify that, at the range of pH used in the present experiments—pH 6.5 to 7.5—, nearly a l l the nicotinic or pantothen ic acid molecules would exist in the ionized state. This follows from a consideration of the equation for the ionization constant of an acid: At pH 7, an acid with an ionization constant of 2 x 10-5 has 2.00 x 10-5/2*01 x lO" 5 or 0.995 of its molecules in the ionized form. This equation can be rewritten as: of the electrical gradient. The analyses of the B vitamin data to be made will consider, therefore, the effect of the electrical gradient— that i s ; as it was done in the analyses of the amino acid data. Effect of Initial Electrochemical gradient on Rate of Absorption.- It was pointed out previously for the data of the amino acids (pp. 178 and 179) that, for several reasons, any interpretation of the relation found for an amino acid between its i n i t i a l electrochemical gradient and its rate of absorption might be of doubtful significance. The same doubt exists in the present analyses of the data for the B vitamins—and for the same reasons. The analyses will be made, nevertheless, in the hope of gaining some information and to point out the way for future experi mentation. Table 34 presents, for eaoh the three B vitamins measured, the estimated i n i t i a l electrochemical potential differences, the absolute decreases of concentration, and the decreases of concentration relative to the estimated i n i t i a l electrochemical potential differences. The calculation of the electrochemical gradients for the B vitamins was made in the same manner as for the amino acids: the 30 millivolt number of Dobson and Phillipson (98)) was utilized for the electrical gradient; the i n i t i a l ruminal solution concentrations of the B vitamins that are recorded in Tables 14a to 16b (pp. 71, 73 , 77 , 78, 81, and 82) and the plasma concentrations of the same B vitamins that are recorded as a part of Table 35 were utilized in the calculation of the chemical gradient* For riboflavin and nicotinic acid, the plasma data given for man were "unfortunately, plasma concentrations of riboflavin and nicotinic acid are not available:for the goat or other ruminants. As a substitute, the plasma concentrations of riboflavin and nicotinic acid in man are used. The similarity of the blood concentrations of each these two B vitamins in man and sheep (see the last two columns of Table 35) suggest that the use of the plasma concentrations of man as a substitute for the plasma concentrations of goat is probably valid. TABLE 34. - Ruminal f i s t u l a experiments Wl, W2, W4, W5, and W6i a tabulation, for eaoh the three B-vita* mins whose concentrations were measured, of estimates of the i n i t i a l eleotrochemioal-potential differences (millivolts), of the absolute deoreases of concentration .(miorogram-moles per 100 m i l l i l i t e r s ) , and of the deoreases of concentration relative to the i n i t i a l electroohemioal potential differences (per oent) as • •H p., s • © Riboflavin Concentration decrease 0 to 70 minxes' .a © r H O to 5 © •ri •8 r-i © P4 Variable © r H O to 3 © •s r H Pi Nicotinic aoid • r H •£) (8 • vi P . s . © Concentration deorease 0 to 70 minutes 6 © r H O to 3 © •8 r H © Variable^. o to © is •8 r H S Pi Pantothenic aoid •3 •H P> © Concentration deorease 0 to 70 minute sa o to •8 r H © Pi Variable* © r H O 10 © •8 r H © 188 199 199 218 1.5 0.8 (0 to 125 minutes) 1*9 (0 to 120 minutes) 1.0 1*0 1.7 0.5 0.8 5.1 r 2.3 (0 to 150 minutes) 90 to 139 107 to 156 135 to 184 8.0 5*8 to 8.9 (0 to 120 minutes) 5.1 3.4 3.3 to 4.8 1.9 to 2.5 9.9 5.4 to 7.3 (0 to 150 minutes) 128 to 140 133 to 145 144 to 155 0.4 3.0 0.0 1.9 to 2.1 1.6 1.1 to 1.3 (0 to 120 minutes) TABLE' 34 - oontinued Ex pe ri me nt  Riboflavin Niootinio acid ' Pantothenic acid Ex pe ri me nt  • r-l H) * i •ri 0, s . © Concentration decrease Concentration deorease • rH cs • •ri CU . © Concentration deorease Ex pe ri me nt  0 to 70 minutesa Variabl e^ - rH t> CS • •ri Cu s-. © 0 to 70 minutes6 Variable^- . 0 to 70 minutes8 Variable 1* Ex pe ri me nt  © r-l o CO © •5 •s • r-l © © * r-l © ca . © > •ri •8 rH © © •s r-l O W •8-r-t © © 18 rH O O © .5 •8 i-i © © •S' rH . O to A Re la ti ve  © CO Re la ti ve  W4 222 4,5 2*0 4.0 (0 tc mini 1.3 > 150 rfces) • • . • . . ... 161 to 173 5.9 3*4 to 3.7 4*9 (0 tc minu 1 2*8 to 3*0 150 tes) ^The concentration decreases that are recorded for each experiment are those decreases which occurred in the first 70 minutes of the experiment. ^The concentration decreases that are recorded for each experiment are those decreases which occurred during the experimental time intervals that are indicated by the data in the parentheses* TABLE. 35;. - Ruminal fistula experiments Wl, W2, W4, W5, and W6» a comparison, for each the three B vitamins whose concentrations were measured, between the lowest-found concen trations in the ruminal solutions and the concentrations in the plasma and blood (all concentrations are expressed as miorogram-moles per 100 milliliters of solution) Vitamin Rumin in e al solution xperiment* Plasma15 Blood b Wl W2 W4 W5 W6 Sheep Man Sheep Man Riboflavin 2.7 2.3 14.9 4.5 10.7 •«• 0.007 0.06 0.04 to to to . - 0.01 0.08 0.16 nicotinic 5.7 . • • 24.0 58.1 . • • 0.20 3.25 1.62 acid to to to 1.22 11.37 7.31 Pantothenic 3.8 . • • 13.6 7.0 8.4 0.09 0.03 0.09 0.07 aoid . to to to to 0.14 0.16 0.23 0.21 aThese data, the lowest-found concentrations, are taken from Tables 14a to 16b (pp. 71, 73, 77, 78, 81, and 82). These data, from Albritton 1s Standard Values in Blood (8), are the lower and upper limits of the 95 per oent range of concentrations that have reported in the literature. used; for pantothenic acid, the plasma data given for sheep were used. In the case of riboflavin, because of range of concentrations of this vitamin in the plasma is so narrow and so close to one unit, a concen tration of 1 x 10~2 micromoles per 100 milliliters was assumed for the plasma concentration. For pantothenic and nicotinic acids, the 1owe st and the highest-found plasma concentrations were utilized in order to calculate the upper and lower limits of the possible concentration differences; hence, in Table 34 two numbers are given for the electro chemical gradients acting on these two vitamins. The data in Table 35 for the absolute rates of decrease of B vitamin concentrations were corrected for the concentration changes that occurred in the control solutions. This correction procedure was done in the same manner as for the amino acids (see p. 185). Examination of the riboflavin data in Table 34 does not reveal any uniform effeot of the i n i t i a l electrochemical potentials on the rates of absorption of riboflavin. The lack of a consistent effect in these data in part may be due to animal variability, but also to the fact that the differences among the i n i t i a l electrochemical potentials were not large enough to produce a striking difference among the rates of absorption. The nicotinic acid data in Table 34 likewise do not permit any definite conclusion to be made about the effect of i n i t i a l electrochemical potentials on the rates of absorption. In these data, there are over- lappings of the ranges of the possible i n i t i a l electrochemical potentials These overlappings make i t difficult to draw any conclusions. On the other hand, the pantothenic acid data in Table 34 demonstrate the following relation between the i n i t i a l electrochemical potentials and the rates of absorption of pantothenic acids the absolute rates of 245 absorption are proportional to the i n i t i a l electrochemical potential differences* These just-described results for the three B vitamins do not yield too much information* The data for riboflavin and nicotinic acid do not provide any evidence to elucidate their manner of absorption from the rumen* The pantothenic acid data do indicate, however, that the ruminal absorption of this vitamin is at least partly due to the effeot of the chemical potential difference--assuming the electrical potentials to have been approximately the same in each the experiments—and that* therefore, simple diffusion forms at least a part of the mechanism concern ed in this absorption* Other than this, the present analysis permits nothing more to be stated as to the manner of absorption of B vitamins from the rumen* Comparison Among the Three B Vitamins of their Rates of Absorption— The data of Table 34 have been rearranged in Table 36* Table 36 also includes the molecular weights of the three vitamins* Examination of the data in the table indicates a probable influence of the molecular size of the molecules on their rates of absorptiont in most the experiments, the smaller molecules usually demonstrate more rapid absorption, even although the i n i t i a l electrochemical gradients are often such as to favour the larger molecules. These results suggest that the process of simple diffusion plays an important role in the absorp tion of the B vitamins from the rumen. Whether or not there also is metabolic assistance by the lining epithelial oells of the ruminal wall can not be stated from the present data* Further experimentation, with more appropriately designed experiments, is required* Transfer of the B Vitamins Against an Electrochemical Gradient.- As i t already has been pointed out for the amino acids (see p* 200), the TABLE 36. - Ruminal fistula experiments Wl, W4, W5, and W6i a tabulation of data that are utilized in the text (p.245 ) to determine the influence of the moleoular size of each the three vitamins on their rates of ruminal absorption Experi B-vitamin Moleoular Estimated i n i t i a l eleotro chemioal potential difference (millivolts) Concentration decrease0 ment* weight0 Absolute (miorogram- moles per 100 milliliters) Relative (per oent) Riboflavin 376.4 199 1*9 1*0 Wl (120 min.) Pantothenic acid 219.2 128 to 140 1*6 1.1 to 1*3 Nicotinic acid 123*1 90 to 139 8.0 5*8 to 8.9 W4 ( 70 min.) Riboflavin Pantothenic acid 376*4 219.2 222 161 to 173 4*5 5*9 2*0 3.4 to 3*7 Riboflavin 376*4 199 1*0 0*5 W5 ( 70 min.) Pantothenic acid Nicotinic acid 219*2 123.1 133 to 145 107 to 156 0*4 5.1 0*3 3*3 to 4*8 TABLE 36 - continued Experi ment6 B-vitamin Molecular weight15 Estimated i n i t i a l electro- ohemical potential difference . (millivolts) Concentration decrease0 Absolute (mi crog ram- moles per 100 milliliters) Relative (per cent) Riboflavin 376.4 218 1.7 0.8 W6 ( 70 min.) Pantothenic acid 219.2 144 to 155 3.0 1.9 to 2.1 Nicotinic acid 123.1 135 to 184 3.4 1.9 to 2.5 W6 (150 min.) Riboflavin Niootinio acid 376.4 123.1 218 135 to 184 5.1 9.9 2.3 5.4 to 7.3 ^The data in parentheses designate the time intervals during which the reoorded concentration de creases occurred. DThese data, rounded off to one decimal plaoe, are from the Handbook of Chemistry and Physics ( 167 )• °These data are taken from Table 35 on page 196. 248 demonstration of a net movement of a substance against an electrochemical gradient would be considered as strong evidence for the existence of one or more other forces being concerned in the movement of the substance* Unfortunately, the B vitamin concentrations that were used in the present experiments were so high that in no case was there any possibility of testing for movement of the vitamins against an electrochemical gradient. Table 35 records the lowest-found concentrations of the three B vitamins in the ruminal solution of each experiment, along with the blood and plasma, concentrations reported in the literature for man and sheep. In every case,' the ruminal solution concentration was so high relative to the plasma concentration that, along with the fact that the electrical gradient also was orientated from ruminal solution to plasma, the absorp tion of the B vitamins always was with the eleotrochemical gradient. The present data, therefore, yield no evidence as to whether or not the lining epithelial oells of the ruminal wall are able to move the B vita mins against an electrochemical gradient. Comment.- The purpose of the previous analyses was to determine i f any evidence.existed in the data of the ruminal fistula experiments as to the manner of absorption of the B vitamins from the rumen. Un- fortunately, these analyses yielded l i t t l e information, other than to suggest that the process of simple diffusion is involved. The nature of the data was not such as to yield evidence for or against the involvement of metabolio energy—sueh^ as might be utilized by phosphory- continued on p. 249 -249. lating enzymes—in the absorption of the B v i t a m i n s 0 0 As for the amino acids, further experimentation is required to elucidate the manner of absorption of the B vitamins from the rumen* B Vitamin Absorption from the Rumen Under Normal Feeding Conditions Introduction The same question can be asked for the B vitamins that was asked for the amino acids: is there any evidence to support the concept that the absorption of B vitamins occurs from the rumen under normal feeding conditions* To be able to answer this question, there is required a knowledge of the fulfillment or non-fulfillment of the following condi tions : 1) the demonstration of the presence of the B vitamins in the rumen.; i 2) the demonstration of the relation of the concentration of the \ individual B vitamins in the ruminal liquor to their concentration in the blood plasmas a rumen to plasma, downward electrochemical gradient would permit the possibility of B vitamin absorption without requiring direct participation of metabolic energy, but would not rule out the possibility of metabolic energy also participating—as, for example, in a phosphory lation of the vitamins—; a plasma to rumen, downward electrochemical It is pertinent to note here that Phillipson and Reid (302) found nearly a l l the extracellular thiamine present in ruminal contents to be already in the phosphorylated form: thiamine pyrophosphate. How, as Kon and Porter (personal communication cited in 302) have demonstrated that thiamine is absorbed from the rumen, i t is to be wondered whether the thiamine is absorbed as such, as the pyrophosphate, or in both forms. Of interest to this question is the suggestion of Snell (357) that the "cell wall" of some microorganisms is impermeable to thiamine pyro phosphate. On the other hand, Rosenberg (327) states that thiamine can be absorbed from the small intestine in the free or in the phosphorylated form. 250. gradient would limit any B vitamin absorption to that accomplished directly or indirectly by the force of metabolic energy; 3) the demonstration of the absorption of the individual B vitamins from an ingesta-filled rumen, the concentration of each the individual B vitamins at which this absorption oocurs being of the same order of magnitude as that found in the fluid of the ruminal ingesta ordinarily. Each of these points will be discussed in turn. Condition l i Demonstration of B Vitamins in the Rumen The B-complex of vitamins is present in the ruminal ingesta in considerable quantities. For many of the B vitamins, columns 2 and 4 of Table 37 record the concentrations of these vitamins that have been found in the ruminal ingesta of the cow and sheep. The concentrations are recorded as micrograms per gram of ruminal ingesta (dried weight). Columns 3 and 5 of Table 37 record again the data of columns 2 and 4, but this time the data are recalculated so as to express the concen trations in terms of the original ruminal ingesta (wet weight). In making this recalculation, the dry matter content of the original ruminal ingesta was assumed to be 15 per cent (404, 405) and a specific gravity of 1 also was assumed. The recalculated data are expressed as micro grams per 100 milliliters of solution. It is not known what proportion of these wet weight concentrations are present as free vitamins in the ruminal liquor. Other than for thiamine and riboflavin, the present author was unable to find any data recorded in the literature, for the concentrations of B vitamins in the ruminal liquor. Phillipson and Cuthbertson (299) do suggest, however, that M i n a l l probability much of the B vitamin content of the rumen is in true solution • • • •'• This TABLE 37. - Concentrations of the B-vitamins in the ruminal ingesta and plasma of the sheep and oow (the plasma concentrations for man are included, since in most oases a knowledge of the plasma concentrations of the B-vitamins in the ruminants is laoking) Ruminal ingesta* Plasmab Sheep Cow Original Calculated Original Calculated data data data data Sheep Cow Man B^vitamin (micrograms (micrograms (micrograms (micrograms per per per per (micrograms gram of 100 milliliters gram of 100 milliliters per 100 milliliters) dry matter) of wet matter) dry matter] of wet matter) (1) (2) (s) (4) (5)' (6) (7) (8) Riboflavin 53 493 4 60 « . . • a • 2.6 to to t 0 r 1 32 478c»° 3.7d , Niootinio ... • . • 15 224 ... • . • 25;: acid to to to 92 13736* 9 150f»S Pantothenic 70 1045 3 45 20 • • • 6 aoid to to to to 56 836 30 35 Thiamine 7 104h 0.12 1.8 3.81 . . . 1 to to to 5 75 9i Pyridoxine 10 149 2 30 . . . • • • 1 to to to 10 149 18J TABLE 37 - continued B vitamin (1) Ruminal ingesta a Plasmab Sheep Cow Original data (micrograms per gram of dry matter) (2) Calculated data (micrograms per 100 milliliters of wet matter) (3) Original data (micrograms per gram of dry matter) (4) Calculated data (micrograms per 100 milliliters of wet matter) (5) Sheep (miorograi (6) Cow is per 100 m: (7) Man .llilitera) (8) Biotin. Folio acid )2.6 • • • >39 • • • 0.08 to 0.31 0.24 to 2.3 1.2 to 4.6 3.6 to 34 ... • • • ... 0.05k 1.0 to 1.7 0.05k ^he "original data" recorded here are selected from data that were found by the author in a search of the literature (6, 177, 1.79, 192, 193, 194, 202, 208, 243, 244, 245, 246, 247, 404, 405). Only those data that had been determined by a biological method—that i s , microbiological, chick, or rat assay—were utilized. The data for the cow represent the lowest and the highest coneentrations.that were found to be recorded. These data, from Albritton 1s Standard Values in Blood (8), are the lower and upper limits of the 95 per cent range of the concentrations that have been reported in the literature. Except for the data of riboflavin, biotin, and perhaps pyridoxine, a l l the data that are recorded were determined by the microbiological method. Riboflavin and biotin were determined ohemically. The method of determination of pyridoxine is not stated. cIn a preliminary assay, for which the microbiological prooedure of Barton-Wright (30) was utilized, the present author found the concentrations of riboflavin in three samples of ruminal liquor from a goat to be 41, 45, and 53 micrograms per 100 milliliters. c'just recently, the author oame across the data of Conrad (76) for the concentration of riboflavin in ruminal liquori for six samples, the concentration ranged between 62.5 and 165.8 with an average of 112.8 micrograms per 100 milli l i t e r s . Although the data of Conrad are too few to draw any valid con clusions, in some of his samples a considerable'portion of the riboflavin apparently was in the free state in the ruminal liquor (see also footnote h). dIn a microbiological assay (procedure of Barton-Wright (30)} of three plasma samples from one goat and of 4 plasma samples from another goat, the present author found the concentrations of riboflavin to range, respectively, between 2 to 5 and between 1 to 4 micrograms per 100 milliliters. eIn a preliminary assay, for which the microbiological procedure of Barton-Wright (30) was utilized, the present author found the concentrations of nicotinic aoid in three samples of ruminal liquor from a goat to be 350, 430, and 470 micrograms per 100 milliliters. 6 Conrad's data (76) for the analyses of two composite samples of ruminal liquor are 395.0 and 563.0 micrograms per 100 milliliters. His data do not indicate what proportion of these concentrations were present as free vitamin in the ruminal liquor. Total nicotinic acid activity. Sln a microbiological assay (procedure of Barton-Wright (30)) of three plasma samples from one goat and of nine plasma samples from another goat (see Table 20, p. 110), the present author found the concen trations of niootinio acid to range, respectively, between 34 to 48 and between 34 to 72 micrograms per 100 milliliters. ^Recently, Phillipson and Reid (302) have recorded the thiamine content in 266 samples removed from a total of 49 sheep that were fed different rations. The concentrations of thiamine ranged between 23 atd 189 micrograms per 100 grams of rumen sample. Although the authors found appreciable concentrations of thiamine in the small food particles and microorganisms of the ruminal samples, most of the thiamine was extracellular and existed as thiamine pyrophosphate in solution in the ruminal liquor, ^cmtrary to these findings, Conrad and Hibbs (76, 78) state that the B-complex vitamins in ruminal liquor largely are bound in the microbial oells. The data in Table 35 of Conrad's thesis (76) do not appear to provide the necessary basis for suoh a statement: only thiamine and riboflavin were measured and even for these two vitamins the data are too few and too variable. In fact, in one sample (#968) the contents of thiamine and riboflavin in the microorganism-free filtrate were greater than the contents of the vitamins in the ruminal bacteria, the content of thiamine in the filtrate being almost double the content in the bacteria. The author makes no comment on this, xTotal thiamine, Rosenberg (327) states that there seems to be a constant level of free thiamine— i»e., non-phosphorylated thiamine—of about 1 microgram per 100 milliliters of plasma. ^These concentrations are for the plasma of monkey. kFree folic acid 2 5 5 . statement appears to be valid for thiamine t in footnote h of Table 37 there is indicated that most of the content of thiamine in the ruminal ingesta is extracellular and present as thiamine pyrophosphate in the ruminal liquor. For riboflavin and nicotinic acid, in footnotes c and e of Table 37 there are recorded the present author's own preliminary data for the concentrations of these two B vitamins in ruminal liquor. Unfortunately, as the ruminal liquor in the author's experiments was a filtrate that resulted from filtering the ruminal ingesta through four layers of cheesecloth, the liquor s t i l l contained solids such as small fragments of food and mioroorganisms* This means that, as some of the measured riboflavin or nicotinic acid content of the ruminal liquor could have been moleoular constituents of these solids, the data do not yield a knowledge of the free concentration of riboflavin and nicotinic acid in the ruminal liquor. The data of Conrad (see footnote c' in Table 37) do indicate, however, that possibly a considerable proportion of the riboflavin may be in the free form. More work is required, however, on this point* Condition 2 ; Demonstration of an Electrochemical Gradient For many of the B vitamins, columns 6, 7, and 8 of Table 37 record the concentrations of these vitamins found in the plasma. Where avail able, the data for ruminants are given, otherwise the data for man are utilized. A comparison in Table 37 between the concentrations of the B vitamins in the ruminal ingesta (wet weight) and in the plasma reveals that the vitamin concentrations in the ingesta are usually much greater than the concentrations in the plasma. If any considerable proportion of these vitamin concentrations that are recorded for the ruminal ingesta are present in the ruminal liquor in the free state,, then these data could be evidence for the existence of downward concentration gradients for the B vitamins in the direction rumen to plasma* Such may be the case for thiamine, as the information contained in footnote h of Table 37 indicates that most of the thiamine in the ruminal ingesta is extra cellular and present in the free state as thiamine pyrophosphate. For riboflavin and nicotine acid there exist the goat data in footnotes c, d, e and g of Table 37* Comparison of the found-concentrations of ribo flavin in the ruminal liquor (41 to 53 micrograms per 100 milliliters) with the found-concentrations in the plasma (l to 5 micrograms per 100 milliliters) reveals that a strong, downward concentration gradient from rumen to plasma probably existed for riboflavin. The same type of comparison for the found-conoentrations of nicotinio acid in the ruminal liquor (350 to 470 micrograms per 100 milliliters) with the found-concen trations in.the plasma (34 to 72 micrograms per 100 milliliters) reveals that a strong, downward concentration gradient from rumen to plasma probably existed for nicotinic acid as well. Unfortunately, as i t already has been noted previously, i t is not known how much of the measured concentrations of riboflavin and nicotinic acid in the ruminal liquor were in the free state. Except for thiamine i t is not possible, there fore, to make any statement as to the direction of the electrochemical gradient across the ruminal wall for any of the B vitamins until more data becomes available on the free concentrations of the B vitamins in the ruminal liquor. Condition 5> Demonstration of Absorption from an Ingesta-Filied Rumen The only data that are available to examine on this point are the 257, results presented in Table 20 (p. 110) for nicotinic acid. These results failed to demonstrate the absorption of nicotinic acid from an ingesta- filled rumen. It is to be noted, however, that the method of detection of nicotinic aoid absorption was by the making of a comparison of the concentrations of nicotinic acid in the portal and peripheral vessel plasmas. The lack of sensitivity of this procedure for the deteotion of substances absorbed continuously and in small amounts has been comment ed upon previously (see p. 116). By contrast, when there was utilized the more sensitive procedure of sampling at periodic intervals a solu tion placed in an empty rumen--that i s , as in the ruminal fistula experiments—, absorption of the B vitamins was demonstrated. Although the data that are assembled in Table 38 reveal that the vitamin concen trations in these ruminal fistula experiments were usually higher than the concentrations reported in the literature for the ruminal ingesta (wet weight), the data also reveal that in many of the experiments the concentrations in the ruminal fistula experiments were of the same order of magnitude as those reported for the ruminal i'ngesta. Comment The previous discussion indicates that many of the B vitamins are present in the rumen in appreciable amounts. These amounts may be large enough that rumen to plasma, downward electrochemical gradients oan exist for many of the B vitamins. Further measurements must be made, however, to determine i f such gradients really do exist. If they are found to exist, then these gradients should permit the B vitamins to be absorbed from the rumen, even i f the ruminal epithelium does not possess the ability to absorb B vitamins against an electrochemical gradient. As well, there TABLE 38* - Ruminal fistula experiments Wl, W2, W4, W5, and W6s a comparison, for each the three B-vitamins whose concentrations were measured, between the lowest- found concentrations in the ruminal solutions and the oonoentrations in the ruminal ingesta (all concentrations are expressed as mlorogram-moles per 100 milliliters of solution) Vitamin Ruminal solution in experiment* Ruminal ingesta0 Wl W2 W4 W5 W6 Sheep Cow Riboflavin 2.7 2.3 14.9 4.5 10.7 1.3 0 .1 to 1*5 Niootinio aoid 5.7 ... ... 24.0 58.1 ... 1*8 to 11.2 Pantothenic aoid 3.8 ... 13.6 7.0 8*4 1 4.8 0.2 to 3.8 aSee footnote a of Table 35 on page 243. bThese data are from Table 37. Their unit of measurement has been changed, however, from miorograms to miorogram-moles per 100 milliliters of ruminal ingesta. s t i l l remains the possibility that a l l or some of the B vitamins can be absorbed from the rumen, even against an unfavourable electrochemical gradient. Finally, although the previous discussion failed to present evidence of B vitamin absorption from the normal ruminal contents, evidence is presented which indicates that ruminal absorption of the vitamins did occur from ingesta-free rumens at concentrations which possibly were of the same order of magnitude as the concentrations that have been reported for ruminal ingesta* This finding suggests that, with suitable methods of experimentation, i t should be possible to demonstrate the absorption of the B vitamins from the ruminal contents. One such suitable method of experimentation might be to use radioactive- labelled vitamins. The Effect of Ruminal Absorption on the Fate of B Vitamins Present in the Rumen Introduction Figure 23, modelled after the figure previously presented to summar ize the principal reactions of nitrogen compounds in the rumen (Fig. 22, P« 214), is proposed by the present author to summarize the principal reactions of the B vitamins in the rumen. Figure 23 suggests that the fate of the B vitamins that enter or are formed in the ruminal ingesta is manifoldt 1. the B vitamins are utilized in the synthesis of microbial enzyme systems, or they are degraded to simpler moleculesj 2. the B vitamins pass with the rest of the constituents of the ruminal ingesta into the more distal parts of the alimentary tract; 3. the B vitamins are absorbed from the ruminal ingesta by passage DO O In saliva In food Entering rumen In rumen lieaving rumen to abomasum and intestines Vit comp amin lexes Free vj Vitamin .tamins precurso rs < Vit comp r \ i rs amin < rree V J lexes \ \ \ \ ^ Vitamii \ microorgj \_ \ precurso v >v 'r \ is of \ inisms \ \ Fooc L protein Vitamii microorgi r \ l is of Free vitarai anisms and vitami precurso3 .ns .n -s .Absorbed through rumen wall? .Absorbed through rumen wall .Absorbed through rumen wall? Fig. 23. Fig. 23. - The principal reactions of the B vitamins in the rumen ("based on information con tained in references 203, 267, 357, 388)• ro through the ruminal wall into the portal circulation. As with the amino aoids, i t is to he expected that the distribution of each B vitamin among the various possible pathways just described w i l l be influenced by the particular set of environmental conditions prevail ing in the rumen, by the known influence of the diet on the composition of the population of microorganisms in the rumen, by the physical and chemical nature of the dietary constituents, by the relative stability of the B vitamins in the ruminal ingesta, and by the physiological state of the animal. Each of these influencing factors previously was discussed in the section dealing with the amino acids (see pp. 216 to 231)• Much of that discussion can find application here; hence, i t is felt that i t is not necessary to present here what largely would be a repetition of the discussion that was presented for the amino acids. The reviews of or reports of Lannen and Tanner (388), Lardinois et al (208), Snell (357), Najjar and Barrett (267), Kon and Porter (203), Hunt e t al (177, 180) and Agrawala et al (6), Conrad and Hibbs (78), Phillipson and Eeid (302), and Hollis et al (169) provide many examples of how variations in differ ent environmental conditions can influence the activities of the popula tion of ruminal microorganisms in relation to B vitamin synthesis and utilization. Only the influence of the diet on the composition of the mioroorganism population wil l be discussed again, as there do exist several interesting examples of how this influence will help to determine the fate of the B vitamins in the rumen. Influence of the Diet on the Composition of the Population of Micro- organisms in the Rumen and, therefore, on the Fate of B Vitamins in the Rumen Snell (357) has described the effects produced on the vitamin re-quirements of individual microorganisms when certain constituents of the cultural medium are varied* As these results could have application and he of value in gaining an understanding of the conditions in the rumen, some details are presented here of the description given by Snell for the "sparing action" produced by certain compounds on the vitamin requirements of microorganisms* For example, the requirement of Aoetobaoter  suboxydans for para-aminobenzoio acid in a cultural medium free of purine bases is about ten times as high as in a medium which contains purine bases* Again, the amount of pyridoxine required for the growth of many lactic acid baoteria depends upon the number and type of amino acids present in the cultural medium* Lactobacillus arabinosus, for example, can grow in the absence of pyrodoxine, i f the medium contains hydrolyzed casein or a matching mixture of synthetio amino acids; i f , however, anyone of a number of amino acids—such as cystine, histidine, phenylala nine, tyrosine, or arginine—is not present, then the microorganism can not grow in the absence of pyridoxine* The production by other moleoular oompounds of the same type of "sparing action" has been demonstrated for folic acid, biotin, and nicotinic acid* In most these cases, the "spar ing action" is suggested as indicating that an individual vitamin is concerned in the microbial synthesis of the particular compounds whose presence "spares" the vitamin* Apparently, when the "sparing" compound is present preformed in the cultural medium, the compound is assimilated directly* Under these circumstances, an otherwise essential function of the spared vitamin is made non-essential and the requirement of the micro organism for the vitamin is reduced or eliminated* The implication of these results with respect to the action of the ruminal population of microorganisms on the B vitamins is clean variation in the quantity and quality of the molecular constituents of the diet should influence the requirements of the microorganisms for the B vitamins, thus producing variation in the distribution of the B vitamins among the three possible pathways that previously were suggested for the B vitamins that enter or are synthesized in the ruminal ingesta. Of more immediate application to the question of the effect of diet on the fate of B vitamins in the rumen are experiments that have been concerned with the effect of diet on the population of ruminal micro organisms as i t exists in the ruminant. The population of ruminal micro organisms is known to be capable of synthesizing a l l the members of the B vitamin complex (see p. 9 ). The extent of this synthesis, under different dietary conditions, is not, however, clear. This situation more readily is appreciated when the method that often was utilized to demonstrate B vitamin synthesis by the ruminal population of micro organisms is considered. The method of demonstration often was to feed to the experimental animals diets that were deficient or almost devoid in their content of the B vitamins. With such diets, i t was possible to demonstrate larger concentrations of B vitamins in the ruminal ingesta as compared to the concentrations in the feed. Several early groups of workers, utilizing this method of demonstration, were able to demonstrate increased microbial synthesis of many of the B vitamins in response to an increased content in the diet of carbohydrate (76, 177, 178, 179, 180, 208), silage (372), urea (76, 208), or thiamine (201, 404, 405). When, however, diets are fed that already are rioh in B vitamins, it is difficult to demonstrate larger concentrations of the B vitamins in the ruminal ingesta as compared to the concentrations in the feed (6, 202, 203). In fact, two groups of workers (6, 203) found that the ooncentra-265. tions of the B vitamins were as high i n the ruminal ingesta of animals that were fed a ration which was poor i n i t s content of the B vitamins as in the ruminal ingesta of animals that were fed a ration which was rich ih i t s content of the B vitamins. The microbial synthesis of the B vitamins in the animals that were fed the ration which was poor in i t s content of these vitamins apparently was sufficient to increase their concentrations to the higher concentrations that were present in the ration which was rich i n i t s content of the B vitamins. From these results, i t appears that when a B vitamin-rich ration i s fed, the amount of microbial synthesis of the B vitamins i s not extraordinary. It is only when a B vitamin-poor ration i s fed, that the amount of B vitamin synthesis becomes extraordinary—and easily demonstratable. Coates et a l (72) comment on these results as follows: These findings indicate a balance between the extent of synthesis and absorption of vitamins by the microflora and ... the concentration (jpf the vitamins] in the rumen. A possible explanation of these results can be deduced from the known effect that a change of diet can have on the proportions of the different species of mioroorganisms present in the rumen (see p. 220)• A ruminant subsisting on a B vitamin-rioh diet would be able to support a popula tion of mioroorganisms which either absorbs i t s B vitamin requirements from the environment or which synthesizes i t s own required B vitamins. Many of the microorganisms which synthesize their required B vitamins probably would decrease or even eliminate, however, the synthesis of these vitamins under the condition of high concentrations of the B v i t a  mins in the d i e t . Now, changing the B vitamin-rich ration of a ruminant to a B vitamin-poor ration would produce a new nutritional environment in the rumen. This new environment would favour the growth and multipli-266 cation of those microorganisms that can synthesize their own B -vitamin requirements, as opposed to those microorganisms that obligatorily require preformed B vitamins in order to grow and multiply. This depend ence of the character of the population of ruminal microorganisms on the character of the diet of their host means that, when a B vitamin-poor ration is fed, the greater proportion of the total B vitamin content of the ruminal ingesta will be that which has been formed by microbial syntheses; on the other hand, when a B vitamin-rich ration is fed, the greater proportion of the total B vitamin content of the ruminal ingesta • will be that which has been ingested with the ration. This variation in, the source of the B vitamins in the ruminal ingesta, with the attendant variation in the metabolic activities of the ruminal microorganisms, most certainly will influence the distribution of the B vitamins among the three possible pathways previously described (see p. 259). Comment •• Keeping in mind the discussion previously presented for the amino acids (see pp. 216 to 231), it becomes apparent from the discussion just presented for the B vitamins that their distribution in the rumen among the various possible pathways will depend upon the influences of many factors. The proportions of the individual B vitamins that are absorbed from the rumen will be greater when the interactions of the many factors are such as to produce higher concentrations of the B vitamins in the ruminal ingesta. Such higher concentrations of B vitamins, occurring together with a metabolically-active ruminal wall and a high rate of flow of blood and lymph through the ruminal wall> should lead to an 267. increased amount of B vitamin absorption. This increased absorption of the B vitamins would be of great nutritional significance to the ruminant (see below)• Part Cj The Possible Nutritional Implications of the Absorption of Amino Acids and B Vitamins from the Rumen The standard thinking of nutrition is so often applied to the ruminant in a way that suggests " i f we could only stop a ruminant being a ruminant what a much better animal it would be". • • • The ruminant is not likely to be badly adjusted to its digestive apparatus and as animal husbandry experts we would be well advised to approach the ruminant with some humility bearing this in mind. To suc cumb to the temptation of forcing the ruminant into the standard mold of nutritional thought which is based largely on the rat is to display a lack of proper appreciation of what is known about ruminant digestion. A. T. Phillipson and D. P. Cuthbertson (299). Introduction If i t be assumed that at least some amino acids and B vitamins can be absorbed from the rumen, then the question of the nutritional implica tions of this absorption can be raised. It is probable that the implica tions are many. Most of the present discussion will be limited, however, to the making of two suggestions as to the possible nutritional signifi cance of the absorption of amino acids and B vitamins from the rumen. The remainder of the discussion will be devoted to an application of the concept of ruminal absorption to explain certain results obtained in nitrogen retention trials with ruminants. The Time Faotor Several workers (107, 130, 131, 366; see 105 for citations of earlier work) have demonstrated the importance of the time factor in the utilization by an animal of its ingested nutrients. It has been found. for example, that the administration to an animal of an amino acid mix ture which otherwise would maintain the nitrogen balance of the animal will not do so i f one or more of the component amino acids is omitted and administered several hours later (107). The explanation of this result apparently is that the oapaoity of the liver to store amino acids for any length of time is limited* This limited capacity becomes mani fested when there arrives at the liver a mixture of amino acids that is lacking in one or more of the amino acids that constitute the proteins synthesized in the live r j the liver deaminates the incoming amino acids and the resulting ammonia rapidly is excreted. Only when the mixture of amino aoids is not lacking in any one of the required amino acids does the liver utilize the mixture of amino acids in the synthesis of protein (257, 389, 390). This behaviour of the liver towards amino aoids is epitomised by Geiger's (130) statement that "protein synthesis occurs only when a l l the essential building stones are available simul- taneously." A seoond example of the importance of the time factor in the utilization by an animal of its ingested nutrients is that noted by Ellman (107). He and others have found that a reciprocal relation exists between amino acids and carbohydrates in that their simultaneous administration to an animal results in better utilization of both amino acids and oarbohydrates~as compared with the degree of utilization of these two olasses of nutrients when each is administered separately* Ellman suggests that the explanation of this reciprocal effect is that "the simultaneous entrance of carbohydrate and amino acids into the metabolic pool enhances the storage or oxidation of carbohydrates or both." To be more speoific, i t is suggested here that the simultaneous presence of amino acids during the utilization of carbohydrates may 269. increase the efficiency of this utilization because of the fact that amino acids are necessary components of the enzymes concerned in the metabolic oxidation of oarbohydrates. The presence of these amino acids at the time of carbohydrate utilization, along with an adequate concentration of the necessary B vitamin components of the enzymes, would help to ensure an optimal concentration of the enzymes concerned in this utiliza tion. Further, it is suggested here that the simultaneous presence of carbohydrate during the utilization of amino acids may increase the efficiency of this utilization because of the fact that the presence of carbohydrate would reduce or eliminate the need of the animal to utilize amino acids as an energy source. The elimination of this need would permit the animal to utilize the amino acids for protein synthe sis—that i s , the well-known "sparing action" of carbohydrates on protein 101 or amino acid utilization. Other examples of the importance of the time factor in nutrition could be presented, but the two examples just given should be sufficient to illustrate the point. With the previous information as background, the first suggestion can be made as to the nutritional significance of the absorption of amino acids and B vitamins by the ruminal wall. As most the volatile fatty acids that are formed in the rumen by cellulose fermentation are known to pass through the ruminal wall into the portal blood, the simultaneous passage of amino acids and B vitamins through the ruminal wall into the portal blood would permit a l l these compounds to arrive at •^The article of Geiger (131) should be referred to for a more detailed consideration of the reasons as to why the simultaneous presence of carbohydrate during the metabolism of amino acids promotes their utilization for protein synthesis. the liver at approximately the same time* Following from the discussion on the importance of the time factor in the utilization by the animal of its nutrients, the simultaneous arrival at the liver of the volatile fatty acids, amino acids, B vitamins, and inorganic ions should promote more efficient utilization of each these nutrients (131)* Such an ad vantageous timing of the arrival of a l l these components at the liver (or elsewhere in the body) would not occur i f the absorption of the amino acids and B vitamins occurred only from the small intestine* The Competitive Factor Introduction* - The second suggestion to be made for the nutritional significance of amino acid and B vitamin absorption from the rumen con cerns the relation of this absorption to a competition that probably exists between the ruminant host and its contained microbial population* The competition referred to is that which probably exists for the amino acids and B vitamins that are ingested as a part of the host*s ration (202, 302, 368, 369, 373) or that are liberated 1 0 2 into the ruminal liquor by some members of the ruminal population. Before suggesting the character of the effect of ruminal absorption of amino acids and B vitamins on this competition, the probable roles played by the host and microbial popu lation in the competition first will be delineated briefly. Competitive Actions of the Microbial Population for Amino Acids and  B vitamins*- The competitive actions of the microbial population would be the result of some of its members either absorbing or inactivating Some of this liberation of amino acids and B vitamins would be by autolysis of microorganisms* In addition, however, there is evidence that microorganisms also liberate amino acids and B vitamins while s t i l l living (54, 104, 132, 375). the amino acids and B vitamins* It is well-known that many microorganisms utilize amino acids for protein and nucleic acid synthesis or enzymatioally degrade amino acids through decarboxylation or deamination. Likewise, Van Lannen and Tanner (388) cite experiments which demonstrate microbial inactivation of the B vitamins thiamine, nicotinic acid, and biotin. Finally, considerable evidence exists that certain microorganisms absorb B vitamins in relatively large amounts (197, 357, 388). Of special interest in this latter respect is the behaviour of some microorganisms towards thiamine and vitamin Bg: "luxury consumptionV-the accumulation within the microorganisms of these vitamins in amounts considerably greater than the amounts sufficient for normal growth (357)• Competitive Actions of the Host for Amino Aoids and B Vitamins.- The competitive actions of the host ruminant for the available amino acids and B vitamins is exerted primarily through the digestive and absorptive capacities of its alimentary canal. The digestive action of the alimentary canal, limited to the abomasum and small intestine, not only liberates amino acids and B vitamins from those feed constituents that have escaped the fermentative action of the microbial population in the ruminant stomach, but also liberates amino acids and B vitamins from the cells of the microbial population as they pass into the digestive parts of the alimentary canal. This digestive action would permit the absorptive action of the alimentary canal to function in the abomasum, small intestine, and large intestine. In addition, the possibility also exists that the absorptive action of the alimentary canal occurs in the first three compartments of the ruminant stomach. - In this case, the absorptive action would be limited to those compounds that are ingested in the absorbable state or to those soluble compounds that are formed by 272 the fermentative action of the microbial population. The combined effect of these digestive and absorptive actions provides to the ruminant host a means of obtaining its requirement of amino acids and B vitamins. The Beneficial Effect of Ruminal Absorption to the Host in its  Competitive Actions for Amino Acids and B Vitamins.- The absorption of amino acids and B vitamins by the ruminal wall is of benefit to the ruminant host in that this absorption conserves for the host amino acids and B vitamins that otherwise would be inactivated by the microbial population. This conservation should be of considerable nutritional significance when feeds are given that are relatively rich in their content of free amino aoids and B vitamins. There remains, however,, a possibility that ruminal absorption may provide an additional benefit of conservation to the host of many of the amino acids and B vitamins that otherwise might be contained within the protoplasm of ruminal micro organisms. Now, i t is accepted quite generally that the amino aoids and B vitamins, contained in one form or another in the microbial proto plasm, later become available to the host after the microorganisms pass •with the ingesta into the digestive parts of the alimentary canal (18, 21, 23, 154, 200, 228). Indeed, several authors (21, 22, 23, 228, 304), by microscopic examination of the alimentary tract ingesta, have provid ed indirect evidence that the digestive enzymes of the abomasum and small intestine can digest ruminal microorganisms. The extent of this digestion remains in doubt, however, because as yet there exists no data to indicate what proportion of the ruminal microorganisms are digested (33, 92). One author (198), on the basis of comparative direct bacterial counts between the ruminal and,intestinal ingesta, even has suggested that l i t t l e of the protein of the ruminal mioroorganisms is digested. Cuthbertson (86) has commented as follows (see also the discussion by- McDonald (237)): The question of disintegration of bacteria in the a l i  mentary tract i s important, for the liberation of their i n t r a  cellular products,- i . e . polysaccharides, as well as the proto plasmic nitrogen, presumably depends on this . . The hypothesis that rumen bacteria are digested as they pass through the abomasum and small intestine rests on circumstantial evidence. It i s . . . not likely that a l l bacteria are k i l l e d by the change in pH Cthat occurs when the ingesta passes from the omasum to the abomasum with i t s low pH 3 * by the action of pepsin, or by the subsequent action of the duodenal and intestinal juices. Again, Pounden et a l (304), for example, have found that not a l l types of ruminal microorganisms can be digested in the abomasum or small intestine. Their observations demonstrate that, although some of the ruminal microorganisms undergo rapid disintegration i n the abomasum or small intestine, "others manage to hold their form until they reach the more posterior parts of the digestive tract, while s t i l l others can withstand a l l the post-ruminal digestive ac t i v i t i e s of the animals." This evidence, that some of the ruminal microorganisms are passed out of the animal i n the feces, indicates that amino acids and B vitamins are being lost by the host. Even that microbial protein which does become exposed to the digestive enzymes of the small intestine probably does.not yield a l l i t s constituent amino acids to the host. This i s so because the ruminal microbial protein i s not digested completely when i t is fed to rats: true d i g e s t i b i l i t i e s have been demonstrated 103 of about 74 for bacteria and of about 90 for protozoa. I f the Several authors (21, 186, 249) have suggested that the ruminal protozoa ingest ruminal bacteria and, by converting the bacterial protein to protozoan protein, thereby increase the d i g e s t i b i l i t y of the microbial protein. As yet, however, no evidence has been found that the ruminal protozoa actually do ingest bacteria (86). digestibilities of miorobial protein are the same in the small intestine of the ruminant as in the small intestine of the rat, then this lack of complete digestibility of the microbial protein is another cause for the loss of amino acids to the host. Further, as i t has been pointed out by McDonald ( 237), because bacteria have a considerable proportion of their 104 nitrogen in the form of nucleic acids and because most of these nucleic aoids probably are not utilized by the host, then the amino acids that have been converted in the rumen into these nucleic acids also are being lost to the host. The host, however, by removing amino acids and B vita mins- from the ruminal liquor before they can be absorbed or utilized by the ruminal microorganisms, conserves many of these nutrients. Further, because of this continual removal of the amino acids and B vitamins from the ruminal liquor, i t is probable that, under conditions of medium or low content of amino acids and B vitamins in the feed, the ruminal population of microorganisms would contain an increased proportion of those types of microorganisms that can synthesize their own amino acids and B vitamins. This, in turn, should lead to an increased liberation of these nutrients into the ruminal liquor from the mioroorganisms—either by excretion, or by autolysis of the cells. In summary, the ruminal absorption of amino acids and B vitamins not only conserves for the ruminant host many of those amino acids and B vitamins that otherwise would be inactivated or be passed out of the animal in the feces, but' also, under certain feeding conditions, probably promotes an increased microbial synthesis and, hence, an increased liberation of these nutrients into the ruminal liquor. The total effect of this ruminal absorption is • Not less than ten per cent of the total nitrogen in ruminal microorganisms occurs as nucleic acids ( 2 3 9 ) . 275. to help to assure for the ruminant host an optimal supply of the required amino acids and B -vitamins. Special Commentt The Application of the Concept of Ruminal Absorption of  Amino Acids to Explain Certain Results of Nitrogen Retention Trials in  Ruminants One of the ways in which the ruminant differs from the non-rumihant is that part of the ruminant1 s dietary protein and amino acids is exposed to the fermentative action of ruminal microorganisms. As a result of this fermentative action, a significant proportion of the dietary protein and amino acids may be utilized for the synthesis of microbial protein (103, 140, 143, 170, 188, 237, 241, 248, 260, 287, 352, 369). Among a large number of different types of microorganisms—including ruminal microorganisms—, the qualitative and quantitative amino aoid constitution of this microbial protein has proved to vary within relatively narrow limits (40, 58, 104, 121, 170, 184, 364). 1 0 5 This means that the conver sion of dietary protein and amino aoids to microbial protein, i f oonsider- 3 06 able, would tend to equate the digestibilities and biological values 105 The degree that changes in the environment of microorganisms can influence their amino acid constitution is something that needs further investigation (104, 121, 184, 318, 364), especially relative to an understanding of protein metabolism in the rumen. 106 The biological value of a protein is dependent on the qualitative and quantitative amino acid constitution of the protein. The closer the amino acid constitution of a protein is to an animal's balanced require ment of amino acids, the more efficiently is the protein utilized by the animal (see 4l). Holmes et al (170) have compared the amino acid composition of ruminal bacterial protein with that of egg protein—a protein that is regarded as possessing an almost ideal balance of essential amino aoids for producing optimal growth in the rat. By this comparison they found isoleucine and methionine each to be quite low in' concentration and, therefore, to be the "limiting" amino acids in the biological value of ruminal bacterial protein. It-is, perhaps, of interest of the dietary proteins and amino aoids, irrespective of their digestibilities and biological values when fed to the non-ruminant (185, 186, 250). Yet several workers (216, 411j see also 61 and 62 for general commentaries), feeding cattle and sheep different readily-soluble proteins at concentra tions CL2 per cent or less) in the diet that should have ensured that at least a major portion of each protein was hydrolyzed by the ruminal 107 microorganisms, found that the type of protein which was fed s t i l l 108 determined the nitrogen retention of the animal. At least a partial explanation for this variation in nitrogen retentions is to be found in the present experimental results which demonstrate the ability of the ruminal wall to absorb amino acids* The ruminal absorption of amino acids that are present in the feed, or that are formed during the micro- to note here that several workers (249, 251, 318; also see the reviews in 109, 250, 282) have attempted to determine the biological value of ruminal microorganisms for the rat* For example, values of 78-80 (318) and 81 or 88 (249, 251) have been found* 107 The situation would be different, of course, i f the protein that was fed were relatively insoluble or were fed in larger amounts than the ruminal population of microorganisms could handle (see 412)* In this case, some of the protein would pass into the abomasum and small intestine and, presumably, this part of the protein would have about the same biological value as when i t is fed to a non-ruminant* Consequently, when insoluble or very large amounts of protein are fed, absorption from the small intestine of dietary amino acids would be an additional factor to consider in explaining differences among animals in their nitrogen retention when they are fed different types of proteins (see also 143, 240, 241, 250, 282, 411). 108 The nitrogen retention of an animal is one index that is used to compare the digestibilities and biological values of proteins. Those proteins whose digestibilities and amino acid constitutions are such as to undergo maximal utilization yield maximal nitrogen retention—that is there is maximal absorption of the amino acids from the alimentary canal and minimal deamination of the amino acids in the liver, with a consequent minimization of the nitrogen excretion in the feces and urine, respectively. £[77, bial fermentation of the dietary protein, provides to the ruminant a different array of amino aoids than would he the case i f a l l the amino acids provided to the animal were consequent upon the digestion of micro bial protein that had passed into the abomasum and small intestine. This absorptive behaviour of the ruminal wall should favour increased or decreased nitrogen retention, depending on whether the dietary proteins and amino acids are of greater or lesser biological value than the microbial protein. It is not unexpected, therefore, that the type of protein fed to a ruminant produces variation in its nitrogen retention. This variation of nitrogen retention implies, in turn, that the digestibil ities and biological values of proteins are not necessarily equated when fed to a ruminant. The following quotation from Reid (319? see also 216) serves to summarize the previous discussions In view of the wide range in the biological value of nitrogen for ruminants, i t seems possible that, under certain conditions and particularly those in which certain nitrogen sources are fed, a portion of the nitrogen utilized may come directly from the dietary protein and the remainder may be provided as bact erial protein. Of course, this partition would depend upon the amount of required amino acids absorbed directly from the rumen or other parts of the digestive tract but, nevertheless, esoaping bacterial action. The relatively high biological value of the proteins of various feeds fed to ruminants may be partially explained on this basis. CONCLUDING COMMENT The stated objective of the present investigation was to determine whether ruminal absorption of amino acids and B vitamins does or does not occur* The results of the investigation appear to support the conclusion that ruminal absorption of amino acids and B vitamins does occur. In the General Discussion, an attempt has been made to determine the manner of this absorption. Although the attempt was only partly successful, enough information was gained to make the following statement of Phillipson (297) an especially appropriate comment at this point: The concept has slowly developed . . . that the epithelium lining the alimentary tract has a greater metabolic importance than is usually appreciated and that its metabolism besides contributing to the total metabolism of the animal regulates not only the absorption of molecules and ions but also prevents loss of molecules and ions from the body into the gut. Transport through the cell is presumably intimately related to the metabolism of the cell and a study of one is incomplete without a study of the other. The possible nutritional implications of the ruminal absorption of amino acids and B vitamins also v/ere discussed. It may well be that the present investigator has placed too much stress upon the possible importance of the ruminal absorption of amino acids and B vitamins. It is quite true that too often one or a few aspects of a problem become overstressed with a concomittant neglect of other aspects of the problem. It i s , however, the hope of the author that, overstressed or not, the present investigation will have raised the question of the quantitative nutritional significance of the ruminal absorption of amino acids and B vitamins and, therefore, will stimulate future research. APPENDIX Ruminal Fistula Operation The purpose of a ruminal f i s t u l a operation is to provide the experimentalist with easy access to the interior of the rumen and i t s contents. Stoddard et a l (363) and Phillipson and Innes (300) cite the early workers who f i r s t developed the technique of making a ruminal f i s t u l a . In recent times many groups of workers have modified the early technique (see, for example, 14, 272, 300, 312, 331, 336). The present author, i n adapting the operation to the goat, likewise has modified the general prooedure. The modified procedure, developed according to a 109 suggestion of Dr. P. A* Klavano that the operation be divided into two parts, i s described below. It since has been discouvered that Rankin (315), i n 1940, devised a similar modification for making ruminal fistulae i n sheep* The site of the operation i s located i n the l e f t paralumbar fossa (see F i g . 24). This area was clipped and shaved the day before the operation. On the day of the operation, the skin i n the area was well- lathered and washed. Two applications of tincture of iodine were made to the operative area. Although occasionally general anesthesia** 0 was used, more frequently the operation was performed under local anesthesia. A 2 to 4 per cent solution of procaine was i n f i l t r a t e d f i r s t oranially 109 •"•Associate Professor and Chairman of the Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Washington State University. **°General anesthesia was induced by intravenous administration of sodium pentobarbital and maintained by inhalation administration of ethyl ether. Fig. 24. - A drawing to show the position of the ruminal fistula A t ruminal fistula in the left paralumbar fossa; B, tuber coxae; C, transverse processes of lumbar vertebrae; D, thirteenth rib; E, rumen; F, abomasum; the inverted I (—•—• —.— designates the lines of infiltration that were used for producing local anesthesia of the left lumbar flank during the ruminal fistula operation* and then dorsally to the site of the incision (Fig. 24). The procaine solution was injected intradermally, subcutaneously, and intamuscularly in the different muscle layers. Suoh a procedure blocks the medial and lateral branches of the ventral divisions and the lateral branches of the dorsal divisions of the last thoracic and first two or three lumbar nerves. If carried out properly, this procedure provides complete desensitization of the skin, fascia, muscles, and parietal peritoneum in the operative area. To locate the position of the i n i t i a l Incision, the last rib and the ends of the transverse processes of the lumbar vertebrae were palpated. The in i t i a l incision was made 1 to 2 inches caudal to the last rib (Figs. 24 and 25). The dorsal limit or commissure of the incision was about 1 inch below a frontal plane through the ends of the transverse processes. From its dorsal limit, the incision was extended ventrad about 4 inches. This size of incision permitted the formation of a fistula that was large enough to allow the insertion of the experi mentalist1 s hand into the rumen. Fascia, abdominal muscles, and parietal peritoneum were incised to expose the dorsal sac of the rumen (Fig. 26). Pushing the fascia, abdominal muscles and parietal peritoneum aside, the wall of the dorsal sac of the rumen was sutured to the deep surface of the free margin of the skin around the incision. Care was taken in the suturing not to penetrate to the interior of the rumen. Using monofila ment nylon^"^" of size 0 or 1, continuous sutures were made along each side of the inoision. Interrupted sutures were placed at the dorsal and ventral commissures. This attachment of the ruminal wall was done in Ethicon, Ethicon Suture Laboratories, New Brunswick, New Jersey. F i g . 25 - The i n i t i a l skin incision such a manner as to expose an oval shaped portion of the ruminal wall (Fig. 27). At the finish of the suturing, the operative area was dusted lightly with an antiseptic powder. Seven to ten days later, the sutures were removed. This completed the first stage of the operation. Two or more days after the removal of the sutures, the second stage of the ruminal fistula operation was performed. An oval-shaped section of the ruminal wall was excised, the incision being made about l/4 inch inside the newly-formed junotion between the skin and the ruminal wall (Fig. 28). After the excision of the section of ruminal wall was completed, the edge of the new incision was dusted with a powder having antiseptic and astringent properties. A fistula plug (see description below) was then inserted to close the new opening into the rumen (Fig. 29). About l/2 hour was required to complete this second stage of the fistula operation. Once a ruminal fistula is formed, i t usually i s necessary to keep it closed by means of a fistula plug. Such a plug prevents leakage of ruminal contents and probably also helps to maintain anaerobic conditions in the rumen. Different types of plugs have been devised by different workers (25, 74, 187, 315, 331, 403; see also 312 and 363). Some of these plugs have been simple, others have been complex. For the fistu- lated goats that were used in the present experiments, a simple plug of soft neolite was designed. It consisted of circular inner and outer flanges, with an intermediate pad of sponge rubber between them. The three parts were held together by two ordinary screws and nuts. The intermediate pad was of such size and shape as to f i t snugly inside the fistula opening. Although the plug was not effective completely in preventing leakage of the ruminal ingesta, the plug was inexpensive, 2 8 5 F i g . 27. - The ruminal w a l l has been F i g . 28. - This photograph shows sutured to the deep surface of the free the animal several weeks l a t e r . The l i n e margin of the s k i n . of e x c i s i o n just has been made, p r i o r t o the removal of the exposed ruminal w a l l * F i g . 29. - A f t e r removal of the exposed ruminal w a l l , the f i s t u l a plug i s i n s e r t e d . Only the external flange of the plug i s v i s i b l e * easy to make, and simple to remove and reinsert* In addition, the soft texture of the sponge rubber center pad minimized irritation at the edge of the fistula* Carotid Loop Operation' The term "carotid loop operation" refers to the permanent exteriori zation of a section of the common carotid artery* Graham et al (137), using the goat, were the first to develop this type of exteriorization* McClymont (236) has adapted the operation to the cow and Schambye (332) has modified the operation for use in sheep and calves. The value of such an exteriorization is in the provision of a blood vessel from which frequent arterial blood samples oan be obtained with ease and with little.disturbance to the animal* Other approaches that have been proposed for obtaining arterial blood are sampling from the radial artery (36), the heart, the internal i l i a c artery by rectal puncture (136), the internal pudendal artery by vaginal puncture, and the middle coccygeal artery of the t a i l (329)• Except for the method of sampling the middle coccygeal artery, a l l these other methods have the disadvant age of either exciting the animal or allowing only infrequent sampling* Further, although applicable to the cow, not a l l the above methods are applicable to the goat beoause of its smaller size* The carotid loop operation was chosen as the method for obtaining arterial blood in the present investigation because of its applicability to the goat as a method that permits frequent sampling, with small or no disturbance of the animal* General anesthesia always was employed for the carotid loop opera tion* Either one of two methods of anesthesia was employed* One method was an adaptation of a procedure developed in England by Longley (217, 218) for general anesthesia of the horse. Longley rapidly administered sodium thiopental intravenously to induce anesthesia and utilized ether by intravenous drip to maintain anesthesia. Conner (75) has utilized a similar procedure to produce general anesthesia in the bovine. In the other method, the present author administered sodium pentobarbital to induce anesthesia and utilized ether by inhalation to maintain anesthesia. Providing that care was taken to maintain the goat in light anesthesia, both methods of anesthesia proved to be applicable to the goat. It is to be noted that care was taken to pillow the neck of the goat in such a manner that the head was tilted toward the floor. This position of the head permitted regurgitated ruminal ingesta or saliva to drain out of the mouth, thereby reducing the possibility of ingesta being aspirated into the trachea and lungs—an event that could cause suffocation of the animal immediately or foreign body pneumonia later. The site of the operation was located on either the left or right side of the neck, and about midway between the base and apex of the neck. The preparation of the operative area followed the same pattern that already has been described for the ruminal fistula operation (see p. 279). The in i t i a l incision, 3 inches in length, was made just lateral to the trachea. By disseoting deeply between the sternomandibularis muscle and the trachea, the oommon carotid artery was located (Fig. 30). The artery and its associated vagosympathetic nerve trunk were disseoted free from the fascia of the carotid sheath and brought to the surface (Fig. 31). The oommon carotid artery then carefully was disseoted free from the nerve trunk (Fig. 32), the dissection extending slightly beyond the oranial and caudal limits of the i n i t i a l skin incision. It sometimes Fig. 30 - A scalpel is inserted between the sternomandib- ularis muscle and the trachea to show the approach for finding the common carotid artery. Fig. 31 - The common carotid artery and the vagosympathetic nerve are shown before being dissected free from one another. They are held in view by a glass rod, with the point of a probe lying between the nerve above and the artery below. Fig. 32 - The common carotid artery has been dissected free from the other structures of the carotid sheath. Fig. 33 - A second skin incision has been made 1 1/2 inches lateral to the first skin incision to form the band of skin passing over the glass rod. 290 was necessary, depending upon the location of the i n i t i a l skin incision, to ligate and cut a small branch of the common oarotid artery. A second skin incision now was made parallel and about 1 l/2 inches lateral to the first skin incision (Fig. 33). The band of skin so formed was dis sected free from the muscles and fascia lying deep to i t . Next the oommon oarotid artery was placed in the band of skin (Fig. 34). The edges of the skin band were sutured together with either size 0 monofilament nylon or stainless steel. This procedure formed a tube of skin with the common carotid artery passing through the tube (Fig. 35). The two free edges of skin left on the neck now were sutured together (Figs. 35 and 36). Figure 37 demonstrates the relative position of the oompleted carotid loop on the neck of the goat. The operation was completed in 1 to 1 l/2 hours. Although for the f i r s t few animals extensive bandaging was appl ied to protect the newly-formed carotid loop, i t later was learned that this was unnecessary. The sutures were removed about 2 weeks after the operation was completed. Cannulation of Ruminal Veins Several workers have reported on the cannulation of blood vessels in man and animals. Ralston et al (314) have reviewed much of the litera ture on this technique and, in addition, have reported on the cannula tion of the external jugular veins in the dairy cow. In 19 experiments, they found the length of time that the cannulation tubes remained patent was 2.1 hours to 14.5 days. Gresson and Glen (83) reported on 24 portal vein oannulations in dogs. These workers concluded that when large veins are cannulated with polyethylene tubes of small caliber, blood samples may be obtained over an average period of approximately 3 weeks. F i g . 35 F i g . 37 Fig. 34* - The common carotid artery has been placed in the band of skin* The band was twisted only for the purpose of taking the photograph* Fig* 3 5 * - The edges of the skin band have been sutured to gether to form a skin tube containing the common carotid artery* The first suture has been made to join the free edges of the skin that are left on the neck after the formation of the skin tube* Fig. 36. - The suturing has been completed. A glass rod is inserted between the skin tube and the neck* Fig. 3 7 . - The photograph shows the position of the skin tube or "carotid loop" on the neck of the goat« 293. Annison et al (17) and Schambye (335) have reported on techniques for cannulating the portal vein in sheep. In the present cannulation experi ments of the ruminal veins, the longest time that the oannulas were maintained patent was 7 days. The procedure for the cannulation of the ruminal veins is outlined below. The site of the operation was located in the left paralumbar fossa. The preparation of the operative field was the same as for the ruminal fistula operation (see p. 279)• General anesthesia was employed with sodium pentobarbital as the induction anesthetic and inhalation ether as the maintenance anesthetic. The method of entering the peritoneal cavity was as for the ruminal fistula operations, except that the incision usually was placed midway between the last rib and a tra